Pathogenesis

of
Human
Pulmonary
Tuberculosis
Insights from the Rabbit Model
This page intentionally left blank
Pathogenesis
of
Human
Pulmonary
Tuberculosis
Insights from the Rabbit Model

Arthur M. Dannenberg, Jr., M.D., Ph.D.

Center for Tuberculosis Research

Departments of Environmental Health Sciences, Molecular Microbiology
and Immunology, and Epidemiology, Bloomberg School of Public Health
Department of Pathology, School of Medicine
Johns Hopkins University, Baltimore, Maryland 21205

Washington, D.C.
Max B. Lurie, M.D. (1893–1966)
Frontispiece: photo of Max B. Lurie courtesy of the late Peter Zappasodi, Henry Phipps Insti-
tute, School of Medicine, University of Pennsylvania, Philadelphia

Back cover: photo of Arthur M. Dannenberg, Jr., courtesy of Frederick W. Dubs, Photography
Laboratory of the Dept. of Pathology, Johns Hopkins Medical Institutions

Cover illustration by Robert Margulies © 1993 Hospital Practice

Copyright © 2006 ASM Press

American Society for Microbiology
1752 N Street, N.W.
Washington, DC 20036-2804

Library of Congress Cataloging-in-Publication Data

Dannenberg,Arthur M.
Pathogenesis of human pulmonary tuberculosis : insights from the rabbit model / Arthur
M. Dannenberg.
p. ; cm.
Includes bibliographical references and index.
ISBN-13: 978-1-55581-373-4
ISBN-10: 1-55581-373-9
1. Tuberculosis—Animal models 2. Tuberculosis—Pathophysiology I. American
Society for Microbiology. II.Title.
[DNLM: 1. Lurie, Max B. (Max Bernard), 1893-1966. 2. Tuberculosis, Pulmonary—
physiopathology. 3. Models,Animal. 4. Mycobacterium bovis—pathogenicity. 5.
Rabbits. 6. Tuberculosis, Pulmonary—microbiology WF 300 D188p 2006]

RC311.D362 2006
616.9
9507—dc22
2006021123

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 Dedication
To Aileen Hart Dannenberg, my treasured wife of 58 years (to date), whose love
and continuous support have made my professional career and this book possible
 CONTENTS

Preface xi
Introduction 1
Major contributions of Max B. Lurie and Arthur M. Dannenberg, Jr.

SECTION 1. PATHOGENESIS OF TUBERCULOSIS

1. Overview 7
Childhood and adult tuberculosis, bacillary virulence, host resistance, contagion,
and prevention
2. Stages in the Pathogenesis of Human and Rabbit
Tuberculosis 22
3. Types of Human Pulmonary Tuberculosis 34
4. Liquefaction of Caseous Foci and Cavity Formation 65

SECTION 2. IMMUNOLOGY OF TUBERCULOSIS

5. Delayed-Type Hypersensitivity, Cell-Mediated Immunity,
and Antibodies in Tuberculosis 97
Their local and systemic natures; innate immunity
6. Macrophages and Other Cells in Tuberculous Lesions 120
Including dendritic cells and lymphocytes

SECTION 3. TUBERCULOUS LESIONS

7. Structural Components of Tuberculous Lesions 155
8. Microvascular Density in Tuberculous Lesions 161
In developing and healing BCG lesions and in tuberculin reactions

vii
viii 䡵 CONTENTS

9. Early Pulmonary Lesions in Rabbits 170

Produced by an intravenous injection of tubercle bacilli
10. Macrophage Turnover, Division, and Activation in Tuberculous
Lesions 177
In developing, peak, and “healed” BCG lesions
11. Lurie’s Pulmonary Tubercle-Count Method 196
To assess bacillary virulence, genetic resistance of the host, and vaccine efficacy

SECTION 4. TUBERCULOSIS IN RABBITS AND OTHER

COMMON LABORATORY ANIMALS
12. Natural Airborne Infection 215
Resistance to the establishment of tuberculosis and to its progress
13. Response of Rabbits to Inhaled Tubercle Bacilli Including
BCG 230
Response to inhaled bovine- and human-type tubercle bacilli and to inhaled BCG
14. Characteristics of Resistance and Susceptibility to Tuberculosis
in Lurie’s Inbred Rabbits 235
15. Comparisons of Tuberculosis in Rabbits, Mice, and Guinea
Pigs 246

SECTION 5. EFFECTS OF HORMONES AND X-IRRADIATION

ON TUBERCULOSIS
16. Effects of Cortisone and Adrenocorticotropic Hormone
on Tuberculosis 273
17. Effects of Estrogen, Chorionic Gonadotropin, and Thyroid
Hormones on Tuberculosis 285
18. Effects of Whole-Body X-Irradiation on Tuberculosis 292

SECTION 6. CYTOKINES AND VASCULAR ADHESION

MOLECULES IN TUBERCULOUS LESIONS
19. Cytokine Production in Primary BCG Lesions 301
Nonspecific and antigen-specific cytokine production in developing and healing
primary BCG lesions
20. Cytokine Production in Reinfection BCG Lesions and
in Tuberculin Reactions 312
Effects of immunization on cell composition and cytokines
21. Vascular Adhesion Molecules in Tuberculous Lesions 327
ICAM-1,VCAM-1, and ELAM-1 in developing and healing rabbit
dermal BCG lesions
 CONTENTS 䡵 ix

SECTION 7. TUBERCULOSIS VACCINES

22. Principles and Guidelines for Developing Better Tuberculosis
Vaccines 341
23. Characteristics of Rabbit BCG Lesions and Efficacies of BCG
and Mycobacterium microti Vaccines 354

SECTION 8. PAST, PRESENT, AND FUTURE

24. Summary and Conclusions 367
25. Suggested Future Research and Unanswered Questions 373
Research on the host; research on the bacillus; vaccines and immunotherapy;
development of new drugs

APPENDIXES
A. Award of the Trudeau Medal for 1955 385
The Trudeau Medal of the National Tuberculosis Association for 1955 awarded to
Max B. Lurie
B. Obituary of Max B. Lurie, M.D. (1893–1966) 387
by Esmond R. Long, M.D., Ph.D., Sc.D.
C. Publications of Max B. Lurie 389
D. Publications of Arthur M. Dannenberg, Jr. 399
E. Guidelines for Preventing the Transmission of Mycobacterium
tuberculosis in Health-Care Settings, 2005
(Table of Contents) 417
Department of Health and Human Services, Centers for Disease Control and
Prevention,Atlanta, Georgia
F. Collected Abstracts of Chapters in This Volume 419
G. Acknowledgments 429

Glossary 431
Index 439
This page intentionally left blank
 PREFACE

This book is a review and update of my contributions to the experimental pathol-

ogy of tuberculosis, as well as those of my mentor Max B. Lurie (see Frontispiece).
The book describes 40 years of his scientific contributions, which were followed
by 40 years of mine. I was in his laboratory during the first 11 years that I worked
in this field.

BACKGROUND
Max B. Lurie, M.D.
Tuberculosis has afflicted many of the past workers in the field or their close fam-
ily members. Lurie probably caught tuberculosis from his mother, who died from
it. He became ill with the disease during his final year at Cornell Medical School,
and after graduating in 1921 he went to the National Jewish Hospital for Con-
sumptives in Denver, Colorado, for treatment.While there, he began his career in
tuberculosis research (with H. J. Corper as his mentor) and continued working on
the host-parasite relationships of this disease for the rest of his life.
When he had recovered from tuberculosis in 1926, he joined the research staff of
the University of Pennsylvania’s Henry Phipps Institute for the Cure and Preven-
tion of Tuberculosis, where he spent the rest of his career, attaining the rank of full
Professor (in Experimental Pathology). Lurie’s publications are listed in Appendix C.
Most of Lurie’s experiments are summarized in his book Resistance to Tuberculo-
sis: Experimental Studies in Native and Acquired Defensive Mechanisms (1). It almost seems
that his determination to complete that book kept him alive during the last few years
of his life. He died of an acute myocardial infarction in 1966, at the age of 73.

Arthur M. Dannenberg, Jr., M.D.

My mother’s first husband died of tuberculosis 6 months after they were married.
As a result, she became a social worker at the Henry Phipps Institute (where Lurie
worked). There she met my father, a practicing pediatrician, who devoted each

xi
xii 䡵 PREFACE

Tuesday afternoon to the care of tuberculous children in the Institute’s clinic.After

clinic, my father would go upstairs to Lurie’s laboratory, where he learned of his
latest experiments.
Lurie was an extremely enthusiastic person, and my father came home quite
inspired.Therefore, it was only natural that he would introduce me to Lurie when,
in 1948, I was deciding whether to pursue a clinical or a research career. I did not
realize at that time that Lurie was one of the world’s leading experimental pathol-
ogists of tuberculosis.
I was associated with Lurie for about 11 years—first as a postdoctoral fellow from
1948 to 1952, and then as an assistant professor from 1956 to 1964. During this time,
I had so many scientific discussions with him that his knowledge of tuberculosis has
became an integral part of my own thinking. In fact, I cite Lurie’s principles every
time I write or lecture.
Tuberculosis was (and is) one of the major diseases of humankind, especially in
developing countries. It kills about 2 million people in the world each year, more
than any other infectious disease (2). If I were going to pursue a research career
instead of a clinical one, I wanted to contribute to the elimination of a disease impor-
tant to mankind.
Fortunately, Lurie outlined what I should pursue in tuberculosis research at the
beginning of my career, specifically, the role of the macrophage, especially its
hydrolytic and metabolic enzymes. Macrophages are the host’s main defense against
the bacillus.The role of lymphocytes was just beginning to be recognized at that
time. More insight was needed from the disciplines of immunology and biochem-
istry into how these cell types function. In addition, the histopathology of the dis-
ease, as observed under the microscope, needed to be correlated with immunological
and biochemical cell functions.
Therefore, I learned the techniques of these (and other) basic sciences and have
used them in different stages of my career to understand the role of mononuclear
cells in tuberculosis lesions (see my publication list in Appendix D). Concurrently,
the whole discipline of histochemistry has evolved.This discipline enables scien-
tists to visualize structural and enzymatic cell characteristics in tissue sections.We
in my laboratory, therefore, used histochemistry to gain deeper insight into the patho-
genesis of tuberculous lesions and how the host controls them.

THE RABBIT MODEL OF TUBERCULOSIS

Lurie favored the rabbit model of tuberculosis because the disease in this labora-
tory animal most closely resembles that found in humans (1, 3–5). Caseous necro-
sis, liquefaction, and cavity formation with bronchial spread of the disease can be
readily produced in rabbits.Tuberculous lesions in mice, on the other hand, are gran-
ulomas that slowly progress with little or no caseation, and cavities never form. How-
ever, mice are an excellent model in which to study cell-mediated immunity in
tuberculosis. For such studies, many reagents are commercially available.
Tuberculosis in guinea pigs progresses rapidly with much caseation and hematoge-
nous and lymphatic spread of the disease. It resembles the susceptible form found
in infants and immunosuppressed persons, but not the slowly progressing fibrotic
cavity form found in immunocompetent humans and rabbits. Guinea pig lesions
occasionally cavitate, but significant bronchial spread is rare. Because of these
species differences, Lurie began rather early in his career to work with rabbits, after
working initially with guinea pigs. I have continued to use the rabbit model.
 PREFACE 䡵 xiii

DERMAL BCG LESIONS

Many of my own studies in the rabbit model were made on developing, peak, and
healing dermal BCG lesions. In rabbits, these lesions resemble pulmonary lesions
produced by human-type tubercle bacilli in that they develop and then regress. Der-
mal BCG lesions begin with numerous bacilli injected into one site. Therefore,
caseation, liquefaction, and ulceration readily occur. However, for the study of nat-
urally occurring tuberculosis, there is no substitute for aerosol infection, where a
single unit of 1 to 3 bacilli in rabbits produces lesions that closely resemble those
occurring in human populations.
For each stage of pulmonary lesion development and healing, a rabbit must be
sacrificed (euthanized). In contrast, multiple dermal BCG lesions of various ages can
be produced in a single rabbit, or multiple lesions started at the same time can be
biopsied at various times under local anesthesia. However, the results of such stud-
ies must be confirmed in rabbits with no previous biopsy and in rabbits with all
lesions of the same age. Nevertheless, the use of multiple lesions on the same rab-
bit in part compensates for the marked difference in cost of purchase and mainte-
nance between rabbits and mice.

PURPOSE AND USE OF THIS BOOK

The purpose of this book is to present in one place our current understanding of
the pathogenesis of tuberculosis derived from Lurie’s and my own research.The book
is written for both clinicians and laboratory researchers. Clinicians will read parts
of it to gain insight into the pathogenesis of tuberculosis as a guide for the care and
treatment of their patients. Laboratory researchers will read parts of it to plan some
of their experiments. For such researchers, I have sometimes provided more details
and references.
I have used Lurie’s original publications as references for many of the statements
made herein and used his book as a reference when it contributed additional infor-
mation. Statements that I heard personally from him are also referenced to his book.

ORGANIZATION OF THIS BOOK

This book was organized for several types of readers, as follows.
(i) Readers wanting a quick understanding of the pathology and immunology
of tuberculosis can read chapter 24,“Summary and Conclusions,” and if more detail
is desired, they can read the chapter abstracts, which are assembled as Appendix F.
(ii) Readers wanting information on the individual subjects can read the perti-
nent chapters.At the head of each chapter, I have provided both an abstract and a
list of the main headings within the chapter.These should enable the reader to more
rapidly find the information he or she is looking for.The book was written so that
each chapter is complete within itself, which results in occasional duplication of text,
figures, and references but is a necessity for the reader of only individual chapters.
Finally, (iii) some readers may want to read the entire book for the various ori-
entations presented.
Many chapters contain more experimental details than others. These chapters
describe types of tuberculosis research that are usually not performed in other lab-
oratories and are therefore unfamiliar to many readers. Full details on the technol-
ogy, however, only appear in the original publications cited.
The “see” before a reference number in this book signifies that the reference con-
tains additional information.This information is pertinent but does not necessarily
xiv 䡵 PREFACE

support the statement to which the reference is attached.Without the “see,” the ref-
erence is supportive.
Note. Lurie (1) used the words “native” and “acquired” resistance.Today, because
of Janeway’s extensive studies (6), the words “innate” and “adaptive” immunity have
replaced Lurie’s terminology. In this book, we mostly used Lurie’s terminology,
because it seemed more appropriate for tuberculosis in his inbred rabbits.

OTHER BOOKS ON TUBERCULOSIS

The classic books on tuberculosis are those of Rich (Johns Hopkins University; 1951)
on the immunopathology (7), Canetti (Pasteur Institute; 1955) on the human
pathology (8), Lurie (1964) on the experimental pathology (1), and Iseman (2000)
on clinical tuberculosis (9). Multiauthored texts by authorities in each respective field
are those edited by Bloom (1994) (10), Reichman (2000) (11), Schlossberg (2006)
(12), Cole et al. (1995) (13), and Rom and Garay (2004) (14).These multiauthored
texts provide more details on the various subjects reviewed herein, but very few stud-
ies in the rabbit model of tuberculosis are included.

REFERENCES
1. Lurie, M. B. 1964. Resistance to Tuberculosis: Experimental Studies in Native and Acquired
Defensive Mechanisms. Harvard University Press, Cambridge, Mass.
2. Dye, C., S. Scheele, P. Dolin,V. Pathania, and M. Raviglione. 1999. Global burden of
tuberculosis. JAMA 282:677–686.
3. Lurie, M. B., and A. M. Dannenberg, Jr. 1965. Macrophage function in infectious dis-
ease with inbred rabbits. Bacteriol. Rev. 29:466–476.
4. Dannenberg, A. M., Jr. 1994. Rabbit model of tuberculosis, p. 149–156. In B. R. Bloom
(ed.), Tuberculosis: Pathogenesis, Protection, and Control.ASM Press,Washington, D.C.
5. Dannenberg, A. M., Jr., and E. M. Collins. 2001. Progressive pulmonary tuberculosis is
not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due
to a continuous host response to mycobacterial products. Tuberculosis 81:229–242.
6. Janeway, C. A. 2002. A trip through my life with an immunological theme. Annu. Rev.
Immunol. 20:1–28.
7. Rich, A. R. 1951. The Pathogenesis of Tuberculosis, 2nd ed. Charles C Thomas, Springfield, Ill.
8. Canetti, G. 1955. The Tubercle Bacillus in the Pulmonary Lesion of Man. Springer Publishing
Company, Inc., New York, N.Y.
9. Iseman, M. D. 2000. A Clinician’s Guide to Tuberculosis. Lippincott Williams and Wilkins,
Philadelphia, Pa.
10. Bloom, B. R. (ed.). 1994. Tuberculosis: Pathogenesis, Protection, and Control. ASM Press,
Washington, D.C.
11. Reichman, L. B., and E. S. Hershfield. 2000. Tuberculosis: a Comprehensive International
Approach, 2nd ed. Dekker, New York, N.Y.
12. Manabe,Y. C., and A. M. Dannenberg, Jr. 2006. Pathophysiology: basic aspects, p. 18-51.
In D. Schlossberg (ed.), Tuberculosis and Nontuberculous Mycobacterial Infections, 5th ed.
McGraw-Hill, New York, N.Y.
13. Cole, S.T., K. D. Eisenach, D. N. McMurray, and W. R. Jacobs, Jr. (ed.). 2005. Tuber-
culosis and the Tubercle Bacillus.ASM Press,Washington, D.C.
14. Rom,W. N., and S. M. Garay. 2004. Tuberculosis, 2nd ed. Lippincott Williams & Wilkins,
Philadelphia, Pa.
 INTRODUCTION
Contributions of Max B. Lurie 1
Contributions of Arthur M. Dannenberg, Jr.
In the early 1900s, experimental tuberculosis was
produced in rabbits and guinea pigs.About the
middle of the 20th century, René Dubos at the
Rockefeller Institute (now University) intro-
duced the mouse model of this disease. Because
of the cost and size of mice and the availability
of specific reagents, the mouse model has been
used ever since by most of the scientists in the
field. However, Max B. Lurie (until his death in
1966) and I continued to use rabbits to study this
disease.
All stages of human pulmonary tuberculosis
(including liquefaction and cavity formation)
can be readily produced in rabbits. Guinea pigs
develop the susceptible childhood form of the
disease that only rarely forms cavities, and mouse
granulomas show little or no caseous necrosis
and never develop cavities.
The rabbit model of tuberculosis has been
occasionally used by other laboratories (1-12), but
the majority of the studies with this model have
been made in Lurie’s and my laboratories. Our
principal contributions are summarized below.
CONTRIBUTIONS OF MAX B. LURIE
Most of Lurie’s experiments are summarized in
his book Resistance to Tuberculosis: Experimental
Studies in Native and Acquired Defensive Mecha-
nisms (13). His most significant contributions
include the following.
1. Insights into the pathogenesis of
tuberculosis (chapter 2). (i) Intracellular
tubercle bacilli are nontoxic and initially grow
well within macrophages without injuring these
cells. (ii) A mature epithelioid cell is a
macrophage that has developed the power to kill
or inhibit the tubercle bacilli that it ingests. (iii)
Tubercle bacilli only kill macrophages (and the
1
2
surrounding tissues) after the host becomes sen-
sitive to their tuberculin-like products. (iv)
Caseous necrosis (and all of the tissue destruc-
tion found in tuberculosis) is due to host
delayed-type hypersensitivity to products of the
bacilli, especially tuberculin-like products. (v)
The extracellular growth of tubercle bacilli in
pulmonary cavities can overwhelm good native
and acquired host resistance by the bronchial
spread of large numbers of bacilli. Although
caseation, liquefaction, and cavity formation
have been well recognized ever since tubercu-
losis was described as a disease, the roles of these
processes in its pathogenesis were significantly
clarified by Lurie’s studies in the rabbit model.
2. Development of inbred rabbit strains
that produced the childhood and adult
forms of this disease (chapter 13). Lurie’s
susceptible rabbits developed the childhood
form of tuberculosis with hematogenous spread
of the disease and did not produce cavities. His
resistant rabbits developed the adult form with
cavities and bronchial spread of the disease.
3. Identification of the characteristics
of native (genetic) and acquired (adap-
tive) resistance that affect the pathogenesis
of tuberculosis (chapter 14). When com-
pared to his inbred susceptible rabbits, Lurie’s re-
sistant rabbits showed (i) greater maturation of
epithelioid cells (now known as “activated
macrophages”) that are capable of destroying
or inhibiting tubercle bacilli, (ii) greater destruc-
tion of bacilli in the primary and secondary
lesions, (iii) greater interstitial inflammation
(containing macrophages and lymphocytes), (iv)
increased lymphatic drainage from pulmonary
lesions to the hilar lymph nodes (resulting from
the increased interstitial inflammation), and
2 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
(v) liquefaction with cavity formation and
bronchial spread.
4. Demonstration of similarities in the
host reaction to virulent bovine-type
bacilli, virulent human-type bacilli, and
BCG (chapter 13). Although the host
responds to all tubercle bacilli in a similar fash-
ion, its ability to arrest the disease varies inversely
with the virulence of the bacilli. Both live and
dead virulent bovine-type tubercle bacilli are
harder for rabbits to destroy and eliminate than
are virulent human-type bacilli.
5. Demonstration of the efficacy of UV
light in preventing tuberculosis.
6. The pulmonary tubercle-count
method (chapter 11). Lurie developed this
method to measure bacillary virulence, host re-
sistance to tuberculosis, and the efficacy of BCG
immunization.
7. Studies on the effects of hormones on
the pathogenesis of tuberculosis. Lurie
demonstrated that estrogens and chorionic
gonadotropin in did not appreciably affect host
resistance to tuberculosis (chapter 17). How-
ever, glucocorticoids and adrenocorticotropic
hormone (ACTH) in pharmacological doses
decreased the host resistance, and ACTH in
physiological doses sometimes increased such
resistance (chapter 16).Thyroid hormones some-
times increased host resistance, but thyroidectomy
(or propylthiouracil administration) decreased it
(chapter 17).
8. Correlation of bacillary growth in
the host with the histopathology of tuber-
culous lesions (chapter 2). Lurie identified
(i) an initial bacillary inhibition, presumably by
pulmonary alveolar macrophages; (ii) a loga-
rithmic (symbiotic) growth phase in mac-
rophages entering the lesions from the blood;
(iii) a stationary growth phase after the host
becomes tuberculin-positive and during which
caseous necrosis is present in rabbits, in guinea
pigs, and undoubtedly in humans; (iv) extra-
cellular bacillary growth associated with liq-
uefaction and cavity formation; and (v) a longer
symbiotic bacillary growth phase reaching
higher titers (before the stationary phase)
whenever the inhaled tubercle bacilli were
more virulent.
Other contributions. Lurie’s other con-
tributions are described in the list of his publi-
cations presented in Appendix C.
CONTRIBUTIONS OF ARTHUR M.
DANNENBERG, JR.
The major contributions of my laboratory group
to experimental tuberculosis include the fol-
lowing.
1. Deeper insight into macrophage acti-
vation and heterogeneity (chapter 6).
2. Studies on the local nature of ac-
quired resistance to tuberculosis (chapter
5). An expanded, recirculating, antigen-specific
lymphocyte population is systemic. However,
these lymphocytes produce delayed-type hyper-
sensitivity (DTH) and acquired cellular resistance
reactions only at sites where tubercle bacilli
and/or their antigens are located. The local
nature of tuberculous lesions explains the many
variations found in the clinical disease.
3. Clarification of the interplay of
delayed-type hypersensitivity (DTH) and
cell-mediated immunity (CMI) (chapter
5). DTH kills macrophages containing many
tubercle bacilli, because these bacilli produce
high local concentrations of tuberculin-like
products. Tubercle bacilli do not grow in the
resulting solid caseous tissue. CMI activates local
macrophages because of cytokines from antigen-
specific lymphocytes, so that the macrophages
can inhibit the intracellular growth of ingested
tubercle bacilli. Both DTH and CMI are needed
to arrest tuberculosis. In the absence of either
DTH or CMI, the host cannot stop the progress
of the disease.These concepts explain all aspects
of Lurie’s bacillary growth curves in the host
(outlined in item 8 in the section above).They
also explain why DTH developed throughout
evolution: DTH is necessary to stop the intra-
cellular multiplication of tubercle bacilli when-
ever CMI (i.e., acquired cellular resistance) can-
not stop it.
4. DTH is a major factor in controlling
the growth of tubercle bacilli (chapter 5).
Mice develop poor DTH and their tissues con-
tain large numbers of tubercle bacilli (chapter
15). Guinea pigs develop poor DTH and their
tissues contain smaller numbers of these bacilli.

Yet mice are considered the more resistant
species, because in mice much less tissue destruc-
tion occurs than in guinea pigs.The DTH:CMI
ratio might be altered for the benefit of the
host by an appropriate vaccine (chapter 22).
5.The beneficial role of antibodies (chap-
ter 5). Hosts that have been immunized to tuber-
culosis usually have circulating antibodies as well
as an expanded population of antigen-specific
Th1 lymphocytes.After virulent tubercle bacilli
are inhaled by such hosts, a local antigen-antibody
reaction immediately produces the C5a compo-
nent of complement and probably other chemo-
taxins.This results in a rapid local accumulation
of macrophages and antigen-specific T lympho-
cytes at sites of bacillary lodgement. Therefore,
even though circulating antibodies apparently
have little or no direct effect on tubercle bacilli,
they increase the rate at which existing DTH and
CMI control local bacillary growth. In this man-
ner, antibodies play an important role in host
defense against endogenous and exogenous rein-
fection with tubercle bacilli.
6. Bacillary latency and dormancy
(chapter 10). Macrophages in tuberculous
lesions have a high turnover rate. Therefore,
nonactivated macrophages from the bloodstream
continually infiltrate tuberculous lesions. Even
in seemingly arrested tuberculous lesions, some
of the nonactivated macrophages probably ingest
occasional tubercle bacilli that escape from the
caseous center. If so, such bacilli will start to mul-
tiply intracellularly until they are again inhibited
by DTH and CMI as described above. Com-
plete bacillary dormancy within arrested tuber-
culous lesions may be rare and may occur only
extracellularly in solid caseous necrotic tissues.
7. Cytokine dynamics (chapters 19 and
20). Immunized hosts rapidly produce cyto-
kines, especially chemokines, which in turn
cause a rapid accumulation of macrophages and
lymphocytes at sites of bacillary lodgement.
After a sufficient number of these defense cells
have accumulated locally, the production of
chemokines is rapidly downregulated. What
determines the amount of local cell infiltration
is worthy of further investigation. Such infiltra-
tion was most pronounced in reinfected rabbits,
as well as in Lurie’s resistant rabbits.
INTRODUCTION 䡵 3
8. Causes of liquefaction and cavity for-
mation (chapter 4). Liquefaction of the
lesion’s solid caseous center perpetuates tuber-
culosis in humans because it enables extracellu-
lar growth of tubercle bacilli and cavity forma-
tion. We showed that proteases, DNases, and
RNases (and probably other hydrolases) are
involved. Yamamura and coworkers (6, 7, 9)
showed that DTH to the tuberculin-like prod-
ucts of tubercle bacilli is involved. Much more
research is needed to understand the cause of
liquefaction and cavity formation and how to
inhibit them (see chapter 25). (The rabbit is
the only common laboratory animal in which
the human form of chronic fibrosing tubercu-
lous cavities can be readily produced.)
REFERENCES
1. Ratcliffe, H. L., and W. F.Wells. 1948.Tubercu-
losis of rabbits induced by droplet nuclei infec-
tion. I. Initial response to infection. J. Exp. Med.
87:575–584.
2. Ratcliffe, H. L., and W. F.Wells. 1948.Tubercu-
losis of rabbits induced by droplet nuclei infec-
tion. II. Response to reinfection. J. Exp. Med.
87:585–594.
3. Weiss, C., and M. L. Boyar-Manstein. 1951. On
the mechanism of liquefaction of tubercles. I.The
behavior of endocellular proteinases in tubercles
developing in the lungs of rabbits. Am. Rev.Tuberc.
63:694–705.
4. Weiss, C., and F. M. Singer. 1953. Mechanism of
softening of tubercles. II. Behavior of desoxyri-
bonuclease in tubercles developing in the lungs of
rabbits. Arch. Pathol. 55:516–530.
5. Weiss, C., J. Tabachnick, and H. P. Cohen.
1954. Mechanism of softening of tubercles. III.
Hydrolysis of protein and nucleic acid during
anaerobic autolysis of normal and tuberculous lung
tissue in vitro. Arch. Pathol. 57:179–193.
6. Yamamura, Y., S. Yasaka, M. Yamaguchi, K.
Endo, H. Iwakura, S. Nakamura, and Y.
Ogawa. 1954. Studies on the experimental tuber-
culous cavity. Med. J. Osaka Univ. 5:187–197.
7. Yamamura,Y. 1958.The pathogenesis of tuber-
culous cavities. Adv.Tuberc. Res. 9:13–37.
8. Kruml, J., L.Trnka, R. Urbancík, and J. Kuska.
1965. Histologic differences between experimen-
tal cavernous tuberculosis in rabbits and human
cavitary disease. Am. Rev. Respir. Dis. 92:299–302.
9. Yamamura, Y., Y. Ogawa, H. Yamagata, and
Y. Yamamura. 1968. Prevention of tuberculous
cavity formation by immunosuppressive drugs.
Am. Rev. Respir. Dis. 98:720–723.
4 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
10. Yamamura,Y.,Y. Ogawa, H.Yamagata, and Y.
Yamamura. 1974. Prevention of tuberculous cav-
ity formation by desensitization with tuberculin-
active peptide. Am. Rev. Respir. Dis. 109:594–601.
11. Kitano, M., S. Kume, and T. Kambara. 1982.
Evaluation of delayed hypersensitivity skin reactions
in rabbits immunized with live BCG. Bull. Ginkyo
Coll. Med.Technol. 6:1–10.
12. Kambara,T.,T. Hiraoka, and I. Kukita. 1985.
Neutral proteases in lymph node cells of rabbits
immunized with heat-killed tubercle bacilli.
Kumamoto Med. J. 38:153–163.
13. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive
Mechanisms. Harvard University Press, Cambridge,
Mass.
 Section 1.

PATHOGENESIS OF TUBERCULOSIS

5
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 1
OVERVIEW

Types of human disease 9

Contagiousness: contracting tuberculosis by the respiratory route 11
Size of infectious particles 12
Virulence of bacillary strain 13
Factors influencing the establishment of a pulmonary tuberculous lesion
(recognized by the conversion of the tuberculin skin test) 15
Factors controlling resistance to tuberculosis 16
Prevention of clinical disease 17

Abstract. Tuberculosis is still one of the major diseases of the world, especially in devel-
oping countries. It kills over 2 million people each year, more than any other infectious
disease.
Childhood-type pulmonary tuberculosis is a disease of susceptible hosts, such as infants
and immunosuppressed individuals. From the primary parenchymal lesion, the bacilli fre-
quently spread via lymphatics and cause caseous lesions of the hilar lymph nodes.The bacilli
may also spread via the bloodstream and cause lesions elsewhere in the host.The primary
lesion, as well as the metastatic lesions, often progresses until the host succumbs.
Adult-type pulmonary tuberculosis is a disease of innately resistant hosts, a category that
includes most immunocompetent persons.The active parenchymal lesion (often subapi-
cal) frequently forms a cavity, in which the bacilli may multiply extracellularly. If so, the
bacilli may spread via the bronchial tree to other parts of the lung. In such a resistant host,
many metastatic microscopic lesions do not progress, but those that do may form new cav-
ities.Also, caseous bronchopneumonia may occur when an appreciable amount of lique-
fied caseum enters the bronchial tree. Cavity formation perpetuates tuberculosis in
humankind because coughing spreads bacilli from the lungs into the environment, where
they may infect other people.
Contracting clinical tuberculosis depends on (i) the size and physiological state of the
bacillary particle, (ii) its virulence, and (iii) the native and acquired resistance of the host.
How to protect personnel against tuberculosis is also discussed in this chapter. UV lights
(shielded to protect people’s eyes) or HEPA-filtered air purifiers should be used more fre-
quently in hospital areas where tubercle bacilli are likely to be present.

Tuberculosis remains one of the major infectious
diseases in the world.Tuberculin testing indicates
that about one-third of the world’s population
is infected with Mycobacterium tuberculosis (about
1.9 billion people), mostly in developing coun-
tries (1).The disease kills more people than any
other infectious disease, and in recent years the
combined infection of human immunodefi-
ciency virus (HIV) and M. tuberculosis has accel-
erated the death rate. Ninety percent of those
infected with virulent M. tuberculosis have an
inapparent disease. About 5% develop clinical
disease during the 2 years after infection,
and about 5% develop it later due to debili-
tation in old age, immunosuppression, and other
causes of decreased host resistance. About
30% of HIV-infected patients die of tuber-
culosis. In fact, HIV-infected patients have a
yearly risk of developing clinical tuberculosis
equivalent to the lifetime risk of most non-
HIV-infected patients. This chapter presents
an overview of the pathogenesis of the
human disease. More details are presented in
chapter 3.

7
8 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 Rapidly progressing miliary tuberculosis in an 11-month-old infant.The

rabbit counterpart of this type of tuberculosis is shown in Fig. 3. Most of the multiple
caseous tubercles are of hematogenous origin.The primary lesion is marked by an arrow.
Note the large size of the homolateral caseous hilar lymph nodes (which seeded the
blood entering the lungs; see text). From the collection of the late professors A. R. Rich
and W. G. MacCallum, Department of Pathology, Johns Hopkins School of Medicine,
Baltimore, Md.
 1. OVERVIEW 䡵 9

TYPES OF HUMAN DISEASE and cell-mediated immunity (CMI) several

Humans inhaling occasional virulent tubercle
bacilli over a period of months develop only one
primary pulmonary tubercle for the following
reasons. (i) Relatively few bacilli are in the air
that people breathe, even in households with
active cases of tuberculosis. Therefore, often
weeks may pass before another inhaled bacillus
begins to multiply in the lung. (ii) Pulmonary
alveolar macrophages are a highly activated cell
population, which in humans probably destroy
most inhaled tubercle bacilli before they mul-
tiply (see chapters 2 and 3). (iii) The host devel-
ops both delayed-type hypersensitivity (DTH)
weeks after a primary microscopic tubercle is
established. DTH and CMI keep other devel-
oping tubercles from reaching a grossly visible
size (see chapters 5 and 11).
Childhood-Type Tuberculosis
In childhood-type tuberculosis, the bacilli in
the primary tubercle drain via lymphatics to
the hilar nodes, where they produce caseous
lesions (Fig. 1, 2, and 3). Bacilli from both the
primary lung lesion and the hilar nodes then
enter the bloodstream and may cause miliary
lesions throughout the body.

FIGURE 2 Miliary tuberculosis of the lungs in a 19-year-old young adult.The tubercles appear
smaller than those in Fig. 1 merely because the lungs of this individual are so much larger than
those of the infant. Caseous hilar lymph nodes were the source of most of these miliary tuber-
cles. In this patient, caseous plaques (not shown) in branches of the pulmonary veins seeded tuber-
cle bacilli into the general circulation and caused miliary tubercles in many other organs. From
the collection of the late professors A. R. Rich and W. G. MacCallum, Department of Pathology,
Johns Hopkins School of Medicine, Baltimore, Md.
 FIGURE 3 Organs of Lurie’s inbred susceptible rabbit F4-33, which died of a gen-
eralized progressive tuberculosis of 3.3 months’ duration.This rabbit had a large, sin-
gle, completely caseous nonliquefied primary lesion in the middle of the left lung,
massive enlargement and caseation of the homolateral hilar lymph nodes, and
hematogenous spread of the disease to the lungs, kidney, pleura, and knee joint.The
rabbit was naturally infected in a room containing airborne tubercle bacilli from rab-
bits with tuberculous kidneys (see chapter 12). Reproduced with permission from
reference 3.
Note that the disease in this susceptible inbred rabbit was similar to the disease
in humans shown in Fig. 1 and 2.The tubercles shown in these three figures could
be almost the same size, because the photographs of the human specimens are so much
smaller than the actual specimens.

10

The secondary lesions in the lung usually
originate from bacilli in the hilar lymph nodes,
because the efferent lymph from these nodes
enters the venous blood. Bacilli in the venous
blood enter the right side of the heart and then
the lungs via the pulmonary arteries.
The secondary lesions in the rest of the body
usually originate from bacilli in primary (and
secondary) lung lesions. Such bacilli enter the
pulmonary veins and then the left side of the
heart to be distributed to sites throughout the
body via the aorta.These bacilli only go back to
the lungs if they pass through peripheral capil-
lary beds and enter the venous blood.
In summary, in childhood-type tuberculosis
most of the secondary tubercles found in the
lungs usually come from caseous lesions in the
hilar nodes, and most of the secondary tubercles
in the other organs usually come from caseous
lesions in the lungs themselves.
Infants and immunosuppressed individuals,
including those with HIV/AIDS infection, often
develop hematogenously spread childhood-type
disease.The Ghon complex is the typical form
of this type of tuberculosis. It is characterized by
a caseous lesion in the lung parenchyma (usu-
ally not in an apical location) with enlarged
caseous hilar lymph nodes.
Adult-Type Tuberculosis
Immunocompetent adults often develop adult-
type tuberculosis, whether or not they had the
disease in childhood or even converted their
tuberculin skin test from an inapparent infection.
Adults who develop this type of tuberculosis as
a primary infection have rather good native re-
sistance and can develop rather strong acquired
resistance after infection.
Adult-type tuberculosis is characterized by
minimal involvement of the hilar lymph nodes
and few, if any, progressive secondary lesions of
hematogenous origin.The disease usually begins
as a single subapical lesion that often cavitates.
From the cavity, the bacilli spread into the air-
ways and then into other parts of the lungs and
to the outside environment.
In this type of tuberculosis, tubercle bacilli
reach the hilar nodes, but because of the high
1. OVERVIEW 䡵 11
native and acquired resistance of the host, bacil-
lary growth is usually inhibited.The hilar nodes
remain small with relatively little caseation, and
few bacilli enter the efferent lymph draining into
the great veins to return to the lungs. If they do
so, they usually do not cause progressive sec-
ondary pulmonary lesions, because of the strong
immunity developed by the host during the
primary infection.
In such immunocompetent hosts, the disease
may progress if the solid caseous center lique-
fies and forms a cavity (Fig. 4, 5, and 6). In the
liquefied caseum, the bacilli may grow extra-
cellularly (for the first time during the course of
this disease) and (after the cavity forms) may
reach such large numbers (Fig. 7) that even the
high native and acquired resistance of the host
is overwhelmed.
Bacilli from the cavities are often
coughed into the environment where they
may infect other people.Therefore, lique-
faction with cavity formation is a major
reason why tuberculosis is perpetuated in
humankind (2).
Lurie developed inbred rabbit families that
mimicked these two types of human disease
(3–5). His susceptible rabbits developed the child-
hood type of rapidly progressing disease with
hematogenous spread (Fig. 3). His resistant rab-
bits developed the adult type of slowly progress-
ing disease with cavitary formation and bronchial
spread (Fig. 6).The principles established exper-
imentally with these inbred rabbits have direct
application to the human disease, which is why
I have continued to use the rabbit model of
tuberculosis throughout my research career.
Chapter 3 describes and illustrates the types of
human tuberculosis in more detail.
CONTAGIOUSNESS: CONTRACTING
TUBERCULOSIS BY THE
RESPIRATORY ROUTE
Several factors influence the infectiousness of
patients with tuberculosis: (i) the frequency
and force of coughing and whether the patient
coughs into the environment or into cupped
hands, (ii) the number of viable bacilli in the
patient’s sputum, (iii) the consistency of the
12 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 Bilateral tuberculous cavities in the upper lobes of a 39-year-old diabetic woman.
Below each cavity are areas of caseous lung tissue.Although infected, the hilar lymph nodes are
not markedly enlarged. In the lung on the right, an applicator stick marks the communication
between the cavity and the bronchus.The rabbit counterpart of this type of tuberculosis is shown
in Fig. 6. From the collection of the late professors A. R. Rich and W. G. MacCallum, Depart-
ment of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

sputum (it is more easily aerosolized when it is
more fluid), (iv) the amount of albumin-like
protein present (which aids bacillary survival in
air), (v) the size of the aerosolized particles
(see below), and (vi) the virulence of the bacilli
(see below). When patients are receiving
antimicrobials, these drugs get concentrated
as the fluids in the aerosolized droplets evap-
orate. Drug-resistant bacilli survive such
increased concentrations better than do drug-
susceptible bacilli. Moisture in the air protects
tubercle bacilli from drying out, as do their
wax-like coats. Finally, UV light, including that
from direct sunlight, is lethal to tubercle bacilli
(6–11). See reference 12 for a more complete
discussion of factors influencing the transmis-
sion of tuberculosis.
SIZE OF INFECTIOUS PARTICLES
When inhaled, only fine particles containing 1
to 3 tubercle bacilli are capable of initiating the
infection, because they remain suspended in the
airstream that enters the alveolar spaces (4; see
further discussion in references 13 and 14).The
heavier bacillary particles (containing more than
3 bacilli, often with bits of caseous material)
impinge upon the mucosal surfaces of both the
nasopharynx and the bronchial tree.These bacil-
lary particles are moved up the bronchial tree by
cilia and are eventually swallowed.The mucosal
 1. OVERVIEW 䡵 13

FIGURE 5 An apical cavity of moderate size in an adult patient. Below the cavity is an area of
caseous consolidation of pneumonic origin. Several caseous foci are also present in the other lung,
probably also pneumonic in origin.The hilar lymph nodes (not shown) contained a few caseous
foci but were only slightly enlarged. From the collection of the late professors A. R. Rich and
W. G. MacCallum, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

surfaces of the respiratory and gastrointestinal
tracts are rather resistant to tuberculosis and
develop lesions only when exposed to large
numbers of tubercle bacilli.
VIRULENCE OF BACILLARY STRAIN
The virulence of the bacillus causing tuberculo-
sis depends on the host’s ability to prevent and
control the disease that the bacillus produces (see
reference 14). Bacillary virulence and the innate
and acquired (adaptive) immunity of the host
affect each other in opposite directions. This
book is about the host’s response to the bacillus,
but I discuss below some of the attributes of vir-
ulence because of their importance to the devel-
opment of new antimicrobials. Such attributes
have recently been discussed in detail (15–19).For
a review of the older literature, see reference 20.
In brief, tubercle bacilli may vary in virulence,
both genetically and phenotypically. Genetically,
Mycobacterium bovis BCG (an attenuated bovine-
type bacillus),virulent human-type tubercle bacilli
(e.g., H37Rv), and virulent bovine-type tuber-
cle bacilli (e.g., Ravenel) are increasingly patho-
genic for rabbits (4, 5). Phenotypically, a viable
bacillus that has dried out or been exposed to
direct sunlight would often be too weak to start
an infection. However, a viable bacillus kept moist
in a dark place would be quite infectious if
aerosolized by air currents.
The pathogenicity of a specific strain of tuber-
cle bacillus is due to both its genotype and the
response of the host.The most common measures
of virulence are (i) the number of bacilli in a par-
ticular organ, (ii) the amount of pathology pro-
duced, and (iii) the time of death of the host.
14 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Organs of Lurie’s

inbred resistant rabbit A2-6, which
died of tuberculosis 9 months after its
tuberculin skin test converted.A sin-
gle primary encapsulated pulmonary
cavity was present in the right lung,
from which tubercle bacilli spread
(via the bronchial tree) to the larynx
and the intestines. No grossly visible
tuberculous lesions were found in
the hilar lymph nodes or in the kid-
neys. Nonprogressive secondary
tubercles were present in both lungs
(probably bronchogenic in origin).
Like the susceptible rabbit depicted
in Fig. 3, this resistant rabbit was nat-
urally infected in a room containing
airborne tubercle bacilli from rab-
bits with tuberculous kidneys (see
chapter 12). Reproduced with per-
mission from reference 3.
Note that the disease in this
inbred resistant rabbit was similar to
the cavitary disease in humans shown
in Fig. 4 and 5.

The virulence of H37Rv can sometimes be
reduced by repeated subculturing (2), which
demonstrates that its genotype is not completely
stable. The virulence of clinical isolates of M.
tuberculosis has also been found to be variable:
several strains isolated from humans with active
tuberculosis in India, Southeast Asia, and Hong
Kong were reduced in virulence for guinea pigs
(21–23).
The entire genome of M.tuberculosis (which has
now been sequenced [24]) has changed relatively
little over the years: it has had a low rate of syn-
onymous and nonsynonymous base substitutions
(25, 26). However, some deletions and insertions
have caused changes in the virulence of various
members of the M. tuberculosis complex (19, 27).
For example, despite the 99% genomic identity
with virulent mycobacterial strains, the absence
of certain genes in BCG and Mycobacterium microti
caused them to be attenuated for humans (19, 28,
29).The attenuation of BCG was produced by
a serial passage of a virulent M. bovis in media
containing ox bile (30, 31; M.A. Behr and P. M.
Small, Letter, Nature 389:133–134, 1997).
 1. OVERVIEW 䡵 15

FIGURE 7 Tubercle bacilli growing profusely in the liquefied caseum in a pulmonary cav-
ity of a rabbit that inhaled about 340 virulent bovine-type tubercle bacilli 33 weeks previously.
Such profuse growth occurs only in some lesions with liquefied centers, presumably where the
composition of the liquefied caseum is most favorable or the adaptation of the bacillus to extra-
cellular growth is most complete. Similar bacillary growth has been found in many human cav-
itary lesions (20). Bacilli were stained with carbol-fuchsin and counterstained with methylene
blue. Magnification, ⫻600. Photograph reproduced with permission from reference 59.

The virulence of various mycobacterial strains
for rabbits and mice may differ, because these
strains probably contain or produce slightly dif-
ferent antigens (32–34). Virulent human-type
tubercle bacilli (e.g., H37Rv) are virulent in
mice and humans but are partly attenuated in
rabbits. Recently, the HN878 strain of human-
type tubercle bacilli (which caused a miniepi-
demic in Houston, Tex.) was found to have
increased virulence for both rabbits and mice
(33).Among virulent human-type strains of M.
tuberculosis, the Erdman strain is more virulent
than the H37Rv strain, which is in turn more
virulent than the CDC1551 strain (34; Y. C.
Manabe et al., submitted for publication).
Several attributes of virulence have recently
been extensively investigated: (i) the entry of
tubercle bacilli into monocytes/macrophages (35,
36), (ii) their survival in these cells (37–39) (see
chapter 6), and (iii) their survival in a variety of
other adverse environments (including solid
caseum), which is facilitated by sigma factors (16,
40) and the synthesis of lipids (18).There are 13
sigma factors among the 190 transcriptional reg-
ulators that have been described in the M. tuber-
culosis genome (40). These factors are carefully
controlled by anti-sigma factors, as well as anti-
anti-sigma factors (40). See chapters 6 and 25 for
additional comments on virulence and chapters
6 and 10 for additional comments on dormancy.
FACTORS INFLUENCING THE
ESTABLISHMENT OF A PULMONARY
TUBERCULOUS LESION
(RECOGNIZED BY THE CONVERSION
OF THE TUBERCULIN SKIN TEST)
Number of Tubercle Bacilli Inhaled
The inhaled population of virulent tubercle
bacilli is not uniform. It contains bacilli that
are too weak to start any infection, as well as
16 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

robust bacilli that are always ready to do so. If

many bacilli are inhaled, a robust bacillus is
likely to be among them.

Microbicidal Power of the Pulmonary

Alveolar Macrophages
Alveolar macrophages vary in their capacity to
destroy the bacillus (4, 5). Some alveolar
macrophages are rich in enzymes (41) and
microbicidins, while others are poor in both.The
ratio of “rich” to “poor” alveolar macrophages
seems to be determined by the native genetic re-
FIGURE 8 An example of an effective disposable
sistance of each individual as well as by pheno- mask.This mask is made of HEPA-filtering material with
typic factors. an exhaust valve in the center. It is backed by a ring of
foam rubber that fits tightly against one’s face (but not
Establishment of the Infection quite as tightly as the less flexible mask shown in Fig.
9). Similar masks are available from Lab Safety Supply,
In humans and rabbits,pulmonary infection (with 401 S.Wright Road, Janesville,Wis. 53546.Tel: 1-800-
virulent human-type tubercle bacilli) begins only 356-0783. http://www.labsafety.com.
after a strongly endowed bacillus is ingested by a
weakly endowed alveolar macrophage. No one
knows exactly how many fine particles contain- On the host side, infants are rather suscepti-
ing human-type tubercle bacilli must be inhaled ble to tuberculosis, apparently because their
by humans before such a combination is reached. immune system is still developing. Susceptibil-
The mean number is probably between 5 and ity is also greater during adolescence in some
200, whereas the mean number for commercial human populations, but not in others. Persons
New Zealand White rabbits is between 500 and debilitated by old age and other conditions are
1,000 (see chapter 11). also more susceptible.
One report (42) indicated that the alveolar Both native and acquired resistance to tuber-
macrophages of people of African descent (i.e., culosis is lowered by immunosuppressive drugs,
blacks) were less effective in destroying inhaled glucocorticoids, and immunosuppressive viruses
tubercle bacilli than the alveolar macrophages of (e.g., HIV); silicosis, uremia, diabetes, alcoholism,
people of European descent (i.e., whites). Blacks
converted to tuberculin positivity more frequently
than whites after similar exposures to tubercle
bacilli in a nursing home.Among the converters,
however, the incidence of clinical disease was
the same (42), indicating that both groups pro-
duced a satisfactory immune response. See chap-
ter 12 for a more complete discussion of the
establishment and progress of tuberculosis.

FACTORS CONTROLLING
RESISTANCE TO TUBERCULOSIS
Some strains of tubercle bacilli may be inher-
ently more virulent than others. Such genetic
(and/or phenotypic) differences in bacillary FIGURE 9 3M 7200S Half-Facepiece Respirator
with 7255 Easi-Air High Efficiency Filter (approved by
virulence would affect the course and type of the National Institute of Occupational Safety and Health
tuberculosis that these strains produce (see ref- [NIOSH]). Similar masks are available from Lab Safety
erence 43). Supply, Janesville,Wis. (See legend to Fig. 8.)
 1. OVERVIEW 䡵 17

and certain neoplastic diseases; gastrectomy; par-
asitic infection (e.g., with hookworm); starvation
and other nutritional deficiencies; and physical
misery, e.g., in the concentration camps of World
War II (44) (see chapters 3 and 16). Two or
more of these conditions are often synergistic.
The anti-inflammatory action of glucocorti-
coids may mask the clinical symptoms of reac-
tivated tuberculosis.
However, most adult human beings are rather
resistant to tuberculosis, some more so than
others (4, 45). In addition, children in develop-
ing countries often develop some acquired
immunity from the environmental tubercle
bacilli that they ingest.
By killing (or inhibiting) tubercle bacilli,
effective antimicrobial agents, such as isoniazid,
rifampin, pyrazinamide, ethambutol, and strep-
tomycin (46), greatly aid the macrophages of the
host in (i) arresting the progress of the disease,
(ii) preventing its complications, (iii) healing
small foci of infection, and (iv) promoting
fibrotic encapsulation of larger foci of infec-
tion. However, even after effective chemother-
apy, residual tuberculous foci may reactivate if the
resistance of the person becomes depressed.
Antimicrobial therapy is discussed further in
chapter 25.
Immunotherapy with killed Mycobacterium
vaccae in combination with chemotherapy shows
promise (47). (M. vaccae is a nonvirulent acid-fast
bacillus isolated from the soil.) Immunotherapy
may also prove useful in the treatment of
multidrug-resistant tuberculosis.
PREVENTION OF CLINICAL DISEASE
People contract pulmonary tuberculosis mainly
by inhaling small airborne particles of 1 to 3
bacilli. Such particles are most often produced
when a patient coughs or sneezes. Rooms where
tuberculous animals are housed may contain
infectious particles if the animals cough, sneeze,
or shed the bacilli in their feces or urine.Vacuum
cleaners and air conditioners do not filter small

FIGURE 10 3M Air-Mate High Efficiency Particle (HEPA) Powered Air Purifying Respira-
tor (PAPR)—HEPA 10 Head Cover and HEPA 12 Hood—with the HEPA PAPR assembly
shown in Fig. 11 (NIOSH approved).Available from Lab Safety Supply, Janesville,Wis. (See leg-
end to Fig. 8.) Photograph is reproduced with permission from the 3M Occupational Health and
Environmental Safety Division.
18 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 11 Components of the 3M Air-Mate HEPA 10 Head Cover and Air-Mate HEPA
12 Hood with the HEPA PAPR assembly shown in Fig. 10 (NIOSH approved).Available from
Lab Safety Supply, Janesville,Wis. (See legend to Fig. 8.) Photograph is reproduced with permission
from the 3M Occupational Health and Environmental Safety Division.

infectious particles of 1 to 3 tubercle bacilli out Necropsies on tuberculous patients or animals

of the air.To our knowledge, only a few exist-
ing vacuum cleaners and none of the common
air conditioners contain HEPA filters in their
exhaust systems. Machines without HEPA filters
only filter out large noninfectious airborne par-
ticles of tubercle bacilli and may actually dis-
seminate smaller infectious particles. Large bacil-
lary particles are not infectious, because during
respiration they do not reach the pulmonary
alveoli where tuberculous lesions begin, but
impinge on the mucosal surfaces of the airways
(which are quite resistant to infection by tuber-
cle bacilli).The efficacy of air ionizers in remov-
ing infectious particles remains to be tested.
should, if possible, be performed under laminar
flow hoods, and electric bone cutters (and other
devices that readily aerosolize bacilli) should
not be used in open spaces. UV lights shielded
to irradiate only the upper air of rooms (away
from the eyes of personnel) are the most prac-
tical and inexpensive way to reduce the num-
ber of viable bacilli in local areas (8–12, 48). UV
lights can be used over doorways if they are
shielded to produce a narrow beam of light
directed only to the floor and to the ceiling.
Taking the following measures would reduce
the incidence of new cases of clinical tubercu-
losis (49–55; J. A. Schaefer, unpublished data).

(i) Tuberculous patients should cough and
sneeze into tissues or cupped hands. (ii) Attend-
ing personnel should wear tight-fitting effective
masks (Fig. 8 and 9) or HEPA-filtered forced-
air head covers or hoods (Fig. 10 and 11)
(Schaefer, unpublished). Putting a mask on the
patient may be of some help but is not as effec-
tive as putting one on the attending personnel,
because the patient’s coughing forces fine par-
ticles of bacilli through the interstices of the
mask. (iii) If they are likely to be exposed to
tubercle bacilli, tuberculin-negative persons
should be vaccinated with an effective BCG
strain, or if developed in the future, a more
effective vaccine. Finally, (iv) the airborne bacilli
in patients’ rooms should be reduced by an
exhaust fan, by a portable HEPA-filtered air
purifier (56), or by UV lights (6, 8–12, 48)
aimed at the ceiling and shielded to protect
the eyes of personnel.
Both portable HEPA-filtered air purifiers
and shielded UV lights are effective in remov-
ing viable tubercle bacilli from air in a room.
HEPA-filtered air purifiers are more expensive,
but the filters usually do not need to be
changed for years, depending on the accumu-
lation of dust in them. Shielded UV lights are
less expensive, but every few months (when
cool), they need to be washed with water con-
taining a detergent or wiped with alcohol to
remove oily films that accumulate on their
surface and block the transmission of the ger-
micidal UV wavelengths (57, 58). Nardell rec-
ommends replacing the UV lamp yearly (E.A.
Nardell, unpublished data).
These prophylactic measures are especially
important for persons who may be exposed to
antimicrobial-resistant tubercle bacilli. Cough-
inducing procedures (such as endotracheal suc-
tioning and bronchoscopy) aerosolize numerous
tubercle bacilli.The advantages and disadvantages
of the various types of protective respirators are
reviewed by Schaefer (unpublished).
Complete guidelines for preventing the trans-
mission of tuberculosis in health-care settings
were recently published by the Centers for Dis-
ease Control and Prevention, Atlanta, Ga. (55).
They cover every aspect of tuberculosis control,
including early screening of patients with pos-
sible tuberculosis, masks and respirators, diagnosis
1. OVERVIEW 䡵 19
and treatment, room-air purification (UV irra-
diation and HEPA filtration), and disinfection of
the patient’s room and utensils. The table of
contents of this extensive prevention guide (55)
is reproduced in appendix E.
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1. OVERVIEW 䡵 21
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 2
STAGES IN THE PATHOGENESIS OF
HUMAN AND RABBIT TUBERCULOSIS

First stage of tuberculosis: ingestion, and often destruction, of inhaled bacilli

by alveolar macrophages 22
Second stage of tuberculosis: logarithmic “symbiotic” growth of the bacilli
in nonactivated monocytes/macrophages 24
Third stage of tuberculosis: ending of the logarithmic stage of bacillary
multiplication by DTH (and CMI) 25
Fourth stage of tuberculosis: progression or arrest of caseous lesions 28
Fifth stage of tuberculosis: cavity formation and bronchial spread of the
disease 30
Summary 31

Abstract. After the inhalation of tubercle bacilli by rabbits and humans, the disease may
progress through the following stages. In humans, the disease begins with the establishment
of only a single primary pulmonary tubercle.
Stage 1: Ingestion and often destruction of bacilli by pulmonary alveolar macrophages.
Stage 2: Logarithmic growth of bacilli within nonactivated macrophages that entered
the developing tubercle from the bloodstream.
Stage 3:Arrest of the logarithmic bacillary growth by delayed-type hypersensitivity, which
kills the bacilli-laden macrophages and often forms a solid caseous center in the tubercle.
Stage 4a: In hosts with weakly developed cell-mediated immunity, enlargement of the
tubercle and its caseous center with hematogenous dissemination of the bacilli.
Stage 4b: In hosts with strongly developed cell-mediated immunity, stabilization or regres-
sion of the tubercle.
Stage 5: Liquefaction of the caseous center, extracellular bacillary growth, cavity for-
mation, and bronchial dissemination of the bacilli.
These stages are not distinct but blend into each other.Also, stages 3, 4, and 5 may occur
in the same lung and even in different parts of the same lesion, depending on the local con-
centration of bacilli and their tuberculin-like products. Native and acquired resistance is
never absolute, because a large number of tubercle bacilli (which have grown extracellu-
larly in a cavity) can overwhelm even the best-developed host resistance and cause sec-
ondary pulmonary lesions.

FIRST STAGE OF TUBERCULOSIS:

INGESTION, AND OFTEN
DESTRUCTION, OF INHALED BACILLI
BY ALVEOLAR MACROPHAGES
Stage 1 (Fig. 1) begins when a bacillary particle
containing 1 to 3 bacilli is inhaled into an alve-
olus. (Larger particles impinge on the bronchial
mucosa and never reach the alveoli.) (See ref-
erences 1 through 4.) Once there, an alveolar
macrophage will ingest the bacilli and often
destroy them, depending on the inherent micro-
bicidal power of the alveolar macrophage and on
the genetic and phenotypic virulence of the
ingested bacillus. Most alveolar macrophages
are highly activated cells (5). They have been
activated nonspecifically by many stimulating
factors, including the ingestion and digestion of
a variety of inhaled microorganisms and organic
matter (see reference 6), as well as an occasional
extravasated erythrocyte.Therefore, in humans
and in rabbits inhaling human-type tubercle
bacilli, the alveolar macrophages may destroy the
bacilli before they multiply. In such cases, the
tuberculin skin test remains negative.
Seven days after the inhalation of human
tubercle bacilli, the lungs of Lurie’s inbred sus-
ceptible rabbits contained 20- to 30-fold more

22
 2. STAGES OF HUMAN AND RABBIT TUBERCULOSIS 䡵 23

viable bacilli than did the lungs of his inbred re-

FIGURE 1 Stage 1. An alveolar macrophage that
has ingested and killed the two tubercle bacilli in a
phagocytic vacuole.The cytoplasm of this macrophage
is darkly shaded to depict a high degree of activation,
i.e., high levels of lysosomal and oxidative enzymes.
Most alveolar macrophages are nonspecifically activated
by the variety of inhaled particles that they ingest. In
humans, an alveolar macrophage is usually able to kill an
inhaled tubercle bacillus, except when the bacillus is
unusually virulent or the macrophage is poorly activated.
Reproduced with permission from reference 4.
sistant rabbits (7, 8) (Fig. 2).Therefore, the alve-
olar macrophages of the resistant host must have
destroyed or inhibited more inhaled bacilli than
did those of the susceptible host. In other words,
the alveolar macrophages of the resistant rabbits
were evidently nonspecifically activated to a
greater extent than were those of the suscepti-
ble rabbits.That the alveolar macrophages from
the resistant rabbits were able to phagocytize
particles more readily than those from the sus-
ceptible rabbits supports this conclusion (9).
In vitro metabolic studies on mineral oil-
induced peritoneal macrophages confirmed that
macrophages from Lurie’s resistant rabbits could
be nonspecifically activated to a greater extent
(by the oil irritant) than those from his suscep-
tible rabbits (10).

FIGURE 2 Changes in the number of virulent human-type tubercle bacilli

(H37Rv) in the lungs of Lurie’s natively resistant and natively susceptible rabbits
at different intervals after an aerosol infection. By 7 days, the resistant rabbits had
inhibited the growth of the bacilli 20 to 30 times more effectively than did the sus-
ceptible rabbits, but, from then on, the two curves were parallel. At 4 to 5 weeks,
the susceptible rabbits had about 13 times more primary pulmonary tubercles in
their lungs than did the resistant rabbits.The means and their standard errors are
shown. Reproduced with permission from reference 8.
The number of tubercle bacilli in the lungs of the resistant rabbits failed to
decrease during the period illustrated, because liquefaction with extracellular mul-
tiplication of the bacillus readily occurred in these rabbits (7, 8). Liquefaction did
not occur in the susceptible rabbits (7, 8), because their macrophages probably devel-
oped only low levels of hydrolytic enzymes.
Inhaled virulent bovine-type tubercle bacilli (Ravenel S) grew to greater titers
than did H37Rv, but produced similar curves (28) (shown in chapter 15).
24 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
SECOND STAGE OF TUBERCULOSIS:
LOGARITHMIC “SYMBIOTIC”
GROWTH OF THE BACILLI IN
NONACTIVATED
MONOCYTES/MACROPHAGES
Stage 2 (Fig. 3) is called symbiotic (7) because
the number of bacilli in the lesions increases
without apparent damage to the host, and the
number of macrophages also increases without
apparent damage to the bacilli. (See references
1 through 4.) If the original alveolar macrophage
fails to destroy or inhibit the inhaled bacillary
particle that it ingested, the bacilli multiply
intracellularly until the macrophage ruptures.
The released bacilli are then ingested by other
alveolar macrophages and by monocytes/macro-
phages arriving from the bloodstream. Both
types of macrophages are attracted to the site by
chemotaxins released from the bacilli (such as N-
formyl-methionyl-leucyl-phenylalanine [11]),
by chemotaxins from live alveolar macrophages
(such as leukotriene B4 [11] and monocyte
chemoattractant protein 1 [12]), by chemotax-
ins from dying alveolar macrophages (11) (such
as uric acid [13]), and by chemotaxins from
FIGURE 3 Stage 2. An early primary pulmonary
tubercle, in which tubercle bacilli have multiplied loga-
rithmically within macrophages that have immigrated
into the lesion from the bloodstream.These newly arrived
macrophages are nonactivated and incompetent. Their
cytoplasm is unshaded to depict the lack of activation.
In fact, the phagocytic vacuoles in the cytoplasm of
these nonactivated macrophages seem to provide an
ideal environment for mycobacterial multiplication.
Stage 2 is called the stage of symbiosis, because the
bacilli are multiplying, the macrophages are accumulat-
ing, and neither the host nor the parasite is injured by the
other. Reproduced with permission from reference 4.
serum (such as C5a released when complement
is activated [11]) (see chapters 19 and 20).
In time, macrophages from the bloodstream
become completely responsible for the fate of
the early lesion. In such a lesion, the alveolar
macrophages rarely participate, because they
remain peripheral, rather far from the bacilli,
which are almost always located more centrally
(Fig. 4).
The macrophages from the bloodstream are
nonactivated and immature.They readily ingest
the bacilli released from alveolar macrophages.
A symbiotic relationship then develops, in which
the macrophages of the host and the bacilli can-
not injure each other. These nonactivated
macrophages cannot inhibit or destroy the
bacilli, and the bacilli cannot injure the
macrophages (Fig. 3 and 5) because the host is
not yet tuberculin positive. With time, many
macrophages and many bacilli accumulate in
this early lesion, which now has become a
microscopic tubercle.
In this symbiotic stage (between 7 and 21 days
after infection), the bacilli grow logarithmically
at the same rate in both resistant and susceptible
rabbits (Fig. 2) (8). Evidently, intracellular bacilli
inhibit the microbicidal mechanisms (14–16) of
these immature macrophages equally well in both
the resistant and the susceptible hosts (see chap-
ters 6 and 15).
In rabbits, the more virulent bovine-type
bacilli (Ravenel S) and the less virulent human-
type bacilli (H37Rv) also grow at the same rate
in nonactivated macrophages. However, the
more virulent bovine-type bacilli grow for a
longer period of time (see chapter 15). Evi-
dently, acquired (adaptive) immunity develops
more slowly with tubercle bacilli of greater vir-
ulence. Perhaps some of the antigens that cause
acquired immunity are released more slowly
from the more virulent strain, because the
bovine type seems to remain intact longer.
Even though little or no difference existed in
the ability of Lurie’s resistant and susceptible rab-
bits to control the growth of tubercle bacilli in
nonactivated macrophages (Fig. 2), a marked dif-
ference existed in the ability of resistant and sus-
ceptible rabbits to activate macrophages. The
 2. STAGES OF HUMAN AND RABBIT TUBERCULOSIS
resistant rabbits apparently activated their pul-
monary alveolar macrophages nonspecifically to
a greater degree than did the susceptible rabbits
before inhalation of tubercle bacilli (see above),
and the resistant rabbits immunologically activated
to a greater degree the blood-borne macrophages
that entered their tuberculous lesions than did the
susceptible rabbits (see below).
䡵 25
FIGURE 4 An early pulmonary
tuberculous lesion with a caseous
center surrounded by blood-borne
macrophages. The alveolar macro-
phages (recognized by their dark
staining for the lysosomal enzyme -
galactosidase) have accumulated
peripherally in nearby alveoli. More
details on this lesion are given in
Fig. 5 of chapter 9. Magnification,
⫻330. Reproduced with permission
from reference 4.
THIRD STAGE OF TUBERCULOSIS:
ENDING OF THE LOGARITHMIC
STAGE OF BACILLARY
MULTIPLICATION BY DTH
(AND CMI)
Stage 3 (Fig. 6) is the early stage of caseous
necrosis. It occurs in rabbits 2 to 3 weeks after
the inhalation of tubercle bacilli, which is the
time when the antigen-specific immune
FIGURE 5 A 2-week pulmonary
lesion produced in a rabbit by the
inhalation of virulent human-type
tubercle bacilli (H37Rv) (8). The
lesion consists of blood-borne non-
activated macrophages in which
large numbers of bacilli had multi-
plied intracellularly. Note that, in
this stage of symbiosis (i.e., before
DTH develops), even numerous
intracellular bacilli do not injure the
macrophages. In other words, the
bacillus is rather innocuous before
DTH develops, and it is the host’s
own DTH reaction that kills tissues
during this disease. Stained with car-
bol-fuchsin, counterstained with
methylene blue. Magnification,
⫻730. Reproduced with permission
from reference 8.
26 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
FIGURE 6 Stage 3. A tubercle 3 weeks of age with
a caseous necrotic center and a peripheral accumulation
of partly activated macrophages (lightly shaded) and
lymphocytes (small dark cells). The first stages of
caseation occur when the tissue-damaging DTH
response to a high concentration of tuberculin-like
products kills the nonactivated macrophages that have
allowed the bacilli to multiply logarithmically within
them.The dead and dying macrophages are depicted by
fragmented cell membranes. Intact and fragmented
bacilli are present, both within macrophages and within
the caseum. Tubercle bacilli do not multiply in solid
caseum. Reproduced with permission from reference 4.
response becomes established. (See references 1
through 4.) The development of tissue-damag-
ing delayed-type hypersensitivity (DTH) that
kills macrophages harboring more than a few
tubercle bacilli eliminates the favorable intra-
cellular environment for bacillary growth (Fig.
6). Blood vessels are damaged, and solid caseous
necrosis is produced within which the bacillus
cannot grow. In other words, tissue-damaging
DTH to high local concentrations of tuber-
culin-like bacillary products is used by both the
resistant and susceptible rabbits to end the log-
arithmic stage of bacillary growth.
Unexpectedly, Lurie’s susceptible hosts inhib-
ited further increases in the number of viable
bacilli just as efficiently as did the resistant hosts
(Fig. 2). Cell-mediated immunity (CMI) could
not be responsible, because the susceptible hosts
developed weaker CMI. The marked inhibi-
tion of bacillary growth found in both strains of
rabbits must therefore be due to the tissue-
damaging DTH.
The concept that DTH kills the nonacti-
vated macrophages in which many tubercle
bacilli are growing, thereby eliminating the
favorable intracellular environment, was advo-
cated by several research groups (17–23) and had
been predicted many years ago (24, 25). In fact,
Robert Koch described it before DTH and
CMI were in our vocabulary (26). Tubercle
bacilli may survive in this solid caseous material,
but they cannot multiply, probably owing to
the anoxic conditions, reduced pH, and the
presence of inhibitory fatty acids (25, 27).
But why did the susceptible rabbits, which are
known to develop rather weak DTH and CMI
responses (7), stop the logarithmic growth of the
bacillus just as effectively as the resistant strain
of rabbits? The answer is that the large number
of bacilli in the susceptible rabbits produced
high levels of tuberculin-like products that
caused a strong local tissue-damaging DTH
response: their tuberculous lesions had larger
caseous centers than those of the resistant rab-
bits (8, 28).The large number of bacilli in the
susceptible rabbits also enhanced their dermal
tuberculin reactivity.
The greater antigenic stimulus in the sus-
ceptible rabbits did not, however, increase their
CMI to the levels found in the resistant rabbits.
The lesions of the susceptible rabbits contained
many more bacilli but relatively few mature
epithelioid cells. In other words, their macro-
phages were not activated to the degree found
in the resistant rabbits. Perhaps the lympho-
cytes of the susceptible rabbits did not produce
the cytokines required to do so. Perhaps their
macrophages were unable to respond appropri-
ately to such cytokines. Or, perhaps, both
occurred, or other factors were involved.
In brief, in stage 3 (the early stage of caseous
necrosis), both the resistant and susceptible rab-
bits locally destroyed the macrophages that had
allowed the bacillus to grow during the sym-
biotic stage. Only after such control was estab-
lished could CMI (producing highly activated
macrophages surrounding the caseous focus)
prevent the progression of the disease. Since sus-
ceptible rabbits do not develop good CMI, the
susceptible rabbits continued (in stage 4) to use
tissue-damaging DTH to inhibit bacillary
 2. STAGES OF HUMAN AND RABBIT TUBERCULOSIS 䡵 27

growth, but in doing so they destroyed increas-
ing amounts of their own tissues.
Ending the logarithmic (symbiotic) stage of
bacillary growth may be due to several factors, but
killing of bacilli-laden macrophages by tissue-
damaging DTH is the major factor in humans,
rabbits, and guinea pigs. CMI develops simulta-
neously with DTH and stops the growth of
tubercle bacilli within macrophages when they
contain relatively few microorganisms. There-
fore, CMI acts synergistically with DTH to con-
trol bacillary growth.
Tuberculin-positive skin tests develop sev-
eral days after both CMI and DTH are expressed
in the tuberculous lesions where the bacillary
antigens are located. Antigen-specific lympho-
cytes expand and accumulate in the draining
lymph nodes and lesions before such lympho-
cytes are available to enter dermal tuberculin
reactions (see chapter 5).
The role of nonspecific factors in ending the
logarithmic stage is minimal (discussed further
in the last section of chapter 5). The bacilli
release few, if any, endotoxin-like factors while
they are growing intracellularly in nonactivated
macrophages. If they did, the macrophages
would be activated during the first 24 h (see ref-
erence 29). Before DTH and CMI develop,

FIGURE 7 (A) Stage 4a. An established tubercle 4 to 5 weeks of age similar to those found in
Lurie’s susceptible rabbits. It has an enlarging caseous center.The bacilli escaping from the edge
of this center are ingested by poorly activated incompetent macrophages. In such macrophages,
the bacilli again find a favorable intracellular environment in which to multiply.They do so until
the tissue-damaging DTH (to high concentrations of tuberculin-like products) again kills these
new bacilli-laden macrophages and enlarges the caseous necrotic center.This sequence may be
repeated multiple times. Lung tissue is destroyed, and the bacilli are spread by the lymphatic and
hematogenous routes to other sites. Metastatic lesions develop in which the tissue destruction
continues. This pattern of tuberculosis is seen in immunosuppressed individuals, such as HIV/
AIDS patients. In this cartoon, several partly activated macrophages are lightly shaded to indicate
that such susceptible hosts develop only relatively weak CMI. Reproduced with permission from
reference 4. (B) Stage 4b.An established tubercle 4 or 5 weeks of age similar to those found in Lurie’s
resistant rabbits.The caseous center remains small, because the bacilli escaping from its edge are
ingested by the highly activated (competent) macrophages (darkly shaded) that surround the caseum.
In these activated macrophages, the bacilli cannot multiply and are eventually destroyed. Such effec-
tive macrophages were activated by T cells and their cytokines. If the caseous center remains solid
and does not liquefy, the disease will be arrested by this CMI response.This scenario occurs in healthy
immunocompetent humans who show positive tuberculin reactions and yet no clinical and often
no X-ray evidence of the disease. Reproduced with permission from reference 4.
28 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
intracellular tubercle bacilli inhibit macrophage
activation and their microbicidal abilities (see
chapters 1 and 6). After DTH and CMI develop,
the bacilli are killed and many of their compo-
nents are released. Only then can many of these
components activate macrophages nonspecifi-
cally and act synergistically with DTH and CMI
to control the progress of the disease.
FOURTH STAGE OF TUBERCULOSIS:
PROGRESSION OR ARREST OF
CASEOUS LESIONS
Stage 4 (Fig. 7) consists of an interplay of CMI
and tissue-damaging DTH. During this stage,
the disease may become clinically apparent, at
least by radiograph, or it may be arrested with
little visible evidence remaining except for con-
version of the tuberculin reaction.
If CMI is weak (Fig. 7A), as in Lurie’s sus-
ceptible rabbits, virulent tubercle bacilli escap-
ing from the edge of the caseous centers again
multiply intracellularly in poorly activated
macrophages (Fig. 8). Again, these infected
macrophages are killed by tissue-damaging
DTH, and the caseous center enlarges. Lym-
FIGURE 8 Photograph of stage
4a: a caseous tubercle in the lungs of
one of Lurie’s susceptible rabbits 5
weeks after the inhalation of human-
type tubercle bacilli. Bacilli escaping
from the caseous center (lower left)
are ingested by the surrounding
poorly activated macrophages, i.e.,
incompetent immature epithelioid
cells (upper right), where they again
find a favorable intracellular envi-
ronment in which to grow (Fig. 7A).
Magnification, ⫻550. Reproduced
with permission from reference 39.
phogenous and hematogenous spread of the
bacilli occurs, and progressive secondary lesions
develop in the hilar nodes, lungs, and other
organs (see chapter 13).
If CMI is strong (Fig. 7B), as in Lurie’s resis-
tant rabbits, virulent tubercle bacilli escaping
from the caseous center are ingested and
destroyed (or inhibited) by highly activated
macrophages that have accumulated perifocally.
The lesions then are stabilized and even regress.
The bacilli that reach the hilar lymph nodes of
these rabbits are inhibited by the same process.
In rabbits, virulent human-type tubercle bacilli
are much less virulent than the bovine type. (In
humans, both types are of similar virulence.) The
disease produced by virulent human-type bacilli
usually heals in a year or so in both susceptible and
resistant inbred rabbits, but the disease produced
by virulent bovine-type bacilli progresses to death
in both rabbit strains (see chapter 13). Figure 2
shows the bacillary titers in the lungs of Lurie’s
rabbits that had inhaled human-type bacilli. Fig-
ure 1 in chapter 15 shows the bacillary titers for
rabbits inhaling the bovine type.
 2. STAGES OF HUMAN AND RABBIT TUBERCULOSIS 䡵 29

FIGURE 9 Stage 5.A recently formed cavity discharging liquefied caseous ma-
terial into a bronchus. In liquefied caseum, the bacilli may multiply extracellularly,
reaching large numbers. High concentrations of tuberculin-like products are pro-
duced and local tissues are destroyed, including the wall of an adjacent bronchus (illus-
trated here).The liquefied caseous material is then discharged into the airways, and
the bacilli disseminate to other parts of the lung and to the environment. Repro-
duced with permission from reference 4.
The large quantities of bacilli and their antigens in liquefied caseum may over-
whelm a formerly effective CMI, causing progression of the disease in Lurie’s re-
sistant rabbits, as well as in immunocompetent humans.Also, among such large num-
bers of bacilli, mutations causing antimicrobial resistance may occur. Lurie’s
susceptible rabbits do not liquefy caseum or form cavities.

FIGURE 10 Wall of a cavity from one

of Lurie’s genetically resistant rabbits 8
weeks after the inhalation of human-
type bacilli (H37Rv). The liquefied
caseous tissue (right) and liquefying
caseous tissue (left) contain a large num-
ber of (rod-shaped) acid-fast bacilli. Such
bacilli were formerly inhibited in solid
caseous tissue, but they grew profusely in
the liquefied caseum in the wall of this
cavity. Stained with carbol-fuchsin, coun-
terstained with methylene blue. Magni-
fication, ⫻540. Reproduced with per-
mission from reference 8.
30 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Five stages of tuberculosis

Stage 1
Pulmonary alveolar macrophages ingest the inhaled bacilli. In rabbits (and humans), most of the resident alveolar
macrophages are highly activated (5) and destroy or inhibit the bacilli before they multiply appreciably (Fig. 1).
However, if a poorly activated alveolar macrophage ingests an inhaled bacillus, it can multiply intracellularly
and eventually destroy the alveolar macrophage.
Stage 2
The bacilli are released from poorly activated alveolar macrophages and are ingested by immature (nonactivated)
monocytes/macrophages that enter the tubercle from the bloodstream.Within these immature macrophages,
the bacilli grow logarithmically (Fig. 3).This stage is called symbiotic (4, 7) because both tubercle bacilli and
macrophages accumulate in the developing lesion without harming each other.
Stage 3
Caseous necrosis ends the symbiotic stage of bacillary growth in humans, rabbits, and guinea pigs (Fig. 6).
Tubercle bacilli cannot multiply within solid caseum. Caseous necrosis is caused by a tissue-damaging delayed-
type hypersensitivity (DTH) reaction to the high local concentrations of tuberculin-like antigens that occur
whenever the bacilli are numerous within (poorly activated) macrophages (2, 3).Therefore, DTH and the
resulting caseous necrosis play major roles in stopping the progression of tuberculous lesions.
At this stage, the lesion contains a solid caseous center. Surrounding this center are nonactivated macrophages,
which permit intracellular bacillary multiplication, as well as some activated macrophages, which do not.The
activated macrophages are produced by the cell-mediated immunity (CMI) that develops along with the DTH
(see chapter 5).
Stage 4
This stage determines whether the early tubercle becomes clinically apparent. Here, CMI (causing local acquired
cellular resistance) plays a major role (see chapters 5 and 11). If only weak CMI develops (Fig. 7A), the bacilli
escaping from the edge of the caseous necrosis multiply again in nonactivated and partly activated macro-
phages.The tissue-damaging DTH immune response again kills these macrophages, causing enlargement of
the caseous center and progression of the disease.
If a strong CMI develops (Fig. 7B), a mantle of highly activated macrophages is produced that surrounds the
caseous center.These macrophages ingest and destroy (or inhibit) the bacilli escaping from the edge of the
caseous center and often arrest the lesion at a subclinical stage.
Stage 5
The solid caseum liquefies, and a cavity forms. In liquefied caseum, the bacillus may multiply extracellularly (for
the first time) and may reach tremendous numbers (Fig. 9).The high local concentration of tuberculin-like
products released by numerous bacilli causes a tissue-damaging DTH response that erodes a nearby bronchial
wall, so that a cavity forms.The liquefied caseum and tubercle bacilli are discharged into the airways, and may
spread to other parts of the lung and to the outside environment—most commonly during spells of coughing.
Arrest of the disease at this stage depends on whether the antigenic load (of both the bacilli and their products)
remains small enough for the host to control.When numerous bacilli are present, even a host with a well-
developed native and acquired resistance may not stop the progress of the disease.

FIFTH STAGE OF TUBERCULOSIS: The liquefied caseum is frequently, but not

CAVITY FORMATION AND
BRONCHIAL SPREAD OF THE
DISEASE
Stage 5 (Fig. 9) is the stage of the liquefaction
of solid caseum and the formation of cavities.
These events can cause the disease to progress,
even in resistant hosts with good CMI, such as
in Lurie’s resistant rabbits and in adult humans
with a fully competent immune system. Lique-
faction with cavity formation perpetuates the
disease in humankind (4, 24, 30–32, 32a).
always (33), an excellent growth medium for
tubercle bacilli (7, 24, 34). In liquefied caseum,
the bacillus may multiply extracellularly (for
the first time during the course of the disease)
and may reach tremendous numbers (Fig. 10).
Bacilli in large numbers are toxic to tissues
because of the host’s DTH to their tuberculin-
like products. Necrosis of a nearby bronchus
occurs, and a cavity is formed.Then, the bacilli
in the liquefied caseum are discharged into the
 2. STAGES OF HUMAN AND RABBIT TUBERCULOSIS
airways and are distributed to other parts of the
lung and to the outside environment, where
they may infect other persons.
Since liquefied caseum contains tuberculin-
like proteins, it causes an exudate whenever it is
aspirated into the alveolar spaces. Caseous bron-
chopneumonia results when this aspiration is
extensive (see chapter 3).
Furthermore, mutant tubercle bacilli that are
resistant to antimicrobial agents may arise among
the large number of bacilli in the liquefied
caseum. For this reason, tuberculosis is usually
treated simultaneously with two to four antimi-
crobials.
Cavities almost never occurred in Lurie’s sus-
ceptible rabbits (7, 31), and cavities rarely occur
in infants or in immunosuppressed adults (30, 35).
Macrophages do not survive in liquefied
caseous material (7). High concentrations of
tuberculin-like antigens exist in liquefied caseum,
and macrophages may have been passively sensi-
tized to such antigens by cytophilic antibodies.
Also, macrophages may be killed by the toxic fatty
acids present (25, 27).Therefore, even activated
macrophages (produced by well-developed CMI)
cannot control the extracellular multiplication
of tubercle bacilli within cavities.
The cause of liquefaction is largely unknown.
Hydrolytic enzymes (32a, 33, 36) play a role, as
well as DTH to the tuberculin-like products of
the bacillus (37, 38) (reviewed in chapter 4).At
present, no therapeutic agent exists to prevent
liquefaction, but, if developed, such an agent
could greatly aid antimicrobial therapy in
reducing the number of viable tubercle bacilli
and arresting the disease.
SUMMARY
Table 1 summarizes the major characteristics
of each of the five stages of tuberculosis.These
stages are not distinct entities, because one stage
blends into the next one. However, they do
provide a structure for understanding the vari-
ous types of tuberculosis that occur in humans
(see chapter 3).
In secondary lesions during the primary infec-
tion and in lesions of exogenous reinfection,
each stage occurs at an accelerated rate because
of the presence of DTH and CMI. Therefore,
䡵 31
tuberculous lesions in human necropsy (or sur-
gical) material can never duplicate primary
lesions in their early stages.The early develop-
ment of primary lesions can only be studied in
animal models.
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䡵 33
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 3
TYPES OF HUMAN
PULMONARY TUBERCULOSIS

Types of clinical tuberculosis 34

Establishment of the infection 35
The early primary lesion 38
Encapsulated caseous or calcified nodules 38
Proliferative and exudative types of pulmonary lesions 38
Small, discrete tubercles of hematogenous origin and miliary
tuberculosis 40
Liquefied caseous lesions and cavities and bronchogenic spread 42
Progressive, locally destructive lesions 46
Advanced fibrocaseous tuberculosis 46
Tuberculous pneumonia and pleurisy 47
Childhood tuberculosis: lymphatic spread and the Ghon complex 49
Adult tuberculosis: endogenous and exogenous reinfection and subapical
localization 51
Causes of subapical localization 52
Multidrug-resistant tubercle bacilli 57
Recent analyses of human cavitary tuberculous lesions 59
Tuberculosis in the immunocompromised host 59
Inapparent arrested primary tuberculous lesions in healthy humans
compared with rabbits 60

Abstract. Human tuberculosis most frequently occurs as a tiny inapparent lesion that stays
dormant throughout the life of the host. If clinical disease is produced, it varies from the
rapidly progressing, hematogenously spread disease that occurs in infants and immuno-
suppressed individuals to a chronic, slowly progressing cavitary disease that is commonly
found in immunocompetent adults.A brief overview of both the childhood and adult types
of pulmonary tuberculosis is presented in chapter 1.
In this chapter, the gross and histopathological characteristics of each type are described
in more detail. Included are the possible causes of subapical localization in adult-type tuber-
culosis, the location of multidrug-resistant tubercle bacilli in the lungs, the characteristics
of tuberculosis in immunocompromised individuals, and a comparison of inapparent
arrested primary lesions in humans and rabbits. For further discussion of many of the top-
ics in this chapter, see reference 1.

TYPES OF CLINICAL TUBERCULOSIS Active pulmonary tuberculosis, once devel-

In most persons who are infected with the
tubercle bacillus, the disease is arrested as a sin-
gle, inapparent primary pulmonary lesion that
is not large enough to be detected in a chest
X-ray but is sufficient to produce a positive
tuberculin reaction. The progression of such
inapparent primary lesions is usually stopped
by the development of delayed-type hypersen-
sitivity (DTH) and cell-mediated immunity
(CMI) (see chapters 2 and 5). Some primary
lesions, however, do not heal while they are
tiny, but progress to a larger size.
oped in humans, is a locally controlled disease
(1–5). Each lesion is handled by the host almost
as if the other lesions did not exist.Thus, lesions
in one area of the lung may liquefy and progress,
while lesions in another area of the same lung
may stabilize or even regress. Even parts of a sin-
gle lesion may progress, while other parts remain
stable or regress. Also, tuberculous lesions may
change over time from one type to another or
may be a composite of several types. Finally, the
disease as a whole may fluctuate between peri-
ods of exacerbation and remission.

34
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 35

TABLE 1 Basic types of pulmonary tuberculosisa
Types of lesions
Encapsulated caseous, liquefied, or calcified nodules
Proliferative type of pulmonary lesions
Exudative type of pulmonary lesions
Cavities
Types of disease
Small discrete tubercles of hematogenous origin;
focally localized or scattered diffusely throughout
both lungs (miliary tuberculosis)
Liquefied caseous lesions with cavity formation and
bronchogenic spread
Progressive, locally destructive lesions
a
Modified from Rich (5).
Table 1 and Fig. 1 list the types of lesions and
types of disease found in patients with pulmonary
tuberculosis.Any combination of lesion type and
disease type can exist in different parts of the
lungs of the same patient, depending on the local
concentration of bacillary products and the host’s
DTH and CMI responses to these products.Large
numbers of actively multiplying bacilli in a given
lesion produce large amounts of tuberculin-like
products, which cause caseous necrosis, but small
numbers of bacilli produce small amounts of
tuberculin-like products, which enhance host re-
sistance by attracting and activating both lym-
phocytes and macrophages with little, if any,
necrosis.The amount of tissue damage (or ben-
efit) is determined not only by the number of
bacilli, but also by the amount of sensitivity that
the host develops to the tuberculin-like products.
Details on the clinical types of human tuber-
culosis are published in references 6 and 7.
Details on its various pathological manifestations
are published in references 3 through 5, 8, and
9 and are succinctly summarized in reference 10.
ESTABLISHMENT
OF THE INFECTION
An inhaled particle containing more than 3
tubercle bacilli is too large to reach the alveo-
lar spaces. It impinges on the walls of the

FIGURE 1 Development, arrest, or progression of human pulmonary tuberculosis. Once a tiny

(0.5 to 1.0 mm) pulmonary lesion is established, the person becomes tuberculin positive.The tiny
lesion is usually arrested, but in about 5% of cases, it progresses to form a larger caseous lesion.
Such a lesion may (i) stabilize with bacillary dormancy, (ii) progress with or without hematoge-
nous spread of the disease, or (iii) liquefy and produce a cavity with or without bronchial spread
of the disease.Any stabilized lesion may reactivate years later if liquefaction and cavity formation
occur with extracellular bacillary growth. Antimicrobials can arrest the disease at any stage,
unless the bacillus becomes drug resistant.
36 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

bronchial tree, moves toward the pharynx on the
mucociliary escalator, and is subsequently swal-
lowed.The bronchial tree and alimentary canal
are rather resistant to low numbers of tubercle
bacilli and do not usually produce progressive
lesions.
Small inhaled particles, containing up to 3 vir-
ulent tubercle bacilli, can, however, remain sus-
pended in the airstream and reach the alveolar
spaces (11).About one-third of such particles do
so. (The remaining two-thirds impinge on the
walls of the bronchial tree.) In the alveolar
spaces, the bacillary particle is usually ingested
by an alveolar macrophage (12, 13). Sometimes
it is exhaled from the alveolus, and sometimes
it drains to lymphoid tissues, where it is even-
tually destroyed.
The bacilli in these inhaled particles may be
healthy and genetically and phenotypically vir-
ulent, or they may be injured, weakened, and less
virulent. A tuberculous lesion will, or will
not, develop depending, respectively, on
the capacity of the bacillus for intracellu-
lar growth and the capacity of the alveo-
lar macrophage to inhibit such growth. A
weak bacillus in a strong alveolar macrophage
will never cause a primary lesion.A strong bacil-
lus in a weak alveolar macrophage will always
cause such a lesion.
Within an alveolar macrophage, the bacillus
may be killed immediately, it may multiply, or it
may remain dormant for various periods of
time. If it multiplies, its progeny will eventually
kill the original alveolar macrophage, which
then releases the bacilli into the alveolar spaces.
Tubercle bacilli are highly chemotactic and
attract to the site other alveolar macrophages, as
well as many monocytes/macrophages from the
bloodstream. The newly arrived blood-borne
macrophages ingest the bacilli but are initially
incapable of limiting their intracellular growth.
(They must first be activated, whereas most

FIGURE 2 A 10-day lesion, produced in a rabbit by the intravenous injec-

tion of BCG.Alveolar macrophages, staining darkly for -galactosidase (our
marker for macrophage activation), have accumulated in the surrounding alve-
olar area (rather far from the bacilli in the small caseous center).Around the
caseous center are viable, young, -galactosidase-negative macrophages from
the bloodstream, which control the fate of the tuberculous lesion.Within the
caseous center are disintegrated -galactosidase-negative macrophages and
more than 10 faintly stained tubercle bacilli (not easily seen in this photo-
graph). Stained with 5-bromo-4-chloro-3-indolyl--D-galactoside, carbol-
fuchsin, and hematoxylin. Magnification, ⫻330. Reprinted with permission
from reference 33.
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
alveolar macrophages are at least partly acti-
vated.) The developing lesion soon becomes
almost entirely composed of blood-borne
macrophages (Fig. 2), many of which contain
multiplying bacilli.
Therefore, alveolar macrophages determine
whether or not a tuberculous lesion becomes
established, but have little effect on its subse-
quent course.They usually remain at the periph-
䡵 37
ery of the lesion, far from the more centrally
located bacilli (Fig. 2). However, the alveolar
macrophages may prevent the development of
secondary lesions when small numbers of bacilli
are discharged into the airways from a tubercu-
lous cavity.
Genetic resistance seems to play a major role
in determining whether an inhaled bacillary
particle establishes a primary lesion. Lurie’s
FIGURE 3 Left lung of a 57-
year-old woman showing an arrested
Ghon complex. A calcified arrested
primary tuberculous lesion is present
subpleurally in the lower lobe, and an
inactive caseous lymph node is pre-
sent at the hilus. The pulmonary
lesion was probably acquired dur-
ing childhood. From the collection
of the late professors A. R. Rich and
W. G. MacCallum, Department of
Pathology, Johns Hopkins School of
Medicine, Baltimore, Md.
38 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
genetically resistant rabbits had alveolar
macrophages that were more adept than those
of his susceptible rabbits at destroying inhaled
tubercle bacilli or inhibiting their growth (11,
14, 15). In other words, when compared with
alveolar macrophages of the susceptible rabbits,
the alveolar macrophages of the resistant rabbits
were activated to a greater degree. (Such
macrophages are activated nonspecifically by
the ingestion of dust, molds, bacteria, and other
inhaled particles.) In established tuberculous
lesions, the macrophages of the resistant rabbits
were also activated to a greater degree (11) by
antigen-specific lymphocytes and their cytokines
(see chapters 2 and 14).
Humans resemble Lurie’s resistant rabbits
(infected with human-type tubercle bacilli) in
that their macrophages can usually be activated
immunologically to arrest the development of
tuberculous lesions. Therefore, human pul-
monary alveolar macrophages should also be
able to be activated nonspecifically to destroy or
inhibit inhaled tubercle bacilli.
THE EARLY PRIMARY LESION
The earliest clinical evidence of a tuberculous
lesion somewhere in the body is a positive
tuberculin skin test.The test becomes positive
when the tubercle bacilli in the early lesion
have multiplied sufficiently in the primary lesion
to produce a sensitizing antigenic load; in
humans this is usually 1 to 2 months after the
bacilli are inhaled. At that time, the lesion is a
small tubercle with a caseous center.This cen-
ter is composed of incompletely autolyzed
macrophages and pulmonary tissue, bacillary
breakdown products, and dead and live (often
dormant) intact bacilli. Surrounding it are the
blood-borne macrophages that control the sub-
sequent fate of the lesion (Fig. 2).
Usually, only one primary lesion is found in
humans, not multiple primary lesions. This is
because (i) only an occasional unit of 1 to 3 vir-
ulent bacilli is inhaled at one time, (ii) an esti-
mate of 20 to 200 bacillary units must be inhaled
to establish the infection (a one-time exposure
of humans during a long airplane flight is
described in references 16 and 17), and (iii)
subsequent inhalations of bacillary units (that are
capable of establishing another lesion) usually
occur weeks after the initial lesion has begun. By
that time, the host has acquired sufficient immu-
nity to prevent the new lesion from reaching
grossly visible size (11).
ENCAPSULATED CASEOUS
OR CALCIFIED NODULES
If a primary lesion reaches 4 to 12 mm in diam-
eter, it may be detected in a chest X-ray. In most
individuals, a lesion of this size is arrested and
encapsulated by fibrous tissue. Its caseous center
may remain caseous, or it may liquefy, calcify, and
occasionally ossify. During calcification, fine,
dustlike particles or more solid masses of calcium
carbonate and calcium phosphate are deposited
within caseous material, making the lesion more
visible in the chest X-ray. Such an encapsulated
nodule usually remains inactive for the life of the
patient (Fig. 3). See the last section of this chap-
ter for the properties and fate of lesions smaller
than 4 mm in diameter.
Roentgenographically, a diffuse distribution
of calcium deposits is characteristic of infec-
tious granulomas, such as those caused by tuber-
culosis, histoplasmosis, and coccidioidomyco-
sis, whereas eccentric calcification is common in
malignancies. In addition, the edges of infectious
granulomas look smooth, whereas the edges of
malignant lesions usually look irregular.
PROLIFERATIVE AND EXUDATIVE
TYPES OF PULMONARY LESIONS
Proliferative lesions (Fig. 4 and 5), sometimes
called “hard” tubercles, are more common when
small quantities of bacilli and their tuberculin-
like products are present in a host with high re-
sistance to tuberculosis. The cellular infiltrate
consists of macrophages, lymphocytes, plasma
cells, and fibroblasts. Mature epithelioid cells
are common, and Langhan’s giant cells are some-
times present. Connective tissue fibers, inter-
spersed among the cells, contribute to the com-
pact nature of this type of lesion. Bacillary
multiplication is minimal because of the good
defense reaction produced by the host.
In humans,the classic orientation (i.e.,a caseous
center surrounded by viable tuberculous granu-
lation tissue containing epithelioid cells) is com-
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 39

FIGURE 4 A proliferative type of miliary tubercle in the liver of an 8-month-old male

infant. Parts of four Langhans’ giant cells are seen. Stained with hematoxylin and eosin. Mag-
nification, ⫻130. From the collection of the late professors A. R. Rich and W. G. MacCallum,
Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

mon in young lesions, such as those in miliary
tuberculosis. In older lesions, this classic orienta-
tion is frequently indiscernible;instead,epithelioid
and giant cells are intermixed with caseous ma-
terial and fibrous tissue. Proliferative lesions may
become chronic, with some areas undergoing
caseous necrosis and other areas undergoing fibro-
sis. Such lesions may eventually progress and give
rise to satellite tubercles that coalesce, or they
may stabilize with encapsulation by collagenous
connective tissue that is often hyalinized and
sometimes contains calcium deposits.
Exudative lesions (Fig. 6 and 7) are more com-
mon when the host is highly sensitive to tuber-
culin, especially when large quantities of bacilli
and their tuberculin-like products are present at
the local site. Mononuclear cells and some gran-
ulocytes accumulate in the alveolar spaces as a
rather loose exudate containing variable amounts
of fibrin. In this exudate, the bacilli multiply
readily, especially in a host with weak defenses.
These lesions vary from small, loose “soft” tuber-
cles (which are frequently confluent) to small or
large areas of tuberculous pneumonia (Fig. 6 and
7). Caseation frequently occurs. Exudative lesions
may resolve almost completely, leaving only
microscopic residues, but, in the absence of
antimicrobial agents, they usually progress.
Lesions exhibiting both proliferative and
exudative features (Fig. 8) are more common
than those of one type alone. Also, as men-
tioned above, proliferative lesions may occur in
one part of the lung, and exudative lesions may
concurrently occur in another part, depending
on the concentration of bacilli and the tuber-
culin-like components in each place.The lesions
may be limited or extensive in scope and some-
times change from one predominant form to the
other in response to shifts in the delicate balance
between host and bacillus. The most striking
change in this balance occurs when tubercle
bacilli proliferate luxuriantly and extracellularly
during liquefaction and cavity formation (see
“Liquefied Caseous Lesions and Cavities and
Bronchogenic Spread” below). Only then,
because of such large quantities of bacilli and
their products, does an exudative response occur
in a relatively resistant host.
40 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 5 A well-encapsulated fibrocaseous pulmonary lesion formed from confluent tuber-

cles in a 5-month-old infant dying of tuberculous meningitis.The central area of necrosis is sur-
rounded by smaller satellite necrotic areas. One Langhans’ giant cell is visible.This was a small
proliferative tubercle that exacerbated as the patient became terminally ill. Stained with hema-
toxylin and eosin. Magnification, ⫻123. From the collection of the late professors A. R. Rich and
W. G. MacCallum, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

SMALL, DISCRETE TUBERCLES
OF HEMATOGENOUS ORIGIN
AND MILIARY TUBERCULOSIS
Most tuberculous lesions, particularly those that
are progressing, shed at least a few bacilli into the
bloodstream from time to time. Pulmonary
lesions shed bacilli into local capillaries and
venules. Lesions in the hilar lymph nodes shed
bacilli into the efferent lymphatics, which trans-
port these bacilli into the bloodstream. Caseous
lesions in a hilar lymph node may occasionally
erode a blood vessel and shed bacilli directly into
the bloodstream. Such lesions may also erode an
adjacent part of the bronchial tree and shed
bacilli into the airways.
Wherever the bacilli are deposited, they are
ingested by macrophages and often form a
microscopic tubercle. Usually, the macrophages
are rapidly activated, the bacilli are destroyed, and
the lesion resolves. Occasionally, the bacilli are
not destroyed, and one or more grossly visible
tubercles form in the lungs, kidney, spleen, liver,
bone, testes, ovary, or brain. In the brain, a semi-
solid or liquefied caseous tubercle near the sub-
arachnoid or ventricular space may rupture and
discharge its contents into the cerebrospinal
fluid, causing tuberculous meningitis.
Miliary tuberculosis occurs when a massive
dose of tubercle bacilli is discharged from a
caseous or liquefied focus into the bloodstream
and the resistance of the host is inadequate, as in
early infancy, in old age, and in immunologically
depressed persons of any age. In this type of
tuberculosis, many small tubercles of uniform size
and hematogenous origin occur simultaneously
in the lungs (Fig. 9) and/or in the liver, spleen,
or kidneys.A miliary tubercle is 2 to 4 mm, the
size of a millet seed (hence the name “miliary”).
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 41

FIGURE 6 An exudative type of tuberculous lesion in the lung of a 47-year-old man.

Depicted is an area of tuberculous pneumonia.A necrotic exudate fills the alveolar spaces, and
the alveolar walls are thickened by infiltrating cells. Stained with hematoxylin and eosin. Mag-
nification, ⫻266.

Tubercle bacilli are distributed to extrapul-
monary organs after entering the left side of the
heart. These bacilli usually originate from
caseous pulmonary lesions that discharge bacilli
into pulmonary veins carrying oxygenated
blood from the lungs to the left side of the
heart.The discharging lesions may even be in the
walls of pulmonary veins themselves. Relatively
few bacilli pass through the peripheral capillary
beds and return to the lungs.
Tubercle bacilli are usually distributed into
both lungs after entering the right side of the

FIGURE 7 An exudative lesion

similar to that shown in Fig. 6 but
stained for tubercle bacilli, some of
which may be discerned in this pho-
tograph (arrows). Stained with Ziehl-
Neelsen, counterstained with meth-
ylene blue. Magnification, ⫻450.
From the collection of the late pro-
fessors A. R. Rich and W. G. Mac-
Callum, Department of Pathology,
Johns Hopkins School of Medicine,
Baltimore, Md.
42 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 8 Part of a typical pulmonary tubercle that shows both proliferative and exudative
features and a Langhans’ giant cell. It was from a 12-month-old infant with miliary tuberculo-
sis. Stained with hematoxylin and eosin. Magnification, ⫻300.
Langhans’ giant cells form when macrophages surrounding bits of caseum fuse to each
other.The nuclei of Langhans’ giant cells are in the periphery and may continue to divide. In
tuberculous lesions, Langhans’ giant cells are a sign of chronicity. From the collection of the late
professors A. R. Rich and W. G. MacCallum, Department of Pathology, Johns Hopkins School
of Medicine, Baltimore, Md.

heart. These bacilli most often originate from
caseous hilar lymph nodes. The hilar lymph
nodes drain the bacilli (via efferent lymphatics)
into the great systemic veins, which carry them
(via the right side of the heart) to pulmonary
arteries carrying nonoxygenated blood to the
lungs. Less frequently, endogenous bacilli reach
the lungs from tuberculous lesions elsewhere in
the body, because such lesions also release bacilli
into the venous side of the circulation. Miliary
tubercles will occur in only one lung if a tuber-
culous lesion (in the pulmonary hilus) discharges
bacilli directly into a branch of the main pul-
monary artery.
Miliary tubercles may be of two types,
depending on the resistance of the host: (i) a
compact “hard” tubercle with epithelioid cells
and occasional giant cells, i.e., the proliferative
type of lesion with or without caseous centers
(Fig. 4 and 5), or (ii) a loosely formed exudative
type, or “soft” tubercle (Fig. 6). (Both are
described in the preceding section.) The exuda-
tive type contains more bacilli (Fig. 7), which
continue to multiply, at least for a while. This
type usually undergoes early and complete
caseation and progresses rapidly. Tubercles with
mixed hard and soft characteristics are com-
mon (Fig. 8).
LIQUEFIED CASEOUS LESIONS AND
CAVITIES AND BRONCHOGENIC
SPREAD
The liquefaction of caseous foci perpetuates pul-
monary tuberculosis in the human population,
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 43

FIGURE 9 Miliary tuberculosis of the lung in a 19-year-old man. Caseous hilar lymph nodes
are present. From caseous plaques (not shown) in the branches of the pulmonary veins, tubercle
bacilli seeded the general circulation and were carried to other organs in the body. From the col-
lection of the late professors A. R. Rich and W. G. MacCallum, Department of Pathology, Johns
Hopkins School of Medicine, Baltimore, Md.

because it results in cavity formation. From the
cavity, the bacilli can spread via the bronchial
tree to other parts of the lung and to the outside
environment (18).
Before a caseous focus liquefies, tubercle
bacilli multiply primarily intracellularly within
poorly activated macrophages, which readily
ingest every bacillus they encounter (12, 13).The
extracellular bacilli in solid caseous tissue rarely
multiply. However, after a caseous focus lique-
fies (Fig. 10), the extracellular bacilli may mul-
tiply tremendously (Fig. 11), especially after a
cavity forms because of the additional oxygen
provided by direct contact with the air in the
bronchial tree (Fig. 12).When faced with such
large numbers of bacilli and their antigenic
components, even persons with high degrees of
acquired resistance to tuberculosis often may
not be able to stop progression of the disease.
(See chapter 4 for a discussion of the causes of
liquefaction and cavity formation.)
Among the large numbers of bacilli within a
liquefied or cavitary lesion, mutants may be
present that are resistant to one or more of the
antimicrobial agents. For this reason, tuberculosis
is usually treated with multiple antimicrobials
given together over many months.
A cavity is formed when a caseous focus rup-
tures through the wall of a nearby bronchus
and discharges its contents into the air passages
44 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 10 Early liquefaction in a small tubercle from a 12-month-old infant who died of
miliary tuberculosis.The lesion is partly surrounded by fibrous tissue. Stained with hematoxylin
and eosin. Magnification, ⫻108. From the collection of the late professors A. R. Rich and W. G.
MacCallum, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

(Fig. 13 and 14).The walls of most cavities con-
sist of an external zone of collagen (the cavity’s
capsule, which occasionally is hyalinized) and a
caseous (often liquefying) internal zone, where
the high oxygen content from the ambient air
nurtures the growth of bacilli (Fig. 12). By
coughing, the patient aerosolizes this infectious
material, disseminating bacilli to other parts of
the lung and to the outside environment.
Between the external and internal zones of the
cavity wall is an intermediate zone of granula-
tion tissue rich in capillaries, granulocytes,
macrophages, lymphocytes, and fibroblasts. At
times, it contains typical tubercles. The three
zones are of variable thickness and not clearly
demarcated from one another. With newly
formed cavities, the internal caseous zone is
thickest.With older, still-active cavities, the exter-
nal capsule is thickest.Around the capsule, usu-
ally between the pleura and the cavity, an area of
atelectasis is often present. This atelectatic area
may prevent perforation into the pleural spaces.
Erosion of incompletely thrombosed vessels
in the intermediate zone leads to hemorrhage
into the wall of the cavity (Fig. 15).There, blood
may pool and give rise to some hemoptysis.
Massive hemoptysis, which is sometimes fatal, is
usually due to the leakage or rupture of a fully
patent blood vessel located in the wall of the
cavity or traversing its lumen.
When a liquefied caseous lesion in the lung or
hilar lymph node discharges its contents into the
air passages, the resulting lesions range from small
pneumonic foci, to larger areas of pneumonia, to
complete lobar pneumonia.The extent of the dis-
ease is determined by the amount of liquefied
caseum aspirated into the bronchial tree, as well
as by the number of (live and dead) bacilli and the
quantity of tuberculin-like products that this liq-
uefied caseum contains.
Local deposits containing small quantities of
bacilli and their components commonly cause
scattered compact proliferative-type tubercles
or (more rarely) confluent proliferative tubercles
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 45

FIGURE 11 A rabbit pulmonary tuberculous lesion showing tubercle bacilli that had grown
profusely in the liquefied caseum.This lesion was probably beginning to cavitate, because some
of the spaces in the liquefied caseum are larger than usual. Such profuse growth occurs only in
some of the lesions with liquefied centers, presumably where the composition of the liquefied
caseum is most favorable and/or the adaptation of the bacillus to extracellular growth is most
complete. Similar bacillary growth has been found in many human cavitary lesions. Stained with
carbol-fuchsin, counterstained with methylene blue. Magnification, ⫻600. Reprinted with per-
mission from reference 34.

with small foci of encapsulated caseous pneu-
monia (Fig. 16). However, local deposits con-
taining large quantities of bacilli and their com-
ponents cause exudative-type lesions that soon
caseate, producing small or large areas of tuber-
culous pneumonia (Fig. 6 and 7).
Whether there is healing or progression
depends on the extent of the disease, as well as
the number of bacilli and their rate of multipli-
cation. Small scattered foci readily heal or are
encapsulated, but areas of tuberculous pneu-
monia may progress into adjacent alveoli and
bronchioles.
Cavitary tuberculous lesions may never heal
during the life of the patient.They may enlarge
or shrink or remain stable. Spontaneous healing
results from a gradual collapse of the cavity,
progressive fibrosis from without, and/or
obstruction of the bronchocavitary junction
followed by absorption of the air within the cav-
ity. Fibrosis is often incomplete, and variable
amounts of caseous and fibrocaseous material
may remain within the fibrotic lesion. Calcium
deposition is common, and occasionally ossifi-
cation occurs.A “healed” cavity, like a “healed”
caseous focus, is seldom completely free of
tubercle bacilli, which may persist for years,
often in a dormant state.
With effective antimicrobial therapy, “open
healing” of a cavity may occur. Inflammation
decreases after the antimicrobial agents have
eliminated most of the bacilli, and the necrotic
contents drain through a still-patent bron-
chocavitary junction. Such drainage is facili-
tated if the cavity is located in an upper lobe (the
usual place). Metaplastic bronchial epithelium
(squamous with no cilia) may eventually line the
cavity.The open-healed cavity may persist as one
with a thick or thin fibrotic wall, or as an
emphysematous bleb or bulla.
46 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 12 The wall of a pulmonary cavity in a rabbit showing tubercle bacilli that had
grown profusely in the liquefied caseum.The bacilli are more numerous near the lumen of
the cavity (on the left), presumably because the oxygen tension there is highest. Such profuse
growth occurs only in the walls of some, not all, cavities. Stained with carbol-fuchsin, coun-
terstained with methylene blue. Magnification, ⫻250. Reprinted with permission from ref-
erence 34.

PROGRESSIVE, LOCALLY host resistance is low. Such lesions grow periph-

DESTRUCTIVE LESIONS
Tuberculous lesions may progress slowly or
rapidly, depending on the number of extracel-
lular and intracellular bacteria, and how easily
the host can destroy them. Slowly progressing
caseous or cavitary foci are the most common
type of progressive tuberculosis in adults (Fig.
13).These foci have compact walls that contain
epithelioid and giant cells, and show consider-
able reparative fibrosis (Fig. 17). Progression is
intermittent.The advancing edge of the lesion
contains fresh tubercles, or fresh or organizing
pneumonia in the adjacent alveolar spaces (Fig.
14). Encapsulated caseous foci and tubercles,
embedded in fibrous tissue, are prominent; hence
the term fibrocaseous (Fig. 5). Satellite lesions are
frequent, and cavitation and extension via the
bronchial passages sometimes occur.
Rapidly progressing caseous foci with incon-
spicuous fibrosis (Fig. 18) are often present when
erally as a loose exudate extending into nearby
alveolar spaces. The exudate (which soon
becomes necrotic) contains young macrophages,
granulocytes, fibrin, erythrocytes, and serum
protein. This type of tuberculosis commonly
occurs in young children. It is rare in adults,
except when they are immunosuppressed, or
when they aspirate large numbers of bacilli from
a cavity. Rapidly formed cavities may occa-
sionally occur (Fig. 19).
ADVANCED FIBROCASEOUS
TUBERCULOSIS
The most common form of advanced tubercu-
losis seen in adults is the fibrocaseous type,
which may progress until death. In this form of
the disease, different lobes of the lung may con-
tain confluent cavities, confluent caseous foci,
areas of caseous pneumonia, or combinations of
these conditions (Fig. 20 and 21). Because of the
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 47

FIGURE 13 Bilateral tuberculous cavities in the upper lobes in a 39-year-old diabetic woman.
Caseous areas surround the cavities. Although infected, the hilar lymph nodes are not markedly
enlarged. In the lung on the right, an applicator stick marks the communication between the cav-
ity and the bronchus. From the collection of the late professors A. R. Rich and W. G. MacCallum,
Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

large numbers of bacilli that ascend the bronchial
tree and are subsequently swallowed, lesions
may occur in the bronchi, trachea, larynx, and
intestinal tract—all of which are rather resistant
to small numbers of bacilli.The patient whose
lungs are shown in Fig. 20 had lesions in each
of these locations. Figure 22 shows an example
of tuberculous laryngitis, which, however, rarely
occurs under effective chemotherapy. Occa-
sionally, an infected bronchus may undergo cica-
tricial stenosis and become obstructed, produc-
ing atelectasis of the lung distal to that site.
Despite the extensive fibrocaseous pulmonary
disease, the hilar nodes of adults usually show
minimal involvement. This is in contrast to
childhood tuberculosis, in which large caseous
hilar nodes often play a major role in the pro-
gression of the disease, usually by seeding bacilli
into the bloodstream via efferent lymphatics.
TUBERCULOUS PNEUMONIA
AND PLEURISY
Tuberculous pneumonia is an exudative response
in the alveolar spaces caused by the presence of
numerous live and dead tubercle bacilli and
their antigenic products. Small or extensive areas
of pneumonia commonly occur when a lique-
fied caseous focus in the lung (or in a hilar
lymph node) discharges its contents into the
airways (Fig. 6, 19, 20, 21, and 23). Since the
48 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 14 An apical cavity of moderate size in the left lung of an adult. Caseous pulmonary
consolidation of pneumonic origin surrounds the cavity. Several caseous foci are also present in
the other lung.The hilar lymph nodes contain a few caseous areas but are only slightly enlarged.
From the collection of the late professors A. R. Rich and W. G. MacCallum, Department of Pathol-
ogy, Johns Hopkins School of Medicine, Baltimore, Md.

caseous material is impregnated with tubercu-
loprotein and other bacillary components, it
causes a pneumonic exudate, even if the num-
ber of intact bacilli is small.This exudate under-
goes caseous necrosis if high amounts of tuber-
culin-like products are present. The initial
exudate contains mononuclear cells, granulo-
cytes, and a coagulum of precipitated protein and
fibrin. In time, epithelioid cells, lymphocytes,
plasma cells, fibroblasts, and, occasionally, giant
cells appear.
The confluence of progressing exudative
lesions results in consolidation of small or large
segments of the pulmonary lobe.At one end of
the spectrum, the pneumonia leads to death.At
the other end of the spectrum, the pneumonia
undergoes resolution with minimal scarring,
especially if only small numbers of viable bacilli
are present and the lung structure is preserved.
Pleurisy results when subpleural caseous lesions
in the lung (or in the hilar lymph nodes) leak or
rupture, discharging bacilli and their compo-
nents into the pleural cavity (Fig. 20). (Similarly,
tuberculous pericarditis can develop from a rup-
ture of such a lesion into the pericardial sac.)
Clinically, the pleurisy is manifested in many
forms: mild, uncomplicated roughenings of the
pleural surface, large or small numbers of miliary
tubercles on the pleural surface,or serous effusions
from large or numerous surface lesions. In more
than half of the patients with tuberculous pleurisy,
the serous effusion is bacteriologically sterile,
because it is only a response to tuberculin-like
products released into the pleural cavity.
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 49

FIGURE 15 A tissue section of a rabbit pulmonary cavity wall. On the left are disintegrat-
ing mature epithelioid cells (see Fig. 15 in chapter 4). On the right is a small blood vessel extend-
ing into the cavity’s lumen. Such exposed vessels are the source of blood in the sputum. In
humans, when a larger blood vessel is similarly exposed in a cavity and ruptures, massive
hemoptysis and sometimes fatal hemorrhage may occur. Glycol methacrylate-embedded
tissue section stained with Giemsa. Magnification, ⫻400. Reprinted with permission from
reference 35.

Tuberculous empyema was a serious compli- sometimes calcification. Depending on the

cation before the antimicrobial era. It consisted
of a purulent exudate in the pleural cavity, usu-
ally surrounded by a fibrocaseous capsule, and
the exudate was frequently superinfected with
pyogenic bacteria.
The type of pleural disease is determined by
(i) the number of live and dead bacilli, (ii) the
quantity of their tuberculin-like products in the
caseous material, (iii) the level of tuberculin
sensitivity of the host, (iv) the friction of the
roughened visceral pleura against the parietal
pleura, (v) the number and types of cells in the
exudate, (vi) the inherent ability of the host to
localize and fibrose new sites of tuberculous
infection, and (vii) the presence or absence of
superinfection with other bacteria.
Tuberculous pleurisy may resolve completely
or may heal with fibrosis, focal adhesions, and
amount of exudate in the pleural space, a large
or small portion of the lung may collapse. Orga-
nization of the exudate and increasing fibrosis
tend to maintain this collapse. References 6 and
7 provide more details on tuberculous pleurisy.
CHILDHOOD TUBERCULOSIS:
LYMPHATIC SPREAD AND THE
GHON COMPLEX
Tuberculosis in children differs from tuberculo-
sis in adults in several respects: (i) young children,
especially infants, usually have lower native and
acquired resistance to this disease; (ii) their hilar
lymph nodes more commonly become enlarged
and caseous (Fig. 18, 23, and 24); (iii) blood-
borne dissemination occurs more readily (Fig. 18;
also see Fig. 9) but is still an exception rather than
the rule; (iv) calcification of caseous foci is more
50 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 16 A cavity in the base of

the upper lobe of the left lung of a 17-
month-old infant.The apical portion was
adherent to the chest wall and solidified
with confluent proliferative tubercles and
encapsulated caseous pneumonic foci.
From the collection of the late professors
A. R. Rich and W. G. MacCallum, Depart-
ment of Pathology, Johns Hopkins School
of Medicine, Baltimore, Md.

common; and (v) the primary pulmonary lesion
in children is usually subpleural in well-ventilated
regions of the lung (Fig. 24), whereas such lesions
in adults are usually subapical (see below).
Tubercle bacilli, either free or within imma-
ture macrophages (or granulocytes), spread via
the lymphatics from the primary lesion to the
hilar lymph nodes. There, in children (and in
adults with low resistance), the bacilli frequently
cause large caseous lesions.The combination of
one or more enlarged caseous hilar nodes plus
a tuberculous focus in the lung periphery con-
stitutes the Ghon complex (Fig. 24), which was
named after Anton Ghon (1866–1936), a Prague
pathologist.
Sometimes, a tuberculous hilar node obstructs
a bronchus by external compression or by exten-
sion of the disease process into the bronchial wall.
The obstruction causes segmental or lobar col-
lapse and pneumonitis, which is called the right-
middle lobe syndrome, as it frequently occurs in
that lobe. Sometimes, such a node ruptures into
a bronchus (Fig. 18 and 23), causing distally focal
or diffuse pneumonic lesions. Such lesions can be
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS 䡵 51

FIGURE 17 A portion of a large cavity in the lung of a rabbit, 18 weeks after the inhala-
tion of about 5,000 virulent bovine-type tubercle bacilli.The liquefied caseum (below) is sur-
rounded by a thick fibrous capsule. In this capsule are many fibroblasts, as well as some
macrophages, lymphocytes, and plasma cells. At the right is an oval metaplastic alveolus next
to a small blood vessel. Glycol methacrylate-embedded tissue section stained with Giemsa. Mag-
nification, ⫻250. Reprinted with permission from reference 1.

minimal or extensive, depending on the con-
centration of bacilli and their antigenic products
and on the host’s reaction to them. Both situa-
tions occur mainly in infants, since older children
and adults only rarely have extensive involvement
of the hilar nodes.
ADULT TUBERCULOSIS:
ENDOGENOUS AND EXOGENOUS
REINFECTION AND SUBAPICAL
LOCALIZATION
As a rule, tuberculosis progresses more slowly in
adults than it does in infants, because the resis-
tance of adults is usually higher. Secondary
endogenous tuberculous foci of lymphatic or
hematogenous origin occur less often in adults,
because most tubercle bacilli are soon destroyed
after they are carried in the lymph to the hilar
nodes or carried in the blood to sites elsewhere
in the body. However, the resistance of immuno-
competent tuberculous adults may be over-
whelmed when a liquefied caseous focus rup-
tures and seeds large numbers of bacilli directly
into a bronchus or into a blood or lymphatic
vessel of appreciable size.
Reinfection-type or adult-type tuberculosis
consists of a small to large pulmonary lesion that
is not accompanied by any marked enlargement
of the hilar lymph nodes.The pulmonary lesion
is most often located in the subapical region. In
this type of tuberculosis, the native and acquired
resistance of the host is sufficient to destroy or
inhibit most of the bacilli that reach the hilar
nodes.Adult-type tuberculosis may occur as a pri-
mary infection or reinfection in either adults or
children if their resistance is high enough to
restrict bacillary multiplication in the hilar nodes.
It may be caused by either endogenous or
52 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 18 Rapidly progressing disseminated tuberculosis in an 11-month-old infant. Both

hematogenous and bronchogenic spread from caseous hilar lymph nodes occurred in the lungs.
Most of the caseous foci are hematogenous in origin.At the hilus in the lung on the right is an
area of caseous pneumonia caused by the rupture of a tuberculous lymph node into a nearby
bronchus. From the collection of the late professors A. R. Rich and W. G. MacCallum, Depart-
ment of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

exogenous tubercle bacilli (see reference 11). In CAUSES OF SUBAPICAL

other words, adult-type tuberculosis may be
caused by a reactivation of a previously arrested
disease or by a primary infection with virulent
tubercle bacilli inhaled from the environment.
The active subapical lesion frequently consists
of a localized area of bronchopneumonia con-
taining macrophages, epithelioid cells, lympho-
cytes, giant cells, and fibroblasts.The lesion usu-
ally heals, and the resulting fibrosis may thicken
the overlying pleura. Sometimes, a cavity forms
either at the time of the initial subapical infec-
tion or, more commonly, years later if reactiva-
tion of a formerly dormant subapical lesion
occurs.Then, the disease may progress into one
of the more serious types described above.
LOCALIZATION
Source of Tubercle Bacilli
Exogenous bacilli, giving rise to adult-type
tuberculosis, would be inhaled directly into the
subapical pulmonary regions, while endoge-
nous bacilli would be carried to these regions by
the bloodstream (see Fig. 13, 14, 20, and 21).The
endogenous tubercle bacilli (originating from
the hilar nodes) could reach the apical region at
the time of the primary infection and remain
dormant there until reactivation occurs (see ref-
erence 19).Alternatively, these bacilli could have
been dormant in the hilar lymph nodes and
could have seeded the subapical region at the
time of reactivation of the disease. Scars in the
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
apical region, the so-called Simon foci (20),
could have been caused by either exogenous or
endogenous tubercle bacilli.
Physiology: Apical versus Basal
Pulmonary Regions
In humans in the upright position, the apical
region of the lung differs from the basal region
in the following manner.
Apical alveoli are more distended owing to
less compression from the weight of the rest of
the lung, which is mostly due to the hydrosta-
tic pressure from the blood it contains.
䡵 53
FIGURE 19 A large, rapidly formed
tuberculous cavity in the upper lobe.
Caseous pneumonia is present throughout
the rest of the lung. Fibrosis is minimal.
The hilar node is markedly enlarged and
mostly caseous. From the collection of
the late professors A. R. Rich and W. G.
MacCallum, Department of Pathology,
Johns Hopkins School of Medicine, Bal-
timore, Md.
The lower regions of the lung are ventilated
more than the upper regions mainly because of
the descent of the diaphragm during inspiration.
Also, when the person is in the upright position,
the lower region has a much greater blood flow
(i.e., perfusion) within its vasculature.A greater
perfusion occurs there because the higher
hydrostatic pressure both dilates the lower vas-
culature and increases the flow of blood within
this vasculature (21).
In the lower regions of the lung, more O2 is
absorbed and more CO2 is released than in the
upper regions, because both the ventilation of
54 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 20 Advanced fibrocaseous tuberculosis in an 11-year-old girl, who died before the
advent of the antimicrobial era.The bilateral cavities in the upper lobes have caused adjacent
areas of caseous bronchopneumonia. On the right is fibrous tissue from pleural adhesions.The
hilar lymph nodes contain several caseous foci. From the collection of the late professors
A. R. Rich and W. G. MacCallum, Department of Pathology, Johns Hopkins School of Med-
icine, Baltimore, Md.

air and the perfusion of blood are increased. In
the upper regions, therefore, less O2 is absorbed
and less CO2 is released.
The lymph flow from the apex is also
reduced, because with decreased blood entering,
less lymph is produced. Lymph forms intersti-
tially from fluids extravasated from the blood-
stream and is moved into the terminal lym-
phatics by the pumping action of the lungs as
they expand and contract.This pumping action
is greater in the lower, more ventilated part of
the lungs.
Effects of the Subapical Localization
on the Establishment of Tuberculous
Lesions
In both the upper and lower alveoli, pulmonary
alveolar macrophages are activated nonspecifi-
cally by dust and inhaled microorganisms. In
humans (and rabbits), such activation enables
the majority of alveolar macrophages to destroy
inhaled virulent human-type tubercle bacilli or
inhibit their growth (see chapter 11). More
inhaled particles enter the lower alveoli, because
they receive a greater proportion of the tidal air
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
than the apical alveoli receive (22).Therefore, in
humans, the alveolar macrophage population
in the lower alveoli might (but not necessarily)
be more highly activated than those in the upper
alveoli. If so, the alveolar macrophages in the
lower alveoli would prevent initial establish-
ment of tuberculous lesions more readily than
those in the apical regions.This possibility, how-
ever, remains to be investigated.
Effects of the Subapical Localization
on the Progress of Tuberculous
Lesions
The apical location should have marked effects on
the progress of an established microscopic lesion
䡵 55
FIGURE 21 Caseous broncho-
pneumonia originating from a large
subapical cavity. From the collection
of the late professors A. R. Rich and
W. G. MacCallum, Department of
Pathology, Johns Hopkins School of
Medicine, Baltimore, Md.
into clinically apparent disease. In early micro-
scopic lesions, the alveolar walls are infiltrated
with macrophages, dendritic cells, and lympho-
cytes from the bloodstream, and an exudate con-
taining these cells enters the adjacent alveoli. In
a person in the upright position,the apical regions
are perfused with less blood than the basal pul-
monary regions (because of gravity), their supply
of O2 is limited (because of less ventilation), and
CO2 (from the cells in the lesion) is less rapidly
removed (see above).The reduced availability of
O2 should decrease the ability of the infiltrating
macrophages to produce reactive oxygen and
nitrogen intermediates, which kill (or inhibit)
intracellular tubercle bacilli, and the reduced
56 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
FIGURE 22 Tuberculous laryngitis in a rabbit that
had inhaled 300 virulent bovine-type tubercle bacilli 33
weeks previously.The lung of this rabbit had 14 lesions,
4 of which had formed cavities.The cavity in the right
lower lobe of the lung had apparently discharged so
many bacilli into the bronchial tree that the larynx
became infected.This laryngeal lesion partly obstructed
the airway. In humans, tuberculous laryngitis has been
rare ever since antimicrobials for this disease became
available. Magnification, ⫻5.4. Reprinted with permis-
sion from reference 35.
removal of CO2 might possibly enable more
tubercle bacilli to survive intracellularly (see
below).Also, in the apical regions, the decreased
flow of lymph would carry fewer bacilli to the
draining lymph nodes and leave more bacilli
within the developing lesion. The immune
response begins in draining lymph nodes.There-
fore,the immune response to inhaled bacilli in the
apical regions would be less rapid (and therefore
less effective) than the immune response to
inhaled bacilli in the basal regions.
Lurie (unpublished) found that small amounts
of CO2 enhanced the in vitro growth of tuber-
cle bacilli. Aerosol samples of virulent tubercle
bacilli were collected in a Rosebury impinger
and were cultured on a modified Lowenstein’s
solid egg medium (11, 23). (The aerosol samples
contained bacillary units of mostly 1 to 3 tuber-
cle bacilli.) When low levels of CO2 were added
to the air in the electric incubator, many more
mycobacterial colonies developed on the culture
medium than when no CO2 was provided. Units
of 1 to 3 tubercle bacilli suspended in air are eas-
ily desiccated and need the most favorable con-
ditions to survive and multiply.The presence of
low levels of CO2 evidently enhances their abil-
ity to survive.
In basal pulmonary regions of an individual
in the upright position, more ventilation occurs
and more blood is perfused through the alveo-
lar capillaries (21), so the early tuberculous lesion
would have more O2 available, and less CO2
would accumulate. In the basal regions, the
increased perfusion of blood would supply
defense cells more rapidly, and the increased
respiratory excursion would increase the flow of
interstitial fluid into the lymphatics.Therefore,
tubercle bacilli (usually within macrophages and
dendritic cells) and their (secreted) antigens
would be carried more rapidly to the draining
lymph nodes, where acquired (adaptive) immu-
nity takes place (see chapters 5 and 6).Therefore,
when a tuberculous lesion is located in the basal
region, a good immune response would develop
sooner and prevent an early tuberculous lesion
from reaching clinically apparent size.
Another possible explanation of why tuber-
culous lesions of endogenous origin develop
apically is that small particles of caseous tissue
containing tubercle bacilli are preferentially dis-
tributed there by the bloodstream. Both tuber-
cle bacilli and the caseous tissue containing
them are rich in lipids and tend to float. Caseous
hilar lymph nodes (via efferent lymphatics)
would drain such bacilli into the great veins.
When the person is upright, the bacilli in these
veins enter the heart and then would probably
enter the upper branches of the pulmonary
arteries, where they would be distributed to
the upper parts of the lungs. (Human infants and
rabbits, however, spend a large part of the day in
the horizontal position, and their primary lung
lesions are usually located in the middle and
lower lobes, rather than subapically.)
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
All of the factors listed above probably play
a role in the subapical localization of clinically
apparent tuberculous lesions in adults, but how
they interact and the relative importance of
each remain to be determined.
MULTIDRUG-RESISTANT
TUBERCLE BACILLI
Multidrug-resistant tubercle bacilli usually arise
in bacillary populations that are multiplying
within cavities (24).The bacilli then ascend the
bronchial tree and may infect other parts of the
䡵 57
FIGURE 23 Extensive confluent
caseous pneumonia in a 13-month-old
infant, caused by the rupture of a large
liquefied caseous hilar lymph node into
an adjacent bronchus. From the collec-
tion of the late professors A. R. Rich and
W. G. MacCallum, Department of
Pathology, Johns Hopkins School of
Medicine, Baltimore, Md.
patient’s lung and also other persons.The bacil-
lary population that is tested for drug resistance
in sputum samples almost always comes from a
cavity in the patient’s lungs. In fact, tubercle
bacilli in noncavitary lesions usually do not
develop drug resistance.
Analyses of bacilli from cavitary lesions by
IS6110-based RFLP (restriction fragment
length polymorphism) technology showed that
different types of antimicrobial resistance could
develop independently by mutations within
different cavitary lesions located in the same
58 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 24 A progressive Ghon complex in the mid portion of a lung from a 6-year-old boy
who died of tuberculous meningitis.At the lower edge of the specimen is the primary subpleural
caseous lesion. Near the center is a markedly enlarged caseous hilar lymph node. Many tubercles
of hematogenous origin are also present. From the collection of the late professors A. R. Rich and
W. G. MacCallum, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Md.

lungs (24). In other words, the initial infecting
mycobacterial strain that developed (or had)
drug resistance in the first cavity can acquire re-
sistance to additional drugs in subsequent cav-
ities arising in the same patient (24). Con-
versely, at least some arrested noncavitary lesions
in the lungs of such patients should contain
drug-susceptible tubercle bacilli if such lesions
were arrested before drug resistance developed
in the cavities.
Tubercle bacilli in arrested caseous foci seem
to be quite stable.The genome of Mycobacterium
tuberculosis bacilli collected from patients during
the 1990s was found by IS6110-based RFLP
technology to be identical to the genome of
freeze-dried M. tuberculosis bacilli collected from
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
FIGURE 25 Confluent, multidrug-resistant, tuber-
culous pneumonia in a patient with AIDS. The lung
parenchyma was replaced by pale nodular zones of
caseation necrosis. Reprinted with permission from
reference 1.
the same patients during the 1960s (25). This
finding suggests that the patients in the 1990s
had reactivation of dormant tuberculous lesions
acquired 30 years earlier. The persistence and
dormancy in tubercle bacilli have been thor-
oughly reviewed (26).
RECENT ANALYSES OF HUMAN
CAVITARY TUBERCULOUS LESIONS
When the patient’s sputum contained many
tubercle bacilli, many tubercle bacilli were also
present extracellularly in the liquefied caseum
of the cavity wall (see references 3 and 5).This
correlation was recently confirmed by Kaplan et
al. (24) with surgically removed lung specimens
from five patients with cavitary lesions. (The
surgery was performed because the disease in
䡵 59
these patients was refractory to antimicrobial
therapy.) In three patients, both the sputum and
the cavitary lesions contained numerous tuber-
cle bacilli. However, in two patients both the
sputum and the cavitary lesions contained no
visible or culturable bacilli. In these two patients,
the surface of their cavities was epithelialized, and
no solid or liquefied caseous necrosis was present.
The cavity walls of the three sputum-positive
patients had an inner liquefied area containing
macrophages and many tubercle bacilli, sur-
rounded in turn by an area of acellular caseous
necrosis with few, if any, visible bacilli, followed
by an outer granulomatous fibrotic area con-
taining epithelioid macrophages, Langhans’ giant
cells, and lymphocytes (24).
In the area containing lymphocytes and
macrophages,the mRNAs of interleukin-2,inter-
leukin-12, gamma interferon, and inducible nitric
oxide synthase were measured by quantitative
reverse transcription-PCR. These markers of
good acquired resistance to tubercle bacilli were
found in ample quantities, which provides direct
evidence in humans for what Lurie observed in
his resistant inbred rabbits (11), namely, that hosts
with cavitary tuberculosis usually have good CMI
(acquired cellular resistance) but cannot control
the large numbers of bacilli that grow extracel-
lularly in the cavity lesions.
Many of the tubercle bacilli on the luminal
surface of the cavity walls resided in macrophages
(24). These macrophages probably were aspi-
rated into the cavity during spells of coughing,
because many macrophages enter the bronchial
tree from the alveolar spaces. Macrophages could
not have entered the cavity by migrating through
the adjacent area of caseous necrosis.
TUBERCULOSIS IN THE
IMMUNOCOMPROMISED HOST
HIV is a major cause of the overall increased inci-
dence of tuberculosis in the world today (20, 27).
In 1 year, the incidence of active disease in HIV-
infected tuberculin-positive persons is the same
as the incidence of active disease in immuno-
competent tuberculin-positive persons during
their entire lifespan. In the immunocompro-
mised patient, tuberculosis is most often due to
a reactivation of a latent focus acquired many
60 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 26 A tissue section from the lung depicted in Fig. 25. Caseosuppurative necrosis
can be seen on the left, and poorly organized tuberculous granulation tissue is on the right.
Stained with hematoxylin and eosin. Magnification, ⫻150. Reprinted with permission from
reference 1.

years previously, but primary tuberculosis also
occurs frequently—possibly because some pul-
monary alveolar macrophages are infected with
HIV and therefore have a decreased ability to
destroy inhaled tubercle bacilli. In such patients,
tuberculosis is usually more severe and more
rapidly progressive than in immunocompetent
patients. Their mortality rates are higher, and
extrapulmonary involvement is more common
(20, 27). In HIV/AIDS patients, the disease of
reactivation often resembles childhood tuber-
culosis with hilar adenopathy, frequent lower
lobe involvement, and the absence of both cav-
itation and extensive fibrosis (Fig. 25 and 26).
In all immunosuppressed patients, the mor-
phology of tuberculous lesions reflects poor host
resistance. For example, in corticosteroid-treated
patients (Fig. 27), tuberculous lesions show abun-
dant caseation with little or no encapsulation or
granulomatous reaction (Fig. 28A). Large num-
bers of bacteria are usually present within the
lesions (Fig. 28B). These changes are morpho-
logically similar to those found experimentally in
corticosteroid-treated tuberculous animals (11).
In patients with HIV/AIDS, the histologic
pattern of tuberculosis generally correlates with
the degree of immunosuppression. In pre-
mortem biopsies, granuloma formation is more
proliferative (and less exudative) than in
necropsy-derived tissues. As the number of
peripheral CD4 lymphocytes decreases, the pat-
tern of necrosis changes from caseous to caseo-
suppurative, i.e., caseous necrosis containing
neutrophils and more undigested nuclear debris
(Fig. 26). Also, the number of mycobacteria
within lesions increases (28). See reference 29 for
a complete overview of the types of tuberculo-
sis found in HIV/AIDS patients.
INAPPARENT ARRESTED PRIMARY
TUBERCULOUS LESIONS IN
HEALTHY HUMANS COMPARED
WITH RABBITS
Inapparent Lesions in Humans
Lindgren described a series of unselected necrop-
sies (made in Finland), among which were indi-
viduals who had been vaccinated with Mycobac-
terium bovis BCG and individuals who had not
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
(30–32).The cases were evaluated at a time when
the majority of the Finnish population had
become tuberculin positive because of inhaling
occasional virulent tubercle bacilli over many
years. In the BCG-vaccinated group, an average
of 11.2 years elapsed between the time when
BCG was given and when the necropsy was
performed (31).
These studies included X-ray examinations of
air-inflated lungs to detect calcified lesions
(down to 0.5 mm in diameter), many of which
were not identified by other methods.Almost all
䡵 61
FIGURE 27 Progressive miliary
tuberculosis and tuberculous pneu-
monia in the left upper lobe of an
immunocompromised 42-year-old
woman receiving corticosteroids for
dermatomyositis.An infarct is present
in the lower lobe. Reprinted with
permission from reference 1.
of the lesions detected were inactive and had
been completely arrested for years. Only one of
the patients in the necropsied series died of
active tuberculosis.
BCG vaccination did not prevent the estab-
lishment of detectable primary pulmonary
lesions caused by virulent human-type tuber-
cle bacilli. However, the lesions in the BCG
group were smaller than those in the unvacci-
nated control group, and the involvement of
the regional lymph nodes was substantially
reduced (30–32). In fact, the nodes of the
62 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 28 (A) Tissue section of an exudative tuberculous lesion from the lung shown in
Fig. 27, showing extensive caseous necrosis without granuloma formation or Langhans’ giant cells.
Within the adjacent alveoli (on the right) is an exudate of fibrin and caseous debris. Stained with
hematoxylin and eosin. Magnification, ⫻170. (B) Numerous intracellular and extracellular acid-
fast bacilli found at the edge of a necrotic area in the lung from the same patient. Stained with
Ziehl-Neelsen, counterstained with methylene blue. Magnification, ⫻1,700. Reprinted with per-
mission from reference 1.

vaccinated group often showed no visible evi-
dence of disease.
Comparison of Inapparent
Lesions in Humans and Rabbits
Lindgren’s studies clearly demonstrated that
humans are more susceptible than rabbits to
virulent human-type tubercle bacilli. In tuber-
culin-positive humans, each primary pulmonary
lesion developed to a size that is detectable at
necropsy. These lesions had a caseous center
and were often calcified (32). In tuberculin-
positive rabbits infected by aerosol, many micro-
scopic lesions apparently regressed before they
became visible (see chapter 11).
Humans and rabbits are both rather resistant
to tuberculosis caused by human-type tubercle
bacilli, because 95% of the lesions in humans do
not progress to clinically active disease, but
remain inactive unless host resistance is lowered.
Nonetheless, humans are slightly more suscep-
tible to tuberculosis than rabbits, because 5% of
humans do not arrest the infection without
antimicrobial therapy, but almost all rabbits do
so (11).
The difference between rabbits and humans
in the development of visible pulmonary lesions
is at least in part due to the tissue-damaging
DTH reaction that these species develop.Tuber-
culous humans are about 100 times more sen-
sitive than rabbits to tuberculin. Therefore,
caseous necrosis and eventual calcification more
readily occur in humans.
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Diseases and Disorders, 3rd ed., vol. 2. McGraw-Hill
Book Co., New York, N.Y.
2. Dannenberg, A. M., Jr. 1999. Pathophysiology:
basic aspects. I. Pathogenesis of tuberculosis. II.
Immunology of tuberculosis, p. 17–47. In D. Schloss-
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terial Infections, 4th ed. The W. B. Saunders Co.,
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3. Canetti, G. 1955. The Tubercle Bacillus in the Pul-
monary Lesion of Man, p. 130. Springer Publishing
Company, Inc., New York, N.Y.
4. Poole, J. C. F., and H.W. Florey. 1970. Chronic
inflammation and tuberculosis, p. 1183–1224. In
H. W. Florey (ed.), General Pathology, 4th ed. The
W. B. Saunders Co., Philadelphia, Pa.
 3. TYPES OF HUMAN PULMONARY TUBERCULOSIS
5. Rich,A. R. 1951. The Pathogenesis of Tuberculosis, 2nd
ed. Charles C Thomas Publisher, Springfield, Ill.
6. Iseman, M. D. 2000. A Clinician’s Guide to Tuber-
culosis. Lippincott Williams & Wilkins, Philadel-
phia, Pa.
7. Hopewell, P. C., and R. M. Jasmer. 2005.
Overview of clinical tuberculosis, p. 15–31. In S.T.
Cole, K. D. Eisenach, D. N. McMurray, and W. R.
Jacobs, Jr. (ed.), Tuberculosis and the Tubercle Bacillus.
ASM Press,Washington, D.C.
8. Medlar, E. M. 1955.The behavior of pulmonary
tuberculous lesions.A pathological study. Am. Rev.
Tuberc. 71:1–244.
9. Wolinsky, E. 1989. Tuberculosis, p. 465–519. In
G. L. Baum and E.Wolinsky (ed.), Textbook of Pul-
monary Diseases, 4th ed., vol. I. Little, Brown & Co.,
Boston, Mass.
10. Tomashefski, J. F., Jr. 1994, Tuberculosis and
atypical mycobacterial infections, p. 451–463. In
M. J. Saldana (ed.), Pathology of Pulmonary Disease.
Lippincott Williams & Wilkins, Philadelphia, Pa.
11. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defense Mech-
anisms. Harvard University Press, Cambridge, Mass.
12. Schlesinger, L. S. 1993. Macrophage phagocyto-
sis of virulent but not attenuated strains of Mycobac-
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in addition to complement receptors. J. Immunol.
150:2920–2130.
13. Schlesinger, L. S., C. G. Bellinger-Kawahara,
N. R. Payne, and M. A. Horwitz. 1990. Phago-
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human monocyte complement receptors and com-
plement component C3. J. Immunol. 144:2771–
2780.
14. Dannenberg, A. M., Jr. 1991. Delayed-type
hypersensitivity and cell-mediated immunity in the
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15. Dannenberg, A. M., Jr. 1993. Immunopatho-
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28:33–40 (Office edition, 51–58).
16. Kenyon, T. A., S. E. Valway, W. W. Ihle, I. M.
Onorato, and K. G. Castro. 1996.Transmission of
multidrug-resistant Mycobacterium tuberculosis during
a long airplane flight. N. Engl. J. Med. 334:933–938.
17. Wenzel, R. P. 1996. Airline travel and infection.
N. Engl. J. Med. 334:981–982.
18. Grosset, J. 2003. Mycobacterium tuberculosis in the
extracellular compartment: an underestimated
adversary. Antimicrob.Agents Chemother. 47:833–836.
19. Balasubramanian, V., E. H. Wiegeshaus, and
D. W. Smith. 1994. Mycobacterial infection in
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20. Hruban, R. H., and G. M. Hutchins. 1993.
Mycobacterial infections, p. 331–350. In D. H. Dial
and S. P. Hammar (ed.), Pulmonary Pathology.
Springer-Verlag, New York, N.Y.
䡵 63
21. West, J. B. 2000. Respiratory Physiology.The Essen-
tials, 6th ed., p. 50–59. Lippincott Williams &
Wilkins, Philadelphia, Pa.
22. Bennett,W. D., M. S. Messina, and G. C. Smal-
done. 1985. Deposition (normalized for lung vol-
ume) is generally greater in basal region of lung than
in apical region. Effect of exercise on deposition and
subsequent retention of inhaled particles. J. Appl.
Physiol. 59:1046–1054.
23. Lurie, M. B., A. G. Heppleston, S. Abramson,
and I. B. Swartz. 1950. An evaluation of the
method of quantitative airborne infection and its use
in the study of the pathogenesis of tuberculosis. Am.
Rev.Tuberc. 61:765–797.
24. Kaplan, G., F. A. Post, A. L. Moreira, H.Wain-
wright, B. N. Kreiswirth, M. Tanverdi, B.
Mathema, S.V. Ramaswamy, G.Walther, L. M.
Steyn, C. E. Barry III, and L.-G. Bekker.
(2003) Mycobacterium tuberculosis growth at the cav-
ity surface: a microenvironment with failed immu-
nity. Infect. Immun. 71:7099–7108.
25. Lillebaek,T.,A. Dirksen, E.Vynnycky, I. Baess,
V. O.Thomsen, and A. B. Andersen. 2003. Sta-
bility of DNA patterns and evidence of Mycobac-
terium tuberculosis reactivation occurring decades
after initial infection. J. Infect. Dis. 188:1032–1039.
26. Zhang,Y. 2004. Persistent and dormant tubercle
bacilli and latent tuberculosis. Front. Biosci. 9:1136–
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27. Barnes, P. F., A. B. Bloch, P.T. Davidson, and
D. E. Snider, Jr. 1991.Tuberculosis in patients with
human immunodeficiency virus infection. N. Engl.
J. Med. 324:1644–1650.
28. Hill, A. R., P. Somasundaram, S. Brustein, K.
Vaidya, S. Powell, P. Li, and B. Suster. 1991.
Disseminated tuberculosis in the acquired immu-
nodeficiency syndrome era. Am. Rev. Respir. Dis.
144:1164–1170.
29. Lucas, S., and A. M. Nelson. 1994. Pathogenesis
of tuberculosis in human immunodeficiency virus-
infected people, p. 503–513. In B. R. Bloom (ed.),
Tuberculosis: Pathogenesis, Protection, and Control.
ASM Press,Washington, D.C.
30. Lindgren, I. 1961. Anatomical and roentgeno-
logic studies of tuberculosis infection in BCG-
vaccinated and non-vaccinated subjects, with bio-
physical investigations of calcified foci. Acta Radiol.
209(Suppl.):1–101.
31. Lindgren, I. 1965.The pathology of tuberculous
infection in BCG-vaccinated humans. Adv.Tuberc.
Res. 14:202–234.
32. Sutherland, I., and I. Lindgren. 1979.The pro-
tective effect of BCG vaccination as indicated by
autopsy studies. Tubercle 60:225–231.
33. Shima, K., A. M. Dannenberg, Jr., M. Ando,
S. Chandrasekhar, J. A. Seluzicki, and J. I.
Fabrikant. 1972. Macrophage accumulation,
64 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
division, maturation, and digestive and micro-
bicidal capacities in tuberculous lesions. I. Studies
involving their incorporation of tritiated thymidine
and their content of lysosomal enzymes and bacilli.
Am. J. Pathol. 67:159–180.
34. Converse, P. J., A. M. Dannenberg, Jr.,T. Shi-
genaga, D. N. McMurray, S. W. Phalen, J. L.
Stanford, G. A. W. Rook, T. Koru-Sengul,
H. Abbey, J. E. Estep, and M. L. M. Pitt. 1998.
Pulmonary bovine-type tuberculosis in rabbits: bacil-
lary virulence, inhaled dose effects, tuberculin sen-
sitivity, and Mycobacterium vaccae immunotherapy.
Clin. Diagn. Lab. Immunol. 5:871–881.
35. Converse, P. J., A. M. Dannenberg, Jr., J. E.
Estep, K. Sugisaki,Y.Abe, B. H. Schofield, and
M. L. M. Pitt. 1996. Cavitary tuberculosis pro-
duced in rabbits by aerosolized virulent tubercle
bacilli. Infect. Immun. 64:4776–4787.
 4
LIQUEFACTION OF CASEOUS
FOCI AND CAVITY FORMATION

Overview 65
Part I. Liquefaction of caseous tissue and cavity formation: a review of the
literature 66
Role of delayed-type hypersensitivity 66
Role of hydrolytic enzymes 66
Caseation and liquefaction are two distinct processes 67
Bronchoscope production of cavities in rabbits 68
Dermal BCG lesions in pilot studies on caseation and
liquefaction 68
Effects of large numbers of tubercle bacilli on liquefaction 68
Part II. Tuberculosis produced in rabbits by aerosolized virulent
M. bovis 71
Background 71
Low-dose Experiments I and III 71
Unique events in the late disease of the low-dose
experiments 74
High-dose Experiment II 74
Tuberculin sensitivity 78
Histopathology 79
Part III. Recent experiments attempting to reduce liquefaction and cavity
formation 84
Studies with Mycobacterium vaccae 84
Preliminary studies with the proteinase inhibitor Ritonavir 86
Summary of cavity formation and suggestions for its prevention 87
A proposed method to study liquefaction and cavity formation 90

Abstract. Part I is a review of the literature on liquefaction and cavity formation. Liq-
uefaction seems to be a delayed-type hypersensitivity reaction to the tuberculin-like
products of the bacillus. It seems to be carried out by hydrolytic enzymes from the sur-
rounding host cells and possibly by enzymes within the caseum after inhibitors have dis-
sipated. Liquefied caseum and cavities occur frequently in rabbits inhaling virulent bovine-
type tubercle bacilli.They also occur occasionally in rabbits inhaling virulent human-type
tubercle bacilli. Part II describes experiments of long duration in which pulmonary cav-
ities were produced in commercial New Zealand White rabbits by aerosolized virulent
bovine-type bacilli (Mycobacterium bovis, Ravenel strain).After the inhalation of a low num-
ber of these bacilli, liquefied caseum and cavities occurred in 6 to 10 weeks.After the inhala-
tion of a high number of these bacilli, liquefied caseum and cavities occurred sooner. Details
on the gross pathology and histopathology of these pulmonary lesions are presented. Part
III describes two attempts to reduce liquefaction and cavity formation in tuberculous rab-
bits. One was immunotherapy with Mycobacterium vaccae.The other was therapy with Riton-
avir, a proteinase inhibitor. Neither treatment (as administered) had any observable effect.

OVERVIEW Liquefaction of solid caseous foci in the lung

The chronic human-type tuberculosis with liq- perpetuates tuberculosis in humans because such
uefaction and cavity formation is easily pro- liquefaction enables the extracellular growth of
duced in rabbits, but it is not as easily produced tubercle bacilli and the formation of cavities
in guinea pigs and cannot be produced in mice. that discharge such bacilli into the airways and

65
66 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
into the environment. In liquefied caseum, espe-
cially that in the cavity walls, tubercle bacilli
sometimes grow to tremendous numbers, which
makes the development of antimicrobial resis-
tance more likely.
Chapter 7 describes the pathophysiology of
liquefaction and cavity formation. Chapter 3
presents details on the human disease. Refer-
ences 1 through 7a below review various aspects
of cavitary tuberculosis.
PART I. LIQUEFACTION OF CASEOUS
TISSUE AND CAVITY FORMATION:
A REVIEW OF THE LITERATURE
Role of Delayed-Type Hypersensitivity
Yamamura’s group published the most significant
studies on the causes of liquefaction (8–10; see
also reference 11). Pulmonary cavities were pro-
duced in 2 to 4 weeks by the injection of vari-
ous materials (emulsified in paraffin oil and lano-
lin) through the chest wall directly into the lungs
of rabbits. Both living and dead tubercle bacilli,
paraffin-oil extracts of heat-killed tubercle bacilli,
and the particulate and proteolipid fractions of
Mycobacterium bovis BCG were all effective if the
rabbits were previously made tuberculin positive
by subcutaneous injections of heat-killed tuber-
cle bacilli in adjuvant. Rabbits not previously sen-
sitized did not develop cavities as frequently, and
those that did had become tuberculin positive
from the intrapulmonary injection. Similar results
were obtained in guinea pigs. Desensitization of
tuberculin-positive rabbits with tuberculoproteins
(9) or a tuberculin-active peptide (10) prevented
cavities from forming.
Azathioprine, an immunosuppressive drug,
also prevented cavities (12), probably by reduc-
ing the amount of delayed-type hypersensitivity
(DTH) (6).When live tubercle bacilli (H37Rv)
were injected (through the chest wall) directly
into the lungs of rabbits, no cavities formed in
the antimicrobial-treated rabbits simultaneously
treated with azathioprine, but cavities readily
formed in the antimicrobial-treated controls that
were not treated with azathioprine (12).
References 2, 3, 4, and 13 describe additional
studies supporting the premise that DTH is
required for solid caseum to liquefy. DTH
apparently does so in the following manner. By
means of cytokines (including chemokines) (14,
15), DTH increases the number of macrophages
surrounding the caseous center. Then, cell-
mediated immunity (CMI) (and DTH) acti-
vates these cells (16), thereby raising their level
of hydrolytic enzymes.Also, DTH increases the
blood flow through the lesions, which probably
lowers the level of the enzyme inhibitors in
the solid caseum. An accompanying antigen-
antibody reaction may play a role by activating
complement, because C3a, C4a, and C5a cause
vasodilation, and the chemotaxin C5a also
attracts leukocytes into the site (15).
Role of Hydrolytic Enzymes
Hydrolytic enzymes play a major role in lique-
faction and cavity formation (6). Products result-
ing from the hydrolysis of proteins and other
macromolecules increase the osmolarity of the
caseum.Water is absorbed, and the fluidity of the
caseum is increased.The most significant stud-
ies on the hydrolytic enzymes involved in liq-
uefaction were performed in the laboratories of
Weiss (17–19) and Dannenberg (20–23).
Weiss and associates produced tubercles in
rabbits by airborne infection with virulent M.
bovis. Caseous centers were present after 7 weeks,
and liquefied centers were present after 12
weeks. By 20 weeks, the cavities were large and
coalescing. Samples of normal lung, tuberculous
granulation tissue from the walls of caseous and
liquefying foci, solid caseum, and completely liq-
uefied caseum were homogenized and assayed
for proteases and nucleases by two different
methods.
In the first method, substrates, e.g., benzoyl-
argininamide, leucinamide, DNA, or RNA,
were added to the homogenates, and their
hydrolytic products were measured. In the sec-
ond method, the homogenates were allowed to
autolyze under toluene at 35°C for 1 to 6 days,
after which the breakdown products of pro-
tein, DNA, and RNA were measured. Both
methods yielded similar results. Tuberculous
granulation tissue had high protease and nucle-
ase activity. Normal lung tissue and recently
caseating tissue had moderate activity. Older
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION
caseous tissue and liquefied tissue had little or no
activity.
Using histochemical techniques, including the
Daoust substrate film technique (24–27), our
laboratory (20–23) provided direct visual evi-
dence for the findings of Weiss and associates.
Proteinase (cathepsin D), DNase, RNase,
hyaluronidase, and other enzymes, e.g., acid phos-
phatase, -galactosidase, -glucuronidase, esterase,
succinic dehydrogenase, and cytochrome oxi-
dase, were most active in live and dead macro-
phages surrounding the caseous and liquefied
centers (7a).The activity of these enzymes dimin-
ished to near zero in fully liquefied areas. Most of
these studies were evaluated in rabbit dermal
BCG lesions with liquefied caseous centers, but
some were evaluated in rabbit cavitary pulmonary
lesions caused by virulent bovine-type tubercle
bacilli (Ravenel) (7a, 20). Immunohistochemical
methods confirmed that cathepsin D was present
in epithelioid cells at the edge of the liquefied
caseum of a pulmonary cavity (20).
In pulmonary tuberculous lesions of
humans, guinea pigs, mice, and rabbits, Oshima
and coworkers evaluated lipases by histo-
chemical methods (28).The Gomori calcium-
lead method was used with polyoxyethylene
sorbitan-monostearate and Tween 80 as sub-
strates. The caseous material in these lesions
showed no lipase activity. Other studies on
lipases and esterases in tuberculosis are reviewed
in references 3 and 18.
Enzyme inhibitors from the bacilli and the
necrotic host tissues probably exist in caseous
material.Their presence may be the reason why
solid caseum does not liquefy for at least a num-
ber of weeks (3, 18, 19).To date, such inhibitors
have not been characterized.
Pepstatin is a nontoxic pentapeptide from
Actinomycetes sp. (29) discovered by Umezawa’s
group. Pepstatin is a highly effective inhibitor of
pepsin (29–31) and rabbit macrophage cathepsin
D (32). However, in two pilot studies, pepstatin
had no observable effect on rabbit dermal BCG
lesions when administered either subcutaneously
or directly into these lesions (32), so these stud-
ies were not continued. Pepstatin also did not
affect the proteinase activity of macrophages or
䡵 67
BCG lesions when studied in tissue sections by
gelatin-substrate film techniques (23).
The level of -galactosidase, our classic lyso-
somal marker for macrophage activation, was
always highest in macrophages surrounding liq-
uefied caseum and was decreased in areas farther
away (11). The high levels of -galactosidase
and other hydrolytic enzymes in macrophages
immediately surrounding the caseous and liq-
uefied centers were possibly due to their inges-
tion and digestion of the necrotic material.“A
good meal in the belly of macrophages” increases
their content of digestive enzymes (33).
Once started, the liquefaction of solid caseous
material seems to be autocatalyic.The ingestion
of liquefied caseous material by the surround-
ing macrophages increases the level of their
hydrolytic enzymes, and the higher level of
these enzymes hydrolyzes solid caseous tissue
more efficiently. Table 1 summarizes the main
causes and results of liquefaction.
Caseation and Liquefaction
Are Two Distinct Processes
References 11 and 34 through 36 review the lit-
erature on caseous necrosis and cavity formation.
However, the distinction between these two
processes is not clearly addressed in references 34
and 35. Solid caseum is caused by the killing of
viable tissue, whereas liquefied caseum is caused
TABLE 1 Causes and results of liquefactiona
Causes
Delayed-type hypersensitivity (DTH) to tuberculin-
like bacillary products
Hydrolytic enzymes, especially proteases, DNases, and
RNases, and probably lipases
Results
Extracellular bacillary multiplication (that is
sometimes tremendous), which may allow
antimicrobial drug-resistant mutants to develop
High concentrations of tuberculin-like products that
cause caseous necrosis of the bronchial wall and a
cavity to form
Spread of bacilli through the air passages to other
parts of the lungs and to the environment, where
they may infect other people
a
Adapted from reference 6.
68 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
by the hydrolysis of solid caseum after it has
formed. Caseous necrosis may lead to liquefac-
tion and cavity formation, but in both rabbits
and humans the majority of caseous lesions do
not liquefy or form cavities.The caseum merely
inspissates, may be walled off by fibrosis, and in
humans often calcifies (see chapter 3). Follow-
ing the inhalation of fully virulent bovine-type
bacilli, Lurie’s susceptible rabbits produced pul-
monary tuberculous lesions with considerable
caseous necrosis, but these lesions never lique-
fied and cavitated (4).
Tuberculous lesions in guinea pigs show con-
siderable caseous necrosis, but liquefaction and
cavities only occur in some guinea pigs and
then only if 1 or 2 pulmonary lesions are pre-
sent (see chapter 15). Reference 36 is a rather
complete review of caseation and cavity for-
mation in rabbits, mice, guinea pigs, humans, and
nonhuman primates. Cavities are readily pro-
duced in rabbits and humans, but they are not
easily produced in guinea pigs and never occur
in mice.
However, like Lurie’s resistant rabbits (4),
commercial New Zealand White rabbits (20, 37)
often form cavities after inhaling virulent
bovine-type tubercle bacilli if given sufficient
time. After the inhalation of virulent human-
type tubercle bacilli, these hosts may also form
cavities (4, 36, 38, 39), but they do so less fre-
quently than after the inhalation of virulent
bovine-type bacilli. Cavity formation in com-
mercially available New Zealand White rabbits
with aerosolized virulent bovine-type tubercle
bacilli is described in part II of this chapter.
Bronchoscope Production of Cavities
in Rabbits
Rabbits were sensitized to tuberculin by inject-
ing subcutaneously 107 heat-killed virulent
bovine-type tubercle bacilli (Ravenel strain) in
a mineral oil-lanolin adjuvant on four different
occasions 3 or 4 days apart (see reference 35).At
21 days after the last injection, 2 ⫻ 104 live vir-
ulent bovine-type tubercle bacilli (Ravenel)
were instilled with a bronchoscope into the
right lower lung. Pulmonary cavities occurred
in every rabbit within 3 to 5 weeks.The instal-
lation of bovine-type tubercle bacilli seemed
to produce cavities more readily than did the
instillation of virulent human-type bacilli. If
the rabbits were not sensitized to tuberculin, cav-
ities were not produced as easily, which con-
firmed Yamamura’s findings (described above).
This bronchoscopic method has the advan-
tage of not requiring an aerosol apparatus to
infect the rabbits.Also, it produces cavities more
quickly, i.e., in 3 to 5 weeks rather than in 10
weeks. However, the bronchial instillation of a
large number of tubercle bacilli does not match
the normal way of developing pulmonary tuber-
culous cavities, in which each pulmonary tuber-
culous lesion develops from a single inhaled
unit containing only 1 to 3 bacilli.
Dermal BCG Lesions in Pilot Studies
on Caseation and Liquefaction
A simpler way to produce liquefaction with
large numbers of bacilli in one location would
be to inject BCG intradermally (see proposed
method beginning on p. 90). The resulting
lesions caseate, liquefy, and ulcerate, discharging
the liquefied caseum, similar to tuberculous
lesions produced bronchoscopically in the lungs.
In rabbits already sensitive to tuberculin, BCG
lesions ulcerate more quickly (called the Koch
phenomenon).
Cathepsin D is a major protease of mac-
rophages (21, 40–43). It exists at high levels in
macrophages surrounding the liquefied caseous
centers of BCG lesions (21).This distribution
of cathepsin D is similar to that found in the
walls of pulmonary cavities produced by
aerosols of virulent tubercle bacilli (20).There-
fore, for pilot studies of liquefaction and cavity
formation, we highly recommend the dermal
BCG model. It does not require an aerosol
apparatus and can be performed in biological
level 2 facilities.
Effects of Large Numbers of
Tubercle Bacilli on Liquefaction
Both bronchoscopically induced pulmonary
lesions and dermal BCG lesions are produced by
injecting large numbers of tubercle bacilli in
one location.The presence of a large number of
bacilli apparently accelerates the liquefying
process (see reference 9). Tubercle bacilli and
4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 69

FIGURE 1 A multiloculated cavity in the lung of a

rabbit (#8, Experiment I) that had inhaled 620 virulent
bovine-type tubercle bacilli (Ravenel S) 19 weeks previ-
ously.The hole at the upper right side of the photograph
is an intact bronchus.The hole at the lower left is part of
the loculated cavity.The center of each is black because the
back was sliced from the specimen to stabilize it for pho-
tography. Note the fibrosis around the large cavity in the
center of the photograph. Magnification, ⫻3. Reprinted
with permission from reference 20.
This is an example of the slowly progressive cavitary
disease frequently found in humans (see chapter 3).
Note:Table 2 of reference 20 lists the necropsy find-
ings on each rabbit that lived 18 or more weeks in the
three experiments that we performed. I did not reproduce
the entire table in this chapter but have listed the rabbit
and experiment numbers in many of the figures, so that
interested readers can easily obtain (from reference 20)
more details on the characteristics of disease in the rab-
bits providing the specimens for these photographs.

FIGURE 2 Multiple cavities in the upper part of the

right lower lobe of the lung of a rabbit (#5, Experiment
III) euthanized 33 weeks after the inhalation of 340 vir-
ulent bovine-type tubercle bacilli.These are mostly sec-
ondary and tertiary cavities that developed locally from
the bronchogenic spread of large numbers of bacilli from
one or two primary lesions. Despite the extensive tuber-
culosis in the upper part of this lobe, no secondary lesions
were grossly visible in the lower part. In addition, the
whole left lung of this rabbit was free of grossly visible
tuberculous lesions. Magnification, ⫻1. Reprinted with
permission from reference 20.
This is an example of the local nature of tuberculosis and
the efficacy of acquired (adaptive) immunity that prevents
the progress of tuberculous lesions arising elsewhere from
relatively low numbers of inhaled bacilli.
70 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 3 Specimen sliced from the right side of the

right lower lobe of rabbit #5, Experiment III (Fig. 2). Note
the absence of functional lung tissue, the large size of the
cavities, the residual semisolid caseous tissue (center), and
the extensive fibrosis. Magnification, ⫻4.

FIGURE 4 Tuberculous lesions in the right upper

lobe of rabbit #5 (Experiment III). (The right lower
lobe of this rabbit is depicted in Fig. 2.) Two large cavi-
ties have been sliced open. Note that most of the lesions
are well encapsulated with fibrous tissue. Fibrous encap-
sulation of tuberculous lesions is common in chronic
human cavitary disease. Magnification, ⫻2.
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 71

their tuberculin-like products are highly chemo-

tactic for monocytes/macrophages and other
cells.These macrophages produce proteases and
other hydrolytic enzymes (see chapter 6), which
can liquefy necrotic tissue.
Tubercle bacilli themselves produce pro-
teinases, nucleases, and lipases (3), which may or
may not play a role in the liquefaction of solid
caseum. Granulocytes also contain such
hydrolytic enzymes, but they are not a promi-
nent feature of most liquefying caseum (9).

PART II. TUBERCULOSIS PRODUCED

IN RABBITS BY AEROSOLIZED
VIRULENT M. BOVIS
Background
Lurie produced liquefaction and cavities in his
inbred resistant rabbits by allowing them to
breathe air containing a very low number of vir-
ulent bovine-type tubercle bacilli (Ravenel
strain) in the animal room over a period of
many months—an experiment not likely to be
repeated today (see chapter 12). Lurie (4, 44), as
well as others (45, 46), also produced cavities by
aerosols of bovine-type tubercle bacilli. In fact,
even aerosols of human-type bacilli, which are
less virulent for rabbits, sometimes produce cav-
ities (4, 38, 39).
Rabbits do not usually develop the extensive
bronchopneumonia that some humans develop
(see chapter 3). Extensive bronchopneumonia in
humans is apparently caused by a DTH reaction
to the tuberculin-like products in the liquefied
caseum that enters the alveoli, and humans are
at least 100 times more sensitive than rabbits to
tuberculin.
Rabbits do not cough.Therefore, the spread
of the disease to other animals housed in the
same room is less frequent than in rooms where
tuberculous monkeys are housed. Rabbits
sneeze, however, which may aerosolize tubercle
bacilli lodged in their nasal passages.
The following is a description of our exper-
iments in which rabbits inhaled aerosols of vir-
ulent bovine-type tubercle bacilli. They show
that cavitary disease is easily produced in this
animal species. Similar experiments were de-
scribed by Lurie in his inbred rabbits (4).
FIGURE 5 Tuberculous laryngitis in a rabbit (#4,
Experiment III) that had inhaled 300 virulent bovine-
type tubercle bacilli 33 weeks previously.The lung of this
rabbit had 14 lesions, 4 of which had formed cavities.
A cavity in the right lower lung apparently discharged
so many bacilli into the bronchial tree that the larynx
became infected. Eventually, the airway became partly
obstructed, so the animal was breathing with difficulty
at the time it was euthanized. Magnification, ⫻5.4.
Reprinted with permission from reference 20.
Before antimicrobials were available to treat tuber-
culosis, a similar tuberculous laryngitis occasionally
occurred in humans. Such patients required a tra-
cheotomy to stop the labored breathing.
Low-Dose Experiments I and III
In our low-dose experiments, commercial New
Zealand White rabbits (20, 37) inhaled 220 to
940 virulent bovine-type tubercle bacilli
(Ravenel S from the ATCC [20]).This strain was
of slightly reduced virulence for rabbits, but
still sufficiently virulent to cause extensive dis-
ease and even death (20, 37). In Experiment I,
two rabbits (each time) were euthanized at 5, 8,
10, and 13 weeks after the aerosol infection.
Their lungs contained 1 to 18 grossly visible or
72 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
FIGURE 6 A caseous tubercle at the bifurcation of
a bronchus near the hilus in a rabbit (#12, Experiment
III) that had inhaled 420 virulent bovine-type tubercle
bacilli (Ravenel S). We euthanized this rabbit at 30
weeks, because it had labored breathing. Magnification,
⫻4.9. Reprinted with permission from reference 20.
Before antimicrobials were available to treat tuber-
culosis, similar lesions occasionally occurred in the
bronchial tree in human beings.
palpable primary pulmonary tubercles. By 5
weeks, the tubercles had developed solid caseous
centers. By 8 weeks, some of the centers had liq-
uefied. And, by 10 and 13 weeks, some of the
lesions contained small cavities. When the
remaining rabbits were euthanized (at 19 and 22
weeks in Experiment I and at 33 weeks in
Experiment III), over half of the solid caseous
foci had liquefied, and many cavities had formed
(Fig. 1 through 4).
The disease varied greatly among the indi-
vidual rabbits. In some, hardly any disease was
present, whereas in others, half of a pulmonary
lobe was destroyed. Most of the rabbits appeared
to have their disease under control, because
their pulmonary lesions were surrounded by
FIGURE 7 A caseous tubercle partly obstructing the
lumen of the trachea (above) and constricting the esoph-
agus (bisected below) (rabbit #12, Experiment III).
Magnification, ⫻4.9.
The esophageal constriction apparently caused such
malnutrition that the resistance of the rabbit was
decreased and many small metastatic pulmonary lesions
developed.
considerable fibrosis. In Experiment III, one
rabbit had four 2- to 3-mm pulmonary lesions
that were almost healed.
Secondary pulmonary lesions were found in
the lungs of several rabbits.They probably arose
by bronchial spread of the disease from one or
more cavities.The secondary lung lesions were
usually much smaller than the primary lesions.
Therefore, most of the primary lesions could be
identified at necropsy.The lymphoid tissue of the
ileocecal junction and appendix of these rabbits
often contained 1- to 2-mm lesions, which
came from the numerous bacilli (growing in
cavities) that ascended the bronchial escalator
and were swallowed (47). One rabbit had two
small (3 to 4 mm) tubercles in one of its kidneys
(probably of hematogenous origin).
The tracheobronchial lymph nodes were
slightly enlarged and occasionally contained
tuberculous granulomas. Similar to immuno-
competent adult humans, rabbits with moder-
ate to high native resistance to tuberculosis do
not develop progressive lesions in their hilar
nodes (4, 5, 47).
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 73

TABLE 2 Data on rabbits dying of pulmonary tuberculosis in the high-dose Experiment II

Approximate A B C
time of death No. of No. of Ratios:
Treatment (or time inhaled visible A/B No. of
(rabbit no.) euthanized virulent cavitiesb Comment
primary for each
when near bacillary lesionsa rabbit
death) (wk) units
Control #2 5 4,800 550 8.7 None Numerous 3- to 4-mm
lesions with minute
caseous centers
M. vaccae #9 6 5,800 210 27.6 Many Over half of the lesions
had cavitated
Control #11 7 4,700 260 18.1 Many Over half of the lesions
had cavitated
Control #10 8.5 3,900 250 15.6 Many Over half of the lesions
had cavitated
Control #4 9 5,300 500 10.6 Many Numerous cavities were
present
Means 4,900 354 13.8
a
Includes primary lesions with cavities. Details on the remaining six rabbits of this high-dose experiment are presented in Table
1 of reference 20. Reproduced with permission from reference 37.
b
Note that, in this high-dose experiment, cavities were present at 6 to 9 weeks after infection.

FIGURE 8 Pulmonary tuberculosis in the left lung of a rab-

bit (#8, Experiment II) that had inhaled 5,500 virulent bovine-
type tubercle bacilli 18 weeks previously.The pleural surface of
the lung is roughened by numerous coalescing tubercles. Mag-
nification, ⫻1.2.
Tuberculous pleurisy frequently occurs in humans due to
similar lesions just below the pleural surface (see chapter 3).
74 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 9 The same lung shown in Fig. 8, bisected horizontally. The lung contains
numerous primary and secondary lesions and many minute cavities. Note how little functional
pulmonary tissue remains in the upper lobe. Magnification, ⫻1.2.
Similar local consolidation occurs in humans (see chapter 3).

Unique Events in the Late Disease of
the Low-Dose Experiments
At 33 weeks, one rabbit showed honeycomb
formations, i.e., multiple coalescing cavities in
both its right upper and right lower lung lobes
(20) (Fig. 2 through 4). Evidently, one or more
primary cavitary lesions had discharged many
bacilli locally.These bacilli created new lesions
nearby, which in turn cavitated. However, despite
the extensive destruction in these two lung
lobes, the rabbit remained in good health, and
the remainder of its lungs showed no grossly vis-
ible evidence of tuberculosis.
Two other rabbits were euthanized because
they showed labored breathing and had lost
weight.The rabbit euthanized at 33 weeks had
extensive tuberculous laryngitis, which nar-
rowed the airways (Fig. 5).The rabbit euthanized
at 30 weeks had two caseous lesions that nar-
rowed the airways (Fig. 6 and 7). One of these
lesions also partly obstructed the adjacent esoph-
agus (Fig. 7).The rabbit euthanized at 30 weeks
was the only rabbit in any of the three experi-
ments that had numerous metastatic caseous
lesions (0.5 to 2.0 mm in diameter) in the lungs,
which were probably caused by a decrease in
host resistance from the malnutrition (1).
High-Dose Experiment II
In Experiment II, New Zealand White rabbits
(20, 37) (inbred by L. F. M. van Zutphen at the
University of Utrecht, The Netherlands)
inhaled 3,900 to 5,800 virulent bovine-type
tubercle bacilli (Ravenel S). Between 5 and 9
weeks, about half of the rabbits had to be euth-
anized because of impaired breathing. Their
lungs contained between 210 and 550 pro-
gressing primary lesions (Table 2). Most of
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 75

FIGURE 10 Pulmonary cavities from two rabbits (in high-dose Experiment II) that had inhaled
about 5,000 virulent bovine-type tubercle bacilli 18 weeks previously. Below each cavity are
encapsulated fibrotic lesions with large semisolid caseous centers that had not discharged into
bronchi.The amount of softening of the caseous material cannot be accurately assessed in for-
malin-fixed tissues, because it is hardened by the fixation process.At necropsy, the consistency
of the caseum varied from that of a “hard” cheese to that of a rather “soft” cheese. Magnifica-
tion, ⫻2.7. Reprinted with permission from reference 20.

these pulmonary lesions had liquefied centers,
and after 6 weeks many cavities were present
(Table 2).
The remaining rabbits were euthanized at
18 weeks. Because they survived so long, their
pulmonary lesions were well encapsulated (Fig.
8 through 11).These “high-dose” rabbits, how-
ever, were less active, more emaciated, and often
had dyspnea upon exertion.All of these rabbits
showed secondary lesions in the appendix and
ileocecal junction, apparently from swallowing
bacilli that ascended the bronchial tree from
the multiple cavities that were present.
Due to the large number of primary pul-
monary lesions and the long duration of the
experiment, we could not always differentiate
secondary lesions from primary lesions in the
lung, but we could do so with about 90%
accuracy.The rabbits in the high-dose exper-
iment averaged 310 primary tubercles, whereas
the rabbits in the two low-dose experiments
averaged only 6.7 and 8.8 primary tubercles,
respectively (Table 3).
When compared with the rabbits in the low-
dose experiments, these rabbits showed larger tra-
cheobronchial lymph nodes with more caseation,
apparently because of increased numbers of
bacilli. However, none of these lymph nodes
showed the extensive caseous necrosis of Lurie’s
susceptible rabbits (4, 5, 7). In other words, an
adult type rather than a childhood type of tuber-
culosis was produced (1, 6) (see chapter 1).
These inbred van Zutphen rabbits and Lurie’s
highly resistant strain III rabbits originated from
the same inbred stock at the Jackson Laborato-
ries in Bar Harbor, Me. (see chapter 14). As
expected, the variation in the number of pri-
mary pulmonary tubercles found among them
seemed to be less than that found among the
commercial rabbits (20, 37), but too few rabbits
were in these experiments to prove this obser-
vation statistically.
 FIGURE 11 Walled-off tuberculous lesions from the lungs of rabbits (in high-dose Exper-
iment II) 18 weeks after the inhalation of about 5,000 virulent bovine-type tubercle bacilli.
In the specimen on the right, there are several semisolid caseous lesions with thick fibrous cap-
sules and one cavity. In the specimen on the left, the amount of fibrosis is so extensive that
little functional lung tissue remains. Magnification, ⫻3.1. Fibrocaseous tuberculosis is a fre-
quent occurrence in humans.

TABLE 3 Ratios: number of inhaled bovine-type tubercle bacilli that produced one grossly visible primary
pulmonary lesion in commercial rabbitsa
A B
Inhaled dose of No. of C
Experiment
viable bovine-type visible primary Ratios: A/B
no.
tubercle bacilli (Ravenel S) pulmonary lesions for each rabbitb
(bacillary units) (including cavities)
Experiment I 523 ⫾ 137 6.7 ⫾ 2.3 107 ⫾ 29
(low dose) (523/6.7 ⫽ 78)
6 rabbits
Experiment II 5,100 ⫾ 186 310 ⫾ 36 18.2 ⫾ 1.7c
(high dose) (5,100/310 ⫽ 16.5)
11 rabbits
Experiment III 418 ⫾ 24 8.8 ⫾ 1.2 63 ⫾ 13
(low dose) (418/8.8 ⫽ 48)
12 rabbits
a
The grossly visible primary lesions of the M. vaccae and control groups were pooled to prepare this table.The means and their
standard errors are listed.The P values for the ratios in column C are as follows: for 107 and 18.2, P ⫽ 0.001; for 107 and 63, P
⫽ 0.124; and for 18.2 and 63, P ⫽ 0.003 (Student’s t test). Reproduced with permission from reference 37.
b
The ratio for each rabbit is the number of inhaled bacilli divided by the number of visible primary pulmonary lesions.The
means in the ratio column were derived from individual ratios.They are not the means in column A divided by the means in
column B, which are given in parentheses.The difference between the two methods is discussed in chapter 11.
c
The difference in ratios between Experiment II and Experiments I and III suggests that the acquired immune response has
a limited capacity to prevent developing primary pulmonary lesions from reaching grossly visible size (see text).

76
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 77

TABLE 4 M. vaccae immunotherapy of rabbits with active tuberculosis produced by the inhalation of virulent
bovine-type tubercle bacilli (Ravenel S)a
High-dose Experiment II Low-dose Experiment III
(survivors euthanized (survivors euthanized
at 18 weeks) at 33 weeks)
M. vaccae Controls M. vaccae Controls
Immunotherapy schedule 5, 9, and 13 weeks 7, 12, 17, 22, and 27 weeks
Rabbit no. 1, 3, 5, 7, 9 2, 4, 6, 8, 10, 11 2, 4, 6, 7, 9, 12 1, 3, 5, 8, 10, 11
No. of inhaled virulent 5,400 ⫾ 270 4,850 ⫾ 230 395 ⫾ 39 440 ⫾ 29
bacillary units P ⫽ 0.149 P ⫽ 0.417
No. of grossly visible 252 ⫾ 26.3 359 ⫾ 56.8 8.0 ⫾ 1.9 9.5 ⫾ 1.5
primary lesions in lungs P ⫽ 0.145 P ⫽ 0.545
(including those with
cavities)
No. of visible primary 3.0 ⫾ 0.9 1.8 ⫾ 0.8
pulmonary lesions P ⫽ 0.341
with cavities
Percentage of visible 35.7 ⫾ 11.9 18.7 ⫾ 9.5
primary lesions that P ⫽ 0.291
cavitated
Dermal tuberculin 2,606 ⫾ 562 mm3; 2,657 ⫾ 396 mm3
sensitivity at 4 weeks
Dermal tuberculin 1,038 ⫾ 191 mm3; 1,283 ⫾ 340 mm3
sensitivity at 33 weeks
No. of metastatic lesions 428 ⫾ 258 320 ⫾ 50 89 ⫾ 47 149 ⫾ 82
in the ileocecal and P ⫽ 0.796 P ⫽ 0.539
appendix lymphoid
tissues
Weight at time of 2.5 ⫾ 0.2 2.7 ⫾ 0.2 4.0 ⫾ 0.3 4.2 ⫾ 0.3
necropsy (in kg) P ⫽ 0.570 P ⫽ 0.578
a
This table summarizes information published in references 20 and 37.The number of primary lesions was derived from all
of the rabbits in each group, including those in Table 2 of this chapter that died early.The other data are from the survivors at the
two times of euthanasia (above). Means and their standard errors are presented. Student’s t test was used to determine significance.
Note that M. vaccae immunotherapy had no apparent effect on the number of grossly visible primary lesions, on the percentage
of those lesions that cavitated, or on the tuberculin sensitivity of the host.

In brief, the high dose of virulent bovine-type
tubercle bacilli probably taxed host resistance to
its limits, so that the hosts with somewhat
weaker resistance succumbed to the disease by
9 weeks. However, most of the hosts with
stronger resistance lived comfortably until they
were euthanized at 18 weeks.
The high dose also had an unexpected effect
on the number of inhaled bacillary particles
required to produce one grossly visible primary
tubercle (called “the ratio” by Lurie [4]).Table 3
shows that, when calculated by the Lurie method
described in chapter 11, only 18 inhaled bacilli
were required to produce one visible tubercle in
the high-dose experiment, whereas 63 and 107
inhaled bacilli were required to do so in the
low-dose experiments. The large number of
inhaled bacilli evidently reduced the local effi-
cacy of the host’s innate and acquired immune
responses, so that 3 to 6 times the number of
microscopic tubercles reached visible size.There-
fore, the host immune system apparently has a
limited capacity, and this capacity was exceeded
in the high-dose experiment. Many factors may
be involved in this dose effect, such as a limited
number of defense cells, changes in Th1/Th2
78 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 12 A glycol methacrylate-embedded tissue section of a tuberculous lesion, from

a rabbit (#8, Experiment I) that had inhaled 620 virulent bovine-type tubercle bacilli 19 weeks
previously.The section was stained histochemically for -galactosidase, which is our marker for
activated macrophages: the darker the blue color produced, the more activated the macrophage
(see chapter 6). This photograph shows portions of three caseous centers, surrounded by
tuberculous granulation tissue containing numerous -galactosidase-positive macrophages
(epithelioid cells). Peripheral to these macrophages are lymphocytes, plasma cells, fibroblasts,
and unactivated (-galactosidase-negative) macrophages.At the lower left are a few alveoli (with
thickened walls) containing accumulations of highly activated alveolar macrophages, which are
easily recognized by their dark -galactosidase-staining cytoplasm. Counterstained with
Giemsa. Magnification, ⫻100. Reprinted with permission from reference 20.

ratios (discussed in reference 37), and increased
glucocorticoid production.
The dose effect did not, however, occur when
virulent human-type tubercle bacilli were
inhaled, apparently because the human type is
less virulent for rabbits than the bovine type
(39). In fact, the dose effect with the human type
seemed to be in the opposite direction: when a
higher number were inhaled, more (not fewer)
human-type bacilli were apparently required to
produce each visible tubercle. See chapter 11 for
a complete discussion of tubercle counts.
Tuberculin Sensitivity
When infected with tubercle bacilli, humans
are about 100 times more sensitive to tuberculin
than are rabbits (see PPD in the glossary).
(Tuberculous guinea pigs are about twice as
sensitive as tuberculous rabbits.) In the rabbits of
low-dose Experiment III with well-controlled
tuberculosis, tuberculin sensitivity decreased
about 50% between 4 and 33 weeks (Table 4)
(20, 37). Humans with well-controlled clinical
tuberculosis may not show as great a reduction
in tuberculin sensitivity. These species differ-
ences may be due to partial desensitization to
tuberculin by the nonpathogenic mycobacteria
in the diet of rabbits (see reference 8).
The studies of Yamamura (8–10, 12) indicate
that tuberculin sensitivity is necessary for cavi-
ties to form in rabbits. In these studies, tubercle
bacilli (or their components) were injected
directly into the lungs through the chest wall
(discussed above in part I).
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 79

FIGURE 13 A glycol methacrylate-embedded tissue section of a tuber-

culous lesion from a rabbit (#8, Experiment I) that had inhaled 620 virulent
bovine-type tubercle bacilli 19 weeks previously.This section was stained for
acid phosphatase, a bright-red color (black in this photograph). Acid phos-
phatase is another marker for macrophage activation (see chapter 6).
The edge of a cavity is at the lower right. Next (proceeding to the upper
left) are unstained macrophages (epithelioid cells), some of which are disinte-
grating, and then an area containing many acid phosphatase-positive
macrophages.The outer layer (upper left) is the surrounding capsule contain-
ing many fibroblasts, a few of which also stain positive for acid phosphatase.
The dark circle in the capsule is a metaplastic alveolus (see Fig. 21–24). Not
all of the mature epithelioid cells (large cells with a rounded outline) stain pos-
itive for acid phosphatase, indicating heterogeneity in this cell population (see
reference 40). Counterstained with Giemsa. Magnification, ⫻330. Reprinted
with permission from reference 20.

In our inhalation experiments, we found no
correlation between the degree of tuberculin
sensitivity at 4 weeks and the percentage of
lesions that cavitated by 33 weeks (37). Cavity
formation is a local phenomenon. Tuberculin
skin testing may not accurately reflect what is
occurring in the lesions themselves. The local
nature of cavity formation and the variability
inherent in outbred rabbits may be why our
inhalation experiments show no correlation
between the initial dermal sensitivity to tuber-
culin and the percentage of lesions that later
formed cavities.
Rabbits with the most cavities at 33 weeks
tended to maintain their tuberculin sensitivity at
higher levels than rabbits with fewer cavities
(20, 37).This trend was probably produced by
the numerous bacilli that grew extracellularly
within cavities and provided a large antigenic
stimulation for maintaining tuberculin sensitiv-
ity (20, 37).
Histopathology
Cavity Formation. Macrophages (epithe-
lioid cells) surrounding caseous and liquefied
sites are often highly activated (Fig. 12 and 13),
i.e., they contain high levels of hydrolytic and
oxidative enzymes (discussed above and in
chapter 6).Their hydrolytic enzymes—DNases,
RNases, proteinases, and probably lipases—
apparently play major roles in the liquefaction
of solid caseous material (7a, 20–23). Cathep-
sin D, which is the main proteolytic enzyme of
macrophages (21), is shown histochemically in
Fig. 14.
80 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 14 A tissue section of the wall of a tuberculous cavity produced in a rabbit (#4, Exper-
iment I) that had inhaled 880 virulent bovine-type tubercle bacilli 22 weeks previously. Immuno-
stained for cathepsin D, a major proteinase of macrophages.
Intact bronchial epithelium is shown at the upper right. At the lower left, this epithelium is
ruptured, allowing the liquefied caseum in the cavity (below) to be discharged into the bronchus.
Cathepsin D (a brown color not easily identified in this black and white photograph) is present
in both live and dead macrophages (epithelioid cells) adjacent to the liquefied caseum.This find-
ing suggests that cathepsin D plays an important role in the liquefaction process. Immunostained
with polyclonal goat antibody to rabbit cathepsin D and the avidin-biotin peroxidase technique.
Magnification, ⫻420. Reprinted with permission from reference 20.

Hemoptysis. In clinical tuberculosis, a fre-
quent occurrence is blood-tinged sputum and
occasionally frank hemoptysis. The cause of
bloody sputum is the erosion of blood vessels in
the cavity wall by the necrotizing process (Fig.15).
Granulated Macrophages. Macrophages
containing granules that stained darkly with
Giemsa, methylene blue, or azure A were occa-
sionally found in the intact tuberculous granula-
tion tissue that surrounded the caseous, liquefied,
and cavitary centers of the pulmonary lesions
(Fig. 16).These macrophages were present both
as isolated cells and as small groups but composed
no more than 5% of the macrophage/epithe-
lioid cell population.The granules were autoflu-
orescent and birefringent and stained darkly with
Sudan Black B,which has an affinity for both lipid
and acidic components. Some of these granules
were even dark in unstained tissue sections.They
did not stain with carbol-fuchsin for acid-fast
material or with Alcian blue or periodic acid-
Schiff reagents for carbohydrates. These dark
granules may contain lipofuchsin, ceroid, or tis-
sue and bacillary debris, but their exact role in the
pathogenesis is unknown (20).
Epithelioid Cells. Large mature epithe-
lioid cells are known to be cells that have
destroyed many or all of the tubercle bacilli
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 81

FIGURE 15 A tissue section of a rabbit pulmonary cavity wall.A small blood vessel extends
into the cavity’s lumen at the right of the photograph. Such exposed vessels are the source of
blood in the sputum. In humans, when a larger blood vessel is similarly exposed in a cavity and
ruptures, massive hemoptysis and sometimes fatal hemorrhage may occur.
Adjacent to the small blood vessel in this photograph are disintegrating mature epithelioid
cells. Lurie (4, 48) showed that such large ovoid cells have destroyed many of the bacilli that
they once contained. However, adjacent to the liquefied caseum in the wall of the cavity, the
concentration of bacillary tuberculin-like products is often too high for such epithelioid cells
to survive very long. The glycol methacrylate-embedded tissue section was stained with
Giemsa. Magnification, ⫻400. Reprinted with permission from reference 20.

they once contained (4, 48). Their histologic
appearance is due to bacillary components being
finely dispersed throughout their cytoplasm (4,
48, 49).We used the histochemical stains for -
galactosidase and acid phosphatase to charac-
terize such cells (16, 22, 40, 50, 51).
Many epithelioid cells were present in the
tuberculous granulation tissue that surrounded
the caseous, liquefied, and cavitary centers of the
lesions (Fig. 12 and 13), but not all of these
cells stained for -galactosidase and acid phos-
phatase.The negative-staining epithelioid cells
may have been activated for other products,
e.g., reactive oxygen and nitrogen intermediates
(discussed in reference 40), or they may have
been deactivated (52) or were dying.
Number of Bacilli. Since we used thin 1-
to 2-m glycol methacrylate-embedded tissue
sections (50, 53, 54) (instead of the usual 7-m
paraffin-embedded sections), the number of
tubercle bacilli seen microscopically appeared to
be less than that described in the literature.With
fluorescent staining, we usually found more
bacilli in liquefied caseum (Fig. 17) than in solid
caseum.With acid-fast staining, we also found
tubercle bacilli within tissue spaces (which were
possibly lymphatics) (Fig. 18), within tissue
macrophages, and within the exudate present in
nearby alveolar spaces.
The number of bacilli that could be cultured
from liquefied caseum showed considerable
variability (Table 5) (20). Some liquefied caseum
82 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 16 An area slightly deeper into the cavity wall than that depicted in Fig. 15. In the
center are macrophages containing dark-staining granules.These macrophages occur in some
places within the cavity wall, but, in most places, nongranulated macrophages predominate.The
nature of these granules is unknown (see text).
Part of the capsule (which surrounds this cavitary lesion) is shown at the left of the photo-
graph. It contains fibroblasts and some lymphocytes and plasma cells. Many of the fibroblasts con-
tain translucent (probably secretory) granules.The glycol methacrylate-embedded tissue section
was stained with Giemsa. Magnification, ⫻600. Reprinted with permission from reference 20.

had tremendous numbers, while other liquefied
caseum in the same rabbit had relatively few (see
rabbits #4A and 4B in Table 5). In other rabbits
(e.g., rabbit #10), no viable bacilli were recov-
ered from the liquefied caseum. The rate of
bacillary multiplication in liquefied caseum
probably was influenced both by the composi-
tion of the liquefied material, including its pH
(3), and by the ability of the bacilli to change
from dormancy in solid caseum to active extra-
cellular growth in liquefied caseum.
In liquefied caseous centers an appreciable
number of acid-fast bacilli were often visible
microscopically (4, 20, 37), but in solid caseous
centers only a few bacilli were visible (4).The
number of bacilli usually increased substantially
after a cavity formed (Fig. 19).
The increased amount of oxygen provided by
connection to the bronchial tree is a stimulus for
bacillary multiplication (4). Figure 20 clearly
demonstrates this phenomenon: the most numer-
ous tubercle bacilli are present in the liquefied
caseum adjacent to the air within the cavity.
Fibroblasts. At 19 and 22 weeks, fibroblasts
were common in the peripheral regions of the
tuberculous granulation tissue, and collagen fibers
formed a capsule around the lesion (Fig. 21 and
22).Were it not for the extracellular multiplica-
tion of tubercle bacilli in the liquefied caseum,the
disease would probably have been arrested.
Lymphocytes and Plasma Cells. From
5 weeks on, the disease appeared to be slowly
progressing. Many lymphocytes and plasma cells
were present in the lesions (Fig. 22 and 23). Dur-
ing the later stages of the disease (at 19 and 22
weeks), occasional lymphoid nodules were
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 83

FIGURE 17 A tissue section of the liquefied caseum in a pulmonary lesion of a rabbit (Exper-
iment II) that had inhaled about 5,000 virulent bovine-type tubercle bacilli 7 weeks previously.
The bacilli were stained with TB Fluorostain (rhodamine B/auramine 0) (Polysciences, Inc.,
Warrington, Pa.) and visualized with an epifluorescent microscope at wavelengths 495 nm (exci-
tation) and 535 nm (emission). Liquefied caseum contains variable numbers of tubercle bacilli,
both microscopically in tissue sections and by culture (see Table 5). Magnification, ⫻400.

found in the granulation tissue surrounding
some of the cavities. Since cavities are con-
nected to the bronchial tree, some of these nod-
ules may have been bronchus-associated lym-
phoid tissue rather than nodules arising de novo
from the infiltrating lymphocytes.
Metaplastic Alveolar Epithelium and
Chemotaxis of Alveolar Macrophages. In
the outer layers of the tuberculous granulation
tissue of lesions 10 or more weeks old, the
epithelium of some of the alveoli and respiratory
bronchioles was cuboidal and formed air-filled
saccules (Fig. 21 through 24). These saccules
probably arose when the alveolar connection to
the airways was at least partly obstructed.
The cuboidal epithelial cells that lined these
saccules could be either metaplastic alveolar
type 1 cells (48) or type 2 cells (which can dif-
ferentiate into type 1 cells) (55). Many of the
epithelial cells shown in Fig. 23 were vacuolated,
suggesting that they were type 2 cells. Type 2
cells secrete surfactant (mostly phospholipids),
and the secretory granules containing these
lipids produce the vacuolated appearance shown
in Fig. 23.
Alveolar macrophages were present within
the metaplastic alveoli (Fig. 23 and 24), as well
as in adjacent alveoli (see Fig. 5 in chapter 9).
The alveolar macrophages frequently had a vac-
uolated appearance (Fig. 23), probably from the
ingestion of surfactant. Chemotactic factors
from both the infiltrating host cells and the
tubercle bacilli themselves had evidently caused
alveolar macrophages to migrate into the sur-
rounding alveolar spaces.The perifocal alveolar
84 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 18 A tissue section of a portion of the cavity wall. Bacilli can be seen in the clear
spaces (possibly lymphatics) found among the intact and disintegrating mature epithelioid cells.
Liquefied caseum is shown in the lower left corner of the photograph.The glycol methacry-
late-embedded tissue section (1 to 2 m thick) was stained with carbol-fuchsin and Giemsa.
Magnification, ⫻1,200.
In rabbits (and humans) with cavitary tuberculosis, tubercle bacilli enter the lymphatics and
the microvasculature. However, the native and acquired resistance of such hosts is usually so
high that metastatic lesions arising by the lymphogenous and hematogenous routes usually
fail to progress. Progressive metastatic pulmonary lesions arise in such resistant hosts when large
numbers of tubercle bacilli enter the airways from the cavity. In this case, many bacilli may
land in one site and may be difficult for the host to control.

macrophages occasionally contained ingested (reviewed in references 37 and 63). Injections of

tubercle bacilli that had entered the bronchial
tree from a cavity.
PART III. RECENT EXPERIMENTS
ATTEMPTING TO REDUCE
LIQUEFACTION AND CAVITY
FORMATION
Studies with Mycobacterium vaccae
M. vaccae is a rapidly growing avirulent acid-fast
bacillus that was originally found free in the pas-
tures of cattle and in their milk (56). When
heat-killed and injected intradermally into
humans, M. vaccae apparently enhanced the CMI
response to virulent tubercle bacilli without
appreciably increasing the DTH response (57–
60). M. vaccae increases the Th1 cytokine
response more than the Th2 response (61, 62)
heat-killed M. vaccae did not convert the tuber-
culin skin test of healthy tuberculin-negative
humans (64).These properties enabled M. vac-
cae to be used as an immunotherapeutic agent
in clinical trials with favorable results (57–59,
63, 65–70), although no effect was found in
some trials (63, 71, 72). To date, M. vaccae
immunotherapy of tuberculosis has shown no
adverse effects. However, further studies are
indicated to determine its best role in the treat-
ment of this disease (63, 72).
In two experiments, we administered M. vac-
cae to rabbits with tuberculosis caused by the
inhalation of virulent bovine-type tubercle bacilli
(37) in order to determine whether M. vaccae
could reduce liquefaction and cavity formation.
Autoclaved M. vaccae (strain NCTC 11659) was
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 85

TABLE 5 Number of tubercle bacilli cultured from the liquefied caseum in selected lesions from rabbits in
low-dose Experiment III at 33 weeksa
No. of viable
No. of primary lesions bacilli in approximately
Rabbit no. (caseous or liquefied) No. of cavities 10 l of liquefied caseum
in entire lung in entire lung from individual noncavitary
pulmonary lesionsb
#1 12 3 85,000
#2A, #B 8 17 A: 450
B: 300
#3 4 0 ⬍10
#4A, #B 10 4 A: 160
B: 600,000
#8 8 1 550
#10 10 0 ⬍1
#11 9 2 300
a
Adapted from reference 20.
b
Note that the number of viable bacilli in 10 l of liquefied caseum varies from ⬍1 to 600,000. Evidently, the bacilli must
change from a dormant state to one of extracellular growth, and the liquefied caseum must be of a composition to favor such growth.

FIGURE 19 A tissue section of a rabbit pulmonary tuberculous lesion (rabbit #4, Exper-
iment III) showing virulent bovine-type tubercle bacilli that had grown profusely in lique-
fied caseum. On the left, the lesion was probably beginning to cavitate, because some of the
spaces in the liquefied caseum seem larger than usual. Such profuse growth occurs only in some
lesions with liquefied centers (see Table 5), presumably where the composition of the lique-
fied caseum is most favorable and/or the adaptation of the bacillus to extracellular growth is
most complete. Stained with carbol-fuchsin, counterstained with methylene blue. Magnifi-
cation, ⫻600. Reprinted with permission from reference 37.
86 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 20 A tissue section of the wall of a rabbit pulmonary cavity (rabbit #5, Experi-
ment III) showing virulent bovine-type tubercle bacilli growing in the liquefied caseum.The
bacilli are growing more profusely near the lumen of the cavity (on the left) where the oxy-
gen tension is highest. Stained with carbol-fuchsin, counterstained with methylene blue. Mag-
nification, ⫻250. Reprinted with permission from reference 37.

injected intradermally into rabbits 3 or 5 times,
4 or 5 weeks apart, and beginning 5 or 7 weeks
after infection, respectively.
We found that M. vaccae had no significant
effect on cavity formation or on any other
observable aspect of tuberculosis, including
tuberculin sensitivity (Table 4) (37). In these
rabbit M. vaccae experiments, no antimicrobial
drugs were given, whereas in the human trials,
antimicrobials were simultaneously given.This
difference and others (such as the number of
individuals evaluated) may have contributed to
our inability to confirm in rabbits the favorable
results of M. vaccae immunotherapy in humans.
Reference 63 is a complete review of studies
employing M. vaccae, including descriptions of
the various clinical trials.
Preliminary Studies with the
Proteinase Inhibitor Ritonavir
Ritonavir (Abbott Laboratories, Inc., Chicago,
Ill.) is an aspartyl proteinase inhibitor used in
the treatment of HIV infection that causes
AIDS. Among the HIV proteinase inhibitors,
Ritonavir is thought to be the most active
against host cathepsin D. Cathepsin D is the
major glutamyl/aspartyl proteinase of rabbit
macrophages (41, 42).
Ritonavir (50 mg/kg) was orally adminis-
tered twice daily to five New Zealand White
rabbits starting 5 weeks after the rabbits had
inhaled sufficient virulent M. bovis (Ravenel) to
produce 50 to 100 primary pulmonary tuber-
cles. Diluent was administered to five control
rabbits that were similarly infected. One Riton-
avir rabbit and one control rabbit were eutha-
nized at 1 month after the beginning of the
therapy, one treated and one control rabbit were
euthanized at 2 months, and the remaining three
treated rabbits and three controls were eutha-
nized at 3 months. At necropsy, rabbits treated
with Ritonavir showed no apparent decrease in
the number of solid caseous tuberculous lesions
that had liquefied. If there was such a decrease,
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION 䡵 87

FIGURE 21 A tissue section of the wall of a rabbit pulmonary cavity (Experiment II) 18
weeks after the inhalation of about 5,000 virulent bovine-type tubercle bacilli.To the right is
the cavity’s air space. Next to it is the adjacent liquefied caseum. In the middle of the photo-
graph is the fibrous capsule.The area at the left contains alveoli (saccules) with metaplastic epithe-
lium and a few large macrophages in the air space. Between these metaplastic alveoli are
macrophages, plasma cells, lymphocytes, fibroblasts, and dilated microvessels (probably venules).
The glycol methacrylate-embedded tissue section was stained with Giemsa. Magnification, ⫻125.
Reprinted with permission from reference 20.

it was obscured by variations in the disease
found among the rabbits.
This preliminary experiment (unpublished)
suggests that a combination of inhibitors of sev-
eral hydrolytic enzymes will be necessary to
inhibit the liquefaction of solid caseous lesions,
e.g., those that inhibit DNases, RNases, and/or
lipases in addition to various proteinases.Whether
or not such therapy would have adverse effects
on host resistance remains to be determined.
An alternative or additional approach would be
to increase the CMI/DTH ratio by an appro-
priate vaccine, such as M. vaccae (described in the
preceding section).
SUMMARY OF CAVITY FORMATION
AND SUGGESTIONS FOR ITS
PREVENTION
An aerosol of bovine-type tubercle bacilli (of
slightly reduced virulence) readily produces a
slowly progressing cavitary form of the disease
in the lungs of commercial New Zealand White
rabbits (20, 37) that was quite similar to the
form frequently found in adult human popula-
tions (1, 2, 73–75).
Tubercle bacilli are inhibited in solid caseous
material before liquefaction occurs, but may
grow to tremendous numbers after it occurs,
especially in cavities.The exact cause of lique-
faction is not known. It seems to be a DTH
reaction to products of the bacilli.Also, the pro-
teinases, nucleases, and lipases from live (76)
and dead macrophages seem to be involved.
These hydrolases apparently diffuse into solid
caseum from the periphery, and inhibitors of
these hydrolases that are present in the solid
caseum apparently disappear (see Fig. 25 and 26)
(1, 3, 7a, 18).
To prevent liquefaction and cavity forma-
tion, we highly recommend that multiple hydro-
lase inhibitors be developed and evaluated for
effectiveness and toxicity in the rabbit cavitary
88 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 22 Another portion of the same cavity as in Fig. 21, shown at higher magnifica-
tion to identify the types of cells present.A thick fibrous capsule (upper half) surrounds the liq-
uefied caseum (below).The capsule contains many fibroblasts, as well as some macrophages, lym-
phocytes, and plasma cells.An oval metaplastic alveolus and a small dilated blood vessel are present
on the right. Upon fixation, liquefied caseum often contains spaces between the coagulated pro-
teins, whereas solid caseum usually does not.
On gross examination, all gradations of caseum fluidity were found among the various lesions.
The gross examination is a more accurate test of fluidity, because fixation solidifies the protein-
rich material. The glycol methacrylate-embedded tissue section was stained with Giemsa.
Magnification, ⫻250.

FIGURE 23 An alveolus (saccule) with metaplastic

epithelium containing alveolar macrophages in the wall of
the same cavity shown in Fig. 21 and 22. Many of the alve-
olar epithelial cells are vacuolated, and the macrophages
within this alveolus are also vacuolated (see text). Nearby
are areas rich in lymphocytes and plasma cells, containing
dilated capillaries or venules. Accumulations of plasma
cells and dilated microvessels are frequent components of
tuberculous granulation tissue.The glycol methacrylate-
embedded tissue section (cut 1 to 2 m) was stained with
Giemsa. Magnification, ⫻350.
Metaplastic alveoli probably arise from partial obstruc-
tion of the connecting bronchus.They no longer func-
tion in the oxygenation of blood.
FIGURE 24 A tissue section
of a rabbit pulmonary cavity wall
(Experiment II) 18 weeks after the
inhalation of about 5,000 virulent
bovine-type tubercle bacilli. The
lumen of the cavity is at the right.
Less fibrosis is present than that in
Fig. 21 and 22, and the metaplastic
alveoli are closer to the cavity
lumen. Several dark acid-phos-
phatase-positive macrophages are
present in the cavity wall, as well as
in the lumen of the metaplastic
alveoli. The glycol methacrylate-
embedded tissue section (cut 1 to 2
m) was stained for acid phos-
phatase and counterstained with
Giemsa. Magnification, ⫻210.

FIGURE 25 A tissue section of a rabbit dermal BCG lesion, 28 days of age, immunostained for cathepsin D, a
major macrophage proteinase.The caseous center has liquefied and ulcerated.The viable and dead macrophages sur-
rounding the liquefied caseum contain high levels of cathepsin D.The dead macrophages within the liquefied caseum
and the viable macrophages in the more peripheral tuberculous granulation tissue contain much-reduced levels of
this proteinase. Stained with goat antiserum to cathepsin D, rabbit anti-goat immunoglobulin (in excess), horseradish
peroxidase–goat antiperoxidase complex, hydrogen peroxide, and 3-amino-9-ethylcarbazole. No counterstain. Mag-
nification, ⫻100. Reprinted with permission from reference 21.
Note how closely this ulcerating dermal BCG lesion resembles the pulmonary cavitary lesion that was produced
by an aerosol of virulent bovine-type tubercle bacilli and shown in Fig. 14. Cathepsin D has a similar distribution
in both lesions. Experiments to study factors involved in liquefaction and cavity formation, as well as drugs to inhibit
such factors, could be performed with dermal BCG lesions and then confirmed with virulent tubercle bacilli in
the lungs.

89
90 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 26 A tissue section of a rabbit dermal BCG lesion, 28 days of age, stained for the
lysosomal enzyme -galactosidase, our marker for activated macrophages. As in Fig. 25, the
caseous center has liquefied and ulcerated.The viable and dead macrophages surrounding the liq-
uefied caseum (in the top third of the photograph) contain the highest levels of -galactosidase.
The dead macrophages within the liquefied caseum contain little -galactosidase, and the viable
macrophages in the peripheral tuberculous granulation tissue (in the lower half of the photograph)
show reduced levels of this hydrolytic enzyme. Stained with 5-bromo-4-chloro-3-indolyl--D-
galactoside, lightly counterstained with hematoxylin. Magnification, ⫻100. Reprinted with per-
mission from reference 21.
This figure demonstrates that macrophages at the edge of liquefying caseum become highly
activated for lysosomal enzymes. Lysosomal proteinases, nucleases, and lipases seem to be major
causes of liquefaction.

model of tuberculosis. Inhibitors of cavity for-
mation would be useful in the treatment of
both drug-susceptible and drug-resistant tuber-
culosis and would reduce the spread of this dis-
ease in human populations.
Also, vaccines that increase the CMI/DTH
ratio, i.e., cause a reduction in DTH with respect
to CMI, may not only prevent clinical tuber-
culosis, but may also reduce the amount of cav-
itation should such clinical disease occur. See M.
vaccae studies (described above) and chapter 22.
The composition of liquefied caseum that
enables tubercle bacilli to multiply extracellularly
is not fully known. Drugs that prevent a com-
position favorable to extracellular bacillary
growth would be a welcome addition to our
control of this disease.
Another approach to the control of tubercu-
losis would be to identify the genes that enable
tubercle bacilli to change from a dormant state
to an actively multiplying state. Then, drugs
could be developed to prevent this occurrence.
A PROPOSED METHOD
TO STUDY LIQUEFACTION
AND CAVITY FORMATION
Research on liquefaction and cavity formation
in tuberculosis has been impeded by the lack
of a convenient method to produce these
phenomena. Here is one suggestion that may
 4. LIQUEFACTION OF CASEOUS FOCI AND CAVITY FORMATION
circumvent this difficulty. It may be a break-
through or may be perfectly worthless, but
because it is simple, it should be evaluated.
Premise. (i) Liquefaction and ulceration in
the skin are surrogates for liquefaction and cav-
ity formation in the lungs. (ii) These phenom-
ena mainly occur after a threshold number of
tubercle bacilli is reached.
Liquefaction and ulceration in the skin could
be used to evaluate drugs that inhibit the host
enzymes and/or the bacillary components that
may cause liquefaction and cavities.An increase
in the number of bacilli required to produce liq-
uefaction in the skin would be a measure of the
potential efficacy of the procedure. If estab-
lished, this intradermal method would be a con-
venient, sensitive, and quantitative measure of the
host’s ability to liquefy caseum and form pul-
monary cavities.
Similarities of ulceration in dermal BCG
lesions and cavity formation in pulmonary lesions
produced by virulent tubercle bacilli are dis-
cussed above (p. 68). Figures 25 and 26 show that
hydrolase distribution is apparently the same in
both pulmonary and dermal cavitary lesions.
Procedure. Live and dead BCG, dead vir-
ulent human-type tubercle bacilli (Erdman or
H37Rv), and dead bovine-type tubercle bacilli
(Ravenel) should be injected intradermally into
rabbits in a range of doses. The lowest dose
required to produce liquefaction and ulcera-
tion at the site of injection could then be deter-
mined. Initially, each bacillary strain could be
used on a single rabbit in a wide range of doses,
and the results could be confirmed in several
rabbits with fewer doses near the threshold con-
centration. The experiment could then be
repeated in a biological level 3 facility with
intradermal live virulent tubercle bacilli, and
finally confirmed in a more analogous system,
namely, in rabbits inhaling aerosolized live vir-
ulent tubercle bacilli.
Caveats. Liquefaction and ulceration of
tuberculous lesions in the skin should reflect liq-
uefaction and cavitation in the lungs because the
same host response occurs in each. However,
䡵 91
variations among rabbits in skin composition
and in their inflammatory and immune responses
may prevent the development of a workable assay
system. Nevertheless, the quantitative dermal
response to tubercle bacilli should be evaluated
because it may greatly hasten the ability to under-
stand and inhibit liquefaction and cavity forma-
tion,which are the major processes responsible for
the perpetuation of tuberculosis in humans.
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36. Helke, K. L., J. L. Mankowski, and Y. C. Man-
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37. Converse, P. J., A. M. Dannenberg, Jr.,T. Shi-
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38. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
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39. Manabe, Y. C., A. M. Dannenberg, Jr., S. K.
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40. Suga, M., A. M. Dannenberg, Jr., and S.
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45. Ratcliffe, H. L., and W. F.Wells. 1948.Tubercu-
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48. Lurie, M. B. 1932. The correlation between the
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49. Yamori, T. 1964. On phagocytes; their structures
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67. Stanford, J. L., C. A. Stanford, J. M. Grange,
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69. Johnson, J. L., R. M. Kamya,A. Okwera,A. M.
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75. Sutinen, S. 1968. Evaluation of activity in tuber-
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 Section 2.

IMMUNOLOGY OF TUBERCULOSIS
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 5
DELAYED-TYPE HYPERSENSITIVITY,
CELL-MEDIATED IMMUNITY, AND
ANTIBODIES IN TUBERCULOSIS

Overview of innate immunity in tuberculosis 98

Delayed-type hypersensitivity in tuberculosis 98
Cell-mediated immunity in tuberculosis 99
DTH and CMI similarities in tuberculosis 99
The most favorable CMI/DTH ratios in tuberculosis 100
CMI and DTH are both required to arrest tuberculous lesions 101
Acquired cellular resistance in tuberculosis 102
Local nature of DTH and CMI; organ resistance 102
Systemic immunity in tuberculosis 104
Macrophage activation 104
Synergism of DTH and CMI in arresting tuberculosis: does true dormancy
exist? 104
The size of the tuberculin skin reaction has no prognostic value 105
The “booster” phenomenon in repeated tuberculin testing 105
Lurie’s eye chamber experiments elucidating the factors involved in
CMI 107
Duration and specificity of ACR and its recall upon reinfection 111
Innate immunity and its relation to acquired (adaptive) immunity 112
Nonspecific and antigen-specific immune responses in innate and adaptive
immunity 114

Abstract. Both delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI)

are T-lymphocyte responses to bacillary antigens presented mainly by dendritic cells. In
tuberculous lesions, DTH kills (nonactivated) macrophages that contain numerous tuber-
cle bacilli when these bacilli release tissue-damaging local concentrations of tuberculin-
like products. In the resulting (solid) caseous necrosis, bacillary growth is inhibited and many
bacilli die because of low oxygen tension and other factors.Therefore, tissue-damaging DTH
has apparently evolved in mammals to stop continuing bacillary growth within the non-
activated macrophages that have permitted such growth.
In tuberculous lesions, CMI activates macrophages so that they can inhibit and destroy
ingested tubercle bacilli. DTH can also activate macrophages if only low local concentrations
of tuberculin-like products are present. In this respect, DTH (the host reaction to tuber-
culin-like products) is part of the CMI response.
Nonactivated macrophages continuously enter every tuberculous lesion and may ingest
tubercle bacilli (see chapter 10).To stop the progression of the disease, macrophages con-
taining a few bacilli must be activated by CMI to prevent further intracellular bacillary mul-
tiplication, and macrophages containing many bacilli must be killed by the DTH process.
The interplay of CMI and tissue-damaging DTH seems to explain the entire spectrum
of the disease found in tuberculous hosts.
Both DTH and CMI exert their control locally.Their main systemic manifestation is
to provide an expanded antigen-specific lymphocyte population to infiltrate local sites of
bacillary lodgement.
Antibodies that aid phagocytosis apparently play little or no role in the destruction
of the tubercle bacillus.The bacillus readily enters macrophages without being opsonized
by antibodies and evidently can multiply intracellularly within nonactivated macrophages
in the presence of antibodies. However, in immunized hosts, antibodies seem to be an
important host defense against the development of clinically apparent tuberculosis.

97
98 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

Antigen-antibody reactions at sites of bacillary lodgement result in the production of

chemotactic factors, including the C5a component of complement. In immunized
hosts, such chemotaxins cause a rapid local accumulation of dendritic cells, macrophages,
and antigen-specific T cells—all of which would accumulate more slowly without
the local antigen-antibody reaction. In other words, in immunized hosts the antigen-
antibody reaction enables the local cell-mediated immune response to occur so rapidly
that the bacillus is often inhibited before it multiplies extensively.

OVERVIEW OF INNATE IMMUNITY of hours and reaches a peak in 1 to 3 days.

IN TUBERCULOSIS
In rabbits and humans, the innate immunity of
pulmonary alveolar macrophages plays an
important role in preventing the establishment
of lesions by inhaled tubercle bacilli.The alve-
olar macrophage population is nonspecifically
(innately) activated. These macrophages can
destroy many inhaled virulent human-type
tubercle bacilli before a tuberculous lesion is
established. However, if the bacillus multiplies,
an early lesion is established. Such a lesion con-
tains dendritic cells, macrophages, and lympho-
cytes. The dendritic cells have innate pattern
recognition receptors (PRR) that induce co-
stimulatory factors for antigen presentation (to
lymphocytes) and direct the type of adaptive
(acquired) immune response that results. Details
on innate immunity and the relation of innate
immunity to acquired immunity are presented
at the end of this chapter.
DELAYED-TYPE HYPERSENSITIVITY
IN TUBERCULOSIS
In tuberculosis, delayed-type hypersensitivity
(DTH) (see glossary) is characterized by an
expanded Th1 lymphocyte population that
responds to extremely small concentrations of the
tuberculin-like bacillary products (1). At sites
where the bacilli lodge, these Th1 lymphocytes
produce cytokines that cause mononuclear cell
infiltration, followed (in humans, guinea pigs, and
rabbits) by caseous necrosis as soon as the local
concentration of tuberculin-like products in-
creases to damaging levels.
When applied to tuberculosis, the terms
DTH and tuberculin sensitivity are used inter-
changeably. In hosts sensitized by a previous
exposure to the antigens of tubercle bacilli, the
intradermal injection of tuberculin produces a
skin reaction that slowly develops over a period
Because of the slow development, this antigen-
specific reaction is called “delayed” (2). A low
concentration of tuberculin (as used in skin
testing) usually causes a local cell infiltration
without necrosis in the sensitized host, but high
concentrations will cause necrosis, especially if
the host possesses high levels of DTH. Because
antigen-specific antibodies are almost always
present in tuberculin-positive hosts, typical cell-
mediated tuberculin reactions usually contain
an Arthus component (see glossary) caused by
antigen-antibody complexes and complement
activation.
Only a rough correlation exists between the
size of the dermal reaction to tuberculin and the
amount of caseation found in the pulmonary
tuberculous lesions. Caseous necrosis in the
lesions is a response to the local concentration
of tuberculin-like products of the bacilli.These
products increase as the bacilli in the lesions
increase.The size of the dermal tuberculin reac-
tion represents the overall systemic sensitivity of
the host to a standard amount of tuberculin.
DTH is an important host mechanism for
stopping the multiplication of tubercle bacilli
within the nonactivated macrophages that con-
tinually enter tuberculous lesions from the
bloodstream. DTH kills macrophages when-
ever they contain more tubercle bacilli than
cell-mediated immunity (CMI) (i.e., the acti-
vation of macrophages) can control. Tubercle
bacilli cannot multiply in the solid caseous
necrosis that forms from the dead macrophages
and the surrounding tissues (see chapter 2).
Therefore, DTH is a fail-safe immune mecha-
nism that apparently developed throughout
mammalian evolution to protect the host when-
ever CMI was inadequate.
The term tissue-damaging DTH is used for
the reaction that causes such necrosis.This form
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS
of DTH develops in tuberculous lesions when-
ever tuberculin-like antigens from the bacillus
reach cytotoxic concentrations (see reference
3).The host could be sensitive to many bacillary
antigens besides those in standard tuberculin
preparations (4–12).Therefore, in this book, I use
the term tuberculin-like to include all antigens
that produce a DTH reaction in the host.The
main characteristic of a tuberculin-like antigen
is its ability to cause macrophage and lympho-
cyte infiltration in very low concentrations and
to cause tissue necrosis at somewhat higher con-
centrations. One tuberculin unit contains
0.00002 mg of purified protein derivative (PPD)
(see glossary).
In brief, DTH and CMI are similar immune
reactions to the antigens of the tubercle bacil-
lus, but the concentrations of the antigens that
elicit DTH are far lower than those that elicit
CMI.
CELL-MEDIATED IMMUNITY IN
TUBERCULOSIS
In tuberculosis, CMI, like DTH, is characterized
by an expanded Th1 lymphocyte population,
but, as stated above, the CMI population is not
as sensitive as the DTH population to bacillary
products. In other words, the local concentra-
tions of antigens that produce CMI rarely get
high enough to cause necrosis (1).
CMI-producing lymphocytes enter sites
where bacilli are located and, via their cyto-
kines, activate macrophages to inhibit or destroy
these bacilli. Gamma interferon (IFN-) and
tumor necrosis factor alpha (TNF-) are major
macrophage-activating cytokines (reviewed in
references 13, 14, and 15). IFN- also induces
interleukin-2 (IL-2) receptors in macrophages
(16–18), after which IL-2 (from T lympho-
cytes exposed to specific antigens) becomes an
additional activating cytokine for these phago-
cytes (17, 18). Activated macrophages produce
reactive oxygen intermediates (19–21) and reac-
tive nitrogen (20–28) intermediates, lysosomal
enzymes (29), and other factors that kill and
digest tubercle bacilli. (See references 30, 31, and
32 for overviews of the role of cytokines in
inflammatory responses.) Chapter 6 discusses
macrophage activation in more detail.
䡵 99
Figure 1 illustrates the basic principles of
CMI. Macrophages are shown in various stages
of activation. Such activation is caused by the
cytokines of antigen-stimulated lymphocytes
(called lymphokines in this figure). Only the
macrophages that are highly activated seem to be
capable of inhibiting or killing fully virulent
tubercle bacilli. Not included in the figure are
antigen-presenting cells (APCs) (dendritic cells),
which process the antigens of tubercle bacilli
and present them to lymphocytes with specific
receptors for these antigens (see chapter 6).
Whether a nonactivated macrophage con-
taining only 1 to 4 bacilli can be activated suf-
ficiently to prevent further intracellular bacillary
growth is not known, but macrophages within
which the bacilli have multiplied to 10 or more
could never do so. In this case, tissue-damaging
DTH is required to kill this macrophage. To
prevent the intracellular multiplication of viru-
lent tubercle bacilli, macrophages in tuberculous
lesions may have to be activated prior to the
ingestion of such bacilli. If so, a major compo-
nent of the CMI response would be the local
accumulation of large numbers of highly acti-
vated macrophages that are ready to ingest
tubercle bacilli released from macrophages killed
by tissue-damaging DTH (Fig. 2 and 3).
DTH AND CMI SIMILARITIES IN
TUBERCULOSIS
DTH is really a form of CMI (2). By conven-
tion, the ability of antigen-specific Th1 lym-
phocytes to cause a more rapid infiltration of
dendritic cells, macrophages, and lymphocytes is
called delayed-type hypersensitivity, and the
ability of Th1 lymphocytes to activate macro-
phages to inhibit and destroy microorganisms is
called cell-mediated immunity.
The relationship between DTH and CMI
in the control of facultative intracellular micro-
organisms was a subject of debate for most of the
20th century (33). DTH and CMI are usually
produced together, albeit in different proportions
depending on the nature of the antigenic stim-
ulus and the genetic characteristics of each host.
CD8 lymphocytes (see glossary) probably play
a significant role in the DTH-mediated killing
of macrophages containing more than a few
100 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 Cell-mediated immunity producing local activation of macrophages (acquired cel-

lular resistance) in a tuberculous lesion. Dendritic cells, macrophages, and antigen-specific Th1
lymphocytes enter the site from the bloodstream. Dendritic cells (not shown) present the bacil-
lary antigens to the lymphocytes (originally in the draining lymph nodes).The lymphocytes pro-
duce lymphokines (LK) (now called cytokines), which activate the macrophages. Only highly acti-
vated macrophages are capable of inhibiting or destroying the virulent tubercle bacilli. Reproduced
with permission from reference 125.

tubercle bacilli (1). And CD4 lymphocytes are
known to play a major role in activating
macrophages so that ingested bacilli do not
grow intracellularly. The percentage of CD4
cells and CD8 cells in tissue sections of pro-
gressing and regressing tuberculous lesions (pro-
duced in rabbits by Mycobacterium bovis BCG) is
given in chapter 6.
Many mycobacterial antigens cause CMI and
DTH via antigen-specific T cells. Some of these
antigens may produce strong DTH (tuberculin
sensitivity) with little CMI (activated macro-
phages), whereas others may produce weak DTH
with strong CMI. In tuberculous lesions, each
antigen has a concentration that best activates
macrophages and a much higher concentration
that causes tissue necrosis.With tuberculin-like
(DTH) antigens, macrophage-activating con-
centrations are low and easily exceeded, so necro-
tizing concentrations are soon attained.
THE MOST FAVORABLE CMI/DTH
RATIOS IN TUBERCULOSIS
To select better vaccines for tuberculosis, we
need to know what antigenic composition pro-
duces the most favorable ratio of CMI to DTH
(5) (see chapter 15). Specifically, we would like
a vaccine to produce increased numbers of
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS 䡵 101

FIGURE 2 Highly activated macrophages surrounding the caseous center of a 12-

day (rabbit) dermal BCG lesion.This figure illustrates an important aspect of effective
cell-mediated immunity and acquired cellular resistance, namely, that large numbers of
activated (-galactosidase-positive) macrophages accumulate around a caseous focus,
so that bacilli released from dead and dying cells will be ingested by competent rather
than incompetent cells. Stained with 5-bromo-4-chloro-3-indolyl--D-galactoside,
counterstained with hematoxylin. Magnification, ⫻90. Reproduced with permission
from reference 45.

antigen-specific lymphocytes that activate
macrophages (CMI) and decreased numbers of
antigen-specific lymphocytes that contribute
to caseous necrosis (DTH). In other words, the
vaccine should increase the inhibition of tuber-
cle bacilli within macrophages (CMI) and
decrease the amount of tissue damage from
their tuberculin-like products.
The same beneficial CMI/DTH ratio cre-
ated by the vaccine would occur in the memory
lymphocytes that infiltrate a developing pul-
monary tubercle caused by the inhalation of
virulent tubercle bacilli.Therefore, the progres-
sion of such a developing pulmonary tubercle
would be stopped with less damage to the tissues
of the host. In other words, the memory T cells,
having the most beneficial CMI/DTH ratio,
would remain in the host after vaccination and
provide the most favorable protection against
the development of clinical tuberculosis.
CMI AND DTH ARE BOTH REQUIRED
TO ARREST TUBERCULOUS LESIONS
From the bacillary growth curves presented in
chapter 3 and a histologic study of the lesions,
it seems that both CMI and tissue-damaging
DTH can inhibit the multiplication of tubercle
bacilli (34–36). However, CMI does so by acti-
vating macrophages to kill or inhibit the bacilli
they ingest, and tissue-damaging DTH does so
by destroying bacilli-laden, nonactivated macro-
phages and nearby tissues, thereby eliminating
the intracellular environment that is so favorable
for bacillary growth (37–39; see also reference
29). Thus, both tuberculin-positive hosts with
good CMI and tuberculin-positive hosts with
poor CMI can arrest bacillary growth. But the
host with poor CMI does so with much dam-
age to its own tissues. Eventually, the host with
good CMI may recover, but the host with poor
CMI will die from excessive tissue destruction.
102 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
FIGURE 3 Macrophages in a 21-day rabbit dermal
BCG lesion stained both for acid-fast bacilli (red) and
for -galactosidase (to show macrophage activation)
(bright blue). (Colors not shown here.) One of these
macrophages shows negligible -galactosidase activity.
It contains numerous bacilli and has ruptured.Another
macrophage (adjacent) shows high -galactosidase activ-
ity. It contains no bacilli, but is apparently ingesting the
bacilli released from the ruptured cell.This figure illus-
trates how acquired cellular resistance, produced by
CMI, stops the local growth of tubercle bacilli, namely,
that highly activated macrophages accumulate at the
local site and ingest (and destroy) the bacilli released from
ineffectual macrophages. Stained with 5-bromo-4-
chloro-3-indolyl--D-galactoside, counterstained with
hematoxylin, then carbol-fuchsin. Magnification, ⫻600.
Reproduced with permission from reference 29.
Tissue-damaging DTH causing local necrosis
stops the initial bacillary growth within non-
activated macrophages (see chapters 2 and 15).
Such cytotoxic DTH can never take the place of
CMI, because the bacilli that escape from the
edge of necrotic areas are ingested by perifocal
macrophages. If the perifocal macrophages have
been sufficiently activated by CMI, they can
destroy the ingested bacilli. If these macrophages
have not been sufficiently activated by CMI, the
bacilli will again grow intracellularly until the
macrophages are killed by tissue-damaging DTH,
thereby enlarging the caseous necrotic area.
ACQUIRED CELLULAR RESISTANCE
IN TUBERCULOSIS
Acquired cellular resistance (ACR) is charac-
terized by a local population of microbicidal
macrophages (Fig. 2 and 4) that have been acti-
vated by the cytokines of accumulating antigen-
specific Th1 lymphocytes. These macrophages
become activated and develop their microbici-
dal ability only where the bacillary antigens are
located (Fig. 4).The greater the local accumu-
lation of highly activated macrophages (Fig. 2),
the greater the host’s ability to destroy or inhibit
the tubercle bacillus.
Since ACR is a local reaction at sites harbor-
ing the tubercle bacillus, it is not technically the
same as CMI, which is systemic. CMI is charac-
terized by an expanded antigen-specific T-cell
population that exists (as memory cells) through-
out the body even after the bacilli and their anti-
gens have been destroyed. In contrast, ACR is
manifested only at sites (containing tubercle
bacilli) where macrophages have been activated
by the cytokines of infiltrating (antigen-specific)
lymphocytes. In this book, I usually include ACR
in the definition of CMI, because ACR cannot
develop locally without the existence of systemic
CMI.Additional properties of ACR are discussed
later in this chapter.
LOCAL NATURE OF DTH AND CMI;
ORGAN RESISTANCE
Marked degrees of macrophage activation take
place locally in tuberculous lesions, where
macrophages become epithelioid cells (see glos-
sary) extremely rich in lysosomal (Fig. 2 through
4) and mitochondrial enzymes (29, 40). The
same degree of activation is not common in
macrophages found elsewhere in the body.
Present in the local lesion are bacillary com-
ponents as well as host defense cells (29), i.e., den-
dritic cells, macrophages, antigen-specific lym-
phocytes, and even plasma cells (41). Because of
the local antigenic stimulus (to lymphocytes)
caused by bacillary components, the macrophages
often become highly activated. Resident cells,
including fibroblasts and vascular endothelial cells
(42), also become activated and produce cyto-
kines. Cytokines are not antigen specific and
stimulate (or suppress) any cell containing the
appropriate receptor.
We demonstrated local macrophage activation
histochemically with Pearson’s indolyl substrate
for -galactosidase (43, 44) (Fig. 2 through 4).
(-Galactosidase is a representative lysosomal
hydrolase.) With the indolyl substrate, we could
stain for both -galactosidase and tubercle bacilli
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS 䡵 103

FIGURE 4 A group of activated macrophages (epithelioid cells) in a 21-day dermal BCG

lesion from a rabbit, stained darkly for the lysosomal enzyme -galactosidase (bright blue). (Color
not shown here.) We use -galactosidase activity as a histochemical marker for activated
macrophages that are capable of destroying tubercle bacilli (29, 40).Although the perifocal tuber-
culous granulation tissue contains thousands of macrophages, only those macrophages located
near tubercle bacilli (and their products) become activated and develop the power to destroy
the bacillus. In other words, CMI is mainly a local phenomenon.The darker the macrophage
is stained for -galactosidase, the more it resembles Lurie’s mature epithelioid cell (see glos-
sary), a cell that has destroyed tubercle bacilli (31, 43). Stained with 5-bromo-4-chloro-3-indolyl-
-D-galactoside, counterstained with hematoxylin. Magnification, ⫻200. Reproduced with per-
mission from reference 29.

in the same tissue section, because the indigo
blue dye produced by -galactosidase was not
removed by the acid-fast staining procedure, in
which the bacilli were stained red by carbol-
fuchsin (29, 40, 45) (Fig. 3).
The local presence of tubercle bacilli was
associated with the formation of partly and
fully mature epithelioid cells. Such cells were
shown by Lurie to have destroyed the tubercle
bacilli that they once contained (34, 46). The
most mature epithelioid cells (i.e., those with
the most rounded appearance) stained the dark-
est for -galactosidase (Fig. 3 and 4). Such cells
did not contain as many bacilli as immature
epithelioid cells, which stained more lightly
(47, 48) (Fig. 3) (see chapter 6). Histochemical
staining for acid phosphatase, -glucuronidase,
cytochrome oxidase, and succinic dehydroge-
nase indicated that many hydrolases and oxi-
dases were markedly elevated in mature epithe-
lioid cells (29, 40).
Highly activated macrophages may be rather
toxic to host tissue because of their high levels of
hydrolases and oxidases. It makes sense that such
activated macrophages are produced only where
the bacilli are located.The host would probably
not survive if such cells were ubiquitous.
The local nature of acquired resistance in
tuberculosis is also evident clinically.A tubercu-
lous lesion in one part of a patient’s lung may
progress while a lesion in another part of the
same lung may regress, depending on the local
concentration of tubercle bacilli at each site and
the host’s reaction to them (discussed more fully
in chapter 3). Such local immunity also occurs
in tuberculous rabbits and guinea pigs (49).
104 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
The importance of the local site in directing
the immune response was clearly brought forth
by Matzinger (50–52).When irritated or injured
by trauma, toxins, or infectious agents, each site
evidently produces or releases factors (including
cytokines) that influence the type of differenti-
ation that occurs in the resident cells as well as
in the infiltrating dendritic cells, macrophages,
and lymphocytes. Such factors first affect APCs
(dendritic cells) so that the “appropriate”
cytokines are produced and the “appropriate”
costimulatory (or coinhibitory) ligands are
upregulated. These ligands then determine
which type of T cell is favored. In each organ
of the host, different coligands may be pro-
duced (53), which could explain why tubercu-
lous lesions in the liver of rabbits rarely progress,
whereas those in their lungs and kidneys usually
do so (see chapters 6 and 7).
SYSTEMIC IMMUNITY IN
TUBERCULOSIS
Individuals infected with tubercle bacilli—
whether the bacilli are virulent or avirulent—
develop greater numbers of T lymphocytes with
specific receptors for mycobacterial antigens in
their blood and lymphoid tissues than do indi-
viduals who have never been infected with such
bacilli (see references 29 and 47). In other words,
such individuals are immunized. When these
individuals inhale exogenous virulent tubercle
bacilli, greater numbers of antigen-specific T
cells will accumulate at sites where the bacilli are
deposited; once there, these T cells will pro-
duce cytokines that hasten the local accumula-
tion and activation of macrophages.The bacilli
are then inhibited or destroyed before they mul-
tiply appreciably, and the developing lesion
remains small and heals rapidly.This acceleration
of the host’s immune response often protects the
immunized person from clinically recognizable
exogenous and endogenous reinfection. The
role of antibodies in accelerating the local accu-
mulation of macrophages and antigen-specific T
cells is discussed below.
In brief, systemic immunity in tuberculosis
is characterized by an expanded antigen-
specific T-cell population that enters each site
where tubercle bacilli are deposited and accel-
erates the local activation of macrophages. Sys-
temic immunity does not prevent the
establishment of a microscopic tubercu-
lous lesion by inhaled tubercle bacilli. It
only stops the progression of such a
lesion by causing a rapid accumulation of
macrophages and antigen-specific lym-
phocytes at that site.
MACROPHAGE ACTIVATION
Macrophages have many functions, e.g., phago-
cytosis, killing microorganisms, cytokine pro-
duction, immunostimulation (as well as
immunosuppression), and some antigen pre-
sentation. Macrophages can be activated for one
or more of these functions and remain inactive
for others (54–57).
Activated macrophages that inhibit and
destroy the tubercle bacillus and its compo-
nents have a greatly increased content of oxida-
tive and hydrolytic enzymes (29, 40, 58). The
actual killing of the bacillus seems to be due to
reactive nitrogen and reactive oxygen interme-
diates (19, 21–28), whereas the breakdown of
bacillary components seems to be due to vari-
ous hydrolases.
We used the lysosomal enzyme -galactosi-
dase as a marker for the type of macrophage acti-
vation that inhibits and destroys tubercle bacilli
(29, 40, 58).This marker was correlated histo-
chemically with other hydrolytic enzymes and
with certain oxidative enzymes (29, 40). See
chapter 6 for a full discussion of macrophage
activation in tuberculosis.
SYNERGISM OF DTH AND CMI IN
ARRESTING TUBERCULOSIS: DOES
TRUE DORMANCY EXIST?
Nonactivated macrophages continually enter
most tuberculous lesions from the bloodstream
(see chapter 10) and may ingest occasional bacilli
that then multiply within these macrophages.
Such bacillary growth is stopped when tissue-
damaging DTH kills the macrophages (see
chapters 2 and 3). In hosts with good CMI, the
bacilli released from the dead macrophages are
often ingested by nearby activated macrophages
that can prevent further bacillary multiplica-
tion (Fig. 3). In other words, even in nonpro-
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS
gressing lesions, at least a few nonactivated
macrophages probably allow some bacillary mul-
tiplication to occur.
The sequence that passes tubercle bacilli from
a nonactivated macrophage into an activated
macrophage suggests that in many “arrested”
tuberculous lesions at least some tubercle bacilli
are not dormant, but are undergoing repeated
cycles of growth and inhibition. These bacilli
would reside at the edge of solid caseous areas,
escape into nearby poorly activated macrophages,
and change from a metabolic state of extracel-
lular dormancy into a state of intracellular mul-
tiplication.To determine (by molecular biology
techniques) which mycobacterial genes are active
in each of these metabolic states, separation of
solid caseum from the surrounding (still viable)
tissues is essential.
Because of such bacillary growth and destruc-
tion, we have concluded that at least some of the
bacilli in arrested tuberculous lesions may not
remain dormant. In fact, in healthy individuals,
tuberculin positivity is probably maintained
because of occasional episodes of bacillary mul-
tiplication and subsequent destruction.
THE SIZE OF THE TUBERCULIN
SKIN REACTION HAS NO
PROGNOSTIC VALUE
The size of the tuberculin reaction depends on
(i) the ability of the host to respond and (ii) the
amounts of the appropriate bacillary antigens
that sensitize the host.
A strong dermal tuberculin reaction could be
caused by a small number of bacilli in a host
innately capable of producing a strong DTH
response, or it could be caused by a large num-
ber of bacilli in a host innately capable of pro-
ducing a weaker DTH response. Lurie’s sus-
ceptible rabbits were genetically poor responders
to many antigens (34, 59, 60). However, they
often developed tuberculin skin reactions just as
strong as those of his resistant rabbits, because
numerous tubercle bacilli were growing in their
bodies (59).
Conversely, a weak dermal tuberculin reac-
tion could signify a poor genetically deter-
mined immune response that allowed many
bacilli to grow in the host, or a good genetically
䡵 105
determined immune response that kept the
bacillary titers low. Therefore, the size of
the dermal tuberculin reaction does not
reflect the amount of disease in the host
or whether it is progressing or regressing
(34). In fact, patients with extensive tuberculo-
sis frequently show tuberculin anergy (61–63),
but after they receive effective antimicrobial
therapy and begin to recover, they again
become tuberculin positive (63). Possible causes
of such anergy are reviewed in references 1
and 62.
A large tuberculin reaction remaining years
after the primary lesion has been arrested prob-
ably signifies that a few viable bacilli are still pre-
sent in caseous foci that are often radiologically
unrecognizable. Evidently, such bacilli are
released from time to time, but the new lesions
that they produce are soon healed.This sequence
boosts the host’s immune system, including the
level of tuberculin sensitivity.
THE “BOOSTER” PHENOMENON IN
REPEATED TUBERCULIN TESTING
A positive second tuberculin skin reaction may
occur after a negative first skin reaction because
of boosting of DTH by antigens in the first test.
Individuals who have been infected with the
tubercle bacillus can, in time, become tuber-
culin negative with or without antimicrobial
treatment. However, in many of these individu-
als a recall of tuberculin sensitivity can be pro-
duced by the antigens in tuberculin (PPD) that
is injected for skin testing (64, 65, 65a). When
retested with intermediate-strength PPD, a per-
son who had a negative skin reaction 1 to 5
weeks earlier may now be tuberculin positive as
a result of the booster effect of tuberculin itself
(reviewed in reference 65).The first injection of
PPD expands the tuberculin-sensitive T-cell pop-
ulation (i.e., the memory T cells) to a level where
the number of these cells is now sufficient to pro-
duce a positive skin reaction with a subsequent
PPD injection.Therefore, the conversion of the
skin test due to boosting by a previous skin test
does not signify that a recent exogenous infec-
tion with tubercle bacilli or even a reactivation
of an arrested endogenous tuberculous lesion
has occurred.
106 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
Improved Diagnostic Tests for Active
Tuberculosis
The tuberculin skin test cannot reliably differ-
entiate between DTH from a progressive tuber-
culous lesion and DTH from environmental
mycobacteria, BCG vaccination, or a nonpro-
gressive arrested lesion. Since the secreted anti-
gen ESAT-6 is produced by virulent human-
type tubercle bacilli and not by BCG or many
environmental mycobacteria, an in vitro diag-
nostic test on human peripheral blood mononu-
clear cells (PBMC) was developed and evaluated
in Ethiopia (66, 67). This test measures the
amount of IFN- produced by PBMC after 5
days in culture and restimulation with ESAT-6.
At the start of this study, the amount of IFN-
 produced by PBMC from household contacts
of persons with active (sputum-positive) tuber-
culosis was determined (68). Two years later
these contacts were evaluated to determine
whether they had clinically active tuberculosis.
The household contacts whose PBMC pro-
duced high levels of IFN- developed clini-
cally active tuberculosis, whereas those contacts
whose PBMC produced low levels of IFN-
remained healthy. Parallel in vitro studies with
tuberculin (PPD) as the antigen showed no dif-
ference between these two groups. Evidently, the
contacts with early progressive tuberculous
lesions could be identified by an increase in the
number of circulating lymphocytes capable of
responding to ESAT-6.
This was the first study in which a laboratory
test could distinguish tuberculin-positive persons
with early progressive tuberculous lesions (who
should be treated with antimicrobials) from per-
sons with nonprogressive lesions (who would
not need antimicrobial therapy). If further stud-
ies confirm these results, the ESAT-6 in vitro test
would be a major addition to the control of this
disease.
Antibodies Enhance Local DTH and
CMI Reactions
Although immunity in tuberculosis is primarily
cell mediated, numerous antibodies circulate in
the hosts (9, 34, 69–74), and plasma cells are
common in tuberculous lesions (41). Circulating
antibodies are present in nearly all tuberculin-
positive patients (reviewed in references 75, 76,
and 77), but, to date, antibody titers are not a
completely reliable way to distinguish between
progressive and inapparent disease (see reference
78). In mice, titers of the serum antibody levels
against mycobacterial glycolipids (diacyltrehaloses
and sulfolipid I) paralleled the bacillary concen-
tration in the host tissues better than did antibody
levels to protein antigens (73). In other words, the
levels of glycolipid antibodies indicated pro-
gression of the disease. In general, Lurie’s resis-
tant inbred rabbit strains produced higher anti-
body titers than his susceptible inbred strains
(59), but exceptions existed (60).
Antibodies have always been thought to play
little or no role in the immunity to tuberculo-
sis, because the passive transfer of immune serum
neither protected the host from contracting the
disease nor appreciably reduced its progression
(75, 77, 79). Recent studies in my laboratory (69)
suggest a role for antibodies that had previously
not been considered; i.e., in hosts with good
cell-mediated immunity, an antigen-antibody
reaction causes a rapid infiltration of DTH-
and CMI-producing cells into sites where tuber-
cle bacilli are located. In other words, anti-
bodies enhance the local cell-mediated
host response (69).
We compared rabbits containing primary der-
mal BCG lesions with rabbits containing rein-
fection dermal BCG lesions (Fig. 5A through
C). The reinfection lesions were begun when
the primary lesions were 24 days of age. In the
first 3 h, a marked infiltration of macrophages and
lymphocytes (and probably dendritic cells)
occurred in the reinfection group while very lit-
tle infiltration occurred in the primary group
(Fig. 5C) (69). It seems that only an antigen-
antibody reaction could produce such an imme-
diate, pronounced antigen-specific chemotactic
effect,because very few lymphocytes are normally
present in the skin. Lymphocytes and antibodies
are the only antigen-specific immunological
defenses that the host possesses.
At 1 to 2 days, a 400- to 500-fold difference
in size between primary and reinfection BCG
lesions was present (Fig. 5C) (69). By 4 to 5 days,
the size of the reinfection lesions had declined,
while the size of the primary lesions had
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS
increased, so that both types of lesion were
grossly similar (Fig. 5A and C).
At 8 days in reinfection lesions and at 12
days in primary lesions, small secondary peaks
occurred in the size of the BCG lesions (Fig.
5A).These peaks were probably caused by the
development of CMI in the primary lesions
and a boosting of the CMI already present in the
reinfection lesions.
In rabbits with primary BCG lesions, skin
tests with Old Tuberculin became positive by 9
days (Fig. 5B), accompanied by a rise in the
levels of antibodies to the secreted antigen PstS-
1 (38 kDa) (Fig. 6A) (69), which is one of sev-
eral antigens secreted by live tubercle bacilli
(80, 81). In rabbits with primary BCG lesions,
antibodies to the constitutive antigens HSP65
and PPD did not increase appreciably until
much later (Fig. 6B and C) (69). Constitutive
antigens are released from tubercle bacilli after
they have been killed.
The almost immediate difference in size
between primary and reinfection BCG lesions
(Fig. 5C) seems to be caused by circulating and
cytophilic antibodies. Circulating antibodies
rapidly combine with the infecting bacilli and
their antigens, causing an Arthus-type reaction
and activating complement (components C1
to C9) (82).The released C3a and C5a compo-
nents cause vasodilation and activate connective
tissue-type mast cells and other cell types,
including vascular endothelial cells (details pre-
sented in reference 69). C5a is a potent chemo-
taxin (83).Antigen-antibody complexes induce
dendritic cell maturation and promote antigen
presentation (84), both of which also hasten the
host’s immune response to reinfection.
Cytophilic antibodies (including IgE) bind to
the Fc receptors on many cell types, e.g., mast
cells, fibroblasts, natural killer cells, macrophages,
and APCs. In the presence of antigen, these
cells become activated and secrete cytokines
(including chemokines) and even the chemo-
taxin leukotriene B4 (85–93). The role of
cytophilic antibodies in tuberculosis has not
been adequately studied.
In brief, in BCG-vaccinated hosts and in
healthy hosts made tuberculin positive by viru-
lent tubercle bacilli,antibodies play an important
䡵 107
role in preventing clinically apparent tuberculo-
sis.Antigens reacting with such antibodies rapidly
bring dendritic cells,macrophages,and antigen-
specific memory T cells to sites of endogenous
and exogenous reinfection.These cells can then
stop many developing microscopic tubercles
from reaching clinically apparent size.However,
the immune effects of antibodies can only
have a beneficial effect in tuberculosis
when an expanded antigen-specific T-cell
population exists to enter developing lesions,
because the passive transfer of antibodies to non-
immune hosts provides little or no benefit.
A variation of the usual passive transfer exper-
iment was recently published (74). SCID mice
were aerosol infected with Mycobacterium tuber-
culosis (H37Rv).After 3 weeks, they were treated
with isoniazid and rifampin for the next 5 weeks.
Then, hyperimmune serum to Mycobacterium
tuberculosis was administered intraperitoneally,
once every 3 days for 4 injections.The mice were
euthanized 2 days after the last injection of
serum.When compared with controls receiving
normal serum, the mice receiving the hyper-
immune serum had markedly reduced bacillary
titers and had reduced tissue destruction by the
recurring disease. Since SCID mice have few, if
any,Th1 lymphocytes, the antibodies may have
had some direct effect on the ability of the
tubercle bacillus to emerge from the dormant
state created by the antimicrobial therapy.
LURIE’S EYE CHAMBER
EXPERIMENTS ELUCIDATING THE
FACTORS INVOLVED IN CMI
Although this book mostly concerns tuberculo-
sis produced in vivo, a description of Lurie’s eye
chamber experiments (performed over 60 years
ago) is included here, because they represent a
combination of in vitro and in vivo studies (34,
94).These experiments showed that antibodies
do not directly affect the ability of macrophages
to inhibit the growth of tubercle bacilli.
In several experiments, Lurie immunized rab-
bits (by the subcutaneous or intravenous routes)
with virulent human- or bovine-type tubercle
bacilli, and, after 1.5 or more months, he injected
acacia (gum arabic) into their pleural cavities.
The resulting mononuclear cell exudate was
108 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 5 (A) Size of primary BCG lesions and reinfection BCG lesions from 3 h to 42 days
in rabbits of Experiment I.The reinfected rabbits had been sensitized intradermally by BCG 24
days previously. Note that the reinfection BCG lesions were many times larger than the primary
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS 䡵 109

collected 4 to 5 days later and washed by cen-
trifugation. The exudate cells were over 85%
mononuclear cells—mostly macrophages, with
some lymphocytes, granulocytes, and probably
dendritic cells.The macrophages were not puri-
fied by density gradient centrifugation or by
adherence to glass.
In the presence of either normal or immune
serum, the macrophages were allowed to ingest
in vitro (for 30 min) carbon particles mixed with
low concentrations of human-type tubercle bacilli
(1 bacillus for 10 or more macrophages). The
carbon particles were used to label these phago-
cytes. Combinations of normal cells with either
normal or immune serum and combinations of
immune cells with normal or immune serum,
were made. One of the four combinations was
injected into the anterior chamber of the right
eye of a normal rabbit, and another combination
was injected into the anterior chamber of the left
eye.After 14 to 20 days, the rabbits in this exper-
iment were sacrificed. Then, (i) the irises with
adherent macrophages (containing intracellular
tubercle bacilli and carbon particles) and (ii) the
anterior chamber fluids (containing tubercle
bacilli, which were mostly within carbon-labeled
macrophages) were cultured to determine the
number of viable bacilli.The irises and cells in the
chamber fluids were also processed and evaluated
microscopically. Relatively few bacilli were found
in the chamber fluids; most of the bacilli were in
the iris preparations (94).
In the eye chambers, the macrophages from
the immunized rabbits inhibited the multipli-
cation of tubercle bacilli in their cytoplasm
much more effectively than did macrophages
from normal rabbits. Both normal serum and
immune serum had no appreciable effects on the
bacillary multiplication. Adherent to (and
within) the irises, many of the carbon-labeled
macrophages from the immunized rabbits had
the appearance of mature epithelioid cells.The
number of carbon particles that the macrophages
contained had not decreased appreciably from
the number originally present. In other words,
the macrophages from immunized rabbits had
apparently undergone little or no cell division
in the anterior eye chambers (see chapter 10).
In the eye chambers, the macrophages from
the nonimmunized rabbits contained numerous
tubercle bacilli and did not form mature epithe-
lioid cells.These macrophages showed a reduc-
tion in the number of intracellular carbon par-
ticles: the numerous tubercle bacilli may have
stimulated these cells to divide, or these bacilli
killed such cells (after local DTH developed) and
several nearby macrophages ingested a portion
of the carbon load.
In the light of modern knowledge, these find-
ings indicate that the macrophages from the

BCG lesions at 3 h, 12 h, and 1, 2, and 3 days (panels A and C), apparently initiated by an
antigen-antibody reaction. Note also that the size of the reinfection BCG lesions reached a sec-
ond peak at 8 days, whereas the primary lesions reached a similar peak at 12 days.These second
peaks were apparently caused by an antigen-specific CMI/DTH reaction. After the second
peaks, the lesions slowly regressed. Each point represents the mean size of the lesions and its stan-
dard error. *P ⬍ 0.05; **P ⬍ 0.01.
(B) Size of 2-day tuberculin reactions in rabbits of Experiment I. In the reinfected host, tuber-
culin sensitivity was highest before challenge.This sensitivity declined thereafter, and no booster
effect from the second BCG injection was apparent. In contrast, hosts with primary BCG infec-
tions developed strong tuberculin sensitivity by 9 days, which tended to remain higher than that
present in the reinfected hosts, possibly because the infecting bacilli were not destroyed as read-
ily. Each point represents the mean size of the tuberculin reactions and its standard error.
(C) Size of primary and reinfection BCG lesions and tuberculin reactions in Experiment II,
each measured from 3 h to 5 days.As in Experiment I, the reinfected rabbits were sensitized intra-
dermally by BCG 24 days previously.
Note that the reinfection BCG lesions and the tuberculin reactions had a similar pattern, and
that the primary BCG lesions remained very small until DTH and CMI started to develop at 4
or 5 days. Each point represents the mean size of the lesions and its standard error. *P ⬍ 0.05;
**P ⬍ 0.01. Reproduced with permission from reference 69.
110 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Antibody levels to the mycobacterial 38-kDa secreted antigens (A) PstS-1,
(B) HSP65, and (C) PPD in rabbits with primary and reinfection BCG lesions. In the rein-
fected hosts, the second injection of BCG enhanced all existing antibody levels. In hosts
with primary BCG infection, the antibody titer to the secreted 38-kDa antigen PstS-1
became substantial by day 9, but the titers to the constitutive antigens HSP65 and PPD rose
more slowly. Each point represents the mean and its standard error. *P ⬍ 0.05; **P ⬍ 0.01.
Reproduced with permission from reference 69.
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS
immunized rabbits were activated specifically by
tubercle bacilli and their products after the
mononuclear cell preparations (containing
macrophages, dendritic cells, and lymphocytes)
were placed within the anterior eye chamber: the
expanded antigen-specific lymphocyte popula-
tion (in the mononuclear cell preparation from
the immunized host) produced cytokines (includ-
ing IFN-) that activated the macrophages pres-
ent, so that these macrophages could inhibit the
intracellular growth of tubercle bacilli during
their 2- to 3-week residence within the anterior
eye chamber.
Similar results were obtained from other eye
chamber experiments in which “immune”
mononuclear cells were collected from the
draining lymph nodes (or bone marrow) 2 days
after a subcutaneous (or intravenous) injection
of virulent tubercle bacilli mixed with carbon
particles. In other words, when compared with
“nonimmune” controls, “immune” mononu-
clear cell preparations that had ingested tuber-
cle bacilli in vivo (in the lymph nodes or mar-
row) inhibited tubercle bacilli in the anterior eye
chamber in a fashion similar to those that had
ingested tubercle bacilli in vitro.
All of these experiments show that circulat-
ing antibodies had little or no direct effect on
the ability of macrophages (from either normal
or immunized hosts) to inhibit the multiplica-
tion of tubercle bacilli.Therefore, immunity in
tuberculosis is cell-mediated by (as we know
today) antigen-specific lymphocytes produc-
ing cytokines that locally activate macrophages.
The many purely in vitro experiments con-
cerning the power of “immune” mononuclear
cells to inhibit the growth of ingested tubercle
bacilli are reviewed by Lurie (34).
DURATION AND SPECIFICITY OF
ACR AND ITS RECALL UPON
REINFECTION
In the 1960s, Mackaness and his associates clar-
ified the role of T lymphocytes and macrophages
in a series of beautifully designed experiments
(95–98, 98a, 99, 100).They injected mice intra-
venously with one type of facultative intracel-
lular bacillus (e.g., BCG, listeria, salmonella, or
brucella) and challenged these mice some time
䡵 111
later with the same or a different type of intra-
cellular bacillus. At various times after chal-
lenge, they determined the number and type of
viable bacilli in the spleens. In this manner, they
could assess the amount of antigen-specific
immunity produced by the primary infection,
because most of the antigens had no cross-
reactivity among the various bacillary types
evaluated.
After an intravenous immunization with
BCG, the acquired resistance (adaptive immu-
nity) of the host will decrease with time. How-
ever, a reinjection of BCG will rapidly recall
high levels of resistance, because the immunized
host retains increased numbers of clonally
expanded (memory) T cells with specific recep-
tors for BCG antigens. After the reinjection of
BCG, these memory T cells rapidly produce
cytokines, which cause local macrophage (and
lymphocyte) accumulation and activation, espe-
cially in the spleen, which is an organ known to
retain a large percentage of intravenously
injected bacteria.
The acquired resistance produced by the first
injection of BCG was not recalled by the
intravenous injection of other types of
facultative intracellular bacilli (e.g., liste-
ria or salmonella). However, after this acquired
resistance (in the spleen) had been recalled by a
second injection of BCG (the specific antigen),
the host could nonspecifically destroy the other
types of facultative intracellular bacilli. In other
words, macrophages in the spleen were rapidly
activated by the expanded antigen-specific T-cell
population that responded to the second injec-
tion of BCG. Once activated, these macrophages
could ingest and destroy the other types of
bacilli.
The specificity of CMI resides entirely in
the T lymphocyte, not in the macrophage.
Macrophages kill intracellular microorganisms
nonspecifically.
In most humans infected with the tubercle
bacillus, the number of antigen-specific T cells in
their blood and tissues decreases over time.Their
tuberculin skin test may even become negative,
especially if the tubercle bacilli and their antigens
have been eliminated. Nonetheless, increased
numbers of antigen-specific (memory) T cells still
112 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
remain, so that upon reinfection with virulent or
avirulent mycobacteria, their tuberculin sensi-
tivity is rapidly recalled, as is their ACR at sites
of bacillary lodgement.
The principles that Mackaness and his asso-
ciates (95–98, 98a, 99, 100) have established can
be briefly summarized as follows (29).
1. ACR (acquired cellular resistance), pro-
duced by many activated macrophages at
sites of bacillary lodgement, is not specific
for the microorganism that induced it.
Once activated, macrophages can kill a
variety of microorganisms.
2. Such local ACR disappears when bacil-
lary antigens are eliminated, which might
take years for many of the antigens of
tubercle bacilli (72; see also reference
101). ACR is rapidly and specifically
recalled locally (in the spleen in Mack-
aness’s experiments) by homologous
bacilli, but ACR is produced by heterol-
ogous bacilli only at the rate found in a
nonimmunized host.
3. The specificity of the rapid recall of ACR
is due to an expanded population of anti-
gen-specific (Th1) lymphocytes, which
circulate as the “memory” cells for DTH
and CMI.
4. The amount of ACR and CMI remain-
ing after recovery from the primary infec-
tion is at least partly dependent on the
persistence and type of bacillus. Mycobac-
teria persist longer than listeria.
5. CMI is relative, not absolute.A large num-
ber of reinfecting bacilli will usually cause
disease. However, the resulting disease is
often less severe than that present in non-
immunized controls.
6. The presence of tuberculin sensitivity
(DTH) means that the host has at least
some CMI, but not necessarily ACR.
However, such a host can develop ACR
locally at a faster rate and to a greater
degree when reinfected with tubercle
bacilli, because the antigen-specific lym-
phocyte population was expanded by the
first infection with tubercle bacilli.
INNATE IMMUNITY AND ITS
RELATION TO ACQUIRED
(ADAPTIVE) IMMUNITY
The late Charles A. Janeway, Jr., initiated the
recent surge of research on innate immunity
(Table 1) (2, 102–108). Such immunity can be
defined as the early host reaction in which the
host recognizes and responds to the invading
microorganisms. Innate immunity is present at
all times and does not substantially increase
with repeated exposure to a given pathogen.
Unlike acquired (adaptive) immunity, it shows
broad specificity in that it recognizes as foreign
many components of microorganisms. In
humans and rabbits (but not mice and guinea
pigs), innate immunity (mainly the alveolar
macrophages) prevents the growth of most
inhaled virulent human-type tubercle bacilli
(see chapters 2 and 15).
Once a tubercle bacillus multiplies in the
lungs, innate immunity is manifested by an
inflammatory response in which local resident
cells release phlogistic, chemotactic, and acti-
vating substances (such as histamine), various
cytokines (including chemokines), and the lipid
mediators: prostaglandins, leukotrienes, and
platelet-activating factor. This initial nonspe-
cific reaction is then continued by the release of
these and other inflammatory mediators from
the cells infiltrating the site.
The innate recognition of invading micro-
organisms falls into two categories: cellular and
serological. Host cells recognize microorgan-
isms by pattern recognition receptors (PRR),
such as Toll-like receptors (109–114). PRR
enable the phagocytes (and dendritic cells) to
recognize (for ingestion) substances in the patho-
gen that are different from those in the host
itself, e.g., various peptidoglycans, lipoproteins,
and lipopolysaccharides (2,103,104).A complete
review of the roles ofToll-like receptors in tuber-
culosis (and leprosy) was published in 2004 (110).
Serological recognition is by the comple-
ment system and various circulating opsonins
(see glossary) (2). Both the alternative pathway
and the mannan-binding lectin (collectin) path-
ways of complement are innately activated by
bacteria. The complement fragments C3a and
 5. INNATE AND ACQUIRED IMMUNITY IN TUBERCULOSIS
TABLE 1 Comparisons of the innate and adaptive immune systemsa,b
Property Innate immune system
Receptors Fixed in genome
Rearrangement not necessary
Distribution Nonclonal
All cells of a class identical
Recognition Conserved molecular patterns
(lipopolysaccharides, lipoteichoic acids,
mannans, glycans)
Self-nonself Perfect: selected over evolutionary time
discrimination
Action time Immediate activation of effectors
Response Costimulatory molecules
Cytokines, such as IL-1 and IL-6
Chemokines, such as IL-8
a
Adapted from reference 106.
b
Lurie (34) used the terms native and acquired to describe resistance to tuberculosis. Because of Janeway’s contributions to
innate immunity (2), the terms innate and adaptive have now replaced Lurie’s terminology. In this book, I have used both desig-
nations interchangeably, with a preponderance of those used by Lurie.
C5a are vasodilatory and increase the local blood
supply, and the chemotaxin C5a brings leuko-
cytes into the local area. C3b opsonizes bacte-
ria for ingestion by phagocytes. (Phagocytes
have C3b receptors.) The terminal components
of the complement cascade create pores in bac-
terial membranes, causing them to lyse. In addi-
tion to complement, serum contains C-reactive
protein and 2-macroglobulin, both of which
are innate opsonins that are increased during
microbial infections.
Surfactant proteins are a third group of innate
host defense, distinct from the cellular and sero-
logical defenses. Surfactant proteins are secreted
into the alveolar spaces by type II pulmonary
alveolar epithelial cells and by Clara cells (115).
SP-A and SP-D are members of the collectin
family. Surfactant proteins enhance phagocyto-
sis of inhaled bacteria by macrophages and neu-
trophils, and can be directly microbicidal for
some bacteria (116, 117).
The host’s innate response catalyzes the adap-
tive (antigen-specific) immune response in the
following manner.After APCs innately recognize
substances unique to microorganisms (often
through their Toll-like receptors [110]), the
APCs upregulate their costimulatory surface
䡵 113
Adaptive immune system
Encoded gene segments
Rearrangements necessary
Clonal
All cells of a class distinct
Details of molecular structure (proteins,
peptides, carbohydrates)
Imperfect: selected in individual somatic
cells
Delayed activation of effectors
Clonal expansion or anergy
IL-2 and others
Effector cytokines, such as IL-4 and IFN-
molecules, e.g., MHC-I, MHC-II, B7.1, and
B7.2 (2, 118), and group 1 CD1 proteins (119).
Costimulatory molecules are necessary for
antigen presentation to T lymphocytes (103,
104). Most self antigens do not cause APCs to
upregulate costimulatory molecules.Therefore,
autoimmune reactions are rare (see references
105 and 111).
APCs also contain coinhibitory surface mol-
ecules that control and limit the antigen-specific
immune response (118). Coinhibition seems to
be defective in some autoimmune diseases (118).
The intricacies of APC,T-cell, and B-cell inter-
actions have been clearly presented by Matzinger
(50, 51) and reviewed extensively in reference 2.
IL-12 is a major cytokine of the host’s innate
defense system (120). It is produced by mac-
rophages and dendritic cells that have ingested
microorganisms and their products. IL-12 acti-
vates both natural killer cells and Th1 lympho-
cytes, causing them to produce IFN-, which,
in turn, increases the microbicidal powers of
macrophages (121). Subsequently, IL-12 induces
IL-10 production in lymphocytes and phago-
cytes, and the IL-10 then inhibits or regulates
IL-12 production (120). IL-10 also activates
Th2 lymphocytes. HIV/AIDS patients have a
114 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
deficiency in their ability to produce IL-12
(120), and this deficiency seems to play a role in
the susceptibility of such patients to tuberculo-
sis (120). The roles of dendritic cells, macro-
phages, and Th1 and Th2 lymphocytes are
described in chapter 6.
In brief, the innate immune response of pul-
monary alveolar macrophages plays an impor-
tant role in preventing the establishment of both
primary and reinfection tuberculous lesions.
Once these lesions are established, the innate
PRR in APCs initiate and direct the acquired
(adaptive) antigen-specific immune response by
upregulating various costimulating factors.The
integration of the innate and acquired immune
responses ensures that most small established
tuberculous lesions do not progress in humans
and rabbits infected with M. tuberculosis.
The basics of innate and acquired (adaptive)
immunity are summarized in Table 1. References
108, 122, 123, and 124 provide more details on
the immunology of tuberculosis.
NONSPECIFIC AND ANTIGEN-
SPECIFIC IMMUNE RESPONSES IN
INNATE AND ADAPTIVE IMMUNITY
Confusion exists in the literature between non-
specific and antigen-specific immunity (acquired
resistance).All antigen-specific immune responses
in tuberculosis have nonspecific components.
In fact, once activated, the macrophage, which is
the main effector cell killing tubercle bacilli,
kills other intracellular bacilli quite effectively (see
Mackaness, reviewed above under “Duration
and Specificity of ACR and Its Recall upon
Reinfection”).
The antigen specificity of each lymphocyte
participating in a tuberculous granuloma has
never been determined. Lymphocytes are non-
specifically attracted into all inflammatory sites
by chemotactic factors, including those from
microorganisms, as well as chemokines from
local cells.The initial accumulation of lympho-
cytes, macrophages, and dendritic cells at sites of
bacillary lodgement is mainly an innate (non-
specific) host inflammatory response to any irri-
tant. However, in the initial stages of pulmonary
tuberculosis, this nonspecific response benefits
the bacillus more than it benefits the host,
because for the first 2 to 3 weeks the tubercle
bacillus multiplies well within the accumulating
nonactivated macrophages by altering their nor-
mal microbicidal functions (see chapter 1).
In contrast, the inflammation caused by the
tuberculin-like products of the bacillus is anti-
gen specific. It is called DTH and causes an
inflammatory reaction that is an order of mag-
nitude greater than that caused nonspecifically
by bacillary irritants in a nonsensitized (non-
immune) host. Details are presented in chapters
19 and 20.
In mycobacteria-infected humans, rabbits,
and guinea pigs, the stopping of the logarithmic
(symbiotic) stage of bacillary growth (at 2 to 3
weeks) seems to be due to an antigen-specific
tissue-damaging DTH reaction at sites where
the bacilli are located (see chapter 2). At this
time, both CMI and DTH develop, many bacilli
are killed, and additional bacillary components
are released.The host reacts both antigen specif-
ically and nonspecifically to the released bacil-
lary components. However, before CMI and
DTH develop, only a few bacillary products are
released.
In other words, the acquired (adaptive) anti-
gen-specific resistance of the host as well as the
local (innate) nonspecific resistance of the host
are both greatly enhanced by DTH and CMI.
Without the antigen specificity of DTH and
CMI, the intracellular multiplication of the
tubercle bacillus would not be controlled, and
a disease would occur that resembles leproma-
tous leprosy, Johne’s disease of cattle, and avian
tuberculosis in birds.
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 6
MACROPHAGES AND OTHER
CELLS IN TUBERCULOUS LESIONS

Antigen-presenting cells 120

Role of the major histocompatibility complex and CD1 proteins in antigen
presentation 122
Phagocytosis of tubercle bacilli by macrophages and their fate within
these cells 123
Mycobacterial dormancy in mouse tuberculous granulomas 124
Macrophage activation 126
Hydrolytic enzymes of rabbit mononuclear and polymorphonuclear exudate
cells, pulmonary alveolar macrophages, and lung homogenates 128
Macrophage enzymes released extracellularly in BCG lesions 128
Macrophage heterogeneity in tissue sections of BCG lesions 129
Causes of macrolocal and microlocal macrophage heterogeneity 131
Epithelioid cells 133
Langhans’ giant cells 133
Mononuclear cell turnover 136
Overview of lymphocytes: their subsets and their functions 137
Th1, Th2, CD4, CD8, Treg, and  T cells 138
Memory T cells 140
NK cells, cytotoxic T lymphocytes, and apoptosis 140
Granulocytes in tuberculous lesions 143
Fibroblasts in tuberculous lesions 144
Lymphatics in tuberculous lesions 144

Abstract. The main types of cells participating in rabbit tuberculous lesions are dendritic
cells, macrophages, natural killer cells, lymphocytes, and granulocytes.The role of most of
these cells is discussed only briefly in this chapter because details are available in textbooks
of immunology.The role of macrophages, however, is presented more fully because their
rates of turnover, their states of activation, their extracellular and intracellular enzymes, and
their heterogeneity have been extensively studied in my laboratory in the rabbit model
of tuberculosis.

Many types of cells and subgroups of cell types
have been identified in inflammatory and
immune responses (1), and all of these cells par-
ticipate in tuberculous lesions.Table 1 lists some
of the major players (see also reference 2). As
stated in the abstract, this chapter briefly describes
the attributes and functions of each of these cell
types. For more details, the reader is referred to
immunology textbooks, e.g., Immunobiology: the
Immune System in Health and Disease (1).
Very few cell functions have been studied in
tissue sections by histochemical techniques to
elucidate how each cell influences its neighbors
in vivo.Cell functions have mostly been studied
in vitro in cell cultures and in vivo in transgenic
or knockout mice.To my knowledge, no histo-
chemical studies (except those in my laboratory)
have been made on rabbit tuberculous lesions.
Information on dendritic cells and various
types of lymphocytes, however, is accumulating
so fast that even current textbooks are not fully
up to date.Therefore, in this chapter, I added a
few items from the recent literature on these two
cell types.
ANTIGEN-PRESENTING CELLS
Dendritic cells (DCs) are the main antigen-
presenting cells (APCs) (1, 3–10) (Table 1).They

120
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 121

TABLE 1 Major cell types involved in specific and nonspecific host defense reactions against the tubercle
bacillusa
Macrophages
Macrophages are the effector cells of the mononuclear phagocyte system.They are produced in the bone
marrow, circulate as monocytes in the bloodstream, and are called macrophages when they emigrate from the
blood into the tissues. Nonactivated monocytes/macrophages allow tubercle bacilli to multiply within them,
whereas highly activated macrophages inhibit or destroy tubercle bacilli. Many pulmonary alveolar
macrophages are highly activated and therefore (in rabbits and humans) often prevent inhaled virulent human-
type tubercle bacilli from starting an infection.
Antigen-Presenting Cells (APCs)
Dendritic cells are the main APCs. Dendritic cells migrate from the site of infection to the draining lymph
nodes, where they initiate an immune response by presenting antigens to the recirculating lymphocytes that
enter these nodes.Within tuberculous lesions, dendritic cells (along with macrophages and B lymphocytes) can
also present antigens to lymphocytes, especially after the host has developed acquired (adaptive) immunity.
Lymphocytes (T Cells and B Cells)
T cells (from the thymus) and B cells (from the bone marrow in mammals and the bursa in birds) provide
immunologic specificity to the host’s defense against tubercle bacilli. In tuberculous lesions, antigen-activated
T cells produce cytokines that activate macrophages to inhibit or destroy the tubercle bacillus.
T cells have been subdivided in a variety of ways based on (i) their surface markers (CD4 and CD8 T cells), (ii)
their receptors (/ and /), (iii) the cytokines they produce (Th1 and Th2 cells), and (iv) their functions
(helper, regulatory, and cytotoxic T cells). See Fig. 11.
Antigen-activated B cells produce antibodies, especially when they differentiate into plasma cells. In tubercu-
lous lesions, antigen-antibody reactions hasten the local accumulation of dendritic cells, macrophages, and
antigen-specific T cells; i.e., antigen-antibody reactions enhance the local cell-mediated immune response
(see chapter 5).
Natural Killer (NK) Cells
NK cells (both local and circulating) provide an important early defense against intracellular microorganisms
(viruses, bacteria, fungi, and protozoa). In tuberculosis, NK cells participate in the killing of bacilli-laden
macrophages and can produce IFN-, which activates macrophages and stimulates a Th1 cytokine immune
response.
a
Adapted from reference 2.

are the “conductor of the whole immunologic
orchestra” (R. K. Gershon, personal communi-
cation modified by Noel R. Rose). DCs that
activateT cells are produced in the bone marrow,
reside in various tissues, and mature by a multi-
step process for antigen processing and antigen
presentation.DCs that activate B cells (called fol-
licular DCs) are cells of uncertain origin.
DCs in the outer paracortex of the lymph
nodes (the most common type) present antigens
to T cells (10).These paracortical DCs are dis-
tinct from the follicular DCs in the germinal
centers of the lymphoid follicles.
Follicular DCs and antigen-activated helper T
cells interact with B cells for antibody produc-
tion.Antigens that cause B lymphocytes to pro-
liferate and produce antibodies are sometimes T-
lymphocyte dependent, whereas other antigens
(e.g., the capsular polysaccharides of bacteria) are
T-lymphocyte independent but nevertheless
seem to require some interaction with T cells
and/or DCs (reviewed in reference 11).
DCs That Activate T Cells
The primary acquired immune response to the
tubercle bacillus is initiated by DCs that activate
T cells. Macrophages and B cells evidently pre-
sent antigens only after antigen-specific T cells
have been expanded by the initial response. Also,
the type of antigen presented by macrophages
and B cells probably differs from that presented
by DCs (1). DCs are 2 to 3 logs more efficient
than macrophages and B cells in presenting anti-
gens to T cells, because mature DCs have high
levels of costimulatory molecules (such as B7.1
and B7.2). DCs also interact with other cells,
122 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
including natural killer (NK) cells and B cells
(12, 13).
DC Kinetics
DCs circulate in the blood (4) and are recruited
to sites of inflammation (14), such as tubercu-
lous lesions. Immature DCs at such sites capture
antigens and carry them to the draining lymph
nodes (1). There, the DCs mature and present
antigens to antigen-specific T cells that recircu-
late through the nodes.
In the draining lymph nodes, these antigen-
specific lymphocytes are activated, stimulated to
proliferate, and stimulated to differentiate into
cytokine-producing effector cells.These antigen-
specific lymphocytes then reenter the circulation
and emigrate into sites of inflammation (includ-
ing tuberculous lesions), where infiltrating DCs,
as well as macrophages and B cells, continue the
antigen-presenting process.
In brief, the primary acquired (adaptive)
immune response to tubercle bacilli is initiated
in the draining lymph nodes but continues at the
sites of infection (see references 12 and 15).
How Individual Organs Affect DCs
The following discussion offers some insight
into why certain organs of the host are more re-
sistant to tuberculosis than other organs (see
chapters 7 and 25).
When injured or irritated, each organ releases
factors that control both innate (nonspecific) and
acquired (adaptive) immune responses. Among
these factors are (i) cytokines (including
chemokines) (12, 16) that interact with one
another, as well as with receptors on nearby
cells (12, 17), and (ii) uric acid that is released
from dying cells and is a strong adjuvant to the
immune response (18). Its use as a potentiator of
vaccines remains to be developed.
Organ specificity determines how local and
infiltrating immature DCs differentiate into
mature subsets, and these subsets determine the
functional characteristics of the infiltrating lym-
phocytes (19–21). Each DC subset produces
different costimulatory molecules for antigen
presentation, which determine the Th1/Th2
lymphocyte balance (7, 8, 22). In other words,
each organ determines the subset into which
immature DCs mature, and therefore deter-
mines the type of local immune response that
occurs in that organ (see chapters 5 and 7).
How microbial components and inflamma-
tory cytokines affect DC phenotypes and func-
tions has been reviewed (12, 21), but much
more information is needed to understand the
process fully.
Tolerogenic DCs
Not only do DCs initiate and enhance the
immune response, but “tolerogenic” DCs exist
that turn off or suppress the immune response
through several mechanisms, including the
induction of regulatory (suppressor) cells (6, 9,
17, 23–26).
DCs as Adjuvants
The use of DCs (expanded in vitro) as adjuvants
to increase the host immune response is begin-
ning to find clinical application (5, 6, 9, 27).
Conventional and Plasmacytoid DCs
DC populations have recently been divided
into conventional DCs and plasmacytoid DCs—
each with differing functions in both antigen
presentation and host defense (28, 29). Plasma-
cytoid DCs produce type I (/) interferons
that increase host resistance to viruses and pos-
sibly other intracellular pathogens.They can be
immunostimulating, immunosuppressive, or even
tolerogenic. Plasmacytoid DCs usually favor a
Th1 antigenic response but have a role in Th2
responses (29). Chronic tuberculous lesions con-
tain many cells that resemble plasma cells (see
chapter 4). How many of these cells are typical
antibody-producing cells and how many are
plasmacytoid DCs remains to be determined.
ROLE OF THE MAJOR
HISTOCOMPATIBILITY COMPLEX
AND CD1 PROTEINS IN ANTIGEN
PRESENTATION
DCs present antigens to most T cells as specific
peptides in the context of the major histocom-
patibility complex (MHC) (1), which means
that a given T cell (with a clonally expressed 
antigen receptor) will recognize the antigen
only when its peptide fragments are bound to
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS
the DC’s MHC molecule. The MHC class I
molecules of DCs present peptides (derived
from proteins that gain access to the cytosol) to
cytotoxic CD8 T cells (1).This pathway recog-
nizes and eliminates host cells containing intra-
cellular microorganisms (reviewed in references
30, 31, and 32).The MHC class II molecules of
DCs present peptides (from exogenous proteins
degraded in endosomes) to CD4 T cells (1).
This pathway produces the delayed-type hyper-
sensitivity (DTH) and cell-mediated immunity
(CMI) that can stop the progression of tuber-
culosis. (CD stands for clusters of differentiation,
specifically, cell surface molecules recognized
by monoclonal antibodies with the designated
number [1].)
MHCs differ from host to host.Therefore, the
response to various antigens also differs from
host to host—in part depending on the affinity
of the host’s own MHC molecules for each
antigenic peptide.This may be one of the rea-
sons why some individuals are more resistant to
tuberculosis than others.
CD1 molecules are an antigen-presentation
system that is similar to (but independent of ) the
MHC system (33–36). CD1 proteins on DCs
have been divided into two groups: group 1
(CD1a, CD1b, CD1c) in rabbits, guinea pigs, and
humans, and group 2 (CD1d) in mice (37).The
characteristics of each group are reviewed in ref-
erences 34 and 35. Insights into how CD1 mol-
ecules function are clearly presented in refer-
ences 36, 38, and 39.
CD1 molecules bind and present (to T cells
with the appropriate receptors) several lipid, gly-
colipid, and lipopeptide components of tubercle
bacilli, such as mycolic acid, lipoarabinoman-
nan, and mycobactin (32, 35, 36, 38–44; see also
reference 45). These T cells are then activated
either to lyse macrophages infected with virulent
Mycobacterium tuberculosis (42) or to produce high
levels of gamma interferon (IFN-) (42), which
is one of the main cytokines increasing the
power of macrophages to inhibit or destroy the
bacillus. DCs expressing CD1 may play an
important role in the host immune response to
tubercle bacilli. In fact, a mycobacterial vaccine
containing an increased amount of lipid was
more effective than the parent vaccine (46).
䡵 123
References 12, 30 to 32, 35, and 47 review the
complexities of antigen processing and pre-
sentation and how they are regulated. Reference
48 reviews DC functions in inflammatory sites
and in organized lymphoid tissue.The section
“Innate Immunity and Its Relation to Acquired
(Adaptive) Immunity” in chapter 5 has addi-
tional information on how antigen-presenting
cells function.
PHAGOCYTOSIS OF TUBERCLE
BACILLI BY MACROPHAGES AND
THEIR FATE WITHIN THESE CELLS
Macrophage Receptors
To recognize a microorganism, macrophages
must first recognize patterns on its surface.The
pattern recognition receptors of macrophages
(and DCs) include mannose receptors, com-
plement receptors, fibronectin receptors, and
Toll-like receptors—all of which are involved in
the host’s innate immune response (1). After
acquired immunity develops, macrophages rec-
ognize antibody-opsonized microorganisms by
Fc receptors with help from complement C3b
receptors (reviewed in references 1, 49, and 50).
Extensive studies have recently been made on
Toll-like receptors (TLRs) (1, 40, 51–53).This
receptor family is composed of at least 10 mem-
bers that recognize various mycobacterial cell
wall components, including lipoproteins and
glycolipids (e.g., lipoarabinomannan). Engage-
ment of human macrophage TLRs by 19-kDa
mycobacterial lipoproteins activates NF-B,
induces interleukin 12 (IL-12) secretion, and
increases the transcription of inducible nitric
oxide synthase (NOS) (54)—all of which par-
ticipate in killing and/or inhibiting tubercle
bacilli.TLR-knockout mice produced conflict-
ing results on the importance of TLRs in resis-
tance to tuberculosis (55–57).
Fate of Mycobacteria within
Macrophages
Because of the receptors just mentioned, M.
tuberculosis is easily bound to macrophages and
internalized into phagosomes. Ordinarily, such
phagosomes fuse with lysosomes, undergo acid-
ification, and receive an abundance of lysosomal
124 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
enzymes that can degrade the microorganisms.
However, M. tuberculosis subverts phagosome-
lysosome fusion and subsequent maturation of
the phagosome into acidic, microbicidal, and
hydrolytic compartments.M.tuberculosis also resists
degradation by lysosomal enzymes.Although the
bacillus inhibits the entry of many microbicidal
factors into the phagosome (58), it allows nutri-
ents (such as iron carried by transferrin) to enter
(reviewed in references 59, 60, 61, and 62).
The tubercle bacillus alters the signal trans-
duction system of macrophages in which it
resides, decreasing the processes that are harm-
ful to it. A mycobacterial eukaryotic-like ser-
ine/threonine protein kinase G modulates sig-
nal transduction pathways for trafficking of
host-cell organelles (63), thereby inhibiting lyso-
some-phagosome fusion.Tetrahydrobenzothio-
phene specifically inhibits protein kinase G and
is a promising candidate for developing another
class of antimicrobials to combat tuberculosis
(63). Also, M. tuberculosis inhibits IFN- tran-
scriptional responses without inhibiting activa-
tion of STAT-1 (64). (IFN- is a major cytokine
that activates macrophages to kill or inhibit
tubercle bacilli.)
In mice, isocitrate lyase is produced by my-
cobacteria that are inhibited within activated
macrophages, but it is not produced by mycobac-
teria that are multiplying within nonactivated
macrophages (59, 65). This lyase enables the
bacillus to subsist on the fatty acids released from
lipids and thereby avoid being destroyed by
the microbicidins of activated macrophages
(reviewed in references 59 and 65). Isocitrate
lyase is present in mycobacteria but not in mam-
malian hosts, and therefore is a potential target
for new antimicrobials.
Virulent tubercle bacilli can persist in a dor-
mancy-like state in solid caseous tissue for many
years, and can persist in such a state within acti-
vated macrophages for a shorter time (see next
section).The accumulation of lipids that enable
such persistence is carried out by a putative tri-
acylglycerol synthase (66). Isocitrate lyase is
involved in the gradual metabolism of such
lipids. This metabolism evidently provides the
small amount of energy required to sustain via-
bility in the dormant state.
Macrophages can inhibit or kill intracellular
tubercle bacilli by means of reactive nitrogen
and oxygen intermediates (RNIs and ROIs), to
which M. tuberculosis is exquisitely susceptible
(67). In mice, chemical inhibition of NOS or
the deletion of the NOS gene exacerbates
tuberculosis (68–70). The proteasomes of the
tubercle bacillus are also involved in its resis-
tance against host RNIs (71, 72). See chapters
1 and 25 for additional information on viru-
lence.
MYCOBACTERIAL DORMANCY
IN MOUSE TUBERCULOUS
GRANULOMAS
Tubercle bacilli can live for months in mouse
granulomas without multiplying and without
dying (73, 74).The bacilli presumably are liv-
ing in activated macrophages (see next section)
that inhibit their multiplication. Rees and Hart
(73) made homogenates of lungs from albino P
strain mice after the mice had been infected
intravenously with virulent human-type tuber-
cle bacilli (H37Rv).These homogenates were
cultured to determine the number of live tuber-
cle bacilli, and smears of these homogenates
were stained by the Ziehl-Neelsen acid-fast
procedure and then counted microscopically to
determine the number of live-plus-dead bacilli.
In these untreated mice, live tubercle bacilli
persisted (during the 20 weeks of this experi-
ment) at only slightly lower titers than did live-
plus-dead tubercle bacilli (Fig. 1) (73).Therefore,
most of the bacilli in the mouse lungs were
viable but not multiplying and being killed,
because, if they had multiplied and then were
killed, the number of live-plus-dead bacilli
would have increased.
To support this conclusion, Rees and Hart
(73) treated mice with pyrazinamide and isoni-
azid from 2 months to about 6 months after
infection (Fig. 1). The antimicrobial treatment
reduced the number of live bacilli in the lung
homogenates over 10,000-fold (and to uncul-
turable levels at 6 months), but the number of
live-plus-dead bacilli was reduced less than 10-
fold (Fig. 1). Therefore, after being killed by
antimicrobials, dead tubercle bacilli remained
intact in the pulmonary granulomas for several
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 125

FIGURE 1 Viable counts and total counts of virulent tubercle bacilli (H37Rv)
in the lungs of mice from 9 to 25 weeks after an intravenous infection.After the
lungs were homogenized, the viable counts were calculated from the CFU devel-
oping on plates containing solid culture medium.The total counts were calcu-
lated from the bacilli observed microscopically on spread-smears after acid-fast
staining. During this time period, one of the four groups of mice received
isoniazid-pyrazinamide (PZA/INH) daily to kill the bacilli.
Note that for untreated mice the average total counts were 0.3 to 0.4 logs
higher (2.0 to 2.5 times) than the average viable counts, and that PZA/INH treat-
ment markedly reduced the viable counts but had relatively little effect on the
total counts.These findings indicate that (i) most of the dead tubercle bacilli per-
sisted in mouse lungs for many weeks; (ii) most of the live bacilli were in a “dor-
mant” nonmultiplying state, because if they had been multiplying and then had
been killed, the total counts (including the dead bacilli) would have increased;
and (iii) the good CMI developed by mice activated macrophages sufficiently
to prevent the intracellular multiplication of most of the tubercle bacilli. How-
ever, at least some of the bacilli were not inhibited by this good CMI, because
the disease progressed until the mice succumbed. In other words, not all of the
bacilli were dormant, and some bacillary multiplication occurred.
Redrawn from reference 73.These results were confirmed in reference 74
using quantitative real-time PCR technology.
126 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
months with few, if any, cycles of multiplication
and destruction.
Other experiments reaching the same con-
clusions were recently performed by the Mc-
Kinney group (74).They used the quantitative
real-time PCR (polymerase chain reaction) to
enumerate the live-plus-dead bacilli.The DNA
of dead tubercle bacilli was found to persist for
months as mycobacterial chromosome equiva-
lents (74), similar to the acid-fast staining
described by Rees and Hart (73).
The above experiments indicate that viru-
lent tubercle bacilli persist in the activated
macrophages of mouse granulomas without
appreciable multiplication or destruction, but
these bacilli grow readily in nonactivated
macrophages (see above). Such persistence can-
not, however, be considered true dormancy,
because the granulomas progress and eventu-
ally kill the mouse (see chapter 15).
Several factors involved in the nonreplicating
persistence of virulent tubercle bacilli in mice
have been identified (75–77). Evidently, the
bacilli upregulate dormancy regulons that enable
them to survive (without multiplying) for weeks
within the phagosomes of macrophages. How-
ever, a small proportion of the bacillary popu-
lation may be in a replicating state (74, 75),
especially if these bacilli have been ingested by
nonactivated macrophages that had recently
entered the granuloma.
Live counts and live-plus-dead counts of
tubercle bacilli should be repeated in tubercu-
lous rabbits and guinea pigs, which develop
strong DTH and readily form caseous necrotic
tissue. In these animals, numerous persisting
tubercle bacilli would probably be found in
solid caseum: some live and dormant and some
dead.Whether tubercle bacilli can persist (as in
mice) for many months within live macrophages
in a dormant state without multiplying or dying
remains to be investigated in rabbits and guinea
pigs. Rabbits may not break down dead tuber-
cle bacilli any better than mice do, because the
Wax D component of tubercle bacilli was found
to persist in rabbit Mycobacterium bovis BCG
lesions for over 56 days (78).
In brief, although nonreplicating dormant
tubercle bacilli evidently can persist in activated
macrophages for many months, dormancy peri-
ods of many years probably only occur in solid
caseous tissue, in which there is no infiltration of
fresh macrophages from the circulation.Appar-
ently, two categories of dormancy exist.The first
category (lasting for months) is a nonreplicating
viable (but inhibited) existence within an acti-
vated macrophage. This category of dormancy
ends whenever the macrophage (containing the
dormant bacillus) dies and the bacillus is ingested
by a nonactivated macrophage. Degenerating
macrophages containing tubercle bacilli are
sometimes found in older mouse granulomas
(see chapter 15).
The second category (lasting for years) is a
nonreplicating existence within solid caseum
that often ends when and if the caseum lique-
fies and the bacillus multiplies extracellularly.
Such caseous necrosis occurs in rabbits, guinea
pigs, and humans, but not in mice.
Several active genes were recently found to be
associated with mycobacterial latency (and per-
haps dormancy) in tuberculous rabbits (Y. C.
Manabe et al., unpublished data). Real-time
reverse transcription-PCR was used to identify
such genes at 5, 10, and 15 weeks after the inhala-
tion of human-type tubercle bacilli (H37Rv), and
also at 15 and 20 weeks in a similar set of rabbits
that were treated with dexamethasone from 10 to
15 weeks to reactivate the healing lesions. Ten
such genes were identified. In rabbits, tuberculous
lesions caused by virulent human-type tubercle
bacilli begin healing at 5 weeks and are often
completely healed by 15 and 20 weeks.
MACROPHAGE ACTIVATION
Macrophages are the major cells of the mononu-
clear phagocyte system (79).This system is com-
posed of promonocytes in the bone marrow,
monocytes in the circulation, and macrophages
in the tissues. Monocytes are called macrophages
after they enter the tissues.
Macrophages in the tissues, especially in sites
of inflammation, differentiate, i.e., become acti-
vated, for a variety of functions (80–84). Such
differentiation is controlled by local conditions,
especially nearby cells and their cytokines. Cer-
tain activated macrophages are highly phago-
cytic; others are highly microbicidal (due to
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 127

RNIs and ROIs as well as other factors); others
are rich in digestive enzymes; others produce
cytokines, e.g., interleukins and tumor necrosis
factor, as well as many other secretory products
(85–87); and still others are releasing and/or
presenting antigens from the tubercle bacilli
within them. Macrophages may combine several
of these functions (82). Therefore, the term
“activated” is generally used rather loosely to
describe macrophages in which one or more of
these activities is enhanced.
How macrophage functions are affected by
local conditions was clearly demonstrated by
the following experiment of Cohn and Benson
(88). Mouse macrophages were cultured in 50%
newborn calf serum for 24 h. After the
macrophages pinocytosed the serum protein
(and presumably digested it), their content of
various hydrolytic enzymes increased to high
levels. On the next day, after the 50% serum was
replaced by 1% serum, the content of these
enzymes decreased to low levels. On the third
day, after the 50% serum was replaced, the con-
tent of these enzymes increased again to high
levels.We confirmed this experiment with rab-
bit macrophages with boiled yeast as the
digestible substrate (89). Therefore, a “good
meal” in the pinocytic or phagocytic vacuoles
of macrophages results in the production of
enzymes to digest that meal. In other words, the
local environment greatly influences how
macrophages differentiate for various functions.
Within the tuberculous lesion, macrophages
also adapt to local conditions (80–82). Macro-
phages must be activated before they can destroy
tubercle bacilli (80, 81, 90). Nonactivated
macrophages provide a very fertile “soil” for
the intracellular growth of tubercle bacilli. In tis-
sue sections of dermal BCG lesions, highly acti-
vated macrophages (staining ⫹⫹⫹ and ⫹⫹⫹⫹
with our activation marker -galactosidase)
contained fewer tubercle bacilli than poorly
activated macrophages (Table 2) (80, 81, 90).This
finding suggests that such highly activated
macrophages had destroyed some of the tuber-
cle bacilli they once contained.
To confirm this conclusion, we produced der-
mal BCG lesions in rabbits by injecting 14C-
labeled BCG (90). In these lesions, we then
determined the percentage of ⫹,⫹⫹,⫹⫹⫹,and
⫹⫹⫹⫹ -galactosidase-positive macrophages
that contained the 14C label without any intact
bacilli, which shows that these macrophages once
contained intact bacilli but had destroyed them.
Table 3 documents our expected findings: highly
activated (⫹⫹⫹ and ⫹⫹⫹⫹ -galactosidase-
positive) macrophages contained a much higher
percentage of the 14C label without intact bacilli
than did poorly activated (⫹ -galactosidase-
positive) macrophages. This is direct proof that
activation of macrophages enables them to destroy
the tubercle bacilli they ingest.
Activation of macrophages can be caused (i)
by the ingestion of necrotic cells and tissues

TABLE 2 -Galactosidase (-Gal) activity and the number of acid-fast bacilli seen in immature and mature
epithelioid cells in nonnecrotic granulation tissue of dermal BCG lesionsa
Intensity of staining for -Gal
(a marker for macrophage activation) ⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹
No. of -Gal-positive cells counted in each tissue 79 ⫾ 12 104 ⫾ 16 68 ⫾ 16 6.7 ⫾ 2.7
section
No. of tubercle bacilli in ⫹⫹, ⫹⫹⫹, and ⫹⫹⫹⫹ 100% 40 ⫾ 9% 22 ⫾ 5% 1.3 ⫾ 0.8%
-Gal-staining cells as a percentage of the no. in
⫹ staining cellsb
a
Cumulative data from six experiments. One tissue section from each of 29 biopsies was evaluated. Every ⫹ to ⫹⫹⫹⫹
-Gal-positive epithelioid cell in a given area of tissue section was counted. Then, for each tissue section, we calculated the
number of acid-fast tubercle bacilli (found microscopically) in 100 ⫹ -Gal cells, in 100 ⫹⫹ -Gal cells, in 100 ⫹⫹⫹ -Gal
cells, and in 100 ⫹⫹⫹⫹ -Gal cells, and listed it as a percentage of the number of bacilli in 100 ⫹ cells. Relatively few ⫹⫹⫹⫹
-Gal cells were present.The means and their standard errors are shown.The P values for the last row were ⬍ 0.001, except
for 40 vs. 22, which was 0.044.This table indicates that as epithelioid cells (macrophages) mature, they stain more strongly for
-galactosidase and destroy tubercle bacilli more efficiently. (Adapted from reference 90).
b
Immature epithelioid cells are partly activated macrophages, and mature epithelioid cells are fully activated.
128 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 3 Macrophage activation and 14C-labeled bacillary components within dermal BCG lesions
% of Macrophages with no
intact bacilli in -Gal categorya
Age of lesions when
Lesions produced by: biopsied (days) ⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹
14
C-labeled bacilli 7 0 0 32.4 21.4
Heat-killed 14C-labeled bacilli 7 0 0 34.6 33.3
a
Percent of macrophages containing only 14C-labeled bacillary components and no intact bacilli in each -galactosidase
(-Gal) category: [14C-pos, but bacilli-neg, cells] divided by [-Gal-pos cells] ⫻ 100).
Note that at 7 days many ⫹⫹⫹ and ⫹⫹⫹⫹ -Gal macrophages contained 14C-labeled bacillary components that were not
attached to intact bacilli, whereas at 7 days all ⫹ and ⫹⫹ -Gal macrophages contained 14C-labeled bacillary components that
were only attached to intact bacilli.
The 14C label produced silver grains in the photographic emulsion covering the tissue section. Ninety percent of the intact
tubercle bacilli (identified by acid-fast staining with carbol-fuchsin) contained silver grains.Therefore, the presence of 14C-labeled
silver grains in macrophages without the presence of intact bacilli indicates that ingested bacilli had been destroyed and broken
down into components that still remained within the macrophages.
In this experiment, heat-killed BCG were not destroyed more readily than live BCG, which is consistent with our finding
that the wax D component of tubercle bacilli can persist in rabbit BCG lesions for at least 56 days (77).
Adapted from reference 90, where complete data and statistics are presented.

and other digestible substances (89), (ii) by the
cytokines released by lymphocytes and other
cells, and (iii) by ligands, such as the mannose
and lipopolysaccharides, combining with specific
macrophage receptors. In general, the greatest
number of activated macrophages is present
when tuberculous lesions reach peak size.
HYDROLYTIC ENZYMES OF
RABBIT MONONUCLEAR AND
POLYMORPHONUCLEAR EXUDATE
CELLS, PULMONARY ALVEOLAR
MACROPHAGES, AND LUNG
HOMOGENATES
Because intracellular hydrolytic enzymes play
major roles in how macrophages digest com-
ponents of tubercle bacilli, we identified and/or
characterized the proteases, esterase, lipase,
lysozyme, DNase, and/or RNase in rabbit peri-
toneal mononuclear and polymorphonuclear
exudate cells, pulmonary alveolar macrophages,
and/or lung tissue. References 84, 89, and 91
through 102 provide specific details.
The proteinase cathepsin D (91, 92, 99), a
chymotrypsin-like esterase (100), and RNase
(102) were purified from rabbit lungs, and anti-
bodies to the purified cathepsin D and RNase
were made (99, 102).Antibodies to rabbit phos-
pholipase A2 were provided by Richard C. Fran-
son, Medical College of Virginia, Richmond
(103). Cathepsin D, RNase, and phospholipase A2
were then visualized immunohistochemically in
tissue sections of BCG lesions (103, 104). The
substrate-film technique was also used to iden-
tify proteinase, DNase, RNase, hyaluronidase,
chondroitinase, and fibrinolytic activities in such
tissue sections (81, 105, 106). Each of these
hydrolases was present in activated macrophages
within the lesions.
In vitro ingested tubercle bacilli downregu-
lated cathepsin G and upregulated cathepsins B
and D in the human macrophage cell line THP-
1 (107).
MACROPHAGE ENZYMES RELEASED
EXTRACELLULARLY IN BCG LESIONS
In addition to their ingestive and digestive func-
tions, macrophages have extracellular functions
(reviewed in references 85, 86, 87, 105, and 108)
that appear to be important in tuberculosis.
Upon activation, macrophages secrete or release
a variety of substances.These include (i) elastase,
collagenase, and plasminogen activator, (ii)
lysozyme, (iii) clotting factors, and (iv) interfer-
ons, as well as (v) colony-stimulating factors,
which enhance monocyte and granulocyte pro-
duction in the bone marrow, and (vi) a variety
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS
of cytokines, e.g., interleukins, chemokines, and
factors that stimulate fibroblasts (see chapters 19
and 20).
Most of these studies have been done in vi-
tro with relatively pure populations of cells.The
studies described below are among the few per-
formed in vivo.They were done before most of
the cytokines were discovered, so only hydrolytic
enzymes were measured.
We placed plastic chambers over dermal BCG
lesions and tuberculin reactions (with the epithe-
lium removed) (108–110). The hydrolytic
enzymes released into the chamber were assayed,
and a quantitative histochemical study was made
of the number of activated (-galactosidase-
positive) macrophages in the chamber beds.
Although a portion of the enzymes assayed
could have come from other cell types,
macrophages were probably the major source,
because they were the dominant cell in the
lesions and contained these enzymes.
At 18 days of age, the BCG lesions were
largest (Fig. 2B), and the activated macrophages
in the chamber beds were most numerous (Fig.
2C and 3). Between 11 and 18 days, the levels
of the five enzymes (assayed in the chamber
fluids) reached their peaks (Fig. 4).At this time,
tuberculin hypersensitivity was well developed
(Fig. 2A), and the bacilli and their components
were still present. Two-week-old polystyrene
lesions (Fig. 5) served as controls.
The enzyme levels in 2-day chamber fluids
from tuberculin reactions (1, 2, and 3 days old)
were not statistically different from the levels in
BCG lesions (Fig. 4).
Active collagenase (an enzyme secreted but
not stored in macrophages) was only detected in
fluids from peak BCG lesions (108). Evidently,
the serum in the chamber fluids was sufficient
to inhibit the lower collagenase levels that were
probably released from smaller BCG lesions and
tuberculin reactions.
These studies demonstrate that, in chronic
inflammatory lesions produced by the tubercle
bacillus, both acid-acting and neutral-acting
hydrolytic enzymes are released extracellularly.
Tissue components would be hydrolyzed locally
when the concentration of these enzymes
䡵 129
exceeded the concentration of their inhibitors,
and the pH was favorable (108).
MACROPHAGE HETEROGENEITY IN
TISSUE SECTIONS OF BCG LESIONS
Color Plates 1 and 2 pictorially demonstrate
macrophage heterogeneity in tuberculous
lesions produced by BCG. For Color Plate 1,
the tissue section was double-stained, first for
cathepsin D (rust color) and then for -galac-
tosidase (blue). Note that macrophages in the
periphery of the lesion contained cathepsin D,
whereas macrophages near the caseous center
contained -galactosidase. We called this type
of macrophage distribution “macrolocal” or
regional (82).
For Color Plate 2, the tissue section was dou-
ble-stained, first for acid phosphatase (red) and
then for -galactosidase (blue). Macrophages
containing acid phosphatase were sometimes
next to macrophages containing -galactosi-
dase.We called this type of distribution “microlo-
cal,” i.e., individual macrophages could be acti-
vated for different functions, even though they
were adjacent to each other in the tuberculous
lesion. Some macrophages (colored purple) were
activated for both acid phosphatase and -galac-
tosidase (Color Plate 2).
We quantitated the macrolocal and microlo-
cal activation of macrophages in developing,
peak, and healing dermal BCG lesions. The
amounts of the four macrophage enzymes eval-
uated were highest when the lesions reached
peak size (Fig. 6). Macrophages staining for -
galactosidase and esterase were always more
numerous near the caseous center of the lesions
than in the periphery (Color Plate 1 and Fig. 7
and 8). In contrast, macrophages staining for
cathepsin D and acid phosphatase were always
more numerous in the periphery than near the
caseous center (Color Plate 1 and Fig. 7 and 8).
In tuberculous lesions, macrolocal and
microlocal heterogeneity should also occur in
macrophage populations producing various
cytokines, RNIs and ROIs, and many other
cell functions, including the upregulation of
different types of receptors.Therefore, the obser-
vations just portrayed clearly establish a general
FIGURE 2 (A) Size of 48-h tuberculin reactions. (B) Size of BCG lesions. (C) Weighted num-
ber of activated -galactosidase-positive macrophages, just below the surface of the chamber beds
of BCG lesions (closed circles) and 72-h tuberculin reactions (open circles). To obtain the

130
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 131

FIGURE 3 Chamber bed of a 32-day BCG lesion. Many darkly staining ⫹⫹⫹ and ⫹⫹⫹⫹
-galactosidase-positive macrophages are present. A thin proteinaceous layer covers the surface
of the chamber bed. Stained with 5-bromo-4-chloro-3-indolyl--D-galactoside, hematoxylin and
eosin. Magnification, ⫻30. Reproduced with permission from reference 108.

pattern for all inflammatory reactions, namely, CAUSES OF MACROLOCAL AND

that the functions of the participating cells are
locally controlled.
Other types of macrophage heterogeneity were
recently reviewed by Mosser (83).There is het-
erogeneity in their receptors,in the cytokines they
produce (including chemokines), and in their
microbicidal abilities. Some macrophages favor
the Th2 response over the usual (IL-12-induced)
Th1 response. Some macrophages downregulate
inflammatory and immune responses by pro-
ducing IL-10 and IL-1-receptor antagonist.
And some macrophages stimulate repair and
healing by producing transforming growth
factor .
MICROLOCAL MACROPHAGE
HETEROGENEITY
How and why such macrolocal and microlocal
distribution of macrophages occurs in tubercu-
lous lesions is a matter of conjecture (82).Vari-
ous possibilities exist, but many need experi-
mental proof.
1. Macrophages probably differentiate locally
for various functions because of interac-
tions with other cells in their environ-
ment.Adjacent (or nearby) lymphocytes,
plasma cells, granulocytes, vascular en-
dothelial cells, fibroblasts, and even other
macrophages may determine the direction

weighted number of -galactosidase-positive macrophages, the number of ⫹ cells was multiplied

by 1, ⫹⫹ cells were multiplied by 2, ⫹⫹⫹ cells were multiplied by 3, and ⫹⫹⫹⫹ cells were
multiplied by 4, and then the products were added together. Reproduced with permission from
reference 108.
132 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 133

in which a given macrophage activates.

Macrophages may express different surface
receptors that determine how these cells
respond to stimuli from adjacent cells.
2. Macrophages may become activated for
one function when the lesion is young
and remain activated for the same function
when the lesion is older. In other words,
two adjacent macrophages can be acti-
vated for a different function at different
times.
3. Macrophages may be activated in one part
of the lesion and then migrate to another
part, possibly due to different chemokine FIGURE 5 Tissue section of a 14-day rabbit skin
lesion produced by the intradermal injection of 60 ⫻
receptors.
106 polystyrene latex particles. This lesion showed no
4. The hydrolytic enzyme content of each inflammation. All of the polystyrene particles were
macrophage may be determined by what within macrophages staining ⫹⫹ to ⫹⫹⫹⫹ for -
it ingests, e.g., the ingestion of lipids may galactosidase. Note that the dermal collagen fibers adja-
stimulate lipase production. If so, in the cent to the macrophages are intact, suggesting that no
effective collagenase was produced by these cells. A
tuberculous lesion, the location of each
small amount of collagenase was found at 48 h in flu-
type of activated macrophage would ids within chambers placed over the polystyrene lesions,
reflect the location of the lipids, proteins, but 2 to 3 times this amount of collagenase was found
carbohydrates and nucleic acids, which in chamber fluids placed over 14-day BCG lesions,
are probably not distributed evenly. where the collagen fibers were hydrolyzed (108). Stained
with 5-bromo-4-chloro-3-indolyl--D-galactoside,
5. The presence or absence of tubercle bacilli
hematoxylin and eosin. Magnification, ⫻350. Repro-
within a macrophage also affects its type duced with permission from reference 108.
of activation (64).

EPITHELIOID CELLS
Epithelioid cells are macrophages in various
states of activation that are sometimes orga-
nized into an epithelium-like pattern due to
adhesion molecules (see chapter 21). In tuber-
culosis, epithelioid cells have large vesicular
(euchromatic) nuclei, suggesting active DNA
transcription for synthetic functions. Mature
epithelioid cells are highly activated cells (Fig.
9 and 10).They are rich in enzymes and micro-
bicidins, and therefore are much more effective
than immature epithelioid cells in destroying
ingested bacilli (80–82, 111, 112). Some epithe-
lioid cells are secretory macrophages.
LANGHANS’ GIANT CELLS
In tuberculosis, Langhans’ giant cells occur more
frequently in human lesions than in rabbit
lesions (see chapter 3).A Langhans’ giant cell is

FIGURE 4 Lysozyme, RNase, DNase, and lactic dehydrogenase (LDH) activities in the cham-
ber fluids of BCG lesions of various ages (closed circles and line graph), 1-day tuberculin reac-
tions (open circles), and normal skin controls (shaded horizontal lines). Lysozyme is both secreted
and stored; RNase and DNase are released on cell death and possibly regurgitated, but not secreted;
and LDH is released only on cell death.
The chambers were glued to the still intact skin around the area where the epidermis was
removed.They were then filled with HEPES culture medium 199, and the fluid within the cham-
bers was collected 48 h later. Note that the highest level of these extracellular hydrolases occurred
when tuberculin sensitivity had developed, the BCG lesions were growing to peak size, and the
greatest number of activated macrophages was in the chamber bed (see Fig. 1). Reproduced with
permission from reference 108.
134 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Estimates of the total amount

of each of four enzymes in tissue sections of
dermal BCG lesions during their develop-
ment and healing. The mononuclear cells
(mostly macrophages) in these tissue sections
were single-stained for acid phosphatase,
cathepsin D, esterase and -galactosidase and
were evaluated microscopically at both 35⫻
and 125⫻ magnifications. For quantitation,
both the distribution and intensity of the his-
tochemically produced color were taken into
account. Then, the total amount of staining
was given a rating on a 0 to 9 scale.The stan-
dard errors of the means are shown.
Note that the total amount of each enzyme
was greatest when the lesions peaked in size
(Fig. 2B), and the levels of each enzyme more
or less rose and fell in parallel during the devel-
opment and healing of the lesions. Repro-
duced with permission from reference 82.

FIGURE 7 Distribution of mononuclear cells (MN) (mostly macrophages) that single-stained

for acid phosphatase, cathepsin D, -galactosidase, or esterase in developing, peak, and healing der-
mal BCG lesions.The percentage of MN staining ⫹ to ⫹⫹⫹⫹ was evaluated microscopically
at the edge of the caseous necrotic center, in the viable tissue near this center, and more periph-
erally in representative high-power fields at ⫻500 magnification.The standard errors of the means
are shown.
Note that macrophages containing -galactosidase and esterase were more frequent near the
caseous center (A and B), and macrophages containing acid phosphatase and cathepsin D were
more frequent in the peripheral regions (C). These findings quantitatively demonstrate the
“macrolocal” distribution of activated macrophages within tuberculous lesions (see text). Repro-
duced with permission from reference 82.
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 135

FIGURE 8 Distribution of activated mononuclear cells (mainly macrophages) in double-stained

tissue sections of developing, peak, and healing rabbit dermal BCG lesions.These sections were
stained histochemically for pairs of enzymes: one red and one blue.The red enzymes were acid
phosphatase, cathepsin D, or red esterase.The blue enzymes were -galactosidase or blue esterase.
The mononuclear cells were counted microscopically at the edge of the caseous necrotic cen-
ter (A), in the viable tissue near this necrosis (B), and in peripheral areas of the lesion (C).
The length of the bars represents 100% of the mononuclear cells in each area.The cells that
stained for only one enzyme of the pair were either red (stippled bars) or blue (hatched bars).
The cells that stained for both enzymes of the pair were purple (black bars).
The black bars in column 3 and the mirror-image patterns C:B:A and A:B:C in columns 1
and 2 show that the same macrophage population contained -galactosidase, red esterase, and blue
esterase. Macrophages containing cathepsin D and acid phosphatase had similar distributions (see
columns 1 and 2), but since they both stained red, they could not be differentiated in the same
tissue section. (The red esterase is from a diazo dye; the blue esterase is from an indolyl dye [82].)
These graphs confirm the microlocal mononuclear cell activation shown in Color Plate 2, i.e.,
that some macrophages almost always stain for an enzyme different from that of the majority of
macrophages in a given area.
Reproduced with permission from reference 82.
136 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 9 Tissue section of a 21-day rabbit dermal BCG lesion stained for -galactosidase,
our marker enzyme for macrophage activation.A group of epithelioid cells with high enzyme
activity is seen in the tuberculous granulation tissue that surrounds the lesion’s liquefied
caseous center (in the lower right corner of the photograph). Mature epithelioid cells (iden-
tified by their rounded appearance) stain the strongest (⫹⫹⫹ and ⫹⫹⫹⫹) for -galactosi-
dase.These activated macrophages cluster in the area of the BCG lesion where the bacilli (iden-
tified by acid-fast staining) are located. Stained with 5-bromo-4-chloro-3-indolyl--
D-galactoside, lightly counterstained with hematoxylin. Magnification, ⫻200. Reproduced
with permission from reference 80.
This picture clearly demonstrates the principle of local immunity, i.e., the bacilli and their
products stimulate local lymphocytes to produce cytokines that activate nearby macrophages.
Highly activated macrophages are known to contain high concentrations of reactive oxygen
and nitrogen intermediates and hydrolytic enzymes that kill or inhibit the tubercle bacillus. Such
highly activated macrophages may be harmful to tissues, especially if they die and release their
contents.Therefore, the host apparently limits the activation of macrophages to local sites of
bacillary lodgement where they are most needed to control the infection.

a multinucleated epithelioid cell. It is formed MONONUCLEAR CELL TURNOVER

when two cells fuse together (sometimes around
a bit of caseous material), or when one cell fails
to divide after duplicating its nucleus, or both
(112). Their nuclei are arranged about the
periphery of the cell, in contrast to the usual for-
eign-body type of giant cell, in which the nuclei
are scattered throughout the cytoplasm. The
presence of Langhans’ giant cells seems to be a
sign of a decrease in the progression of the dis-
ease, as well as a sign of chronicity (111, 112).
These cells do not play a major role in the
pathogenesis of tuberculosis (112).
Macrophages and lymphocytes entering an
established tuberculous lesion do not remain
there very long (113). Within 10 days, more
than 90% of them have “turned over,” i.e., they
have died (because of apoptosis or tissue-dam-
aging DTH) or have left the lesion via the
lymphatics and have been replaced by new
macrophages. With the development of CMI
and DTH, the rate of entry of these cells, their
rate of loss, and their rate of activation are
increased. In the developing lesion, the rate of
entry exceeds the rate of loss, so large numbers
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS
of macrophages and lymphocytes accumulate
and activate. Chapter 10 presents details on
macrophage turnover.
In almost-healed BCG lesions, macrophage
turnover still occurs but at reduced levels.This
suggests that, even in arrested tuberculous lesions,
a few nonactivated macrophages enter and some
intracellular bacillary multiplication may occur
(113).True bacillary dormancy may only exist in
solid caseous tissues and not in the viable tissues
that surround such necrotic foci (113).
OVERVIEW OF LYMPHOCYTES:
THEIR SUBSETS AND THEIR
FUNCTIONS
Lymphocytes (Table 1) are the antigen-specific
effector cells of the host’s acquired (adaptive)
immune response (1).Their role in the patho-
genesis of tuberculosis is described in chapter 5.
Lymphocytes develop clonally expressed anti-
gen-specific receptors by DNA rearrangements.
When exposed to the antigen for which they
FIGURE 10 Tissue section of a 33-day rabbit der-
mal BCG lesion stained for cytochrome oxidase, an
enzyme involved in oxygen metabolism. The large
cells are epithelioid cells, similar to those in Fig. 9.The
most mature ones with the rounded appearance stain
the darkest. In other words, the macrophages most
effective in inhibiting the intracellular growth of the
tubercle bacillus contained the highest levels of both
hydrolytic and oxidative enzymes. The other cells
(which we cannot differentiate) are probably small
macrophages, dendritic cells, lymphocytes, and plasma
cells. Stained with 8-amino-1,2,3,4-tetrahydroquino-
line and p-aminodiphenylamine (84) with no coun-
terstain, so the cell nuclei stain lighter than the cyto-
plasm. Magnification, ⫻470. Reproduced with
permission from reference 81.
䡵 137
Common T-cell subsets
CD4
• Th1 cells (producing IL-1, IL-2, TNF-␣, and
IFN-␥)
• Th2 cells (producing IL-1, IL-2, TNF-␣, IL-4,
IL-5, IL 10, and IL-13)
• Regulatory T cells (CD4⫹/CD25⫹) (produ-
cing IL-10 and TGF-␤)
CD8
• Cytotoxic T cells
• Cytokine-producing cells (IFN-␥ and others)
FIGURE 11 A generalized presentation of the com-
mon types of T cells, i.e., those with  antigen recep-
tors (see reference 1). Note that T cells with the CD4
and CD8 surface markers can produce similar cytokines.
have a receptor, they respond by clonal prolif-
eration, which greatly increases their number.
Antigen-specific B lymphocytes (called B
cells) come from the bone marrow and produce
antibodies. Antibodies enhance the local accu-
mulation of defense cells in tuberculous lesions
(see chapter 5). B cells make greater amounts of
antibody when they differentiate into plasma
cells. Plasma cells are common in tuberculous
lesions (see chapter 4).
Antigen-specific T lymphocytes (called T cells)
come from the thymus.They produce the CMI
and DTH that develop in tuberculosis and other
infections. T cells recognize antigens that are
processed and bound to either MHC (class I or
II) or CD1 molecules (see “Antigen-Presenting
Cells” at the beginning of this chapter).
T lymphocytes have been divided into vari-
ous subsets by their CD4 and CD8 surface anti-
gens, by their functions (T helper [Th] cells, reg-
ulatory T cells, and cytotoxic T cells), and by the
cytokines they produce (Th1 and Th2) (Fig.
11). For additional details, the reader should
consult reference 15 and textbooks of immunol-
ogy (e.g., reference 1). Most of the studies on the
types and functions of lymphocytes in tubercu-
losis have been made in mice (40, 41, 114, 115).
Numerous lymphocytes accumulate in tuber-
culous lesions that do not seem to have any
receptor specific for antigens of tubercle bacilli.
The role of these bystander lymphocytes remains
to be determined. They probably produce
cytokines that participate in this chronic inflam-
matory reaction.
138 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
In the host’s circulation and in lymphoid tis-
sues, the number of naive lymphocytes and the
number of memory lymphocytes seem to be
controlled independently (116). During any
infection, including tuberculosis, antigen-specific
lymphocytes proliferate, and the entire lym-
phoid system adjusts accordingly.
The overall control of the number of lym-
phocytes in the host was recently reviewed in
reference 117.
TH1, TH2, CD4, CD8, TREG,
AND  T CELLS
Th Cells
The Th1 subset produces IL-2 (which causes T-
cell proliferation) and IFN- and tumor necro-
sis factor beta (TNF-), both of which activate
macrophages. IFN- downregulates the Th2
response. By means of reverse transcription-
PCR, we evaluated changes in IFN- in devel-
oping and healing tuberculous lesions produced
by BCG in rabbits. IFN- production seemed
to occur later than the production of the
chemokine MCP-1 (see chapter 19).
IL-12 is a major cytokine that specifically
expands the Th1 population and upregulates its
functions. Macrophages and DCs are the main
producers of IL-12, especially when they con-
tain intracellular microorganisms, such as M.
tuberculosis (118).Therefore, IL-12 plays a major
role in acquired host resistance to tuberculosis.
It also plays a role in innate immunity by acti-
vating NK cells.
The Th2 subset produces IL-4, IL-5, IL-6, IL-
10, and IL-13, all of which promote antibody
production by B cells. IL-4, IL-10, and IL-13
downregulate the Th1 response.
CD4 and CD8 Lymphocytes in
Tuberculous Lesions
The percentage of CD4 and CD8 cells in the
mononuclear cell population of rabbit dermal
BCG lesions was determined (119). During
both the development and healing of the BCG
lesions, CD4 cells were always more numerous
than CD8 cells (Fig. 12 and Table 4).At 2 days,
reinfection BCG lesions and tuberculin reactions
showed a higher percentage of CD8 cells than
did primary BCG lesions (Fig. 12), suggesting
that the presence of tuberculin sensitivity
enhanced the accumulation of cytotoxic T cells,
which could kill macrophages in which tuber-
cle bacilli were growing. At 5 days, reinfection
BCG lesions showed a higher percentage of
both CD4 and CD8 cells than did primary
BCG lesions (Fig. 12). Cytotoxic M. tuberculosis-
specific CD8 T cells preferentially recognize
cells that are heavily infected with tubercle
bacilli (120). CD8 functions in infectious diseases
are reviewed in reference 121.
Regulatory T Cells
Regulatory T cells (Treg), formerly called sup-
pressor T cells (122), downregulate antigen-
specific immune responses, usually by produc-
ing IL-10 (123, 124). These lymphocytes also
stimulate macrophages and other cells to pro-
duce prostaglandin E2 and other suppressor fac-
tors, including lipoxins (1, 87, 125). In addi-
tion, many inflammatory mediators, such as the
cytokines TNF-, IFN-, and transforming
growth factor , as well as oxygen and nitrogen
intermediates, can switch from being pro-
inflammatory to anti-inflammatory (87) and
thereby probably contribute to the regression
and healing of tuberculous lesions. See references
124 and 126 through 130 for details on Treg cell
function in vitro and in vivo.A substantial num-
ber of these cells are CD4⫹CD25⫹ (131, 132).
Treg cells keep the immune response from
becoming excessive and causing tissue damage.
When infections are properly controlled, the
proper balance between effector immunocytes
and Treg cells exists (133).
 T Cells
Most T cells have alpha-beta () antigen recep-
tors (Fig. 11). However, a small population of T
cells possessing gamma-delta () antigen recep-
tors also exists (reviewed in reference 134).
Since  T cells are activated before  T cells,
they have been postulated to play a role in the
early antigen-specific immune response against
tuberculosis (135, 136). Health care profession-
als recently exposed to M. tuberculosis, as well as
patients with active infection, have increased
numbers of  T cells in their blood (137–140;
also see references 141–143).
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS
 T cells seem to have a much wider influ-
ence on the host’s response to microorganisms
than was previously believed, because they affect
functions of NK cells, DCs, B cells, and other T
cells, in part by producing IFN- and other
cytokines (134, 144).At least some of the  T
cells have receptors that combine directly with
intact antigens without the usual processing and
presentation by DCs. In other words, unlike
T cells with  receptors, this  group does
not require processing of antigens and is
neither class I nor class II MHC restricted (1).
䡵 139
FIGURE 12 Percentage of mononuclear
cells (MN) (mostly lymphocytes) immuno-
stained for CD4 (A) or CD8 (B) in primary
and reinfection BCG lesions and in tuber-
culin reactions. At 2 days, the reinfection
BCG lesions and the tuberculin reactions
contained a higher percentage of CD8 cells
than did the primary lesions, suggesting that
tuberculin sensitivity increases the number
of cytotoxic CD8 cells in tuberculous
lesions. Note, however, that CD4 cells are
always much more numerous than CD8
cells (compare the y axes).
Each point represents the mean of four
lesions with its standard error. For reinfec-
tion BCG lesions versus primary BCG
lesions: *P ⬍ 0.05 and **P ⬍ 0.01; for
tuberculin reactions versus primary BCG
lesions: †P ⬍ 0.05; and for reinfection
BCG lesions versus tuberculin reactions:
‡P ⬍ 0.05 and ‡‡P ⬍ 0.01. Reproduced
with permission from reference 119.
 T cells expressing the V2V2 T-cell
receptor constitute the majority of circulating
 T cells in humans and nonhuman primates,
but are absent in mice (32, 145).The V2V2 T
cells recognize small organic phosphate anti-
gens that are present in M. tuberculosis. In
macaques after BCG reinfection, the V2V2⫹
T-cell subset expands 2 to 9 times. Similarly,
BCG-vaccinated macaques have a rapid recall of
V2V2 T cells in bronchoalveolar lavages after
a challenge with virulent M. tuberculosis (146).
These findings indicate that V2V2⫹ T cells
140 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
TABLE 4 Percentage of CD4 and CD8 lymphocytes in the mononuclear cell population of primary BCG
lesions during their development and healinga
Age of BCG % of CD4 % of CD8
lesion (days) cells (A) cells (B)
9 13.2 ⫾ 1.2 8.7 ⫾ 2.1
23 17.8 ⫾ 1.1 5.4 ⫾ 0.9
37 15.7 ⫾ 2.4 6.3 ⫾ 1.6
a
Note that the number of CD4 cells was always higher than the number of CD8 cells (A/B) and that the ratio of CD8 to
CD4 cells (CD8/CD4 ⫽ B/A) was highest in 9-day lesions. At 9 days, many bacilli were multiplying in nonactivated macrophages,
and DTH developed that stopped such multiplication (see chapter 2). (Adapted from reference 119.)
In single-cell suspensions of rabbit tuberculous lungs and their hilar lymph nodes, similar ratios of CD4/CD8 cells were found
by flow cytometry (unpublished data).
are important contributors to the acquired
immune response to tubercle bacilli.
V2V2 T cells are absent in mice (32, 145),
but they have V4⫹  T cells that regulate air-
way hyperreactivity (147).
MEMORY T CELLS
Memory CD4 and CD8 T lymphocytes are of
two types: central memory cells and effector
memory cells (15). Effector memory cells are
located in peripheral tissues and in sites of
inflammation, where they provide immediate
protection against microorganisms (15). Central
memory cells are located in the lymphoid
organs, primarily the lymph nodes and spleen.
There, they can rapidly expand and differenti-
ate to supply effector T cells to peripheral sites
(15).The two types can be recognized by their
CD surface markers (15).Vaccines that greatly
increase the number and availability of memory
cells should provide the best protection against
clinical tuberculosis (see chapter 22).
NK CELLS, CYTOTOXIC
T LYMPHOCYTES, AND APOPTOSIS
The killing of poorly activated macrophages in
which tubercle bacilli are multiplying intracel-
lularly is an important factor in the control of
tuberculosis (1) (see chapter 2). NK cells and
cytotoxic T lymphocytes (CTLs) play major
roles in this host defense reaction.
NK Cells
NK cells (see Table 1) compose 5 to 10% of the
peripheral blood lymphocyte population.They
proliferate in response to both macrophage and
T-cell-derived cytokines. NK cells have the
Ratio CD4/CD8 Ratio CD8/CD4
(A/B) (B/A)
1.5 0.66
3.3 0.30
2.5 0.40
morphology of large granular lymphocytes and
are currently defined as cytotoxic cells that do
not express surface CD3 or T-cell antigen recep-
tors, but do express CD56.
The NK cells have both MHC class I and
non-MHC-requiring receptors, whereas the
cytotoxic T (CD8) cells recognize only specific
antigenic peptides in association with MHC
class I molecules. In fact, engagement of surface
receptors on NK cells with MHC class I mol-
ecules on host cells usually turns off, rather than
activates, the lytic machinery of the NK cells
(148). Cells infected by many viruses and other
intracellular microorganisms have little or no
MHC class I expression and will be lysed by NK
cells, whereas healthy cells (expressing normal
levels of MHC I) will not be lysed. Some viruses
produce MHC class I homologs that prevent
NK cells from lysing host cells that contain
these viruses (148), but similar virulence mech-
anisms have yet to be investigated in mycobac-
teria.
Since NK cells can kill infected host cells
without prior sensitization, they are considered
part of the innate immune system (149). In
tuberculosis, NK cells are an early source of
IFN-, which activates macrophages and en-
hances Th1 immunity. Also, macrophages can
activate NK cells (150).
NK1.1 T cells express  T-cell receptors
that recognize CD1 molecules, rather than
MHC class I or MHC class II molecules (1; also
see references 36 and 151). Since they mostly
respond to lipid and glycolipid antigens, they
probably play a role in the early host response
to tubercle bacilli.
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS 䡵 141

Only a few studies have been made on NK
cells and mycobacteria. Human peripheral blood
mononuclear cells kill more intracellular M.
tuberculosis when cultured with NK cells (152).
Also, human NK cells (stimulated in vitro with
IL-12) activate macrophages to inhibit the intra-
cellular growth of Mycobacterium avium (153).
Macrophages containing intracellular bacte-
ria produce TNF- and IL-12, which activate
NK cells. NK cells, in turn, produce much IFN-
 (and many other cytokines) that (i) activate
macrophages nonspecifically early during an
infection, and (ii) favor a subsequent Th1 spe-
cific immune response over a Th2 response
(154). However, NK cells can also produce IL-
5 and IL-13, which favor a Th2 response (154).
In mice, the removal of NK cells did not
substantially alter host resistance to tuberculo-
sis (155). In humans, however, NK cells are
probably more important, because humans defi-
cient in NK cells are extremely susceptible to
intracellular infections such as that caused by
herpesvirus (156).
NK cells can directly lyse cells in which bac-
teria and viruses are multiplying. Therefore,
NK cells are a major force in early antigen-
independent host resistance to tuberculosis.
NK cells also interact with DCs, presumably in
ways beneficial to the host (157, 158).
NK cell functions are discussed more fully in
Immunobiology (1) and in reference 149.
CTLs
Antigen-specific CTLs come from the thymus,
as do other antigen-specific T cells. Most CTLs
are CD8 cells. They recognize self-peptide

FIGURE 13 Mononuclear cells (MN) and granulocytes (PMN) per mm2 of tissue section in
BCG lesions at various times during their development and healing.The mononuclear cells were
mostly macrophages with some medium and large lymphocytes (and probably some dendritic
cells). In the BCG lesions, only the areas that were densely infiltrated with cells were counted.
These areas were usually found about one-third of the distance from the edge of the caseous cen-
ter to the edge of the lesion. PMN were even more numerous nearer the caseous center. At 37
days, the BCG lesions were much smaller, so the total number of cells present was much reduced.
Reproduced with permission from reference 163.
142 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 14 A tissue section of a 19-day rabbit dermal BCG lesion incubated on a film of RNA
for 1 h at 23°C and stained with toluidine blue. Note the “starry sky” appearance representing
RNase activity in a percentage of the granulocytes.The cells that have digested the substrate film
beneath the tissue section were probably eosinophils, because eosinophils are known to contain
high levels of RNase (181, 182), and because most of the PMN (recognized by their multilobed
nucleus) were inactive. Magnification, ⫻180. Reproduced with permission from reference 81.

antigens in the context of MHC class I. CTLs The perforin-independent (nonsecretory)

require activation by exogenous antigens, as
well as by cytokines (IL-2 and others), to
develop their full cytotoxic activity. Most (but
not all) CD8 cells exhibit a Th1 cytokine pro-
file. References 159 and 160 provide more
details on CTLs.Their role in mouse tubercu-
losis is reviewed in references 40 and 41.
Apoptosis
Both NK cells and CTLs kill their target cells by
perforin-dependent and perforin-independent
apoptotic pathways (1). Perforin is a protein that
can polymerize to form a hole (or pore) in the
target cell’s membrane. Perforin, cytolysin, and
hydrolytic enzymes (called granzymes) are stored
in secretory granules of NK cells and CTLs.
After entry through the perforin-produced hole
in the target cell’s membrane, granzymes (espe-
cially proteases) apparently contribute to the
DNA fragmentation that characterizes apopto-
sis, which is programmed cell death.
pathway of apoptosis occurs by cross-linking
(on the target cell’s surface) Fas (CD95) or
TNF-receptor molecules with the Fas ligand on
NK cells and CTLs. Fas is defined as a cell
receptor that induces apoptosis. (In humans, Fas
is also known as APO-1.) The cross-linking of
Fas molecules on cell membranes initiates pro-
grammed cell death via the sphingomyelin
(ceramide) signal transduction pathway.
Apoptosis keeps the immune response to var-
ious antigens from becoming excessive (161).
Also, the viability of monocytes/macrophages
(and probably activated lymphocytes) depends
on stimulatory cytokines, without which these
cells undergo apoptosis (162).
In mice, apoptosis evidently plays a major
role in killing macrophages containing numer-
ous intracellular tubercle bacilli, because in
mouse tuberculosis very little necrosis occurs
(see chapter 15).The relative roles of apoptosis
and caseous necrosis in controlling the numbers
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS
of bacilli in tuberculous humans, guinea pigs, and
rabbits remain to be determined.
GRANULOCYTES IN
TUBERCULOUS LESIONS
Polymorphonuclear leukocytes (PMN) are pre-
sent in tuberculous lesions (Fig. 13), especially
near their caseous centers (Color Plate 2) (163).
They do not seem to be able to destroy tuber-
cle bacilli and may, in fact, play a role in their dis-
semination (112). Rabbit PMN are eosinophilic,
in contrast to human, mouse, and guinea pig
PMN, which are neutrophilic. Because of their
eosin staining, rabbit PMN should be called het-
erophils, not neutrophils (164). PMN evidently
recruit immature DCs to sites of inflammation
and activate them (165). Platelet chemokines
(e.g., platelet factor 4 and -thromboglobulins)
FIGURE 15 A postcapillary venule in a guinea pig
contact-sensitivity reaction to dinitrochlorobenzene.
Note the preservation of three basophils (arrows): one
just outside the venule, one inside the venule, and one
between the endothelium and its basement membrane.
In this thin GMA tissue section, endothelial cells and
pericytes can be easily distinguished by their location.
In other words, these thin plastic-embedded tissue sec-
tions enable a resolution with light microscopy that
approaches the resolution with low-power electron
microscopy. In guinea pigs, rabbits, and humans, mast
cells and basophils can be easily distinguished by their
shape and staining characteristics (164).The tissue spec-
imen was embedded in GMA, cut at 1 to 2 m, and
stained with Giemsa. Magnification, ⫻790. Reproduced
with permission from reference 164.
䡵 143
are major factors in recruiting PMN to sites of
inflammation (166). The number of PMN in
an inflammatory lesion is determined not only
by their rate of entry, but also by the rate at
which they undergo apoptosis (167).
Eosinophils are also present in tuberculous
lesions (168), but eosinophils are not easily dis-
tinguished from PMN in rabbits. Rabbit eo-
sinophils often have large orange-brown egg-
shaped granules, whereas rabbit PMN usually
have smaller granules and stain a brighter orange
(164). The eosinophils in rabbit BCG lesions
seem to show higher ribonuclease activity than
any other cell present (Fig. 14). However, per-
oxidase staining with diaminobenzidine would
probably distinguish eosinophils from PMN
with more certainty (164). Reference 169
reviews the beneficial and detrimental effects of
eosinophils on cells participating in inflamma-
tory processes. The role of eotaxin in mouse
mycobacterial granulomas was analyzed in ref-
erence 170.
We have made no studies on basophils and
mast cells in rabbit tuberculous lesions, but we
have studied basophils and mast cells in dermal
lesions produced by the chemical irritant dilute
sulfur mustard (164, 171–173). For these studies,
we prepared 1- to 2-m sections from tissues
embedded in glycol-methacrylate (GMA) (Fig.
15 and 16) (164, 174, 175). GMA preserves blue-
purple (i.e., basophilic) granules within these
FIGURE 16 Mast cells in a tissue section of normal
rabbit skin.The tissue specimen was “cold-embedded”
in GMA (174), cut at 1 to 2 m, and stained with
Giemsa. Magnification, ⫻900.
144 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
two cell types.These granules are often destroyed
in paraffin-embedded preparations. In the mild,
slowly developing acute dermal inflammatory
lesions produced by 1% sulfur mustard, basophils
in relatively large numbers (20 to 30% of the
granulocyte infiltrate) entered during the first
2 h in both rabbits and guinea pigs (Fig. 15)
(171).After this time, the percentage of basophils
decreased (171). Perhaps, basophils (and the his-
tamine that they contain) enhance many early
inflammatory responses, including those pro-
duced by the tubercle bacillus.
We also evaluated mast cells in dermal sulfur
mustard lesions produced both in vivo in rab-
bits (Fig. 16) (172) and in vitro in organ-cultured
full-thickness human skin explants (173). The
thin GMA tissue sections enabled us to assess the
amount of mast cell degranulation produced
FIGURE 17 Activated fibroblasts (⫹⫹⫹ to ⫹⫹
⫹⫹) between collagen fibers in the corium of a heal-
ing (6-day) rabbit dermal inflammatory lesion (pro-
duced by the topical application of 1% sulfur mus-
tard). These fibroblasts were stained histochemically
for the lysosomal enzyme acid phosphatase, which pro-
duces the bright red color that appears dark in this
black-and-white photograph. The high activation of
these fibroblasts is indicated by their large size and the
large amount of acid phosphatase that they contain.
Normal rabbit skin has relatively few activated fibro-
blasts (179).Activated fibroblasts produce the new col-
lagen and ground substance associated with healing.
A small blood vessel containing erythrocytes can be seen
in the lower half of the photograph.
Depicted is a 6-m “cold-embedded” GMA tissue
section, stained histochemically with naphthol AS-BI
phosphate and fast red violet LB, and counterstained
with hematoxylin. Magnification, ⫻540. Reproduced
with permission from reference 179.
by the sulfur mustard and to correlate this
degranulation with the amount of histamine in
the organ-culture fluids (173).
At sites of many infections, mast cell cytokines
have been found to enhance the recruitment of
T cells (176, 177).They probably play a similar
role in lesions produced by the tubercle bacillus.
Reference 178 is an extensive review of the
ultrastructure of human mast cells and basophils
and how their organelles function.
FIBROBLASTS IN
TUBERCULOUS LESIONS
We were not always able to distinguish acti-
vated fibroblasts in tissue sections from acti-
vated infiltrating macrophages and have not
studied them in tuberculous lesions, except in
relation to fibrosis and healing (see chapter 4).
However, in tissue sections of rabbit dermal sul-
fur mustard lesions, the number of activated
fibroblasts (Fig. 17) was increased and the num-
ber of activated infiltrating macrophages was
decreased as the lesions healed (179).
LYMPHATICS IN TUBERCULOUS
LESIONS
Lymphatics have rarely been evaluated in tissue
sections, mainly because they are not readily
visible in standard paraffin-embedded sections.
FIGURE 18 A 1-day dermal inflammatory lesion that
was produced in the skin of a rabbit by topical 1% sulfur
mustard in methylene chloride. Note the dilated lym-
phatic vessel and two adjacent small blood vessels con-
taining erythrocytes.This acute chemically induced lesion
was grossly edematous, so the lymphatic vessel was dilated
from the excess tissue fluid that it was removing. Depicted
is a “cold-embedded” (174) 1- to 2-m GMA tissue sec-
tion, stained with Giemsa (164). Magnification, ⫻600.
 6. MACROPHAGES AND OTHER CELLS IN TUBERCULOUS LESIONS
In thin GMA-embedded sections, lymphatics are
readily visible. Figure 18 shows a dilated lym-
phatic in a 1-day rabbit dermal sulfur mustard
lesion that was grossly edematous (180).
Tuberculous lesions are never this edema-
tous. However, lymphatics carry tubercle bacilli
from the lesions to the draining lymph nodes,
and lymphatic endothelial cells probably become
activated and produce cytokines, similar to the
endothelial cells of the microvasculature.
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 Section 3.

TUBERCULOUS LESIONS
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 7
STRUCTURAL COMPONENTS
OF TUBERCULOUS LESIONS
Overview 155
Tuberculous granulation tissue 155
Caseous tissues 155
Liquefied caseum and cavities 157
Healing tuberculous lesions 158
Persisting viable tubercle bacilli 158
Organ and species resistance 158
Resistance to bovine-type and human-type tubercle bacilli
Abstract. This chapter describes the structural components of tuberculous lesions: the sur-
rounding granulation tissue, solid caseous necrosis, liquefied caseum, cavities, and the
fibrosis and calcification of healing lesions.
OVERVIEW
In humans, rabbits, and guinea pigs, typical
tubercles often have a caseous necrotic center.
Surrounding this caseous center is tuberculous
granulation tissue, which is rich in macrophages
and lymphocytes and contains dendritic cells,
plasma cells, polymorphonuclear leukocytes,
eosinophils, fibroblasts, and many capillaries (see
chapter 6).The caseum is initially solid, but in
time may liquefy. In solid caseum, many tuber-
cle bacilli are dead, but some are only dormant.
In liquefied caseum, the bacilli may multiply
extracellularly, sometimes extensively. If they
multiply, their tuberculin-like products can cause
necrosis of a bronchial wall so that a cavity
forms.The tubercle bacilli then spread via the
bronchial tree to other parts of the lung and to
the environment. Healing with fibrosis can occur
if the number of bacilli remains small.
TUBERCULOUS GRANULATION
TISSUE
The active part of all lesions in tuberculosis con-
sists of tuberculous granulation tissue (1). It con-
tains young macrophages, activated macrophages
(now called epithelioid cells), natural killer cells,
antigen-presenting cells (dendritic cells), lym-
phocytes, plasma cells, granulocytes (usually near
areas of caseous necrosis), capillaries, lymphatics,
155
159
reticular fibers, and, in time, fibroblasts and the
collagen and ground substance which they pro-
duce (see chapters 4 and 6). See chapters 8 and
21 for details on the role of the vasculature in
these lesions.
In humans, tuberculous granulation tissue
often contains ill-defined granulomas and areas
of caseous necrosis, along with variable numbers
of tubercle bacilli.The cells in tuberculous gran-
ulomas produce a variety of cytokines, e.g.,
interleukin 1 (IL-1), IL-2, and IL-12, monocyte
chemoattractant protein-1, tumor necrosis fac-
tor, gamma interferon, and transforming growth
factor  (see chapters 19 and 20) (2–4).
CASEOUS TISSUES
In rabbits, guinea pigs, and humans, the necrotic
tissue in tuberculous lesions is described as
caseous, because it resembles cheese, being rather
homogeneous, yellow-white, and rich in lipids
and proteins derived from tubercle bacilli and
dead cells. It is caused by tissue-damaging
delayed-type hypersensitivity (DTH). (See chap-
ters 3 and 4 for photographs of caseous tissue.)
No connective tissue fibers are usually found
within the caseum, because they were hydrolyzed
by the collagenases and elastases of macrophages
and granulocytes in the tubercle before caseation
took place. In time, caseous foci in humans may
156 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
calcify or even ossify.Tubercle bacilli may survive
in solid caseous material, but they are usually
inhibited there by the reduced oxygen tension
and relatively low pH (5), as well as by the local
accumulation of toxic fatty acids (6, 7).
In rabbits, guinea pigs, and humans, caseous
necrosis first occurs at the end of the logarith-
mic (symbiotic) stage of tuberculosis when
tuberculin sensitivity develops (see chapter 2).
The nonactivated macrophages in which the
bacilli reside are killed by the tissue-damaging
DTH reaction, so the intracellular growth of
tubercle bacilli is stopped. Whether or not a
true caseous center develops and enlarges
depends on the sensitivity of the host and the
amount of tuberculin products in the sur-
rounding tuberculous granulation tissue. Con-
siderable caseation is produced when the host
develops high degrees of tuberculin sensitivity
and/or large amounts of tuberculin-like prod-
ucts are released from numerous bacilli.
When macrophages surrounding the caseous
center are poorly activated, the bacillus again
grows intracellularly, and again the DTH reaction
kills such cells. The lesion progresses, and the
caseous center enlarges. However, when most of
the macrophages surrounding the caseous center
are strongly activated, they will inhibit or destroy
the bacilli that they ingest, and the lesion will
regress or at least stabilize. All early lesions in
humans apparently develop a caseous center (see
chapter 3), but only some of the lesions develop
such a center in rabbits infected by aerosol with
virulent (human-type) tubercle bacilli.
Macrophages cannot penetrate very far into
the caseous center.The center is avascular and
the adjacent blood vessels are thrombosed (8).
In addition to the relatively low oxygen tension
and pH mentioned above, the caseous material
probably contains toxic metabolic products and
relatively high concentrations of tuberculin-like
products. Thus, the host’s defense cells cannot
eliminate tubercle bacilli within caseous foci,
but, fortunately, such bacilli cannot multiply
appreciably in solid caseum because of these
same adverse conditions.
What causes the death of macrophages that
have ingested tubercle bacilli and the death of
nearby tissues is not precisely known (Table 1).
Intact bacilli seem rather nontoxic to
macrophages (9). DTH is definitely involved,
because caseous necrosis occurs in tuberculous
lesions at about the same time that the tuber-
culin skin test becomes positive. Table 1 lists
some of the possibilities. (i) Natural killer cells
and cytotoxic T cells seem to kill bacilli-laden
macrophages and may also injure nearby tis-
sues. (ii) Clotting factors from macrophages, as
well as from necrotic cells and tissues, may acti-
vate the clotting system, impairing the local
blood supply and causing additional tissue injury
(8) (see chapter 8). (iii) Reactive oxygen and
nitrogen intermediates (e.g., superoxide and
nitric oxide produced by activated macrophages)
may kill cells and tissues as well as bacilli. (iv)
Certain cytokines produced by macrophages
and T lymphocytes (such as tumor necrosis fac-
tor) are probably toxic at higher than normal
concentrations. (v) Hydrolytic enzymes, e.g.,
proteinases, nucleases, and lipases, released from
live and disintegrating macrophages and gran-
ulocytes may injure tissues directly. (vi) Antigen-
antibody reactions, or even aggregated proteins
in the necrotic tissues, may locally activate the
complement system, which may injure cells.
TABLE 1 Causes of tissue damage and caseous
necrosisa
Natural killer cells and cytotoxic T cells
Involving perforin, apoptosis and other mechanisms
Anoxia
Produced by thrombosis (macrophages produce
clotting factors)
Cell products that may be toxic at high local
concentrations
Reactive oxygen and nitrogen intermediates; certain
cytokines, such as tumor necrosis factor; hydrolytic
enzymes; and complement
Bacillary products that may be toxic at high
local concentrations
Intact tubercle bacilli are nontoxic, but when they are
broken down, toxic products, such as “cord factor”
(trehalose dimycolate), may be released.
Overview
Caseous necrosis is initiated by a tissue-damaging
DTH reaction to high local concentrations of
tuberculin-like bacillary products.Th1-type
lymphocytes impart specificity to this reaction.
a
Reprinted with permission from reference 1.
 7. STRUCTURAL COMPONENTS OF TUBERCULOUS LESIONS
(vii) After tubercle bacilli are fragmented, toxic
components, such as “cord factor” (trehalose
dimycolate), may be released. The most likely
cause of the tissue damage, however, is a com-
bination of many of these possibilities (10).
Regardless of the exact mechanisms involved,
it is generally agreed that almost all of the cell
death and tissue damage found in tuberculosis
are due to DTH, either directly or indirectly, and
that thrombosis of the vasculature is a major
cause of resulting necrosis.
LIQUEFIED CASEUM AND CAVITIES
Liquefaction occurs when solid caseous mate-
rial softens (see chapter 4) (6, 9, 11–14, 14a). In
contrast to pyogenic abscesses, which liquefy
soon after they form, the caseous foci of tuber-
culosis may not liquefy for months, if ever. For
photographs of liquefied caseum and cavities see
Fig. 1 and chapters 3 and 4.
The exact cause of liquefaction is not known,
but the following pathophysiological sequence is
consistent with present knowledge (Table 2).
The antigens of the tubercle bacillus, especially
tuberculin-like antigens (after the host becomes
tuberculin positive) (15, 16), cause an accumu-
lation of macrophages at the site (see chapter 10).
Then, the proteinases and nucleases (14a) and
䡵 157
TABLE 2 Causes and results of liquefactiona
Causes
DTH to tuberculin-like bacillary products
Hydrolytic enzymes, especially proteases, DNases and
RNases, and probably lipases
Results
Extracellular bacillary multiplication (that is
sometimes tremendous), which may allow
antimicrobial drug-resistant mutants to develop
Caseous necrosis of bronchial walls and cavity
formation, caused by the high concentrations of
tuberculin-like products
Spread of bacilli through the air passages to other
parts of the lungs and to the environment, where
they may infect other people
a
Adapted from reference 1.
probably lipases (17, 18) released from these
macrophages liquefy the solid caseous material
(see chapter 4). The breakdown products of
caseous material are osmotically active, so that
fluid is absorbed from the surrounding tissues.
This liquefied material, rich in nutrients, is often
an excellent culture medium for tubercle bacilli
(5, 9, 13, 14), which now may grow profusely and
extracellularly for the first time during the course
of this disease (Fig. 1) (1, 9).The wall of a nearby
bronchus is eroded by the tissue-damaging DTH
FIGURE 1 The wall of a cavity from a
genetically resistant rabbit, 8 weeks after
the inhalation of human-type tubercle
bacilli.The liquefied caseous tissue (right)
and liquefying caseous tissue (left) are
both swarming with (rod-shaped) acid-fast
bacilli. Cavities can be produced by both
virulent human-type tubercle bacilli (illus-
trated here) and virulent bovine-type
tubercle bacilli (illustrated in chapter 4);
the bacilli can grow profusely in cavities
produced by either type. Magnification,
⫻620. Reproduced with permission from
reference 25.
158 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
reaction to the large amount of tuberculin-like
products present. The bronchus ruptures, and
the liquefied caseum, often containing numer-
ous bacilli, is discharged into the air passages,
thereby forming a cavity.Then air enters, and the
high oxygen content provides an additional stim-
ulus for the extracellular bacillary growth.
Current knowledge on the cause of lique-
faction is presented in chapter 4. Detailed char-
acteristics of human and rabbit tuberculous
cavities are presented in chapters 3 and 4,
respectively.
HEALING TUBERCULOUS LESIONS
In humans, caseous centers occur more readily
than in rabbits because of the high human tuber-
culin sensitivity.Arrested calcified caseous lesions
as small as 0.5 mm can still be found years after
they were formed (see chapter 3).
Larger foci may reactivate many years later if
the resistance of the person is lowered, or if liq-
uefaction occurs (see chapters 1, 3, and 4).
Whether calcified foci less than 1 mm in diam-
eter can reactivate remains to be determined.
In rabbits infected with virulent Mycobacterium
tuberculosis, enlargement of lesions with or with-
out solid caseous centers is prevented by the local
accumulation and activation of macrophages (see
chapters 2 and 5) (19, 20).Tuberculous lesions 0.1
to 2 mm in diameter may heal completely and
disappear.Larger lesions (2 to 5 mm) with caseous
centers may eventually be absorbed. Still larger
caseous and liquefied foci (5 to 20 mm) may
undergo fibrous encapsulation and are thereby
isolated within the host (see chapter 4).
In both humans and rabbits, the healing of
small endogenous metastatic tubercles (and small
tubercles of exogenous reinfection) involves
basically the same mechanisms as healing of
primary foci, i.e., the local accumulation and
activation of macrophages at the site of infection.
However, because of the already existing cell-
mediated immunity and DTH in the host,
macrophage accumulation and activation occur
more rapidly in metastatic tubercles (see chap-
ters 5 and 10) and often stop bacillary multipli-
cation in the early stages of lesion development.
Since the interplay of local factors determines
the fate of each lesion (21, 22), hosts with clin-
ical tuberculosis may have progressing and heal-
ing lesions within the same lung (see chapter 3).
PERSISTING VIABLE
TUBERCLE BACILLI
In humans, after even a clinically inapparent
tuberculous infection heals, the lungs may con-
tain small encapsulated, solid, or semisolid caseous
foci. In such foci, tubercle bacilli may persist in
a dormant (almost nonmetabolizing) state (see
chapters 1 and 6) and are therefore resistant to
killing by antimicrobial agents. (Many bacteria,
including the tubercle bacillus, have dormancy
genes that enable such persistence [23].)
This bacillary persistence is the reason why
any infection with virulent tubercle bacilli is
potentially dangerous. The bacilli may remain
viable for decades and may cause active disease
when the host resistance is lowered by advanced
age, corticosteroids, immunosuppressants, HIV,
or other factors.
Persistence of viable tubercle bacilli is prob-
ably the reason why a positive tuberculin reac-
tion is often maintained for life. Each time the
bacillus multiplies in a primary or secondary
focus, the immune system is stimulated.
Although in humans (and rabbits), new metasta-
tic foci usually heal as microscopic lesions, suf-
ficient bacillary antigen is almost always pro-
duced to maintain the tuberculin reaction.
One of the arguments in favor of vaccina-
tion with effective Mycobacterium bovis BCG is
that the host is safer harboring BCG than har-
boring virulent tubercle bacilli.Although both
attenuated and virulent strains increase resis-
tance to exogenous reinfection, hosts harbor-
ing virulent strains have a greater risk of pro-
gressive infection should their resistance be
impaired.
ORGAN AND SPECIES RESISTANCE
Different organs of the host have different sus-
ceptibilities to tubercle bacilli. In rabbits, tuber-
cles in the liver usually remain small and heal,
whereas in their kidneys they enlarge and cause
renal disease (9). In guinea pigs, the reverse
situation occurs: tubercles enlarge in the liver,
but they remain small in the kidneys and usu-
ally heal (9).
 7. STRUCTURAL COMPONENTS OF TUBERCULOUS LESIONS
In each animal species and in each organ,
macrophages from the blood enter the devel-
oping lesion, but once there, they are evidently
influenced by local factors present in the specific
organ and by genetic factors present in the
species. To date, these local factors have not
been identified, but they probably involve
cytokines (produced by resident cells) that affect
the infiltrating dendritic cells, macrophages, and
lymphocytes (see chapters 5 and 6).
RESISTANCE TO BOVINE-TYPE AND
HUMAN-TYPE TUBERCLE BACILLI
In general, bovine-type tubercle bacilli are more
virulent than human-type bacilli in most animal
species, especially in the rabbit, where the dif-
ference is marked (see chapter 13). (In humans,
the difference in virulence is not readily appar-
ent.) In both guinea pigs and rabbits, live as
well as dead bovine-type bacilli are more diffi-
cult for the host to destroy than the human
type, and the bovine type usually produces larger
lesions (9).
Live bovine-type bacilli can apparently sur-
vive at a lower oxygen tension than can human-
type bacilli (9), which explains some of the dif-
ferences found in the disease produced by the
live strains. However, the main difference prob-
ably is in their lipid compositions (see references
5, 9, and 24).
REFERENCES
1. Dannenberg,A. M., Jr., and J. F.Tomashefski,
Jr. 1998. Pathogenesis of pulmonary tuberculosis,
p. 2447–2471. In A. P. Fishman (ed.), Fishman’s Pul-
monary Diseases and Disorders, 3rd ed., vol. 2.
McGraw-Hill Co., Inc., New York, N.Y.
2. Chensue, S. W., K. Warmington, J. Ruth,
P. Lincoln, M.-C. Kuo, and S. L. Kunkel. 1994.
Cytokine responses during mycobacterial and schis-
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3. Sugisaki, K., A. M. Dannenberg, Jr., Y. Abe,
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T.Yoshimura, P. J. Converse, and P. Mounts.
1998. Nonspecific and immune-specific up-
regulation of cytokines in rabbit dermal tubercu-
lous (BCG) lesions. J. Leukoc. Biol. 63:440–450.
4. Shigenaga, T., A. M. Dannenberg, Jr., D. B.
Lowrie, W. Said, M. J. Urist, H. Abbey, B. H.
Schofield, P. Mounts, and K. Sugisaki. 2001.
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䡵 159
molecules and cytokines (chemokines) cause a
rapid antigen-specific cell infiltration at sites of
bacillus Calmette-Guérin reinfection. Immunol-
ogy 102:466–479.
5. Long, E. R. 1958. The Chemistry and Chemother-
apy of Tuberculosis, 3rd ed., p. 163–182. Lippincott
Williams & Wilkins, Baltimore, Md.
6. Poole, J. C. F., and H.W. Florey. 1970. Chronic
inflammation and tuberculosis, p. 1183–1224. In H.
W. Florey (ed.), General Pathology, 4th ed. The
W. B. Saunders Co., Philadelphia, Pa.
7. Hemsworth, G. R., and I. Kochan. 1978.
Secretion of anti-mycobacterial fatty acids by nor-
mal and activated macrophages. Infect. Immun.
19:170–177.
8. Courtade, E. T., T. Tsuda, C. R. Thomas,
and A. M. Dannenberg, Jr. 1975. Capillary
density in developing and healing tuberculous
lesions produced by BCG in rabbits. A quantita-
tive study. Am. J. Pathol. 78:243–260.
9. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive
Mechanisms. Harvard University Press, Cambridge,
Mass.
10. Laskin, D. L., and K. J. Pendino. 1995.
Macrophages and inflammatory mediators in tis-
sue injury. Annu. Rev. Pharmacol.Toxicol. 35:655–
677.
11. Canetti, G. 1955. The Tubercle Bacillus in the Pul-
monary Lesion of Man. Springer-Verlag, New York,
N.Y.
12. Rich, A. R. 1951. The Pathogenesis of Tuberculosis,
2nd ed. Charles C Thomas Publisher, Springfield,
Ill.
13. Converse, P. J., A. M. Dannenberg, Jr., J. E.
Estep, K. Sugisaki, Y. Abe, B. H. Schofield,
and M. L. M. Pitt. 1996. Cavitary tuberculosis
produced in rabbits by aerosolized virulent tuber-
cle bacilli. Infect. Immun. 64:4776–4787.
14. Converse, P. J., A. M. Dannenberg, Jr., T.
Shigenaga, D. N. McMurray, S.W. Phalen, J.
L. Stanford, G. A.W. Rook,T. Koru-Sengul,
H. Abbey, J. E. Estep, and M. L. M. Pitt.
1998. Pulmonary bovine-type tuberculosis in rab-
bits: bacillary virulence, inhaled dose effects, tuber-
culin sensitivity, and Mycobacterium vaccae
immunotherapy. Clin. Diagn. Lab. Immunol. 5:871–
881.
14a. Dannenberg, A. M., Jr., and M. Sugimoto.
1976. Liquefaction of caseous foci in tuberculosis.
Am. Rev. Respir. Dis. 113:257–259. (Editorial.)
15. Yamamura,Y. 1958.The pathogenesis of tuber-
culous cavities. Adv.Tuberc. Res. 9:13–37.
16. Yamamura, Y., Y. Ogawa, H. Maeda, and
Y. Yamamura. 1974. Prevention of tubercu-
lous cavity formation by desensitization with
tuberculin-active peptide. Am. Rev. Respir. Dis.
109:594–601.
160 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
17. Dannenberg, A. M., Jr., and W. E. Bennett.
1964. Hydrolytic enzymes of rabbit mononuclear
exudate cells. I. Quantitative assay and properties of
certain proteases, nonspecific esterases and lipases of
mononuclear and polymorphonuclear cells and
erythrocytes. J. Cell Biol. 21:1–13.
18. Dannenberg, A. M., Jr., and W. E. Bennett.
1963. Hydrolases of mononuclear exudate cells and
tuberculosis. I. Exudate characteristics, esterases,
proteinases and lipase. Arch. Pathol. 76: 581–591.
19. Dannenberg, A. M., Jr. 1991. Delayed-type
hypersensitivity and cell-mediated immunity in the
pathogenesis of tuberculosis. Immunol.Today 12:228–
233.
20. Dannenberg, A. M., Jr. 1993. Immunopatho-
genesis of pulmonary tuberculosis. Hosp. Pract. 28:
33–40 (Off. ed. 51–58).
21. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
genesis of tuberculosis: specificity, systemic and local
nature, and associated macrophage enzymes. Bacte-
riol. Rev. 32:85–102.
22. Suga, M., A. M. Dannenberg, Jr., and
S. Higuchi. 1980. Macrophage functional hetero-
geneity in vivo: macrolocal and microlocal macro-
phage activation, identified by double-staining
tissue sections of BCG granulomas for pairs of
enzymes. Am. J. Pathol. 99:305–324.
23. Gangadharam, P. R. J. 1995. Mycobacterial dor-
mancy. Tuberc. Lung Dis. 76:477–479.
24. Raffel, S. 1961. Immunity, 2nd ed., p. 412-441.
Appleton, New York, N.Y.
25. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
culosis in the light of the host-parasite relationships
in natively resistant and susceptible rabbits. Am.
Rev.Tuberc. Pulm. Dis. 72:297–329.
 8
MICROVASCULAR DENSITY
IN TUBERCULOUS LESIONS

Overview 161
Production of developing and healing BCG lesions and 2-day tuberculin
reactions 161
Capillary density determined by gelatin-colloidal carbon perfusion 162
Histopathology: microvasculature-cell interactions 163
Regulators of angiogenesis 167
Pathophysiology of blood supply 167
Microvascular thrombosis 168

Abstract. The vasculature plays an important role in the pathogenesis of tuberculous

lesions. Blood vessels bring in the host defense cells, and vascular thrombosis is a major
cause of caseous necrosis. This chapter describes the study of microvascular density in
tissue sections of developing and healing dermal BCG lesions and in 48-h dermal tuber-
culin reactions.
Rabbits were placed under deep terminal anesthesia, and their entire vasculature was
perfused (via the aorta) with a gelatin-colloidal carbon suspension.Then, serial 250-µm-
thick tissue sections of the dermal BCG lesions were prepared, and the total length of the
microvasculature in the whole BCG lesion was calculated from measurements made with
a microscope containing an ocular grid.
By 3 days, the vascular density in BCG lesions had increased to roughly 1.6 times that
found in normal skin. It remained at this level for at least 6 to 7 weeks.The vascular den-
sity in tuberculin reactions showed a similar increase.
It was concluded that the local microvasculature increases relatively little during the course
of this slowly healing infection.A greater blood flow through existing capillaries evidently
provides most of the nourishment needed by the infiltrating cells.
These studies also demonstrated that microvascular thrombosis is a major cause of the
caseous necrosis that occurs during the course of this disease.

OVERVIEW The study described in this chapter on der-

The microvasculature plays a major role in the
inflammatory response to infectious agents (see
reference 1). It supplies defense cells, antibodies,
and other blood components so that the offend-
ing agent is destroyed (or detoxified), and
necrotic debris is removed. For convenience, in
this chapter I often use the word “capillaries” to
represent the entire microvascular network (2),
i.e., the precapillary arterioles that control the
amount of blood entering, the capillaries them-
selves, which supply oxygen and nutrients to the
tissues and remove waste products, and the post-
capillary venules through which leukocytes
enter the tissues (2, 3).
mal BCG lesions seems to be the only one
ever done on the total amount of microvas-
culature of tuberculous lesions as they progress
and heal. See chapter 21 for our studies on the
microvascular adhesion molecules in such
lesions.
PRODUCTION OF DEVELOPING
AND HEALING BCG LESIONS AND
2-DAY TUBERCULIN REACTIONS
In Experiment I, each rabbit had either 21-day
or 42-day dermal BCG lesions when eutha-
nized. In Experiment II, each rabbit, when euth-
anized, had 49-, 35-, 15-, and 3-day lesions;

161
162 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

i.e., lesions of different ages were present on
their flanks at the same time (1).
Tuberculin reactions were produced by the
intradermal injection of a 1:10 dilution of Old
Tuberculin and evaluated 2 days later.
CAPILLARY DENSITY DETERMINED
BY GELATIN-COLLOIDAL CARBON
PERFUSION
Procedure
Rabbits under deep terminal anesthesia were
heparinized, exsanguinated, and then perfused
via the aorta with a warm solution of 2% gelatin,
10% Pelikan India ink (a colloidal carbon sus-
pension), and 1% detergent in 0.9% pyrogen-free
NaCl (1).This procedure filled the entire vas-
culature of the animal, including capillaries and
venules, with the gelatinized colloidal carbon
particles. After formalin fixation, serial tissue
sections (250 m thick) of the BCG lesions
and tuberculin reactions were prepared in a
cryostat, stained with hematoxylin, and mounted
serially in Permount.Then, in the nonnecrotic
tissues of the lesions, the average capillary den-
sity was determined in millimeters of capillary
length per mm2 of 250-m tissue section (see
reference 1).
Results and Conclusions
The capillary density in BCG lesions of various
ages increased roughly 1.6 times when com-
pared with the density in the normal skin nearby
(Fig. 1).This increase was evident as early as 3
days, which suggests that vascular proliferation

FIGURE 1 Capillary density in the BCG

lesions of Experiment II and in the normal
skin nearby. In this experiment, the skin of
each of the six rabbits contained 3-, 15-,
35-, and 49-day BCG lesions when they
were perfused with the gelatin-colloidal
carbon suspension. The means and their
standard errors are depicted.
The capillary density of BCG lesions
(in lengths per mm2) was increased as early
as 3 days. This density was highest at 15
days, when tuberculin sensitivity was devel-
oping, and decreased relatively little as the
lesions healed. Reproduced with permission
from reference 1.
 8. MICROVASCULAR DENSITY IN TUBERCULOUS LESIONS 䡵 163

occurred rather rapidly. The perfusion of the HISTOPATHOLOGY:

vasculature with the warm colloidal carbon sus-
pension seemed to dilate every blood vessel in
the entire host.Therefore, none of the microvas-
culature was closed by precapillary sphincters,
and every vessel in both the BCG lesions and
normal skin was apparently filled with the col-
loidal carbon suspension.
In 15-day BCG lesions, the capillary density
tended to be maximally different from that in
normal skin (Fig. 1). At that time tuberculin
sensitivity was developing (1). Otherwise, the
capillary density changed rather little as the
BCG lesions developed and then healed. In
other words, the 60% increase in the density of
the capillary network seemed adequate to con-
tinuously supply the local needs throughout the
course of this infection.
The capillary densities in the 2-day tuberculin
reactions resembled those found in the BCG
lesions (1).
MICROVASCULATURE-CELL
INTERACTIONS
BCG Lesions
The vascular network plays a role in the for-
mation, progression, and resolution of BCG
lesions. Following the injection of tubercle
bacilli, granulocytes and mononuclear cells
(mostly macrophages) emigrate from the sub-
papillary vascular plexus that nourishes the epi-
dermis and hair follicles. The hydrolases from
cells in the exudate are probably responsible for
the dissolution of ground substance, collagen
fibers (4), and the fibrin that had formed (5).
Some of the cells in the exudate died and
formed microabscesses (Fig. 2), which soon
fused to create the caseous center. Presumably
by looping, budding, and anastomosing (6), the
capillary network increased around the hair fol-
licles and formed a plexus around the caseous
center (Fig. 3, 4, and 5). The capillaries were

FIGURE 2 Dermal BCG lesion, 3 days of age.An increased number of vessels are present in
the subpapillary vascular plexus surrounding the hair follicles (sometimes called the peritrichous
plexus). In the center and on the right of the photograph, the hair follicles were damaged by
microabscesses developing between them. In the lower part of the photograph, the deep cuta-
neous vascular plexus is prominent. It supplies the intrinsic muscles of the skin and helps regu-
late body heat. Gelatin-colloidal carbon perfusion, counterstained with hematoxylin. Magnifi-
cation, ⫻16. Reproduced with permission from reference 1.
164 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 3 Dermal BCG lesion, 15 days of age, cut near the edge of the necrotic center. At
this location, the vessels of the subpapillary vascular plexus form a rich anastomosing network
with the vessels of the deep cutaneous vascular plexus. Magnification, ⫻23.
We used an ocular grid to count (at 44⫻ magnification) the lengths of capillaries within
grid squares, each measuring 0.45 by 0.45 mm. In the grid square shown centrally in the inset,
the 23 pieces of microvasculature totaled 8.3 grid widths. Gelatin-colloidal carbon perfusion,
counterstained with hematoxylin. Magnification, ⫻3.7. Reproduced with permission from ref-
erence 1.

thrombosed next to the caseous center. Such
thrombosis is thought to be a major cause of
caseous necrosis.
The capillaries of the deep cutaneous plexus
are situated in the dermis just above the muscle
fibers that move the skin (Fig. 2 and 3).This vas-
cular plexus contains many arterial-venous shunts
that regulate the flow of blood to the skin for
heat exchange. As the caseous center enlarged,
this deep cutaneous plexus also proliferated, sup-
plying branches that surrounded the lower half
of the caseous center and fused with similar
branches from the subpapillary plexus. Some
capillary plexuses became buried in exudate cells
(Fig. 4), some were injured, some leaked (Fig. 6),
and some thrombosed.
After the bacilli were inhibited or destroyed
by the macrophages (7–11), the BCG lesions
regressed and began to heal. Much of the
necrotic material was discharged by ulceration
(Fig. 6), and the rest was gradually ingested,
broken down, or sequestered by the infiltrating
macrophages. The capillaries surrounding the
now shrunken caseous center apparently
remained fully functional and served as the
source of new phagocytes, as well as nutrients for
fibroblasts.The capillary network was reduced
to normal size only after most of the necrotic
material had been removed and new connective
tissue had formed (Fig. 7).
Tuberculin Reactions
The tuberculin reactions usually showed no
necrosis, although microabscesses were occa-
sionally found.The capillaries in these reactions
were mainly from the subpapillary plexus, with
little contribution from the deep cutaneous
plexus (Fig. 8). In other respects, the capillary net-
work resembled that found in the nonnecrotic
tissues of fully developed BCG lesions (1).
FIGURE 4 Dermal BCG lesion, 35 days of age.This section was cut from the outer regions
of the caseous necrotic center (but more central than that in Fig. 3). Here, the cellular infiltrate
is not completely necrotic, and the capillaries are still patent or only partly thrombosed. Gelatin-
colloidal carbon perfusion, counterstained with hematoxylin. Magnification, ⫻22. Reproduced
with permission from reference 1.

FIGURE 5 Dermal BCG lesion, about 21 days of age.This section was cut through the caseous
necrotic center.The intact epidermis is shown above.The liquefied caseous necrotic center is shown
below. Part of this center fell out during the preparation of this tissue section.
Note that in the necrotic area the capillaries were not perfused with carbon, which indicates
that thrombosis and the resulting ischemia are major causes of tissue necrosis. Gelatin-colloidal car-
bon perfusion, counterstained with hematoxylin. Magnification, ⫻23.
165
166 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Dermal BCG lesion, 15 days of age, with ulceration and discharge of some of the
necrotic material.The vascular plexus at the base of the ulcer is richly developed. Some leakage
of the colloidal carbon into the necrotic area is evident. Gelatin-colloidal carbon perfusion, coun-
terstained with hematoxylin. Magnification, ⫻20. Reproduced with permission from reference 1.

FIGURE 7 Healing dermal BCG lesion, 49 days of age. The total vascular network has
decreased, and most of the necrotic material has been discharged or absorbed. Fibroblasts, pro-
ducing new collagen fibers and ground substance, are present.The intrinsic muscles of the skin
(the panniculus carnosis) can be seen at the bottom of the photograph. Gelatin-colloidal carbon
perfusion, counterstained with hematoxylin. Magnification, ⫻17. Reproduced with permission
from reference 1.
 8. MICROVASCULAR DENSITY IN TUBERCULOUS LESIONS 䡵 167

FIGURE 8 A positive 2-day tuberculin reaction.The subpapillary vascular plexus surround-

ing the hair follicles has proliferated, but no necrosis is present. Gelatin-colloidal carbon perfu-
sion, counterstained with hematoxylin. Magnification, ⫻23. Reproduced with permission from
reference 1.

REGULATORS OF ANGIOGENESIS thrombospondin, platelet factor 4, interferons,

Angiogenesis is controlled by a balance between
positive and negative regulators (2). Most of
these regulators are produced by macrophages,
lymphocytes, granulocytes, mast cells, fibroblasts,
pericytes, and platelets (see references 2 and 12
through 21).
The major stimulatory regulators of angio-
genesis are hypoxia, extracellular matrix and
connective tissue components (13), angiogenin,
angiopoietin-1, nitric oxide, fibroblast growth
factors, vascular endothelial growth factor, tumor
necrosis factor alpha, transforming growth fac-
tor , interleukin-8, epidermal growth factor,
granulocyte colony-stimulating factor, platelet-
activating factor, urokinase, plasminogen activa-
tor, and prostaglandins E1 and E2 (12). Metal-
loproteinases degrade the nearby extracellular
matrix, thereby enabling endothelial cell migra-
tion to establish new microvasculature.
The major inhibitory regulators of angio-
genesis are angiostatin, endostatin, vasostatin,
interleukin 12, and tissue inhibitors of metallo-
proteinases (2, 12).
PATHOPHYSIOLOGY
OF BLOOD SUPPLY
An adequate blood supply is necessary for cells,
tissues, and organs to function.The amount of
blood received by a tissue depends both on the
number of microvessels and the amount of blood
flowing through them.Visualization of capillar-
ies by microangiography with colloidal carbon
(or radio-opaque suspensions) demonstrates the
number of functional microvessels, but not the
amount of blood flow through them.
In acute inflammation, blood flow is often at
its maximum within a few hours and is limited
by the number of capillaries already in the tis-
sue. In delayed-type inflammation, blood flow
can be further enhanced by capillary prolifera-
tion.Therefore, even though microangiographic
techniques do not measure total blood flow
168 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
into the tissues, they do measure the total capac-
ity of the local vasculature.The density of this
vasculature in dermal BCG lesions was increased
an average of 1.6 times over that of normal skin
and remained at this capacity as the lesions pro-
gressed and healed.This moderate increase was
apparently adequate to stop the progression of
the BCG infection.
Note that the skin and lungs are endowed
with much more vasculature than is required to
nourish these tissues. The skin has excess vas-
culature to dissipate the excess body heat and
nourish the hair follicles.The lungs have excess
vasculature to supply oxygen and remove car-
bon dioxide from the blood. In each case, the
total vasculature should be ample to supply
most of the needs of acute and chronic inflam-
matory processes without substantial increases.
MICROVASCULAR THROMBOSIS
Our studies also demonstrate the importance of
vascular thrombosis in the formation of caseous
necrosis in tuberculosis (see Fig. 5 and 6).
Thrombosis not only prevents hemorrhage but
also plays a role in killing nonactivated
macrophages in which numerous tubercle bacilli
are growing (see chapters 7 and 21).
The clotting of blood is under tight control
and is subject to many interacting mechanisms
(2), including cytokines (22).
REFERENCES
1. Courtade, E. T., T. Tsuda, C. R. Thomas, and
A. M. Dannenberg, Jr. 1975. Capillary density in
developing and healing tuberculous lesions pro-
duced by BCG in rabbits.A quantitative study. Am.
J. Pathol. 78:243–260.
2. Majno, G., and I. Joris. 2004. Cells,Tissues, and
Disease. Principles of General Pathology, 2nd ed. Oxford
University Press, New York, N.Y.
3. Tanaka, F.,A. M. Dannenberg, Jr., K. Higuchi,
M. Nakamura, P. J. Pula, T. E. Hugli, R. G.
DiScipio, and D. L. Kreutzer. 1997. Chemo-
tactic factors are continuously released by cultured
intact developing and healing skin lesions produced
in rabbits by sulfur mustard. Inflammation 21:251–
267.
4. Wahl, L. M., S. M. Wahl, G. R. Martin, and
S. E. Mergenhagen. 1974. Production of colla-
genase by macrophages exposed to lymphocyte
products. Fed. Proc. 33:618.
5. Unkeless, J. C., S. Gordon, and E. Reich. 1974.
Secretion of plasminogen activator by stimulated
macrophages. J. Exp. Med. 139:834–850.
6. Schoefl, G. I. 1963. Studies on inflammation.
III. Growing capillaries: their structure and per-
meability. Virchows Arch. Pathol.Anat. 337:97–141.
7. Lurie, M. B. 1965. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive Mech-
anisms. Harvard University Press, Cambridge, Mass.
8. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
genesis of tuberculosis: specificity, systemic and local
nature, and associated macrophage enzymes. Bacte-
riol. Rev. 32:85–102.
9. Mackaness, G. B. 1971. Delayed hypersensitivity
and the mechanism of cellular resistance to infec-
tion, p. 413–124. In B. Amos (ed.), Progress in
Immunology (1st International Congress of Immunology).
Academic Press, Inc., New York, N.Y.
10. Ando, M., A. M. Dannenberg, Jr., M. Sugi-
moto, and B. S. Tepper. 1977. Histochemical
studies relating the activation of macrophages to the
intracellular destruction of tubercle bacilli. Am. J.
Pathol. 86:623–634.
11. Lurie, M. B., P. Zappasodi, E. Cardona-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
to the intracutaneous inoculation of BCG as an
index of native resistance to tuberculosis. J. Immunol.
68:369–387.
12. Talks, K. L., and A. L. Harris. 2000. Current
status of antiangiogenic factors. Br. J. Haematol.
109:477–489.
13. Inber, D. E., and J. Folkman. 1989. How does
extracellular matrix control capillary morphogen-
esis? Cell 58:803–805.
14. Folkman, J., and Y. Shing. 1992. Angiogenesis.
J. Biol. Chem. 267:10931–10934.
15. Polverini, P. J., R. S. Cotran, and M. M. Shol-
ley. 1977. Endothelial proliferation in the delayed
hypersensitivity reaction: an autoradiographic study.
J. Immunol. 118:529–532.
16. Cashin, C. H., B. Dodge, and G. P. Harper.
1991. Angiogenesis and chronic inflammation.
Agents Actions 34:332–338.
17. Keatinge,W. R., and M. C. Harmon. 1980. Local
Mechanisms Controlling Blood Vessels. Monographs of the
Physiological Society, vol. 37. Academic Press, Inc.,
London, England.
18. Folkman, J., and M. Klagsbrun. 1987. Angio-
genic factors. Science 235:442–447.
19. Folkman, J. 1995.Angiogenesis in cancer, vascu-
lar, rheumatoid and other diseases. Nat. Med. 1:
27–31.
 8. MICROVASCULAR DENSITY IN TUBERCULOUS LESIONS 䡵 169

20. Hammersen, F., and O. Hudlicka (ed.). 1984.
Angiogenesis. Progress in Applied Microcirculation, vol.
4. S. Karger, Basel, Switzerland.
21. Melter, M., M. E. J. Reinders, M. Sho, S. Pal,
C. Geehan, M. D. Denton, D. Mukhopadhyay,
and D. M. Briscoe. 2000. Ligation of CD40
induces the expression of vascular endothelial
growth factor by endothelial cells and monocytes
and promotes angiogenesis in vivo. Blood 96:3801–
3808.
22. Joseph, L., L. M. Fink, and M. Hauer-Jensen.
2002. Cytokines in coagulation and thrombosis: a
preclinical and clinical review. Blood Coagul. Fibri-
nolysis 13:105–116.
 9
EARLY PULMONARY
LESIONS IN RABBITS

Advantages of producing early pulmonary lesions by the intravenous

injection of tubercle bacilli 170
Activation and division of blood-borne macrophages in
the early lesions 170
Division of activated pulmonary alveolar macrophages 175

Abstract. In rabbits, 1- to 7-day pulmonary tuberculous lesions produced by aerosols are

difficult to find because the inhaled dose of tubercle bacilli cannot be made large enough.
A large intravenous dose, however, readily produces many such tubercles. This chapter
describes their characteristics and provides information on the activation and multiplica-
tion of macrophages within such lesions.
To produce these early lesions, we injected rabbits intravenously with 108 to 109 tuber-
cle bacilli (BCG). The blood-borne macrophages that entered the developing tubercles
became partly activated during the first day.These entering macrophages retained their abil-
ity to divide, i.e., incorporate [3H]thymidine ([3H]TdR), even though they had ingested
tubercle bacilli. In contrast, fully activated macrophages within tuberculous lesions lose their
ability to divide (see chapter 10).
Pulmonary alveolar macrophages did not seem to participate in early pulmonary lesions
produced by the intravenous route, but accumulated in the surrounding alveolar spaces.
However, even though these alveolar macrophages were highly activated, they retained their
ability to divide.

ADVANTAGES OF PRODUCING microscopic lesions.Therefore, early pulmonary

EARLY PULMONARY LESIONS BY
THE INTRAVENOUS INJECTION OF
TUBERCLE BACILLI
In rabbits, 1- to 7-day pulmonary lesions pro-
duced by the inhalation of tubercle bacilli are
usually too few to find microscopically in tissue
sections. The inhaled dose is always too small
compared to the number of alveolar spaces, and
the highly activated alveolar macrophage pop-
ulation often prevents the inhaled bacilli from
establishing lesions.
An intravenous injection of tubercle bacilli
eliminates these difficulties.A much larger bacil-
lary dose can be deposited in the lungs, and the
pulmonary alveolar macrophages are bypassed.
Following an intravenous injection, the bacilli
(lodging in capillaries) are ingested directly by
nonactivated blood-borne monocytes/macro-
phages, in which they grow and produce early
lesions produced by intravenous tubercle bacilli
should resemble lesions produced by inhaled
virulent tubercle bacilli in the early symbiotic
(logarithmic) stage (see chapter 2), and also
resemble beginning metastatic lesions caused
by hematogenously spread tubercle bacilli.
In clinically active human tuberculosis, early
tubercles of hematogenous origin often occur.
They should be similar to those described here,
except that their progress would be acceler-
ated, because they develop in a person who is
already tuberculin positive.
ACTIVATION AND DIVISION OF
BLOOD-BORNE MACROPHAGES IN
THE EARLY LESIONS
In each rabbit an uncentrifuged suspension of
BCG containing 108 to 109 viable bacilli was
injected intravenously (1).The suspensions con-

170
 9. EARLY PULMONARY LESIONS IN RABBITS 䡵 171

FIGURE 1 One-day pulmonary lesion produced by intravenously injected BCG.A small blood
vessel (part of which is cut tangentially) is depicted with an embolus of tubercle bacilli in its cen-
ter (arrow). Some of the macrophages in this lesion stain ⫹⫹ for -galactosidase. In the alveo-
lar spaces (on the left) are six or seven (dividing) [3H]TdR-positive mononuclear cells (proba-
bly lymphocytes and young macrophages). In the lower right corner are three [3H]TdR-negative
alveolar macrophages, identified by their large size and ⫹⫹⫹ staining for -galactosidase. In vi-
tro [3H]TdR, 5-bromo-4-chloro-3-indolyl--D-galactoside, carbol-fuchsin, and hematoxylin. Mag-
nification, ⫻760. Reproduced with permission from reference 1.

tained many units of 2 to 5 bacilli, as well as
some larger units. Many of these units lodged in
the microvasculature of the lung and formed
microscopic lesions (Fig. 1).
To assess the percentage of dividing cells in
the developing lesions, we incubated pieces of
pulmonary tissue (3 to 4 mm in diameter) with
[3H]TdR in vitro under hyperbaric oxygen (1)
(see chapter 10). Then, frozen tissue sections
were prepared, stained for -galactosidase (our
lysosomal marker for macrophage activation [1–
4]) and sometimes for cytochrome oxidase (our
mitochrondrial marker for macrophage activa-
tion [1, 2, 5]), and autoradiographed as described
in reference 1. This in vitro method labeled
only about half as many mononuclear cells as did
an intravenous injection of [3H]TdR during
the first hour (1) (see chapter 10).
In 1-day pulmonary BCG lesions, mononu-
clear cells (mostly macrophages with some lym-
phocytes and probably dendritic cells) accumu-
lated around a central bacillary core (Fig. 1) (1).
At this stage within the lesions, a few mononu-
clear cells showed ⫹ and ⫹⫹ -galactosidase
activity, but [3H]TdR-labeled mononuclear cells
were rarely found there (Fig. 1). Most rabbit
lymphocytes do not stain for -galactosidase.
In 2- and 3-day pulmonary BCG lesions,
more macrophages were activated, i.e., had
increased their levels of -galactosidase. Some of
these macrophages died, and some of them
divided (Fig. 2 and 3A and B). Perifocally, many
new mononuclear cells accumulated, and these
cells divided more frequently (Fig. 2) (1). At
about this time, typical tuberculous lesions
became established. They contained a central
172 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 Two-day pulmonary lesions produced by intravenously injected BCG. A few

acid-fast tubercle bacilli (arrows) can be seen in their small necrotic centers. Mononuclear cells
from the bloodstream surround the centers.The lesions contained bright-blue ⫹⫹ -galactosi-
dase-positive macrophages, some of which were necrotic. In the alveolar areas outside the lesions
are about a dozen young -galactosidase-negative mononuclear cells showing [3H]TdR incor-
poration. In vitro [3H]TdR, 5-bromo-4-chloro-3-indolyl--D-galactoside, carbol-fuchsin, and
hematoxylin. Magnification, ⫻520. Reproduced with permission from reference 1.

core of dead and dying cells and a more periph-
eral region of accumulating and proliferating
cells (Fig. 2).
In 6-day pulmonary BCG lesions, -galac-
tosidase had disappeared from the dead cells in
the necrotic centers, but cytochrome oxidase
sometimes remained there (Fig. 4). By 10 and 12
days, neither enzyme was present in the necrotic
areas (Fig. 5).
In rabbits, 13-day pulmonary lesions pro-
duced by an aerosol inhalation of 5.6 ⫻ 106 vir-
ulent bovine-type tubercle bacilli and 19-day
pulmonary lesions produced by an aerosol
inhalation of 4.2 ⫻ 107 virulent human-type
bacilli showed percentages of [3H]TdR-labeled
macrophages similar to those produced by intra-
venous BCG (1).
In summary, the activation of macrophages in
rabbit pulmonary tuberculous lesions produced
by intravenous BCG began within 24 h. By 6
days, many macrophages had died and formed
the distinct necrotic center of the developing
tubercle. Only recently arriving macrophages,
i.e., those in 2- and 3-day lesions and those in
the peripheral areas of older lesions, seemed to
divide, i.e., incorporate [3H]TdR. Macrophages
close to the necrotic center no longer divided.
They had apparently entered the lesions earlier,
and some of them were probably dying.
Data presented in reference 1 indicate that
intracellular tubercle bacilli do not stimulate
macrophage division and that tissue macrophages
do not divide when they are strongly activated
for -galactosidase (see chapter 10).
 9. EARLY PULMONARY LESIONS IN RABBITS 䡵 173

FIGURE 3 Three-day pulmonary lesions produced by intravenously injected BCG.

(A) In the upper part of the photograph is a [3H]TdR-positive macrophage staining ⫹⫹
for -galactosidase containing over 10 bacilli (upper arrow). Below this cell is a [3H]TdR-
negative, ⫹ -galactosidase-positive macrophage containing four bacilli (lower arrow). A
⫹⫹⫹ -galactosidase-positive macrophage with two bacilli is at the far right.The lowest
“black” cell is probably a [3H]TdR-positive, -galactosidase-negative lymphocyte. (B) A
[3H]TdR-positive, ⫹ -galactosidase-positive macrophage containing two bacilli is marked
with an arrow. Two ⫹⫹⫹ -galactosidase-positive ([3H]TdR-negative) macrophages are
also present. [3H]TdR-positive mononuclear cells containing bacilli were rare and could
be found with assurance only in early pulmonary lesions. In vitro [3H]TdR, 5-bromo-4-
chloro-3-indolyl--D-galactoside, carbol-fuchsin, and hematoxylin. Magnification, ⫻720.
Reproduced with permission from reference 1.
174 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 Six-day pulmonary lesion produced by intravenously injected BCG and stained
for cytochrome oxidase (CO). In the center are epithelioid cells rich in CO. Surrounding them
are young infiltrating macrophages, most of which are still poor in CO. At the periphery are
numerous alveolar macrophages, rich in CO, that were apparently attracted to the site by chemo-
taxins released from the lesion. Some of the epithelioid cells in the center are already necrotic,
but their CO activity is still present. 1,2,3,4-Tetrahydroquinoline and p-aminodiphenylamine;
no counterstain. Magnification, ⫻180. Reproduced with permission from reference 1.
 9. EARLY PULMONARY LESIONS IN RABBITS 䡵 175

FIGURE 5 Ten-day pulmonary lesion produced by an intravenous injec-

tion of BCG. In the caseous center of the tubercle are disintegrating -galac-
tosidase-negative epithelioid cells containing more than 10 faintly staining
tubercle bacilli.Around the center are still-viable young -galactosidase-neg-
ative mononuclear cells, several of which show [3H]TdR incorporation.Alve-
olar macrophages, staining ⫹⫹⫹ and ⫹⫹⫹⫹ for -galactosidase, have
accumulated in the surrounding alveoli below the tubercle.These alveolar
macrophages did not contain tubercle bacilli, but several had incorporated
[3H]TdR (not clearly seen in this photograph). In vitro [3H]TdR, 5-bromo-
4-chloro-3-indolyl--D-galactoside, carbol-fuchsin, and hematoxylin. Mag-
nification, ⫻320. Reproduced with permission from reference 1.

DIVISION OF ACTIVATED highly activated tissue macrophages within the

PULMONARY ALVEOLAR
MACROPHAGES
These in vitro [3H]TdR studies also provided
information on pulmonary alveolar macro-
phages.Alveolar macrophages (as a population)
are highly activated (5) with a high content of
-galactosidase (4) and other enzymes (4–11).
They frequently migrated from neighboring
alveoli into the alveoli surrounding the young
tuberculous lesions (Figs. 1, 4, and 5), but no
tubercle bacilli were found in these alveoli.The
highly activated alveolar macrophages adjacent
to the lesions could limit the local spread of
tubercle bacilli if it occurred.
Alveolar macrophages with ⫹⫹⫹ to ⫹⫹⫹⫹
-galactosidase activity were often labeled with
[3H]TdR (Fig. 5). Therefore, highly activated
alveolar macrophages can still divide, whereas
lesions rarely do so (see chapter 10).
REFERENCES
1. Shima, K., A. M. Dannenberg, Jr., M. Ando,
S. Chandrasekhar, J. A. Seluzicki, and J. I.
Fabrikant. 1972. Macrophage accumulation, divi-
sion, maturation, and digestive and microbicidal
capacities in tuberculous lesions. I. Studies involv-
ing their incorporation of tritiated thymidine and
their content of lysosomal enzymes and bacilli.
Am. J. Pathol. 67:159–180.
2. Dannenberg, A. M., Jr., O. T. Meyer, J. R.
Esterly, and T. Kambara. 1968.The local nature
of immunity in tuberculosis, illustrated histochem-
ically in dermal BCG lesions. J. Immunol. 100:931–
941.
3. Ando, M., A. M. Dannenberg, Jr., M. Sugi-
moto, and B. S. Tepper. 1977. Histochemical
studies relating the activation of macrophages to the
176 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
intracellular destruction of tubercle bacilli. Am. J.
Pathol. 86:623–634.
4. Yarborough, D. J., O. T. Meyer, A. M. Dan-
nenberg, Jr., and B. Pearson. 1967. Histochem-
istry of macrophage hydrolases. III. Studies on
-galactosidase, -glucuronidase and aminopeptidase
with indolyl and naphthyl substrates. J. Reticuloen-
dothel. Soc. 4:390–408.
5. Dannenberg, A. M., Jr., M. S. Burstone, P. C.
Walter, and J.W. Kinsley. 1963.A histochemical
study of phagocytic and enzymatic functions of
rabbit mononuclear and polymorphonuclear exu-
date cells and alveolar macrophages. I. Survey and
quantitation of enzymes, and states of cellular acti-
vation. J. Cell Biol. 17:465–486.
6. Carson, M. E., and A. M. Dannenberg, Jr.
1965. Hydrolytic enzymes of rabbit mononuclear
exudate cells. II. Lysozyme: properties and quanti-
tative assay in tuberculous and control inbred rab-
bits. J. Immunol. 94:99–104.
7. Meyer, O. T., A. M. Dannenberg, Jr., and
K. Mizunoe. 1970. Hydrolytic enzymes of rabbit
mononuclear and polymorphonuclear exudate cells
and pulmonary alveolar macrophages. III. Deoxyri-
bonuclease and ribonuclease: properties and quan-
titative assay in macrophages from tuberculous
and control inbred rabbits. J. Reticuloendothel. Soc.
7:15–31.
8. Rojas-Espinosa, O., P. Arce-Paredez, A. M.
Dannenberg, Jr., and R. L. Kamenetz. 1975.
Macrophage esterase: identification, purification
and properties of a chymotrypsin-like esterase
from lung that hydrolyzes and transfers nonpolar
amino acid esters. Biochim. Biophys.Acta 403:161–
179.
9. Rojas-Espinosa, O., A. M. Dannenberg, Jr.,
L. A. Sternberger, and T.Tsuda. 1974. Role of
cathepsin D in the pathogenesis of tuberculosis.
A histochemical study employing unlabeled anti-
bodies and the peroxidase-antiperoxidase complex.
Am. J. Pathol. 74:1–17.
10. Namba, M., M. Suga, F. Tanaka, A. M. Dan-
nenberg, Jr., A.T. Hastie, and R. C. Franson.
1983. Immunocytochemical demonstration of rab-
bit ribonuclease and phospholipase A2 by the per-
oxidase-antiperoxidase technique in professional
phagocytes (pulmonary alveolar macrophages and
granulocytic and mononuclear peritoneal exudate
cells) and in glycol methacrylate sections of dermal
tuberculous (BCG) lesions. J. Reticuloendothel. Soc.
34:425–435.
11. Tsuda, T., A. M. Dannenberg, Jr., M. Ando,
O. Rojas-Espinosa, and K. Shima. 1974.
Enzymes in tuberculous lesions hydrolyzing pro-
tein, hyaluronic acid and chondroitin sulfate: a
study of isolated macrophages and developing and
healing rabbit BCG lesions with substrate film
techniques; the shift of enzyme pH optima towards
neutrality in “intact” cells and tissues. J. Reticu-
loendothel. Soc. 16:220–231.
 10
MACROPHAGE TURNOVER,
DIVISION, AND ACTIVATION
IN TUBERCULOUS LESIONS

Overview 178
In vitro [3H]TdR methods and their efficacy 178
Percentage of MN labeled in vitro with [3H]TdR in BCG lesions 179
In vitro division of activated macrophages in BCG lesions 179
In vitro division of macrophages containing tubercle bacilli in
BCG lesions 180
In vivo [3H]TdR studies of BCG lesions of various ages 180
MN entry into BCG lesions 180
MN disappearance from BCG lesions 182
Rates of MN division within BCG lesions 185
[3H]TdR-labeled MN in 1-day “tuberculin traps” 185
[3H]TdR-labeled MN in the blood 185
Rates of activation of MN in primary and reinfection BCG lesions 187
Macrophage turnover, activation, and division in healing dermal
BCG lesions 189
MN activation in tuberculin reactions and in lesions caused by nonspecific
irritants 192
Macrophage turnover in mouse tuberculous granulomas 193
Insights into DTH and CMI provided by our kinetic studies in
rabbits 193
Recapitulation of our kinetic studies on macrophages in tuberculous lesions
produced by BCG in rabbits 194

Abstract. In rabbit BCG lesions, the turnover of mononuclear cells was most rapid in BCG
lesions at 2 to 3 weeks, when the lesion size peaked and tuberculin sensitivity and acquired
cellular resistance were well developed. (The mononuclear cells were mostly macrophages,
with some medium and large lymphocytes and probably some dendritic cells.) At this 2-
to 3-week peak, more macrophages entered, more died or left, more remained at the site,
and more became activated than before or afterward. Before this time, the host had neither
delayed-type hypersensitivity nor cell-mediated immunity, so no antigen-specific enhance-
ment of the inflammatory response occurred.After this time, the bacilli and their antigenic
products had decreased, so antigen-specific stimuli for cell infiltration and activation were
reduced. In “healed” lesions, the mononuclear cell turnover still occurred but was decreased.
The continuous entry of live nonactivated macrophages into the viable parts of tuber-
culous lesions provides fresh intracellular sites where tubercle bacilli can multiply before
they are again inhibited by the delayed-type hypersensitivity and cell-mediated immunity
of the host. In tuberculosis, bacillary dormancy of long duration can only be present in
caseous necrotic tissue where no live host cells exist.

To my knowledge, our papers on macrophage
kinetics in rabbits (references 1 through 9) are
the only ones in which macrophage entry, divi-
sion, activation, accumulation, and death were
evaluated in tuberculous lesions as they devel-
oped and healed.This chapter is a synthesis of
these nine papers and provides new insights and
interpretations that were not presented in the
original publications. (Most of the chapter was
published as a review in reference 10.)
I realize that these kinetic studies are complex
and difficult to understand. Yet, they provide

177
178 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
insights into cell turnover within tuberculous
lesions that are not obtainable in any other way.
For a quick review of this chapter and its rele-
vance, see the last two sections.
The mononuclear cells (MN) that we
counted in our Giemsa-stained tissue sections
were mostly macrophages, with some medium
and large lymphocytes and probably some den-
dritic cells. Cells staining for -galactosidase
and cells containing tubercle bacilli were almost
always macrophages.Therefore, we usually used
the term macrophages rather than mononu-
clear cells (or MN) when cells containing -
galactosidase or tubercle bacilli were evaluated.
Lymphocytes do not usually stain for -galac-
tosidase, or stain only weakly.We do not know
the percentage of dendritic cells in the mac-
rophage population that was evaluated, but we
expect that it is relatively small.
OVERVIEW
We performed both in vitro (1) and in vivo
(2–9) experiments. In the in vitro experiments,
biopsy samples of dermal BCG lesions of vari-
ous ages were incubated with tritiated thymidine
([3H]TdR) for 1 h under hyperbaric oxygen.
The in vitro experiments clearly showed that
only a small percentage (1.6 ⫾ 0.1%) of MN
divided locally within the lesions (Table 1).
These results were confirmed by in vivo exper-
iments in which the rabbits were euthanized 30
or 60 min after an intravenous injection of
[3H]TdR (Table 1).
In vivo experiments described herein pro-
vide information on macrophage turnover in
tuberculous lesions. In these experiments,
[3H]TdR was injected intravenously once dur-
ing the development and healing of the dermal
BCG lesions. Macrophages came from a rapidly
dividing precursor population in the bone
marrow (11–13), where they became [3H]TdR
labeled. They then entered the bloodstream
and subsequently entered the BCG lesions
along with unlabeled macrophages. Five days
after the intravenous [3H]TdR pulse, we found
that 19 to 35% of MN were labeled in the
lesions (2).Therefore, in vivo [3H]TdR-label-
ing experiments (in contrast to those in vitro)
enabled us to study the rates of entry of labeled
TABLE 1 Comparison of in vitro and in vivo
[3H]TdR-labeling of MN in dermal BCG lesionsa
In vitro (%) In vivo (%)
1.3 2.6
1.4 3.0
1.4 3.8
1.7 4.1
1.9
2.0
Mean:
1.6 ⫾ 0.1b 3.4 ⫾ 0.3
a
The percentages of in vitro and in vivo [3H]TdR-labeled
MN in rabbit 10-day dermal BCG lesions were compared. For
the in vitro experiments, MN were labeled by incubating thin
BCG lesion biopsy specimens in [3H]TdR (1.0 Ci/ml) for
1 h under hyperbaric oxygen. For the in vivo experiments, the
rabbits were euthanized 30 or 60 min after an intravenous
[3H]TdR pulse (0.5 Ci/g of body weight). The in vitro
[3H]TdR labeled only about half as many MN as the in vivo
[3H]TdR labeled.Adapted from reference 1, where additional
data are presented.
b
P ⬍0.001.
macrophages into the lesions and their disap-
pearance from the lesions, as well as their rates
of division and their rates of activation as the
BCG lesions developed, peaked, and regressed.
IN VITRO [3H]TdR METHODS
AND THEIR EFFICACY
Cross sections (2 to 3 mm thick) of these lesions
were incubated under three atmospheres of
oxygen at 37°C for 1 h in culture medium 199,
containing 1.0 Ci/ml of [3H]TdR in 20%
fetal calf serum (1, 14). Frozen tissue sections of
these explants (prepared in a cryostat) were (i)
stained for the lysosomal enzyme -galactosi-
dase, (ii) autoradiographed to detect [3H]TdR
incorporation, (iii) stained for acid-fast bacilli,
and (iv) counterstained with hematoxylin (1, 2).
We only evaluated MN in the viable areas sur-
rounding the caseous centers (10).At these sites,
some activated MN were usually present, as
were the bacilli and/or their products.The MN
were mostly macrophages (10).
The in vitro method labeled about half as
many MN (1) as did a 1-h in vivo [3H]TdR
pulse (Table 1).This discrepancy may be due to
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION 䡵 179

mild cell injury (from anoxia or trauma) during
the biopsy procedure. It does, however, provide
an approximate measure of the number of cells
undergoing local division within the tubercu-
lous lesion. The in vitro method is much less
costly than the in vivo method, because relatively
little [3H]TdR is used.
PERCENTAGE OF MN LABELED
IN VITRO WITH [3H]TdR IN BCG
LESIONS
In vitro [3H]TdR incubation labeled 0.8 to 4.0%
of the mononuclear cells (Tables 1 and 2) (1).This
low percentage remained fairly constant through-
out the growth and regression of these lesions
(Table 2). This low percentage of in vitro
[3H]TdR-labeled MN should be compared to
the 19 to 35% in vivo [3H]TdR-labeled MN
found in the lesions 5 days after an intravenous
[3H]TdR pulse (2).The [3H]TdR-labeled MN at
5 days were labeled in the bone marrow and
entered the lesions during the 5-day period.
From these studies we concluded that relatively
few MN divide locally in the lesions,and the MN
labeled in vitro with [3H]TdR had arrived
recently from the bloodstream (9).
IN VITRO DIVISION OF ACTIVATED
MACROPHAGES IN BCG LESIONS
In viable areas of BCG lesions that contain
bacilli, many macrophages develop into epithe-
lioid cells, rich in mitochondrial and lysosomal
enzymes (15, 16). Our stain for -galactosidase
is a marker for such activated cells. The more
mature the epithelioid cell, the darker it stains
for -galactosidase (15, 16). Such darkly stained
macrophages/epithelioid cells can effectively
inhibit or destroy tubercle bacilli (15–17).
In the in vitro [3H]TdR experiments, some
macrophages staining ⫹ and ⫹⫹ for -galac-
tosidase incorporated [3H]TdR, but macrophages
staining ⫹⫹⫹ and ⫹⫹⫹⫹ for -galactosidase
did not (1). Apparently, the strongly stained
macrophages had become so completely differ-
entiated for digestive and microbicidal functions
that they no longer continued to divide.
In the in vivo [3H]TdR experiments on der-
mal BCG lesions (described below), some

TABLE 2 In vitro[3H]TdR labeling of dermal BCG lesions indicates that intracellular tubercle bacilli do not
stimulate mononuclear cell (MN) divisiona
Percent Total no. of MN with both
3
Age of lesion Total no. of Percent of of MN [H]TdR label and intracellular
3
when biopsied MN counted [H]TdR- containing tubercle bacilli
(days) (A) positive tubercle
MN (B) bacilli (C) Expectedb Observed
Experiment I
7 14,100 2.0 ⫾ 0.02 3.5 ⫾ 0.8 8.1 0
12 13,800 4.0 ⫾ 1.3 2.7 ⫾ 0.8 7.5 5
19 31,500 2.3 ⫾ 0.6 0.4 ⫾ 0.2 5.7 0
Experiment II
7 17,900 1.0 ⫾ 0.3 3.4 ⫾ 0.7 4.9 0
12 8,300 1.6 ⫾ 0.3 4.4 ⫾ 0.5 4.3 0
19 22,400 0.8 ⫾ 0.2 0.8 ⫾ 0.4 2.4 0
Experiment III
7 27,500 1.6 ⫾ 0.3 4.6 ⫾ 1.2 16.7 5
14 29,600 2.6 ⫾ 0.5 3.5 ⫾ 1.1 24.0 7
a
The observed number of “doubly marked” MN was less than the expected number, which suggests that intracellular tuber-
cle bacilli did not stimulate macrophage division. Means and their standard errors are listed. More details are in footnote a of Table
1.Adapted from reference 1.
b
The expected numbers of “doubly marked” MN were not obtained by multiplying the means listed in columns A, B, and C:
they were obtained by multiplying A ⫻ B ⫻ C from individual rabbits and then averaging them. For example, in Experiment III,
the mean A ⫻ B ⫻ C: 27,500 ⫻ 1.6% ⫻ 4.6% ⫽ 20.2, whereas the average of A ⫻ B ⫻ C from individual rabbits was 16.7. Both
20.2 and 16.7 for the expected “doubly marked” MN were much higher than the 5 that were found.
180 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
[3H]TdR-labeled macrophages staining ⫹⫹⫹
and ⫹⫹⫹⫹ for -galactosidase were found.
These local macrophages had probably incor-
porated [3H]TdR before they became highly
activated for -galactosidase, because at least 5
days (rather than 1 h) had elapsed after exposure
to [3H]TdR.
IN VITRO DIVISION OF
MACROPHAGES CONTAINING
TUBERCLE BACILLI IN BCG LESIONS
[3H]TdR-positive macrophages containing
tubercle bacilli could occasionally be found in
1- to 3-week BCG lesions (Table 2). These
findings indicate that a few macrophages con-
taining tubercle bacilli were still able to divide.
The observed number of MN “doubly
marked” with [3H]TdR and intracellular bacilli
was lower than expected (Table 2). The lower
observed number could have been caused by
the shorter residence of [3H]TdR-labeled
macrophages in the dermal lesions. (Macrophages
labeled in vitro with [3H]TdR were recently
entering cells with little opportunity to ingest
bacilli.) The lower observed number of doubly
marked MN could also have been caused by
the lymphocytes in the [3H]TdR-labeled MN
population. Lymphocytes never ingest bacilli,
but they may divide more frequently than
macrophages within BCG lesions, where the
antigens of the bacillus are located.
From these findings, we conclude that
macrophage division is not appreciably stimu-
lated by intracellular bacilli, and that macrophage
division is probably not a major mechanism by
which these phagocytes reduce their bacillary
load. Lurie came to the same conclusion in his
eye chamber experiments (see chapter 5).
IN VIVO [3H]TdR STUDIES OF BCG
LESIONS OF VARIOUS AGES
The remainder of this chapter describes exper-
iments in which [3H]TdR was injected intra-
venously into rabbits 1 day before or 6, 14, 27,
41, or 63 days after dermal BCG lesions were
begun. These lesions peaked in size at 2 to 3
weeks and then slowly regressed.
The highest percentage of [3H]TdR-labeled
MN was found in tissue sections of the BCG
lesions about 5 days after the intravenous
[3H]TdR injection (2).This injection had appar-
ently labeled most of the dividing MN (mono-
cyte/macrophages) while they were still in the
bone marrow, and 5 days was enough time for
these cells to leave the marrow and accumulate
in the lesions.
[3H]TdR is rapidly incorporated into cells
that replicate their DNA prior to cell division,
and the excess (unincorporated) [3H]TdR is
degraded and/or excreted within a few hours
(18). Once [3H]TdR is incorporated into DNA,
it will remain there for the life of the cell (18)
(unless it is diluted out by cell division).
In these experiments, [3H]TdR (0.5 Ci/g
of body weight) was injected intravenously.
When [3H]TdR of either 2 Ci/mmol or 20
Ci/mmol specific activity was used, no differ-
ences were found in the percentage of MN
labeled or in their [3H]TdR grain counts (7).
[3H]TdR cutoffs of 3 or 5 grains per cell were
used, depending on the background silver grains
produced in each batch of Kodak photographic
emulsion.
The analysis of these in vivo [3H]TdR ex-
periments is complex, so only the main princi-
ples will be presented here. If more details
(including the actual numerical data) are de-
sired, the reader should consult references 1
through 9.
MN ENTRY INTO BCG LESIONS
Figure 1 depicts the total number of [3H]TdR-
labeled MN entering the BCG lesions during
the 5-day period between the [3H]TdR pulse
and lesion biopsy. Large numbers of [3H]TdR-
labeled MN entered primary BCG lesions when
the host became tuberculin positive.
Rabbits with reinfection BCG lesions were
already tuberculin positive.Therefore, the num-
ber of labeled MN in reinfection lesions 4 days
of age was similar to the number in primary
lesions at 11, 19, and 32 days (Fig. 1).
As BCG lesions were developing and healing,
the percentage of [3H]TdR-positive MN (5 days
after the [3H]TdR pulse) remained between 19
and 35% of the total MN population (Fig.2) (data
in reference 2). During the final stages of healing
in BCG lesions 41 and 63 days of age, this per-
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION 䡵 181

FIGURE 1 The total number of [3H]TdR-labeled MN in primary BCG lesions of various

ages 5 days after an intravenous injection of [3H]TdR (3HT in figure). (A, age of BCG lesions
when [3H]TdR was injected intravenously; B, age of BCG lesions when biopsied. For example,
biopsies for the ⫹6 days on the bar graph were taken when the BCG lesions were 11 days of
age.)
Most of the [3H]TdR-labeled MN had incorporated [3H]TdR in the bone marrow before
they entered the lesions.Therefore, the number of [3H]TdR-labeled MN should be proportional
to the number of labeled plus unlabeled MN that entered during the previous 5 days.The num-
ber of [3H]TdR-labeled MN was 19 to 34% of the total number of MN within the lesions (2).
Note that many more MN entered after DTH was present. Reproduced with permission from
reference 2.
The total number of [3H]TdR-labeled MN in BCG lesions was calculated from the lesion
size (when measured with calipers), the number of MN per mm2 (counted microscopically), and
the percentage of these MN that were [3H]TdR labeled. After the caseous center developed in
1 to 2 weeks, the total number of [3H]TdR-labeled MN in the lesions was less than the num-
ber calculated, because caseous necrosis was present (3, 5).
182 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 Rate of appearance and disappearance of [3H]TdR-labeled MN in primary BCG

lesions of various ages. [3H]TdR (3HT in figure) was given as a single intravenous pulse to dif-
ferent groups of rabbits at the times indicated at the top of the graph, i.e., when their BCG lesions
were –1, ⫹6, ⫹14, or ⫹27 days of age. Note that in each case the percentage of [3H]TdR-labeled
MN in the lesions reached its peak about 5 days later, and that about 10 days after the peak these
labeled MN had largely disappeared from the lesions (see text). Reproduced with permission from
reference 3.

centage of [3H]TdR-labeled MN was reduced
but was still about 12% (data in reference 4).
From these studies, we concluded that delayed-
type hypersensitivity (DTH) and cell-mediated
immunity (CMI) caused a high rate of entry of
new MN into BCG lesions throughout their
development and regression.
MN DISAPPEARANCE
FROM BCG LESIONS
The continual entry of new MN into the BCG
lesions must be offset by a continual disappear-
ance of MN from the lesions by dying or by leav-
ing via the lymphatics,because the lesions reached
their peak size in 14 to 24 days and then regressed
(2, 3, 5). In other words, most MN in BCG
lesions had a rather high turnover rate and did not
apparently persist there for long periods of time.
Peak [3H]TdR labeling of MN in the lesions
occurred around 5 days after the [3H]TdR pulse,
and most of these labeled MN had disappeared
from the lesions 10 days later (Fig. 2).The rate
of disappearance of [3H]TdR-labeled MN was
most rapid when both acquired resistance
(DTH/CMI) and bacillary antigens were locally
present (Fig. 2 and 3) (3, 4).
Before DTH and CMI developed, about 40%
of the [3H]TdR-labeled MN remained in the
lesions during the 10 days between 5 and 15
days after the [3H]TdR pulse (Fig. 3). When
the lesions peaked in size, only about 5% of the
MN remained for these 10 days. In the final
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION 䡵 183

FIGURE 3 Percentage of [3H]TdR-labeled MN that survived for 10 days in primary dermal

BCG lesions at various times during their growth and regression, specifically, the number of
[3H]TdR-labeled MN in the lesions at 15 days (after the intravenous [3H]TdR injection) divided
by the number at 5 days, multiplied by 100. (3HT is [3H]TdR.) In this experiment 5 days after
the intravenous [3H]TdR pulse, 11 to 33% of the MN in the lesions were labeled with [3H]TdR
(3).Ten days later many of these cells had disappeared (died).The percentage that survived dur-
ing this 10-day period is plotted in this graph (see text).The P values between the upper and lower
points are statistically significant (see references 3 and 4). Reproduced with permission from ref-
erence 4.
184 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 Changes in [3H]TdR grain counts in [3H]TdR-labeled MN over 22 days.An intra-

venous pulse of [3H]TdR (3HT in figure) was given when the primary BCG lesions were either
6 or 14 days of age.The grain count made 1 day after the intravenous pulse of [3H]TdR was con-
sidered 100%. Halving of this grain count should indicate one cell division.This figure suggests
(i) that MN in BCG lesions divided only once (or twice), and (ii) that the presence of DTH
reduced the time for this single division to occur, but see the text for another interpretation. Repro-
duced with permission from reference 2.
Grain counts on MN in BCG lesions of reinfection (with [3H]TdR given at –1 day) resem-
bled the lower curve. Grain counts on MN in primary lesions (with [3H]TdR given at –1 day)
resembled the upper curve, but the 1-day primary lesions were small with few cells to count. Grain
counts on MN in regressing primary BCG lesions (with [3H]TdR given at ⫹27 days) fell
between these two curves.

stages of healing, when most of the bacilli and
their tuberculin-like products had been
destroyed, about 40% again remained (Fig. 3).
These percentages are only approximate, because
during this 10-day period, MN containing
decreasing numbers of [3H]TdR-labeled cells
were continually entering the lesions from the
bloodstream (Fig. 2).
Nevertheless, this approximation clearly
showed that [3H]TdR-labeled MN disappeared
from primary BCG lesions at an increased rate
when the host first developed DTH and CMI.
Similar studies with BCG lesions of reinfec-
tion confirmed that the rate of disappearance of
[3H]TdR-labeled MN was increased in hosts
possessing DTH and CMI (3).
This disappearance was largely due to cell
death, because the size of the caseous centers
increased after the hosts became tuberculin pos-
itive (3, 5). High local concentrations of tuber-
culin are known to produce necrosis in tuber-
culin-positive rabbits, guinea pigs, and humans.
Spector’s group also found that the highest
macrophage turnover rates occurred in granu-
lomas produced by agents that were most inju-
rious to tissues (19, 20).
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION 䡵 185

RATES OF MN DIVISION
WITHIN BCG LESIONS
To assess MN division, we counted the silver
grains in the [3H]TdR-labeled MN. Halving of
[3H]TdR grain counts indicates one cell divi-
sion after allowance is made for grains in the
background. At 2 and 5 days after the pulse of
[3H]TdR, MN in the 14-day BCG lesions
showed lower [3H]TdR grain counts than did
MN in the 6-day lesions (Fig. 4), which may sug-
gest that DTH and CMI increased the rate of the
local MN division. However, a more likely
explanation would be that by 2 and 5 days a
larger number of young dividing (bone marrow-
derived) MN with lower grain counts apparently
had infiltrated the 14-day lesions than had infil-
trated the 6-day lesions. (MN apparently divide
frequently in the bone marrow and less fre-
quently in the lesions.) The [3H]TdR grain
counts in this experiment did not drop below
50% of the initial grain count, which suggests
that MN remaining in the lesions did not divide
more than once.A comparison of primary and
reinfection BCG lesions confirmed that the
presence of DTH reduces the [3H]TdR-grain
counts of MN in the lesions (Table 3).
[3H]TdR-LABELED MN IN 1-DAY
“TUBERCULIN TRAPS”
Six rabbits were injected intradermally with
BCG in multiple sites, and a single intravenous
[3H]TdR pulse was given 4 days later. At var-
ious times thereafter, a lesion from each rabbit
was biopsied, and the [3H]TdR-labeled MN
were evaluated (5) (Fig. 5). One day before
each BCG biopsy was taken, we injected Old
Tuberculin intradermally as an MN “trap” to
collect unlabeled and [3H]TdR-labeled MN
from the blood. The 1-day traps and BCG
lesions were biopsied at the same time. The
traps monitored the percentage of MN labeled
with [3H]TdR that entered the BCG lesions
from the bloodstream during the 24 h before
the biopsy.
At each biopsy time, the percentage of
[3H]TdR-labeled MN in the 1-day trap was
about the same as the percentage in the BCG
lesions (Fig. 5), which was consistent with a
high turnover rate for MN within the lesions.
The mean percentage of MN labeled with
[3H]TdR in the traps had decreased from 68%
to 5% by 12 days after the [3H]TdR injection
(5), suggesting that the bone marrow’s supply of
[3H]TdR-labeled MN was mostly depleted by
this time.
The [3H]TdR grain counts of MN in the 1-
day traps were also about the same as those
within the BCG lesions (5), which suggests that
the major reduction in MN grain counts
occurred in the bone marrow (and possibly the
blood) before the MN entered the lesions.
[3H]TdR-LABELED
MN IN THE BLOOD
The [3H]TdR-labeled MN in the blood itself
generally resembled those in the traps and in the
BCG lesions—both in the percentage of MN
that were [3H]TdR labeled (Fig. 5) and in the

TABLE 3 [3H]TdR grain counts in [3H]TdR-positive -galactosidase-positive macrophages in primary and

reinfection dermal BCG lesionsa

Days after No. of [3H]TdR grains in macrophages that stained

[3H]TdR ⴙⴙ, ⴙⴙⴙ, and ⴙⴙⴙⴙ for -galactosidase in: P value
injection Primary BCG lesions BCG lesions of reinfection
5 12.9 ⫾ 1.8 9.7 ⫾ 0.5 0.056
8 11.9 ⫾ 1.6 6.7 ⫾ 0.3 0.006
a 3
[ H]TdR was injected intravenously 1 day before the dermal BCG lesions were begun. Note that macrophages activated for
-galactosidase had lower [3H]TdR grain counts in the early reinfection BCG lesions than in the early primary BCG lesions. In
reinfection BCG lesions, there is a more rapid infiltration of macrophages from the bone marrow (where their precursors are rapidly
dividing) and a more rapid activation of these macrophages (for -galactosidase) (see Fig.7).Adapted from reference 2.
186 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 5 Percentage of cells labeled with [3H]TdR (3HT in figure) (A) in blood and (B) in
1-day tuberculin traps and in BCG lesions at various times after a single intravenous pulse of
[3H]TdR when the BCG lesions were 4 days old.The percentages of [3H]TdR-labeled MN in
the blood, traps, and BCG lesions showed similar patterns, which indicates that [3H]TdR incor-
poration occurred before the MN entered the BCG lesions or soon afterward. It also indicates
that the drop in percentage of [3H]TdR-labeled MN in the lesions was at least in part due to a
drop in [3H]TdR-labeled MN that continued to enter the BCG lesions from the bloodstream.
These rabbits became tuberculin positive when the BCG lesions were 9 days old. Reproduced
with permission from reference 5.

reduction in their [3H]TdR grain counts over
time (5).At 12 days and thereafter, the percent-
age of [3H]TdR-labeled MN in the blood was
somewhat higher than it was in the tuberculin
traps (Fig. 5), and probably represented long-
lived lymphocytes that did not migrate into the
inflammatory lesions produced by the 1-day
tuberculin traps.
In brief, these studies indicate that most of the
MN in BCG lesions recently came from the
bone marrow via the bloodstream and that they
have a high turnover rate within these lesions.
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION
FIGURE 5 Continued.
RATES OF ACTIVATION OF MN
IN PRIMARY AND REINFECTION
BCG LESIONS
The activation of MN in BCG lesions was
assessed histochemically with Pearson’s indolyl
method (15, 21, 22) for the lysosomal enzyme
-galactosidase. The levels of this “marker”
enzyme in macrophages increase as they become
mature epithelioid cells (15, 16). Mature epithe-
lioid cells are capable of destroying tubercle
bacilli (17, 23–25).
Rabbits with either primary or reinfection
dermal BCG lesions were given an intravenous
pulse of [3H]TdR 1 day before or 6, 14, or 27
days after those lesions were begun (3). (The
䡵 187
reinfection BCG lesions were started when their
primary BCG lesions were 27 days of age [3]).
Biopsies were made periodically thereafter, sec-
tioned, stained for -galactosidase, and autoradi-
ographed. The approximate time in which the
[3H]TdR-labeled macrophages entered the BCG
lesions was known from the time of the [3H]TdR
injection, and their degree of activation was indi-
cated by their -galactosidase activity.
Figure 6 shows the percentage of [3H]TdR-
labeled MN that stained ⫹⫹ to ⫹⫹⫹⫹ for
-galactosidase 5 and 8 days after a single
[3H]TdR pulse (given when primary BCG
lesions were 6, 14, and 27 days of age). The
188 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Percentage of recently entering MN (i.e., [3H]TdR-labeled MN) in primary der-

mal BCG lesions of various ages that were activated for -galactosidase. MN enter BCG lesions
in an unactivated state.They become activated at a rate that depends on the intensity of the BCG
stimulus and on the sensitivity of the host to this stimulus.
The highest percentage of entering MN became activated when [3H]TdR (3HT in figure)
was injected intravenously into rabbits with BCG lesions 14 days of age. At this time both the
DTH and CMI of the host and the antigenic products of the bacilli were at their height. Means
and their standard errors are shown. Reproduced with permission from reference 3.
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION 䡵 189

FIGURE 7 Percentage of [3H]TdR-labeled MN in early primary dermal BCG

lesions and in early lesions of reinfection. (3HT, [3H]TdR.) A higher percentage of
[3H]TdR-labeled MN staining ⫹⫹ and ⫹⫹⫹ to ⫹⫹⫹⫹ for -galactosidase was pre-
sent in reinfection lesions than in primary lesions, indicating that young (recently enter-
ing) MN (that incorporated [3H]TdR in the bone marrow) were activated more
rapidly when DTH and CMI were present. Reproduced with permission from refer-
ence 3.The -galactosidase substrates used for Fig. 6 and Fig. 7 came from different lots.

largest percentage of MN was activated for
-galactosidase in 14-day lesions, which was
the time when the size of the lesions and the
amount of DTH and CMI were at their peaks.
The [3H]TdR-labeled -galactosidase popula-
tion consisted almost entirely of macrophages,
because few rabbit lymphocytes stain ⫹⫹ to
⫹⫹⫹⫹ for -galactosidase.
At 4 and 7 days of age, the BCG lesions of
reinfection contained about twice the percent-
age of ⫹⫹ to ⫹⫹⫹⫹ -galactosidase–
[3H]TdR-labeled macrophages that the pri-
mary BCG lesions contained (Fig. 7), but by 14
days this difference between the reinfection and
primary lesions was no longer present (3).
MACROPHAGE TURNOVER,
ACTIVATION, AND DIVISION IN
HEALING DERMAL BCG LESIONS
Rabbit dermal BCG lesions 27, 41, and 63 days
of age were in their early, intermediate, and
late stages of healing, respectively. Five days
190 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 8 Total number of [3H]TdR-labeled MN (see Fig. 1) that entered early-healing and
late-healing dermal BCG lesions in 5 days. The number of [3H]TdR-labeled MN in these
lesions, 5 days after the [3H]TdR (3HT in figure) pulse, is proportional to the number of new
MN that entered during these 5 days.This figure shows that, even in the final stages of healing,
an appreciable number of new MN still enter tuberculous lesions.The difference between the
27-day experiment and the 41- and 63-day experiments was statistically significant. Reproduced
with permission from reference 4. (The 27-day experiment presented here was different from the
one presented in Fig. 1.Also, the lesion size here was calculated with a 0.52 factor, not the 0.20
factor used in Fig. 1 [see reference 3].)
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION 䡵 191

FIGURE 9 Percentage of recently entering MN (i.e., [3H]TdR-labeled MN) that were acti-
vated for -galactosidase in 5 and 8 days in dermal BCG lesions during early, intermediate, and
late stages of healing. (3HT, [3H]TdR.) In the late stages of healing, a lower percentage of
[3H]TdR-labeled MN was activated, apparently because most of the antigenic bacillary prod-
ucts had been destroyed. Reproduced with permission from reference 4. (The substrate for -
galactosidase used for Fig. 7 and 9 came from the same lot.)

after a [3H]TdR pulse, the percentage of
[3H]TdR-labeled MN in the 41- and 63-day
lesions was about two-thirds that in the 27-day
lesions (data in reference 4), indicating that
MN turnover in these late lesions was still
appreciable. (The late lesions were about half the
size of 27-day lesions [4].) A reduced number
of [3H]TdR-labeled MN entered these late
lesions (Fig. 8), a reduced percentage disap-
peared between 5 and 15 days after the
[3H]TdR pulse (Fig. 3), and a reduced per-
centage became activated (Fig. 9).These find-
ings indicate that these almost healed lesions
were still quite active and that some bacillary
growth and destruction probably occurred
within them.
192 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 10 Size of the tuberculin reactions (top) and percentage of the MN in these tuber-
culin reactions that stained ⫹⫹ and ⫹⫹⫹ for -galactosidase in primary and reinfection
dermal BCG lesions (bottom). (3HT, [3H]TdR.) Note that (i) Old Tuberculin (OT) itself acti-
vates considerable numbers of MN, and (ii) after DTH develops, the tuberculin reactions in
rabbits with primary BCG lesions are comparable to those in rabbits with reinfection BCG
lesions.
We do not know the amount of tuberculin and the amount of other bacillary compo-
nents within BCG lesions.Therefore, comparisons of the percentages of MN staining for
-galactosidase in these tuberculin reactions with those in the BCG lesions are meaning-
less. Reproduced with permission from reference 6.

MN ACTIVATION IN TUBERCULIN
REACTIONS AND IN LESIONS tuberculin reactions were biopsied 1 and 2 days
CAUSED BY NONSPECIFIC later. On both days rather similar results were
IRRITANTS obtained (6).
Tuberculin reactions were produced with Old Rabbits reinfected with BCG (and rabbits
Tuberculin in rabbits with primary BCG lesions with primary BCG infection after they became
and in those with reinfection BCG lesions.The tuberculin positive) showed large tuberculin
 10. MACROPHAGE TURNOVER, DIVISION,AND ACTIVATION
reactions (Fig. 10). Such antigen-specific reac-
tions contained a greater percentage of both
[3H]TdR-labeled and unlabeled -galactosi-
dase-positive MN than did the small nonspecific
reactions in unsensitized rabbits (Fig. 10).
Vaccination with BCG had little or no effect
on the host’s reaction to nonspecific irritants.
Both tuberculin-positive BCG-vaccinated rab-
bits and nonvaccinated control rabbits showed
similar degrees of cell infiltration and MN acti-
vation in response to the intradermal injection
of dilute phenol and carrageenan (6). In other
words, BCG vaccination enhanced the host
response to the tuberculin-like products of the
bacillus, but not to nonspecific irritants.
MACROPHAGE TURNOVER IN
MOUSE TUBERCULOUS
GRANULOMAS
Tuberculous mice develop only low levels of
DTH.Therefore, mouse granulomas show little
or no necrosis, and the blood supply within
them remains intact (see chapter 15). Like rab-
bits and guinea pigs, mice allow aerosolized vir-
ulent tubercle bacilli to grow logarithmically in
their lungs until DTH and CMI develop. Mice
evidently use apoptosis to stop this logarithmic
growth (see chapter 15).Then, a stationary stage
occurs, in which the number of viable bacilli no
longer increases.
When apoptosis kills macrophages, the sur-
rounding tissues are not killed and no inflam-
matory reaction occurs (26). Comparisons
between the apoptosis found in mice and the
caseous necrosis found in rabbits and guinea
pigs have never been made. In fact, rabbits and
guinea pigs may use both methods to kill
macrophages that contain more than a few
tubercle bacilli.The apoptosis method may be
less efficient, because in C57BL/6 mice (a rather
resistant strain), virulent human-type tubercle
bacilli grow (during the logarithmic stage) to
higher titers than they do in rabbits and guinea
pigs (see chapter 15).
Macrophage turnover in tuberculous granu-
lomas of rabbits and guinea pigs is probably
greater than that found in the granulomas of
mice. Cells undergoing necrosis produce many
chemotaxins (26), and cells undergoing apoptosis
䡵 193
produce none (26).With more necrosis, a greater
number of macrophages should enter and a
greater number should die.
With greater macrophage turnover, more
nonactivated macrophages would enter the
tuberculous granulomas. Such macrophages
would permit tubercle bacilli to multiply within
them until they are controlled by the DTH-
CMI process. In other words, the greater
turnover of macrophages in the lesions of rab-
bits and guinea pigs should allow more growth
and destruction of tubercle bacilli than in the
lesions of mice where the bacilli appear to be
more “dormant” (see chapter 6).
These conclusions must be confirmed exper-
imentally, because macrophage turnover rates in
tuberculous lesions have been studied only in
rabbits.
INSIGHTS INTO DTH AND CMI
PROVIDED BY OUR KINETIC
STUDIES IN RABBITS
Our [3H]TdR-labeling studies clearly estab-
lished that in tuberculous lesions the presence of
DTH and CMI increased the turnover of MN
(mostly macrophages). Prior to these studies,
we knew that the presence of DTH increased
MN accumulation, but we did not know much
about their rates of disappearance, except that
some MN died in the caseous centers.Therefore,
the rates of MN entry and disappearance from
tuberculous lesions were much higher than we
previously thought.
Similarly, we knew that CMI increased the
accumulation of activated macrophages (15, 16),
but we did not know the rate at which enter-
ing macrophages became activated. Our
[3H]TdR studies showed that macrophages
became activated within 5 to 8 days.
We found that relatively little local MN
(macrophage) division took place.The reduction
in their [3H]TdR grain counts seemed to be
mostly caused by the entry (into the lesions) of
new macrophages with reduced grain counts.
In sensitized hosts, the intradermal injection
of low concentrations of Old Tuberculin not
only caused cell infiltration (a classic DTH reac-
tion) but also activated an appreciable number
of macrophages in the infiltrating population (6).
194 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
These findings indicate that the tuberculin-like
products of the bacillus contribute to CMI,
because activated macrophages can inhibit
and/or destroy ingested tubercle bacilli.
High concentrations of tuberculin-like bacil-
lary products are known to cause necrosis in sen-
sitized hosts.To control the growth of tubercle
bacilli in the host, such necrosis (and/or apop-
tosis) is apparently required to kill macrophages
within which numerous bacilli are multiplying
(27–29).Any future tuberculosis vaccine that is
more effective than BCG in human popula-
tions will most likely produce some degree of
DTH—if not to tuberculin itself, at least to
other bacillary components that activate
macrophages in low concentrations and cause
necrosis in high concentrations.
RECAPITULATION OF OUR KINETIC
STUDIES ON MACROPHAGES IN
TUBERCULOUS LESIONS PRODUCED
BY BCG IN RABBITS
In tuberculous lesions produced by BCG in rab-
bits (and probably those in guinea pigs and
humans), macrophages have a continual turnover.
Numerous macrophages enter and many die or
leave via lymphatics. However, despite this high
turnover, many macrophages accumulate in the
lesions and become activated.
In an early lesion (produced by BCG), local
macrophage activation does not develop rapidly
enough to be effective, but after DTH and
CMI develop, the rate of macrophage activation
increases considerably, which enables the host
to control the BCG infection. Activated
macrophages ingest and inhibit the bacilli
released from killed macrophages.
If numerous activated macrophages are pre-
sent, tuberculous lesions will regress and even-
tually heal. However, even in almost-healed
lesions, nonactivated macrophages continuously
enter and may ingest bacilli escaping from the
edges of the caseous center.Therefore, a few of
the bacilli may undergo many cycles of intra-
cellular multiplication before the number of
activated macrophages becomes sufficient to
stop the progression of the disease.
In rabbits, and most likely in humans, the pop-
ulation of tubercle bacilli is not dormant in the
viable tuberculous granulation tissue of large or
even small “arrested” tuberculous lesions, because
a continuing influx of nonactivated macrophages
occurs even in almost-healed BCG lesions.These
nonactivated macrophages provide an opportu-
nity for renewed intracellular bacillary growth.
Populations of tubercle bacilli may only be truly
dormant within solid caseous necrotic tissue
where macrophages cannot survive.
Therefore, solid caseous tissue would be the
best source of bacilli for the identification of
dormancy genes. Such studies were begun in
healing tuberculous lesions of rabbits infected by
aerosol with virulent human-type tubercle bacilli
(H37Rv) (Y. C. Manabe et al., in preparation).
Ten such genes were identified, but the location
(within the lesions) of the bacilli containing
these genes remains to be identified. See chap-
ter 6 for more details.
REFERENCES
1. Shima, K., A. M. Dannenberg, Jr., M. Ando,
S. Chandrasekhar, J. A. Seluzicki, and J. I.
Fabrikant. 1972. Macrophage accumulation, divi-
sion, maturation, and digestive and microbicidal
capacities in tuberculous lesions. I. Studies involv-
ing their incorporation of tritiated thymidine and
their content of lysosomal enzymes and bacilli.
Am. J. Pathol. 67:159–180.
2. Ando, M., A. M. Dannenberg, Jr., and
K. Shima. 1972. Macrophage accumulation, divi-
sion, maturation and digestive and microbicidal
capacities in tuberculous lesions. II. Rate at which
mononuclear cells enter and divide in primary
BCG lesions and those of reinfection. J. Immunol.
109:8–19.
3. Dannenberg, A. M., Jr., M. Ando, and
K. Shima. 1972. Macrophage accumulation, divi-
sion, maturation and digestive and microbicidal
capacities in tuberculous lesions. III.The turnover
of macrophages and its relation to their activation
and antimicrobial immunity in primary BCG
lesions and those of reinfection. J. Immunol.
109:1109–1121.
4. Ando, M., and A. M. Dannenberg, Jr. 1972.
Macrophage accumulation, division, maturation
and digestive and microbicidal capacities in tuber-
culous lesions. IV. Macrophage turnover, lysosomal
enzymes and division in healing lesions. Lab. Inves-
tig. 27:466–472.
5. Tsuda, T., A. M. Dannenberg, Jr., M. Ando,
H.Abbey, and A. R. Corrin. 1976. Mononuclear
cell turnover in chronic inflammation. Studies on
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tritiated-thymidine-labeled cells in blood, tuberculin
traps and dermal BCG lesions of rabbits. Am.
J. Pathol. 83:255–268.
6. Ando, M. 1973. Macrophage activation in tuber-
culin reactions of rabbits with primary BCG infec-
tion and reinfection. J. Reticuloendothel. Soc. 14:132–
145.
7. Dannenberg, A. M., Jr., M. Sugimoto, L. P.
Fay, and A. L. Massaquoi. 1976. In vivo labeling
effectiveness of tritiated thymidine of high and low
specific activities in rabbits. Radiat. Res. 67:98–103.
8. Ando, M.,A. M. Dannenberg, Jr., E. Courtade,
and K. Shima. 1976.Turnover of tritiated-thymi-
dine-labeled mononuclear cells in tuberculous
lesions of rabbits.A comparison of primary dermal
BCG lesions and those of reinfection. Proc. Soc.
Exp. Biol. Med. 151:491–494.
9. Chandrasekhar, S., K. Shima, A. M. Dannen-
berg, Jr., T. Kambara, J. I. Fabrikant, and
W. G. Roessler. 1971. Radiation, infection and
macrophage function. IV.The effect of radiation on
the proliferative abilities of mononuclear phagocytes
in tuberculous lesions of rabbits. Infect. Immun.
3:254–259.
10. Dannenberg, A. M., Jr. 2003. Macrophage
turnover, division and activation within developing,
peak and “healed” tuberculous lesions produced in
rabbits by BCG. Tuberculosis 83:251–260.
11. van Furth, R. 1970. Origins and kinetics of
monocytes and macrophages. Semin. Hematol.
7:125–141.
12. Virolainen, M. 1968. Hematopoietic origin of
macrophages as studied by chromosome markers in
mice. J. Exp. Med. 127:943–951.
13. Volkman, A. 1970. The origin and fate of the
monocyte. Ser. Haematol. 3:62–92.
14. Fabrikant, J. I., C. L. Wisseman III, and M.
J.Vitak. 1969.The kinetics of cellular prolifera-
tion in normal and malignant tissues. II. An in
vitro method for incorporation of tritiated thymi-
dine in human tissues. Radiology 92:1309–
1320.
15. Dannenberg, A. M., Jr., O. T. Meyer, J. R.
Esterly, and T. Kambara. 1968.The local nature
of immunity in tuberculosis, illustrated histochem-
ically in dermal BCG lesions. J. Immunol. 100:931–
941.
16. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
genesis of tuberculosis: specificity, systemic and local
nature, and associated macrophage enzymes. Bacte-
riol. Rev. 32:85–102.
䡵 195
17. Ando, M., A. M. Dannenberg, Jr., M. Sugi-
moto, and B. S. Tepper. 1977. Histochemical
studies relating the activation of macrophages to the
intracellular destruction of tubercle bacilli. Am. J.
Pathol. 86:623–634.
18. Cleaver, J. E. 1967. Thymidine Metabolism and
Cell Kinetics. Frontiers of Biology, vol. 6. North-
Holland Publishing Company, Amsterdam, The
Netherlands.
19. Spector,W. G. 1969.The granulomatous inflam-
matory exudate. Int. Rev. Exp. Pathol. 8:1–55.
20. Ryan, G. B., and W. G. Spector. 1970.
Macrophage turnover in inflamed connective tissue.
Proc. R. Soc. (Biol.) 175:269–292.
21. Pearson, B., P. L. Wolf, and J.Vazquez. 1963.
A comparative study of a series of new indolyl
compounds to localize -galactosidase in tissues.
Lab. Investig. 12:1249–1259.
22. Yarborough, D. J., O. T. Meyer, A. M. Dan-
nenberg, Jr., and B. Pearson. 1967. Histo-
chemistry of macrophage hydrolases. III. Studies
on -galactosidase, -glucuronidase and aminopep-
tidase with indolyl and naphthyl substrates. J. Retic-
uloendothel. Soc. 4:390–408.
23. Lurie, M. B., P. Zappasodi, E. Cardona-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
to the intracutaneous inoculation of BCG as an
index of native resistance to tuberculosis. J. Immunol.
68:369–387.
24. Lurie, M. B. 1932. The correlation between the
histological changes and the fate of living tubercle
bacilli in the organs of tuberculous rabbits. J. Exp.
Med. 55:31–54.
25. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive Mech-
anisms. Harvard University Press, Cambridge, Mass.
26. Majno, G., and I. Joris. 2004. Cells,Tissues, and
Disease. Principles of General Pathology, 2nd ed. Oxford
University Press, New York, N.Y.
27. Dannenberg, A. M., Jr. 1993. Immunopatho-
genesis of pulmonary tuberculosis. Hosp. Pract.
28:33–40. (Off. ed. 51–58.)
28. Dannenberg,A. M., Jr. 2001. Pathogenesis of pul-
monary Mycobacterium bovis infection: basic princi-
ples established by the rabbit model. Tuberculosis
81:87–96.
29. Dannenberg, A. M., Jr., and F. M. Collins.
2001. Progressive pulmonary tuberculosis is not
due to increasing numbers of viable bacilli in rab-
bits, mice and guinea pigs, but is due to a contin-
uous host response to mycobacterial products. Tuber-
culosis 81:229–242.
 11
LURIE’S PULMONARY
TUBERCLE-COUNT METHOD

Tubercle development in humans [197]

Lurie’s tubercle-count method [197]
Advantages of the rabbit model of tuberculosis [199]
Role of alveolar macrophages in the establishment of grossly visible primary
pulmonary tubercles [200]
Tubercle counts to determine bacillary virulence [200]
Tubercle counts to determine the genetic resistance of the host [204]
Tubercle counts to determine the degree of immunization produced by a
given vaccine [204]
A comparison of tubercle counts and “the ratios” [204]
Effect of inhaled dosage on the ratio [206]
Variability of tubercle counts and ratios [206]
Comparison of aerosol mycobacterial infection with intravenous
infection [206]
Pulmonary tubercle counts in guinea pigs and mice [207]
Tubercle counting in mice—methodology [210]

Abstract. Lurie’s tubercle-count method consists of counting the number of grossly vis-
ible primary pulmonary tubercles, present 5 weeks after an aerosol infection of rabbits with
virulent human-type tubercle bacilli. It is a quantitative measure of clinically apparent dis-
ease.At 5 weeks, the grossly visible primary tubercles are easily recognized, and many micro-
scopic tubercles have regressed. Since human-type tubercle bacilli are not fully virulent
for rabbits, the pulmonary-count method has a sensitivity that is not possible with fully
virulent strains.
The number of grossly visible pulmonary tubercles produced by human-type bacilli
decreases (i) when rabbits are infected with bacilli of reduced virulence, (ii) when rabbits
of high genetic (innate) resistance are used, and (iii) when rabbits are effectively immu-
nized, so that they can rapidly activate macrophages and stop the development of early tuber-
cles while they are still microscopic in size.Therefore, the pulmonary tubercle-count method
can be used to assess (i) bacillary virulence, (ii) the genetic resistance of the host, and (iii)
the efficacy of vaccines for tuberculosis.

In rabbits, the number of primary tubercles
present in the lung following the inhalation of
virulent human-type tubercle bacilli is a precise
measure of both the ability of the bacillus to
produce clinically recognizable disease and the
ability of the host to prevent such clinical dis-
ease. To date, tubercle counting in rabbits has
been used in references 1 through 13, as well as
in Lurie’s studies on the effects of adrenal and
thyroid hormones (described in chapters 16
and 17).The method has been used only in rab-
bits, and remains to be developed for mice and
guinea pigs.
The majority of virulent human-type tuber-
cle bacilli inhaled by rabbits (and probably by
humans) are immediately destroyed or inhibited
by pulmonary alveolar macrophages (2) (see
chapter 2). Also, the majority of the bacilli
inhaled by rabbits that do manage to grow (and
form microscopic tubercles) are inhibited after
the host acquires immunity in 2 or 3 weeks. In
other words, the visible pulmonary tubercles

196
 11. LURIE’S PULMONARY TUBERCLE-COUNT METHOD 䡵 197

(found at 5 weeks in rabbits) are produced only
by bacilli that have survived the forces of both
native and acquired host resistance.
When fully virulent bovine-type tubercle
bacilli are inhaled by rabbits, differences in host
resistance will not be readily apparent, because
one grossly visible pulmonary tubercle is pro-
duced for every unit (of 1 to 3 bacilli) that is
inhaled into the alveoli by either resistant or sus-
ceptible hosts (2, 13, 14) (Table 1). However,
when human-type tubercle bacilli (which are
only semivirulent for rabbits) are inhaled, there
is a wide range in the number of bacilli required
to produce one grossly visible tubercle.There-
fore, small differences in host resistance or in
bacillary virulence are easily recognized.
To develop the tubercle-count method for
mice and guinea pigs, a semivirulent strain of
tubercle bacilli must be used. Fully virulent
human strains of tubercle bacilli in these hosts
(like fully virulent bovine strains in the rabbit)
produce one grossly visible tubercle for every
unit inhaled into the alveoli, and therefore would
not easily detect variations in host resistance by
the tubercle-count method (see chapter 15).
TUBERCLE DEVELOPMENT
IN HUMANS
Human pulmonary tuberculosis begins as a sin-
gle lesion, because bacilli are inhaled gradually,
usually over periods of months. Most of the
inhaled bacillary units seem to be ingested by
activated alveolar macrophages and immedi-
ately destroyed (or inhibited) without converting
the tuberculin skin test. However, a small pro-
portion of the inhaled bacilli may multiply in a
weakly activated alveolar macrophage and even-
tually cause a single primary tubercle.The host
then becomes tuberculin positive.The exact num-
ber of inhaled bacilli that produces a detectable
tubercle is not known for humans.A mean of 20
to 200 is a reasonable estimate. Most of these early
tubercles never progress beyond a millimeter or
so in size (see Lindgren’s studies described at the
end of chapter 3).A few, however, may progress
to clinically apparent tuberculosis.
Human tuberculosis (and tuberculosis in rab-
bits and guinea pigs exposed naturally to low
concentrations of tubercle bacilli in room air)
begins as only one primary pulmonary lesion
(see chapter 12). However, multiple grossly vis-
ible primary pulmonary tubercles are usually
produced in laboratory animals following an
aerosol infection. Each of these tubercles devel-
ops whenever (i) an alveolar macrophage allows
intracellular bacillary multiplication and (ii) a
weak acquired immunity does not prevent the
resulting microscopic tubercle from reaching
visible size. Many tubercles in multiple sites
more accurately reflects the overall resistance of
a given host better than does only one tubercle.
LURIE’S TUBERCLE-COUNT
METHOD
With Lurie’s tubercle-count method, rabbits are
infected by aerosol with a known number of

TABLE 1 The number of inhaled bovine-type tubercle bacilli (Ravenel) required to produce one primary
pulmonary tubercle in Lurie’s inbred rabbitsa
No. of bacilli
Rabbit No. of rabbits No. of isolated required to form
Resistance bacilli estimated as
strain exposed one visible tubercle
inhaled (range) (the ratio)
CaC Susceptible 6 40–462 2.8 ⫾ 0.2
AD Intermediate 22 90–688 3.4 ⫾ 0.3
III Resistant 6 216–306 2.9 ⫾ 0.3
C Susceptible 4 29–546 3.3 ⫾ 0.7
a
Note that 3 small units of inhaled fully virulent bovine-type tubercle bacilli (Ravenel) generate one grossly visible primary
pulmonary tubercle in every rabbit strain, despite differences in their genetic resistance. Means and their standard errors are listed.
The mean ratios were calculated from the ratios of individual rabbits by the Lurie method (see text and Tables 2, 3, and 5).Adapted
from references 2 and 13.
198 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

virulent human-type bacilli—Lurie used H37Rv
(1, 3, 15). Five weeks later, the animals are euth-
anized. Then, the grossly visible primary pul-
monary tubercles (Fig. 1 and 2) are counted and
their diameters are measured, either at the time
of necropsy (4, 12) or in formalin-fixed lungs at
a later time (1, 2, 7–9, 15).
In our experiments, an aliquot of the air in the
exposure chamber was collected by means of an
impinger into a 1:10 dilution (in 0.9% NaCl) of
Oleic Albumin Complex (Dubos) (Becton Dick-
inson, Co., Sparks, Md.) containing an antifoam
agent.Then, the impinger fluids were cultured (4,
10–12). Similar methods were used by Lurie to
determine the inhaled bacillary dose (1, 14). He
calculated the inhaled dose for each rabbit from
Kleiber’s formula based on the weight of each
rabbit (1, 14). We calculated the inhaled dose
from the volume of air breathed by each rabbit
in a plethysmograph (3, 4, 10–12).When all of the
exposed rabbits were of similar size, the variations
in the number of bacilli inhaled by each rabbit
were insignificant when compared to the much
larger variations in the number of tubercles caused
by differences in the innate and acquired resis-
tance of each rabbit.

FIGURE 1 Formalin-fixed lungs of a commercial rabbit that inhaled

about 33,000 virulent human-type tubercle bacilli (H37Rv) 5 weeks pre-
viously. Upon dissection, these lungs contained 131 grossly visible pri-
mary tubercles, with no apparent secondary tubercles.The ratio of the
number of bacilli estimated as inhaled to the number of grossly visible
primary tubercles produced was 250. Effective BCG (and other vaccines
for tuberculosis) should increase this ratio at least fivefold (9, 12). Small
areas of caseous necrosis are visible in many of the tubercles. On the left,
this photograph shows the ventral surface of the right upper, middle, and
azygous lobes; on the right, the entire left lung (upper and lower lobes)
is shown.The right lower lobe had been removed for culture.This right
lower lobe contained 23 grossly visible tubercles and 1.35 ⫻ 105 cultur-
able tubercle bacilli. Magnification, ⫻1.1. Reproduced with permission
from reference 3.
 11. LURIE’S PULMONARY TUBERCLE-COUNT METHOD 䡵 199

FIGURE 2 A tissue section of a primary lesion similar to the lesions shown in Fig. 1. From
left to right are (i) one of the small sites of caseous necrosis, (ii) a surrounding area of large epithe-
lioid macrophages, and (iii) an outside area that is densely infiltrated by smaller macrophages, lym-
phocytes, and plasma cells.Azure-eosin stain. Magnification, ⫻350. Reproduced with permission
from reference 3.

ADVANTAGES OF THE RABBIT Cavities never form in mice and in Lurie’s

MODEL OF TUBERCULOSIS
Most scientists in tuberculosis research at the
present time evaluate both bacillary virulence
and vaccine efficacy in mice, and a few scientists
evaluate them in guinea pigs.Aliquots of infected
lungs and spleens are usually cultured, and the
time of death is usually recorded. Although
bacillary titers in the lungs and spleen reflect
both the virulence of the bacilli and the native
and acquired resistance of the host (just as tuber-
cle counts do), certain aspects of host-parasite
interactions are not taken into account by such
measurements.
For example, in humans and in rabbits, the
bacilli may multiply extracellularly to tremen-
dous numbers in liquefied caseum, especially in
the liquefied caseum of the inner part of a cav-
ity wall. Cavities readily form in commercial
rabbits and in Lurie’s inbred resistant rabbits.
inbred susceptible rabbits, and they rarely form
in guinea pigs.The extracellular multiplication
of tubercle bacilli in cavities is not affected by
the native or acquired resistance of the host.
Tuberculous humans usually have strong
tuberculin sensitivity and considerable caseation,
and rabbits have moderate amounts of each,
especially if virulent bovine-type bacilli are used
to infect the animals. Caseation reduces the
number of viable tubercle bacilli that can be cul-
tured from lungs and other organs (see chapters
2 and 5).Tuberculous guinea pigs usually have
good delayed-type hypersensitivity (DTH) and
much caseation, and their lesions contain rela-
tively few bacilli (16) (see chapter 15). Tuber-
culous mice usually have weak DTH and little
or no caseation, and their lesions contain more
bacilli (16) (see chapter 15).Yet, despite lower
numbers of bacilli present, the disease often
200 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
progresses more rapidly in guinea pigs than it
does in mice. In other words, the number of cul-
turable bacilli in the lungs of different species
does not always parallel their native and acquired
resistance.
Tubercle counting in rabbits has additional
advantages over tubercle counting in mice and
guinea pigs. In New Zealand White rabbits
inhaling virulent human-type bacilli (H37Rv),
only 1 in 500 to 1,000 inhaled bacillary units
produces a grossly visible tubercle at 5 weeks
(see below and chapter 14).As stated above, the
vast majority of these bacilli are destroyed or
inhibited by the alveolar macrophages and by
the developing immune response. Therefore,
small changes in host resistance can be readily
detected.
Tubercle counting in mice and guinea pigs
is not as sensitive to such host defenses. In these
hosts, as in rabbits inhaling fully virulent
bovine-type bacilli (Table 1), only about 3
inhaled bacillary units are required to produce
a grossly visible tubercle. Therefore, in mice
and guinea pigs, the tubercle-count method
will not as easily detect small changes in bacil-
lary virulence and host resistance. Tubercle
counting in mice and guinea pigs is discussed
further at the end of this chapter.
Because of the differences in caseation and
cavity formation among rabbits, mice, and
guinea pigs, we recommend that rabbits be
included in experiments assessing (i) the viru-
lence of the infecting human-type tubercle
bacilli, (ii) the genetic resistance of the host,
and (iii) the efficacy of vaccines.
ROLE OF ALVEOLAR MACROPHAGES
IN THE ESTABLISHMENT OF
GROSSLY VISIBLE PRIMARY
PULMONARY TUBERCLES
Only one-third of the bacillary units of 1 to 3
bacilli remain suspended in the airstream and
reach the alveolar spaces (1, 14, 17–19). Units of
more than about 3 bacilli are so heavy that they
do not remain in the airstream, but impinge
upon the walls of the bronchial tree, often at its
bifurcations. They are then carried by the
bronchial mucociliary escalator to the pharynx
and are swallowed. Both the bronchial and
intestinal mucosa are rather resistant to infection
by tubercle bacilli (1).
Alveolar macrophages are the first cells of the
host to ingest the inhaled tubercle bacilli that enter
the alveoli of the lung.The alveolar macrophage
population is highly activated (20–22), apparently
because of the continuous ingestion of dust par-
ticles and microorganisms.A “good meal” in the
“stomach”of macrophages upregulates their diges-
tive and probably their oxidative enzymes (23).The
alveolar macrophages of Lurie’s resistant rabbits
seemed to be more highly activated than those of
his susceptible rabbits (1, 24–29).
In rabbits, and probably in humans, most
inhaled virulent human-type bacilli are soon
destroyed or inhibited by activated alveolar
macrophages and do not multiply. If no bacillary
multiplication occurs, the antigenic load remains
below that required to call forth an immune
response, and the host remains tuberculin neg-
ative. Following the inhalation of virulent
human-type tubercle bacilli, an initial micro-
scopic tubercle becomes established only after a
strong bacillus is ingested by a relatively weak
alveolar macrophage (1, 26–28): weak tubercle
bacilli are usually destroyed by the alveolar
macrophages in rabbits and probably in humans.
A full discussion of the role of alveolar
macrophages in the establishment of tuberculosis
is presented in chapter 12.
TUBERCLE COUNTS TO DETERMINE
BACILLARY VIRULENCE
Overview
Among the laboratory animals commonly used
to study tuberculosis, rabbits are unique.
Immunocompetent rabbits usually recover when
infected with fully virulent human-type tuber-
cle bacilli (1,15, 30), and usually die when
infected with fully virulent bovine-type tuber-
cle bacilli (1, 31). In fact, in the early part of the
20th century, rabbits were used to determine
whether the bovine or the human type of tuber-
cle bacillus had infected a given patient.
Lurie’s tubercle-count method in rabbits can
be used to assess the virulence of mycobacterial
isolates from human cases (4), and to measure the
virulence of human-type bacilli that were altered
 11. LURIE’S PULMONARY TUBERCLE-COUNT METHOD
by molecular biological techniques.The method
provided a quantitative measure of virulence,
namely, “the ratio,” which is the number of
inhaled bacillary units (of 1 to 3 bacilli) divided
by the number of grossly visible primary tuber-
cles present 5 weeks after infection (1, 3), i.e., the
number of inhaled bacilli that produces one
primary pulmonary tubercle.
Virulence of Bovine-Type Tubercle
Bacilli
With fully virulent bovine-type bacilli (Ravenel
S), about 3 bacillary units must be inhaled to
produce one grossly visible primary pulmonary
tubercle (a ratio of 3) (1, 2, 13, 14) (Table 1).
With bovine-type bacilli of somewhat reduced
virulence, this “ratio” varied from 18 to 107
(see Table 3 in chapter 4).The large number of
inhaled bovine-type bacilli seemed to decrease
the host’s ability to prevent grossly visible pri-
mary tubercles (10, 11). (However, a large num-
ber of inhaled human-type bacilli seemed to
increase the host’s ability to prevent grossly vis-
ible primary tubercles [5].)
Virulence of Human-Type Tubercle
Bacilli (H37Rv)
With H37Rv, Lurie’s resistant strain III rabbits
required an average of 1,065 inhaled bacillary
units to produce one visible tubercle, but his sus-
ceptible strain C rabbits required only an aver-
age of 70 bacillary units to do so, i.e., ratios of
1,065 and 70, respectively (Table 2) (1, 2).With
H37Rv in commercial New Zealand White
rabbits, we obtained ratios ranging from 765 to
1,946 (Table 3) (12).These ratios were similar to
those in Lurie’s resistant strain III rabbits. Ratios
for Lurie’s other inbred rabbit strains are listed
in Table 2.
By 1965, Lurie evidently had an even more
virulent H37Rv strain (8).The ratios of four of
his inbred rabbit strains were IIIR (TR) ⫽ 182
⫾ 36, AD ⫽ 56 ⫾ 4, and C ⫽ 48 ⫾ 8. (IIIR
was a more resistant substrain of III rabbits.) In
other words, in each of these inbred rabbit
groups, this more virulent H37Rv strain pro-
duced more tubercles per inhaled dose than
did the H37Rv bacilli used in all of his previ-
ously reported experiments. (Compare these
䡵 201
ratios with those presented in Table 2.) There-
fore, variations in virulence of human-type
tubercle bacilli can have profound effects on
the number of primary pulmonary tubercles
generated.*
Note that the greater the virulence of the
inhaled bacillary strain, the smaller the difference
in tubercle counts between the resistant and
susceptible inbred rabbits. In other words,
genetic (and immunological) differences are
most apparent in the tubercle counts when
aerosolized virulent human-type tubercle bacilli
of somewhat reduced virulence are used to
infect the rabbits.
This principle was recently confirmed with
Thorbecke inbred rabbits. which have reduced
resistance to tuberculosis (see Tables 3 and 5 in
chapter 14).The more virulent Erdman strain of
human-type tubercle bacilli did not produce
statistically different tubercle counts when Thor-
becke rabbits were compared to outbred rabbits,
but the less virulent H37Rv strain did so (6).
Virulence of a Clinical Mycobacterium
tuberculosis Isolate
In 1994 and 1995, a miniepidemic of tubercu-
losis occurred at the Kentucky-Tennessee bor-
der where the Oshkosh clothing factory is
located. The human-type bacillary strain that
caused this epidemic was designated CDC1551
by the Centers for Disease Control and Pre-
vention in Atlanta, Ga. Because it caused many
*Part of the apparent increased virulence of H37Rv
between 1956 and 1965 may have been due to a reduced
ability to grow in vitro after aerosolization. If fewer
bacilli were culturable from the aerosol, the ratios (bacilli
inhaled divided by number of tubercles produced)
would be decreased—even though the ability of these
bacilli to grow in the rabbit host (i.e., virulence) was
unchanged. About 1958, Lurie moved his laboratory
from the University of Pennsylvania’s old Henry Phipps
Institute building on 7th Street to new quarters on the
main university campus on 36th Street.When incubated
in the new location in an electric incubator, the bacilli
obtained after aerosolization may not have been able to
start as many colonies (on Lowenstein’s agar slants) as
they did in the gas-heated incubator in the old location.
Gas burners release CO2 into the air of the incubator.
When Lurie increased the CO2 (using a compressed-
CO2 cylinder), many more colonies developed from the
same inoculum.
 202
TABLE 2 The relative resistance of inbred rabbit strains as determined by their response to quantitative inhalation of human-type bacilli (H37Rv)a

䡵
E G
C No. of bacilli F Average diameter
A B D Variability in of tubercles (mm)

PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

No. of bacilli required to form
Rabbit strain No. of rabbits estimated as Range of tubercles one visible tubercle column E in and number of
and resistance evaluated inhaled (range) generated (the ratio) percent rabbits in which
(Lurie method) SE/ratio ⫻ 100 they were
measured
C (suscept.) 30 1,100–45,000 14–1,500 70 ⫾ 14 20% 4.0 ⫾ 0.27 (11)
(14/70)
FC (suscept.)b 45 700–54,000 8–1,400 79 ⫾ 9 11% 3.9 ⫾ 0.15 (33)
(9/79)
CaC (suscept.) 7 9,200–15,000 100–290 97 ⫾ 12 12% 3.9 ⫾ 0.20 (7)
(12/97)
A (intermed.) 11 14,900–21,000 51–230 230 ⫾ 24 10% 3.8 ⫾ 0.12 (11)
(24/230)
AD (intermed.) 12 25,000–35,000 25–370 362 ⫾ 103 28% 3.2 ⫾ 0.06 (12)
(103/362)
III (resistant)b 71 570–40,000 0–150 1,065 ⫾ 138 13% 3.1 ⫾ 0.11 (52)
(138/1,065)
FC (suscept.) 12 700–7,200 8–200 107 ⫾ 20 19%
(20/107)
Ca (suscept.) 9 520–3,100 3–440 49 ⫾ 19 39%
(19/49)
III (resistant) 8 570–4,300 0–11 ⬎642 ⫾ 107
a
Lurie determined the resistance of each rabbit by calculating the number of inhaled human-type bacilli required to form one grossly visible tubercle (the ratio), i.e., the number of
bacillary units inhaled by each rabbit divided by the number of visible tubercles produced at 5 weeks after infection. He then averaged the individual ratios to obtain the ratios listed above
for each inbred rabbit strain.
The average size of these tubercles also reflected host resistance; e.g., the tubercles of resistant strain III were smaller on average than those of susceptible strains C, FC, and CaC.
Data for strains C, FC,A,AD, and III are from reference 2; data for strain CaC are from reference 1 (p. 241); data for strains Ca and the second FC and III are from reference 15. Means
and their standard errors are listed.
b
The large number of rabbits in the III and FC groups (71 and 45, respectively) would, of course, have smaller standard errors than the 11 strain III and 13 strain FC rabbits listed in
Table 5.
 11. LURIE’S PULMONARY TUBERCLE-COUNT METHOD 䡵 203

TABLE 3 Number of inhaled virulent human-type bacilli (H37Rv) required to produce one grossly visible
primary pulmonary tubercle (the ratio) in commercial outbred New Zealand White rabbitsa
A. Experiment no. (no. of rabbits) I (6) II (6) III (6) IV (8)
B. Mean no. of viable bacilli 370,000 60,000 230,000 38,000
inhaled by each rabbit
C. Mean no. of visible tubercles 278 ⫾ 65 81 ⫾ 21 217 ⫾ 53 65 ⫾ 15
in lungs of each rabbit
D. Mean diameter of these tubercles 2.3 ⫾ 0.1 1.6 ⫾ 0.2 1.6 ⫾ 0.2 2.2 ⫾ 0.2
E. No. of bacilli inhaled by each 1,946 ⫾ 622 1,058 ⫾ 270 1,535 ⫾ 432 765 ⫾ 123
rabbit divided by the number of
tubercles produced by each rabbit:
average of individual ratios
(Lurie method)b
F.Average no. of inhaled bacilli 1,330 740 1060 585
divided by average number of
tubercles produced
(ratios of averages: B/C)
G.Variability in row E in percent: 32% (622/1,946) 26% (270/1,058) 28% (432/1,535) 16% (123/765)
SE/ratio ⫻ 100 (Lurie method)c
H.Variability in row C in percent: 23% (65/278) 26% (21/81) 24% (53/217) 23% (15/65)
SE/no. of tubercles ⫻ 100c
a
Note that the ratios tended to be higher when more bacilli were inhaled, perhaps because acquired immunity developed sooner.
Tubercle size, however, seemed independent of the bacillary dose inhaled. Means and their standard errors are listed. Data are from
reference 12.
b
In row E, the standard errors were calculated from the ratios of each rabbit in a given experiment.
c
Unexpectedly, the standard errors as percent of the means found in these outbred rabbits were not significantly larger than
those found in Lurie’s inbred rabbits (see Table 5), indicating that Lurie’s inbreeding did not appreciably reduce the variability within
each rabbit strain.

clinical cases after short exposure times,
CDC1551 was thought to be more virulent
than most clinical M. tuberculosis isolates.
Using Lurie’s tubercle-count method, we
found that, on the contrary, CDC1551 produced
about the same number of primary tubercles as
did the laboratory strain H37Rv in commercially
available New Zealand White rabbits (4) (Table
4); and, unexpectedly, the tubercles produced by
CDC1551 were significantly smaller and con-
tained fewer viable bacilli (4) (Table 4).There-
fore, the rabbits controlled the progress of the dis-
ease produced by CDC1551 better than they
controlled that produced by H37Rv (4). This
finding was supported by a careful evaluation of
CDC1551 in mice (32). Other studies in mice
also showed that CDC1551 was not more vir-
ulent than other strains of M. tuberculosis, includ-
ing Erdman (33) and H37Rv (33, 34) and sev-
eral clinical isolates (35).
Virulence for Rabbits of Erdman,
H37Rv, and CDC1551 (Oshkosh)
Strains of Human-Type Bacilli
Erdman was more virulent for rabbits than
H37Rv, and H37Rv was more virulent than
CDC1551 (5). Genetic analysis of these strains
identified some of the factors responsible for the
differences (5).
Effect of Freezing and Thawing on
Infectivity
In rabbits, log-phase human-type tubercle bacilli
produced more grossly visible tubercles than a
similar inhaled dose of viable frozen-and-
thawed bacilli of the same strain (5).The frozen-
and-thawed bacilli were probably more easily
destroyed by pulmonary alveolar macrophages.
This finding supports the concept that the phe-
notypic state of a viable tubercle bacillus affects
its ability to initiate the disease: a dormant
204 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 4 Virulence of two strains of human-type tubercle bacilli—H37Rv and CDC1551 (Oshkosh)—
assessed in rabbits by Lurie’s tubercle-count methoda

Strain of aerosolized bacilli No. of visible tubercles produced Average diameter of

5 weeks after aerosol infection tubercles (mm)
H37Rv 8.0 ⫾ 4.0 1.9 ⫾ 0.1
CDC1551 (Oshkosh) 5.2 ⫾ 1.5 1.2 ⫾ 0.1
a
About 8,000 viable units of 1 to 3 bacilli were inhaled by each commercial outbred rabbit.The number of primary tubercles
produced by the strain CDC1551 was similar to that produced by H37Rv, but the CDC1551 tubercles were smaller (P ⫽ 0.007)
and contained fewer bacilli when cultured. Means and their standard errors are shown. Data are from reference 4.

bacillus is probably less infectious than an
actively multiplying bacillus, possibly because its
metabolism is inactive at the time of inhalation.
TUBERCLE COUNTS TO DETERMINE
THE GENETIC RESISTANCE OF THE
HOST
Lurie’s tubercle-count method is useful in assess-
ing the innate (genetic) resistance of the host (1,
2, 7) (Table 2).About 1,000 bacillary units of 1
to 3 H37Rv bacilli were required to produce
one such tubercle in the resistant strain III rab-
bits, whereas only about 50 to 100 such units
were required in the highly susceptible strains C,
FC, Ca, and CaC (1, 2) (Table 2). In addition,
the average diameters of the tubercles in rabbits
of resistant strain III were smaller than those in
the susceptible strains C, FC, and CaC (1, 2)
(Table 2). Among the intermediate strains, A
had relatively large tubercles, and AD had rela-
tively small tubercles (Table 2). Chapter 14
describes the use of the tubercle-count method
to determine the resistance of crossbred resistant
and susceptible rabbits.
After the tubercle-count method was devel-
oped, Lurie used it to select the most resistant and
the most susceptible rabbits for breeding: when
rabbits (at necropsy) showed the desired tuber-
cle counts, their nearest relatives were chosen as
breeders to continue the desired characteristic.
TUBERCLE COUNTS TO DETERMINE
THE DEGREE OF IMMUNIZATION
PRODUCED BY A GIVEN VACCINE
Vaccines have no effect on whether an inhaled
tubercle bacillus establishes a microscopic lesion
in the host,because alveolar macrophages have no
immunological specificity. Effective acquired
resistance is recalled only after the inhaled bacil-
lus has multiplied and produced enough anti-
genic material to react with the memory lym-
phocytes entering the developing tubercle.These
antigen-specific lymphocytes rapidly accumulate
in the lesions and produce gamma interferon and
other cytokines that rapidly activate nearby
macrophages and other cells.Therefore, tubercle
bacilli are killed or inhibited sooner in the immu-
nized host, so fewer tubercles reach grossly visi-
ble size.Chapter 23 describes experiments that use
the tubercle-count method to assess vaccine effi-
cacy.Tables 5 and 6 summarize the experiments
made to date with various live vaccines in Lurie’s
inbred rabbits and in commercial outbred rabbits.
A COMPARISON OF TUBERCLE
COUNTS AND “THE RATIOS”
In general, larger rabbits inhale more bacilli and
therefore have higher tubercle counts than
smaller rabbits do, if both have similar resis-
tance to tuberculosis. Because the supply of
Lurie’s inbred rabbits was limited, both small and
large rabbits of the same inbred strain sometimes
had to be used in a given experiment. Also,
major differences in size existed among the var-
ious inbred rabbit strains themselves, e.g., the
strain III rabbits were much larger than the
strain FC and CaC rabbits.
Lurie compensated for such variations in
inhaled dose by dividing the number of bacilli
inhaled (by each rabbit) by the number of grossly
visible tubercles developed in order to obtain a
ratio. Strains III and FC rabbits had ratios that dif-
fered substantially (Table 5). In addition to adjust-
ing for variations in rabbit size, ratios enable the
pooling of data from several experiments in
 11. LURIE’S PULMONARY TUBERCLE-COUNT METHOD 䡵 205

TABLE 5 Variability in tubercle counts in two of Lurie’s inbred rabbit strainsa

No. of No. of No. of inhaled bacilli Average

Rabbit strain bacilli tubercles required to produce diameter
and numberb inhaled formed one tubercle (ratios) (mm)
(Lurie method)c
III 5⫽27 6,006 27 1,222 3.4
III 5⫽66 6,468 5 1,293 3.9
III 5⫽69 7,014 9 1,779 3.3
III 5⫽76 7,392 6 1,232 2.9
III 5⫽81 7,392 2 3,696 1.5
III 5⫽74 5,273 13 1,406 2.7
III 6⫽2 5,397 18 1,300 3.9
III 6⫽3 5,366 20 1,268 3.8
III 6⫽11 5,366 9 1,596 2.3
III 6⫽14 5,086 4 1,272 2.3
III 6⫽21 4,930 5 1,986 2.3

Mean and SE 6,209 ⫾ 340 10 ⫾ 3 1,004 ⫾ 296 2.9 ⫾ 0.2

Mean ratio from 1,621
the averages listed (6,209/10)

FC 2-9 4,215 107 1,039

FC 3-13 4,700 14 1,050
FC 2-46 4,470 55 1,081
FC 2-47 5,960 63 1,095
FC 3-18 5,960 201 1,030
FC 2-50 6,647 72 1,092
FC 3-29 7,233 32 1,226
FC 3-31 1,796 17 1,106
FC 4-3 2,120 8 1,265
FC 3-37 1,590 36 1,044
FC 3-36 1,440 9 1,160
FC 2-56 1,080 11 1,098

Mean and SE 3,601 ⫾ 695 52 ⫾ 23 107 ⫾ 20

Mean ratio from 69
the averages listed (3,601/52)
a
Variability is seen in the individual values listed.This variability is represented by the SE.The SE can also be represented as
a percentage of the mean: SE/mean ⫻ 100; e.g., for strain III, 296/1,004 ⫻ 100 ⫽ 29%, and for strain FC, 20/107 ⫻ 100 ⫽ 19%.
Commercial New Zealand White rabbits showed a similar amount of variation (see Table 3, row G). Adapted from references 2
and 9.
b
Lurie’s code for labeling his rabbits is best described with the following examples: III 5⫽27 means the 27th rabbit of resis-
tant strain III rabbits in the fifth generation with at least one backcross (double dash) to one of the parents. FC 2-9 means the 9th
rabbit of susceptible strain FC rabbits in the second generation with no backcrosses (single dash) to any parent.
c
The ratio is the number of inhaled human-type tubercle bacilli (H37Rv) required to produce one grossly visible primary pul-
monary tubercle 5 weeks after infection. It is the number of inhaled bacilli divided by the number of visible tubercles produced.
The Lurie method was to divide the dose inhaled by each rabbit by the number of primary tubercles present in each rabbit. He
then averaged the ratios (see text).
206 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
which the number of bacilli in the aerosol was
markedly different.Although ratios have all these
advantages, they increase the difference between
rabbits with few grossly visible tubercles and
those with many tubercles. Specifically, when a
series of tubercle counts is compared with a series
of ratios on the same group of rabbits, the rabbits
with few tubercles show proportionally higher
ratios than do the rabbits with many tubercles. In
other words, the mean ratio, obtained by divid-
ing the number of bacilli inhaled by each rabbit
by the number of primary tubercles in each rab-
bit, is inflated by ratios derived from rabbits with
low numbers of tubercles.
For example, in Table 5 the inflated ratio
obtained by averaging the ratios from individ-
ual strain III rabbits was 1,004, whereas the
ratio obtained by dividing the average inhaled
dose by the average number of tubercles was
621. Similarly, for the FC rabbits, the inflated
ratio was 107, and the ratio of averages was 69.
Similarly, in Table 3, the averages of individual
ratios compared to ratios of the averages were
1,946 versus 1,330, 1,058 versus 740, 1,535 ver-
sus 1,060, and 765 versus 585; i.e., averaging
ratios from each rabbit inflated the means from
131% to 162%.
We recommend that comparisons of all
experimental and control groups be made by
first averaging the dose inhaled by each group
and dividing it by the average number of tuber-
cles produced in all of the rabbits in that group.
When calculated in this manner, the ratios of the
two groups being compared will not be inflated
as much by individual rabbits with fewer pri-
mary tubercles than the mean.
In brief, Lurie’s use of ratios from individual
rabbits exaggerated the differences between his
resistant and susceptible rabbits but did not alter
the conclusions he derived from them.
EFFECT OF INHALED DOSAGE
ON THE RATIO
In rabbits, the dose of inhaled tubercle bacilli
seemed to have an effect on the ratio. When
compared to a low inhaled dose, a high inhaled
dose of virulent bovine-type tubercle bacilli
reduced the effectiveness of the host’s immune
system; i.e., fewer bacilli were required to produce
each grossly visible tubercle (11) (see Table 3
in chapter 4). In contrast, with human-type
bacilli (which are always of reduced virulence
for rabbits), a high inhaled dose increased the
effectiveness of the host’s immune system, i.e.,
a greater number of bacilli were required to
produce each visible tubercle (5). In other
words, the high inhaled dose of the more vir-
ulent bacillus (the bovine strain in rabbits)
reduced acquired (adaptive) host resistance,
whereas the high inhaled dose of the less vir-
ulent bacillus (the human strain in rabbits)
enhanced acquired host resistance (discussed
further in chapter 4).
VARIABILITY OF TUBERCLE
COUNTS AND RATIOS
The standard error of the mean (SE) repre-
sents the variability within a given strain of
rabbits. With Lurie’s strain III rabbits, the
SE/mean for the ratios (expressed as a per-
centage of the mean) was 29% (296/1,004)
(Table 5). (The relatively high variability may be
partly due to the low number of tubercles pro-
duced; low numbers of tubercles exaggerate
ratio.) With Lurie’s strain FC rabbits, the SE was
19% (20/107) (Table 5), and with commer-
cially outbred New Zealand White rabbits, it
was 16 to 32% (Table 3). In other words, Lurie’s
inbreeding did not always reduce the variabil-
ity over that found among outbred New
Zealand White rabbits. However, the recently
available Thorbecke inbred rabbits (see chapter
14) did show less variability than outbred rab-
bits in their resistance to tuberculosis (6; unpub-
lished data).
COMPARISON OF AEROSOL
MYCOBACTERIAL INFECTION
WITH INTRAVENOUS
INFECTION
After an aerosol infection, acquired immunity
develops mostly in bronchus-associated lym-
phoid tissues and the hilar lymph nodes (see
reference 36). After an intravenous infection,
acquired immunity develops mostly in the
spleen. Therefore, the route of infection may
affect measurements of (i) bacillary virulence, (ii)
host genetic resistance, and (iii) vaccine efficacy.
 11. LURIE’S PULMONARY TUBERCLE-COUNT METHOD
Bacillary Virulence
When aerosols of two bacillary strains of dif-
fering virulence are inhaled, the alveolar
macrophage population (which is highly acti-
vated) would immediately destroy or inhibit
greater numbers of the less virulent strain.When
such bacillary strains are given intravenously,
the two strains would initially grow at about the
same rate in the lungs, because they are ingested
by the nonactivated blood-borne macrophages
within the pulmonary vasculature.
For example, C57BL/6 mice injected intra-
venously with wild-type and attenuated tuber-
cle bacilli showed the same bacillary titers at 2
weeks, and then the titers in the attenuated
group gradually declined (37; see reference 38)
(also see chapter 15). Yet, mice receiving the
wild-type bacilli died, and those receiving the
attenuated bacilli lived (38). If the mice had been
infected by aerosol, markedly different bacillary
titers might have been found at 2 weeks, because
the alveolar macrophage population is already
activated and kills or inhibits many attenuated
inhaled tubercle bacilli before one is allowed to
multiply. Therefore, to compare the virulence
of two strains of tubercle bacilli, an aerosol infec-
tion is the most sensitive assay.
Host Genetic Resistance
Lurie’s resistant and susceptible inbred rabbits
apparently differed in their ability to activate
macrophages—both nonspecifically in the pul-
monary alveolar macrophage population and
immune-specifically in their acquired resistance
(see chapters 2 and 5).As with bacillary virulence,
infection by aerosol would measure a combina-
tion of the nonspecific resistance of alveolar
macrophages and the acquired resistance devel-
oped by expanding antigen-specific lympho-
cyte populations. Infection by the intravenous
route would measure only the acquired type.
Vaccine Efficacy
Immunization has little effect on innate immu-
nity, i.e., the activation of the alveolar
macrophage population after the nonspecific
effects have disappeared.Therefore, either aerosol
or intravenous routes of challenge are satisfac-
tory for measuring vaccine efficacy. In each
䡵 207
case, the vaccine increases the number of anti-
gen-specific lymphocytes, so that the acquired
(adaptive) host response develops more rapidly
after challenge with virulent tubercle bacilli.
However, as stated at the beginning of this sec-
tion, the immunity recalled after an aerosol
challenge may be somewhat different from the
immunity recalled after an intravenous chal-
lenge, in which the spleen plays a major role. In
addition, different organs have different resis-
tances to the growth of tubercle bacilli within
them (see chapter 15).
Other Routes of Infection
Bacilli injected by the intraperitoneal route
rapidly enter the bloodstream. They travel
through the diaphragm into major lymphatic
trunks that drain into the great veins.Therefore,
the intraperitoneal route resembles the intra-
venous route. Bacilli injected subcutaneously,
intramuscularly, or intradermally first drain to the
local lymph nodes (where acquired immunity
develops), and only relatively few bacilli rapidly
enter the bloodstream.All of these routes bypass
the alveolar macrophages and measure mainly
the resistance (immunity) acquired by the host
during the course of the infection.
PULMONARY TUBERCLE COUNTS IN
GUINEA PIGS AND MICE
Tubercle counts in guinea pigs and mice will not
be as useful as they are in rabbits. In these two
laboratory species, each inhaled fully virulent
human-type tubercle bacillus reaching the alve-
olar spaces produces a primary microscopic
tubercle that progresses until the host dies (39,
40) (see chapter 15). In guinea pigs and mice, the
pulmonary alveolar macrophages (a highly acti-
vated cell population) slow (but do not stop) the
intracellular multiplication of the inhaled bacilli.
Similarly, macrophages activated by antigen-
specific lymphocytes in tuberculous lesions of
immunized guinea pigs and mice only slow the
progression of pulmonary lesions.
When euthanized at the appropriate time,
immunized guinea pigs had fewer grossly visible
pulmonary tubercles than did nonimmunized
controls, and the tubercles were smaller than
those in controls (39). If the mice had been
 208
䡵
PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
TABLE 6 Vaccine efficacy: number of inhaled human tubercle bacilli (H37Rv) required to produce one grossly visible primary pulmonary tubercle in unvacci-
nated and vaccinated rabbitsa

B C
Unvaccinated Vaccinated
D E
No. of rabbits No. of bacilli No. of rabbits No. of bacilli Vaccine Vaccine
evaluated inhaled by each evaluated inhaled by each efficacy: no. of efficacy: no. of
rabbit divided rabbit divided tubercles in tubercles in F
A by the no. of by the no. of vaccinated vaccinated P values:
Inbred or tubercles tubercles rabbits as rabbits as vaccinated vs.
commercial produced by produced by percent of the percent of the unvaccinated
rabbit and each rabbit each rabbit no. in control no. in control (using ratios in
vaccine used (avg. of (avg. of rabbits from rabbits from column D)
individual individual ratios of averages of
ratios [Lurie ratios [Lurie averages individual
method]) method]) (B/C ⫻ 100)b ratiosb
Inbred rabbits
Inbred resistant
strain III rabbits
BCG (Phipps) 8 (1,642 ⫾ 107 8 2,948 ⫾ 914 22% P ⫽ 0.013
Inbred susceptible
strain FC rabbits
BCG (Phipps) 12 (1,107 ⫾ 20 9 1,124 ⫾ 18 86% NS

(continued)
Commercial rabbits
BCG (Danish) 6 (1,946 ⫾ 622 6 4,841 ⫾ 2,281 54% 40% P ⫽ 0.08
6 (1,535 ⫾ 432 6 2,458 ⫾ 863 69% 62% NS
BCG (Japanese) 6 (1,946 ⫾ 622 6 5,587 ⫾ 1,480 48% 35% P ⫽ 0.06
6 (1,535 ⫾ 432 6 1,813 ⫾ 154 61% 85% P ⫽ 0.08
BCG (Tice) 6 (1,535 ⫾ 432 6 2,167 ⫾ 441 65% 71% NS
BCG (Pasteur) 8 (1,765 ⫾ 123 8 5,870 ⫾ 1,246 13% 13% P ⫽ 0.001

Mycobacterium 6 1,946 ⫾ 622 6 3,257 ⫾ 753 56% 60% P ⫽ 0.07

microti NCO

11.
8712
M. microti ATCC 6 1,058 ⫾ 270 6 1,427 ⫾ 302 62% 74% NS

LURIE’S PULMONARY TUBERCLE-COUNT METHOD

35781
M. microti ATCC 6 1,058 ⫾ 270 6 3,586 ⫾ 1,026 65% 30% NS
35782
M. microti OV 254 6 1,535 ⫾ 432 6 5,233 ⫾ 859 40% 29% P ⫽ 0.03
6 1,058 ⫾ 270 6 3,440 ⫾ 1,732 25% 31% P ⫽ 0.007
8 1,765 ⫾ 123 8 5,254 ⫾ 2,091 22% 15% P ⫽ 0.002
M. microti ATCC 6 1,058 ⫾ 270 6 3,076 ⫾ 969 29% 34% P ⫽ 0.01
11152 8 1,765 ⫾ 123 8 7,805 ⫾ 44,15 20% 10% P ⫽ 0.002
a
In addition to ratios from Lurie’s inbred strain III and strain FC rabbits, this table lists the mean ratios from each experiment containing 6 to 8 vaccinated commercial rabbits and 6 to 8
control commercial rabbits (see text). NS, not significant, using P ⫽ 0.10.We realize that the P values listed between 0.05 and 0.10 are not usually considered significant, but they indicate
a trend. Data are from references 2, 9, and 12.
b
The percentages listed in columns D and E are a comparison of the average tubercle count in each experiment, which is more precise than a comparison of individual ratios (see text).
Insufficient numbers of rabbits were evaluated to compare the efficacy of the various vaccines (see text). However, the vaccines with the best P values seem most promising.

䡵
209
210 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
euthanized at the appropriate time, tubercle
counts could have been made.
In brief, tubercle counting is most valuable for
testing vaccine efficacy in rabbits challenged by
aerosol with human-type tubercle bacilli
(H37Rv),because rabbits have sufficient resistance
to destroy or inhibit most of these bacilli. How-
ever, in guinea pigs and mice, tubercle counting
could be an excellent way to quantitate vaccine
efficacy—especially if they are challenged by
aerosol with tubercle bacilli of somewhat reduced
virulence and they are euthanized before many
(but not all) microscopic pulmonary lesions reach
countable size.
TUBERCLE COUNTING IN MICE—
METHODOLOGY
We did not make pulmonary tubercle counts in
mice inhaling M. tuberculosis, but we did so in
mice inhaling the virulent gram-negative bacil-
lus Burkholderia pseudomallei, which produces
melioidosis (41–44). (This bacillus was formerly
called Malleomyces pseudomallei and Pseudomonas
pseudomallei.) We usually made pulmonary lesion
counts by dissecting the lungs after they were
fixed.When the lesions were too numerous to
count directly, we counted the surface lesions
and prepared a graph that enabled us to estimate
the total number of pulmonary lesions from
the number of surface lesions (41). We also
counted in tissue sections the number of micro-
scopic pulmonary lesions produced by avirulent
B. pseudomallei 2 days after their inhalation,
before they regressed (43).
In brief, the methodology developed in mice
for counting lesions produced by inhaled B.
pseudomallei could be used for counting tuber-
cles produced by inhaled M. tuberculosis.
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Y. C. Manabe. 2003. Susceptibility to tuberculo-
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7. Lurie, M. B., P. Zappasodi,A. M. Dannenberg,
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liposaccharide PmKo from tubercle bacilli as an
index of resistance to tuberculosis. Bull. Johns Hop-
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9. Lurie, M. B., P. Zappasodi, E. Cardona-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
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68:369–387.
10. Converse, P. J., A. M. Dannenberg, Jr., J. E.
Estep, K. Sugisaki,Y.Abe, B. H. Schofield, and
M. L. M. Pitt. 1996. Cavitary tuberculosis pro-
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11. Converse, P. J., A. M. Dannenberg, Jr.,
T. Shigenaga, D. N. McMurray, S. W. Phalen,
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H. Abbey, J. E. Estep, and M. L. M. Pitt. 1998.
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13. Allison, M. J., P. Zappasodi, and M. B. Lurie.
1962. Host-parasite relationships in natively resistant
and susceptible rabbits on quantitative inhalation
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14. Lurie, M. B., A. G. Heppleston, S. Abramson,
and I. B. Swartz. 1950. An evaluation of the
method of quantitative airborne infection and its use
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15. Lurie, M. B., S. Abramson, and A. G. Hep-
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sistant and susceptible rabbits to the quantitative
inhalation of human-type tubercle bacilli and the
nature of resistance to tuberculosis. J. Exp. Med.
95:119–134.
16. Francis, J. 1958. Tuberculosis in Animals and Man.
A Study in Comparative Pathology, p. 293–318. Cas-
sell and Company, Ltd., London, United Kingdom.
17. Brown, J. H., K. M. Cook, F. G. Ney, and
T. Hatch. 1950. Influence of particle size upon the
retention of particulate matter in the human lung.
Am. J. Public Health 40:450–458.
18. Goldberg, L. J., and W. R. Leif. 1950.The use
of radioactive isotopes in determining the retention
and initial distribution of airborne bacteria in the
mouse. Science 112:299–300.
19. Sonkin, L. S. 1951. The role of particle size in
experimental airborne infection. Am. J. Public Health
53:337–354.
20. Dannenberg, A. M., Jr., M. Burstone, P. C.
Walter, and J.W. Kinsley. 1963.A histochemical
study of phagocytic and enzymatic functions of
rabbit mononuclear and polymorphonuclear exu-
date cells and alveolar macrophages. I. Survey and
quantitation of enzymes, and states of cellular acti-
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21. Myrvik, Q. N., E. Soto Leake, and D. Gonza-
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normal rabbit alveolar macrophages and oil-induced
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22. Myrvik, Q. N., E. Soto Leake, and B. Fariss.
1961. Lysozyme content of alveolar and peritoneal
macrophages from the rabbit. J. Immunol. 86:133–
136.
23. Dannenberg, A. M., Jr., P. C. Walter, and
F. A. Kapral. 1963. A histochemical study of
phagocytic and enzymatic functions of rabbit
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24. Dannenberg,A. M., Jr. 1991. Delayed-type hyper-
sensitivity and cell-mediated immunity in the patho-
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26. Dannenberg, A. M., Jr., and G. A. W. Rook.
1994. Pathogenesis of pulmonary tuberculosis: an
interplay of tissue-damaging and macrophage-
activating immune responses—dual mechanisms
that control bacillary multiplication, p. 459–483. In
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27. Dannenberg,A. M., Jr., and J. F.Tomashefski,
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p. 2447–2471. In A. P. Fishman (ed.), Fishman’s Pul-
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28. Dannenberg, A. M., Jr. 1999. Pathophysiology:
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II. Immunology of tuberculosis, p. 17–47. In
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Mycobacterial Infections, 4th ed. The W. B. Saunders
Co., Philadelphia, Pa.
29. Henderson, H. J., A. M. Dannenberg, Jr., and
M. B. Lurie. 1963. Phagocytosis of tubercle bacilli
by rabbit pulmonary alveolar macrophages and its
relation to native resistance to tuberculosis.
J. Immunol. 91:553–556.
30. Heppleston, A. G. 1949. Quantitative air-borne
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31. Lurie, M. B. 1941. Heredity, constitution and
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44(Suppl. 3):1–125.
32. Manca, C., L. Tsenova, C. E. Barry III,
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Musser,V. H. Freedman, and G. Kaplan. 1999.
Mycobacterium tuberculosis CDC1551 induces a more
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33. Kelley, C. L., and F. M. Collins. 1999. Growth
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34. North, R. J., L. Ryan, R. LaCource,T. Mogues,
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mycobacteria in mice as an unreliable indicator of
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35. Orme, I. M. 1999.Virulence of recent notorious
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79:379–381.
36. Strober,W., B. Kelsall, and T. Marth. 1998. Oral
tolerance. J. Clin. Immunol. 18:1–30.
37. McKinney, J. D., K. Höner zu Bentrup, E. J.
Muñoz-Elías,A. Miczak, B. Chen,W.-T. Chan,
D. Svenson, J. C. Sacchettini,W. R. Jacobs, Jr.,
and D. G. Russell. 2000. Persistence of Mycobac-
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38. Glickman, M. S., J. S. Cox, and W. R. Jacobs,
Jr. 2000. A novel mycolic acid cyclopropane syn-
thetase is required for cording, persistence, and
virulence of Mycobacterium tuberculosis. Mol. Cell 5:
717–727.
39. Horowitz, M. A., G. Harth, B. J. Dillon, and
S. Maslesa-Galic. 2000. Recombinant bacillus
Calmette-Guérin (BCG) vaccines expressing the
Mycobacterium tuberculosis 30-kDa major secretory
protein induce greater protective immunity against
tuberculosis than conventional BCG vaccines in a
highly susceptible animal model. Proc. Natl.Acad. Sci.
USA 97:13853–13858.
40. Medina, E., and R. J. North. 1999. Genetically
susceptible mice remain proportionally more sus-
ceptible to tuberculosis after vaccination. Immunol-
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41. Dannenberg,A. M., Jr., and E. M. Scott. 1956.
Determination of respiratory LD50 from number of
primary lesions as illustrated by melioidosis. Proc. Soc.
Exp. Biol. Med. 92:571–575.
42. Dannenberg,A. M., Jr., and E. M. Scott. 1958.
Melioidosis: pathogenesis and immunity in mice and
hamsters. I. Studies with virulent strains of
Malleomyces pseudomallei. J. Exp. Med. 107:153–166.
43. Dannenberg,A. M., Jr., and E. M. Scott. 1958.
Melioidosis: pathogenesis and immunity in mice and
hamsters. II. Studies with avirulent strains of
Malleomyces pseudomallei. Am. J. Pathol. 34:1099–1121.
44. Dannenberg,A. M., Jr., and E. M. Scott. 1960.
Melioidosis: pathogenesis and immunity in mice
and hamsters. III. The effect of vaccination with
avirulent strains of Pseudomonas pseudomallei on the
resistance to the establishment and the resistance to
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J. Immunol. 84:233–246.
 Section 4.

TUBERCULOSIS IN RABBITS AND OTHER

COMMON LABORATORY ANIMALS
This page intentionally left blank
 12
NATURAL AIRBORNE INFECTION

Lurie’s natural airborne infection [215]

Resistance to the establishment of tuberculous lesions produced by virulent
bovine-type bacilli in Lurie’s rabbits [216]
Resistance to the progress of pulmonary tuberculosis produced by virulent
bovine-type bacilli in Lurie’s rabbits [218]
Conversion of the tuberculin skin test with no grossly visible primary
pulmonary lesion at necropsy [223]
Only a single primary tuberculous lesion develops under natural
conditions [223]
Resistance to the establishment of tuberculous lesions produced by virulent
human-type bacilli in Lurie’s rabbits [223]
Resistance to the progress of pulmonary tuberculosis produced by virulent
human-type bacilli in Lurie’s rabbits [224]
Establishment and progress of tuberculosis in mice and guinea pigs [224]
Riley’s experiments with natural airborne infection [224]
Other methods of room air sampling [226]
Comparisons of tuberculosis in rabbits and humans [226]
Effect of immunization on resistance to the establishment and resistance to
the progress of an acute septicemic disease (melioidosis) and a chronic,
slowly progressing disease (tuberculosis) [226]

Abstract. Using natural airborne infection of virulent bovine-type tubercle bacilli over
many months, Lurie showed that resistance to the establishment of tuberculosis and resis-
tance to its progress are separate phenomena: his inbred resistant rabbits converted their
tuberculin skin tests an average of 2.7 months sooner than did his inbred susceptible rab-
bits.Yet, once established, the disease progressed slowly in the resistant rabbits and rapidly
in the susceptible rabbits.The separation of the establishment and progress of tuberculo-
sis is only applicable to experiments in which occasional fully virulent tubercle bacilli are
inhaled over many months. It does not seem applicable to rabbits or humans inhaling
human-type tubercle bacilli, which are never fully virulent in these hosts.
Airborne infection of laboratory animals over many months has, however, established
other concepts directly applicable to tuberculosis in humans. (i) Only a single grossly vis-
ible primary pulmonary lesion will be produced, despite the continuous presence of vir-
ulent tubercle bacilli in the air.The immunity developed in response to the primary lesion
is evidently sufficient to prevent other occasionally inhaled tubercle bacilli from causing
grossly visible lesions. (ii) Some animals (and perhaps a few humans) may convert their der-
mal tuberculin reactions, and yet show no grossly visible primary lesions in their lungs at
necropsy.This occurrence may be due to the early spread of inhaled bacilli out of the lungs
to the hilar lymph nodes, where the growth of tubercle bacilli can be more easily con-
trolled.These concepts are consistent with what Riley found when he exposed guinea pigs
for months to air from a ward containing sputum-positive tuberculous patients.

LURIE’S NATURAL AIRBORNE
INFECTION
Lurie’s natural airborne experiments (1–5) were
performed in a room where the runs contain-
ing the source rabbits were separated by a fine-
mesh metal screen from the cages containing
uninfected inbred rabbits (Fig. 1). When the
infected commercial rabbits (shedding virulent
bovine-type tubercle bacilli in their urine)
moved around in the bedding on the floor of

215
216 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

their runs, the tubercle bacilli became airborne.
The approximate time at which a primary
(microscopic) tuberculous lesion was established
in the lungs of the uninfected inbred rabbits was
indicated by the conversion of their tuberculin
skin tests, which were made every 2 to 4 weeks
(Tables 1 and 2) (1, 3).
Lurie performed three natural airborne
experiments, each with a different concentration
of virulent bovine-type tubercle bacilli in the air
of the room (Table 2) (3). He compared one
genetically resistant strain of rabbits with two
genetically susceptible strains.Table 1 is a com-
posite of all three experiments. It shows the
variations among the individual rabbit strains.
These experiments established two basic prin-
ciples: (i) Resistance to the establishment of a
tuberculous lesion is distinct from resistance to
its progress, and (ii) a single active primary pul-
monary lesion usually produces enough acquired
immunity to prevent subsequently inhaled
tubercle bacilli from causing lesions that reach
grossly visible size (2, 3) (discussed below).
RESISTANCE TO THE
ESTABLISHMENT OF TUBERCULOUS
LESIONS PRODUCED BY VIRULENT
BOVINE-TYPE BACILLI IN LURIE’S
RABBITS
Resistance to the establishment of an infection
is to be contrasted with resistance to its progress
(1, 3, 5). In tuberculosis, lesions are established
when the bacillus initially multiplies in the host,
and lesions progress if such multiplication con-
tinues.
In Lurie’s natural airborne experiments,
almost every unit of virulent bovine-type tuber-
cle bacilli that is retained in the alveoli should
cause progressive disease in both his resistant and

FIGURE 1 A diagram of the rabbit cages that Lurie used for natural airborne infection.Thirty
commercial rabbits, many of which were shedding virulent bovine-type tubercle bacilli (Ravenel)
in their urine, were distributed into the three rabbit runs (rear of the diagram).As the rabbits moved
throughout the runs, they caused tubercle bacilli in the urine-dampened bedding to become air-
borne.
Noninfected inbred resistant and susceptible rabbits were placed in 30 cages (front of the dia-
gram).These rabbits inhaled occasional tubercle bacilli over a period of many months.The cages
were moved daily, so that during the course of 30 days, each cage resided in each position for 1
day. Reproduced with permission from reference 2.
 12. NATURAL AIRBORNE INFECTION 䡵 217

TABLE 1 Resistance to the establishment of tuberculosis and resistance to its progress with a natural airborne
contagion of virulent bovine-type tubercle bacillia
Preallergic period Duration of disease
Rabbit strain Rabbit no.
(months) (months)
Strain A, high resistance A8⫽19 0.9 4.7
A8⫽29 1.3 10.4
A7⫽31 1.8 No visible lesions
A8⫽43 1.8 No visible lesions
A4-4 2.0 No visible lesions
A8⫽51 2.3 4.1
A7⫽26 2.3 5.0
A3⫽3 3.2 11.5
A6⫽21 3.3 No visible lesions
A5⫽3 3.3 12.9
A5⫽4 3.3 No visible lesions
A5⫽21 3.3 10.7
A7⫽5 3.5 No visible lesions
A7⫽36 3.6 8.0
A5⫽2 4.3 No visible lesions
A7⫽10 4.9 No visible lesions
A2⫽11 5.0 7.5
A7⫽4 6.0 No visible lesions
Average 3.1 ⫾ 1.3 8.3 ⫾ 3.1
Strain F, low resistance F4-25 1.9 3.8
F4-30 2.4 3.0
F2-15 3.0 6.2
F5-2 4.6 No visible lesions
F6-25 5.1 3.7
F2-3 6.0 5.0
F5-14 6.0 No visible lesions
F4-33 6.2 3.0
F3-9 6.5 8.0
F6-14 8.3 2.1
F3-7 11.0 3.5
F4-11 11.7 4.0
Average 6.1 ⫾ 3.0 4.2 ⫾ 1.7
Strain C, low resistance C6-9 1.3 3.9
C2-8 2.0 9.5
C6-32 2.1 3.6
C5-50 2.3 3.9
C4R-2 2.7 4.3
C5-30 3.0 3.3
C4S-9 3.3 Tuberc. ⫹ snuffles
C2-6 3.7 3.7
C4S-11 3.8 Tuberc. ⫹ snuffles
C4R-6 4.7 3.8
C4S-30 4.9 No visible lesions

(continued next page)

218 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Resistance to the establishment of tuberculosis and resistance to its progress with a natural airborne
contagion of virulent bovine-type tubercle bacillia (continued)
Preallergic period Duration of disease
Rabbit strain Rabbit no.
(months) (months)
Strain C, low resistance C2-1 5.0 3.0
(continued) C6-1 5.5 No visible lesions
C2-7 7.0 5.0
C6-26 8.3 Tuberc. ⫹ snuffles
C5-14 9.0 2.6
C2-18 11.9 No visible lesions
Average 4.7 ⫾ 2.8 4.2 ⫾ 1.8
a
This table lists data on all of the rabbits with positive tuberculin reactions that were used in Lurie’s natural airborne infec-
tion experiments over a 10-year period. Note that the resistant A rabbits converted their tuberculin reactions sooner, but lived
longer, than did the susceptible F and C rabbits.This table was reproduced in full, because these rabbit strains are now extinct,
which makes these experiments unique. Preallergic period: A vs. F: P ⫽ 0.001; A vs. C: P ⫽ 0.024. Duration of disease: A vs. F:
P ⫽ 0.001;A vs. C: P ⫽ 0.001. Means and standard errors are listed. Data from reference 3.

susceptible rabbits. Fully virulent bovine-type
bacilli may be somewhat inhibited by pul-
monary alveolar macrophages, but these bacilli
are never inactivated (6, 7).
In Lurie’s high-intensity experiment, the re-
sistant rabbits converted their tuberculin skin
tests several months earlier than did the sus-
ceptible rabbits—an average of 2.0 versus 4.7
months (Table 2 and Fig. 2) (3).Therefore, with
virulent bovine-type tubercle bacilli, a primary
lesion was apparently established sooner in the
resistant group.
In the resistant rabbits, could the alveolar
macrophages (which are highly activated [8])
trap inhaled bacilli more effectively than the
alveolar macrophages of the susceptible rabbits?
In one experiment, alveolar macrophages that
were washed out of the lungs of resistant rabbits
phagocytized twice as many tubercle bacilli in
vitro than did those washed out of the lungs of
susceptible rabbits (9). If fewer tubercle bacilli
were phagocytized in vivo in the susceptible
rabbits, then more bacilli would ascend the
bronchial escalator. (The bronchial tree is rather
resistant to tuberculosis.)
Could the bronchial trees of the two rabbit
groups have been of such a configuration that
more bacilli reached the alveoli in the resistant
group? We do not know.The susceptible group
had more overt snuffles (infection with Pas-
teurella sp. or Bordetella bronchisepticum) than did
the resistant group (7). Perhaps, more bacilli in
the susceptible group adhered to the bronchial
walls, because of the increased mucus there, and
did not reach the alveolar spaces.
Did the resistant rabbits breathe more air
than the susceptible ones? The resistant rabbits
were usually larger, but the susceptible rabbits
were usually more active.
In brief, many possibilities exist for why, in
these natural airborne experiments, the resistant
rabbits (on average) converted their tuberculin
reactions sooner than did the susceptible rabbits,
but none of these possibilities has been investi-
gated further.
RESISTANCE TO THE PROGRESS
OF PULMONARY TUBERCULOSIS
PRODUCED BY VIRULENT BOVINE-
TYPE BACILLI IN LURIE’S RABBITS
In the highest-dose natural airborne experiment,
the inbred resistant rabbits lived an average of 6.4
months after they developed tuberculin sensi-
tivity (3) (Table 2, Fig. 2).They died of a slowly
progressing cavitary disease with bronchial spread
(Fig. 3), similar to that found in adult humans.The
susceptible rabbits, in contrast, only lived an aver-
age of 3.5 months (3) (Table 2, Fig. 2) and devel-
oped a hematogenously spread disease (Fig.4) (see
chapters 13 and 14), similar to that found in
infants and immunosuppressed individuals (see
TABLE 2 Effect of the intensity of natural airborne contagion on tuberculosis acquired by rabbits of various levels of natural resistancea
C E
Avg. No. (%) of rabbits
D G
B duration F
A No. (%) of that developed Duration of H
No. of of Preallergic (or
Intensity of rabbits that positive tuberculin fatal Type of
Rabbit rabbits exposure pretuberculous)
contagion developed reactions without tuberculosis tuberculosis
strain exposed before period (months)
tuberculosis demonstrable (months)
death tuberculosis
(months)
A (resistant) Low 7 14.6 0 4 (57%) 4.4 – None
Medium 6 10.4 3 (50%) 2 (33%) 3.7 11.4 Slowly progressive,
localizing
High 8 9.3 5 (63%) 2 (25%) 2.0 6.4 3 intermediate;

12.
2 slowly

NATURAL AIRBORNE INFECTION

progressive,
localizing
C, F
(susceptible) Low 11 9.8 2 (18%) 3 (27%) 5.5 3.0 Rapidly
progressive,
disseminating
Medium 11 8.6 10 (91%) 0 4.3 3.8 Rapidly
progressive,
disseminating
High 7 8.1 6 (86%) 1 (14%) 4.7 3.5 Rapidly
progressive,
disseminating
a
Note that a higher percentage of resistant A rabbits than susceptible C and F rabbits developed a positive tuberculin reaction without evidence of grossly visible tuberculosis at necropsy
(column E). Note also that (as expected) a higher intensity of contagion caused a higher percentage of rabbits to develop tuberculosis (column D). Adapted from reference 3.

䡵
219
220 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 Resistance to attack (the preallergic period) and resistance to the progress (dura-
tion) of tuberculosis in the resistant A and susceptible C and F strains of rabbits.The graph on
the left shows the average time (in months) in which these rabbits converted their tuberculin reac-
tions.The graph on the right shows the average time (in months) that these rabbits lived after
they became tuberculin positive.
The difference in the preallergic period between the resistant A strain and the susceptible
F strain was highly significant, but that between the A strain and the susceptible C strain was not
(3). However, the duration of the disease in the resistant A strain was always significantly longer
than that in both susceptible strains (C and F) (3).
Experiments were performed with relatively low, medium, and high numbers of tubercle bacilli
in the air of the room.This figure represents data from the experiment with the highest num-
ber of tubercle bacilli. Reproduced with permission from reference 3.

chapters 1 and 3).Therefore, the resistant rabbits
lived about twice as long as susceptible rabbits
after infection with virulent bovine-type tuber-
cle bacilli (2, 3), which confirmed that the resis-
tant group in this experiment was truly resistant
and that the susceptible group was truly suscep-
tible. These findings were supported by other
natural airborne experiments (Table 2).
The tubercles in resistant hosts contained
fewer bacilli than those in susceptible hosts (see
chapters 2, 13, and 14), because of the more
rapid development of macrophages into mature
epithelioid cells (1, 10, 11).A mature epithelioid
cell is one that is highly activated, i.e., rich in
oxidative and hydrolytic enzymes (e.g., -galac-
tosidase) and therefore capable of inhibiting the
intracellular growth of tubercle bacilli and
digesting their components (12–14).
In the resistant rabbits, tubercle bacilli spread
to the hilar lymph nodes more readily than they
did in susceptible rabbits,but once in the lymph
nodes,the bacilli are inhibited much more effec-
tively in the resistant rabbits (2,7,10).After infec-
tion with virulent bovine-type tubercle bacilli,
the hilar lymph nodes of the resistant rabbits
remained small and showed little caseation, and
the growth of bacilli was well controlled.In con-
trast, the hilar nodes of the susceptible rabbits
were large and showed considerable caseation,
and bacillary growth was poorly controlled (2).
With the bovine-type infection, the bacilli
invaded the bloodstream in both resistant and
susceptible rabbits, especially after caseous necro-
sis had developed. The resistant rabbits usually
developed relatively few metastatic lesions in
their lungs, spleen, and kidneys, and such lesions
 12.
had relatively few bacilli and relatively small
amounts of caseation (2, 7). In contrast, the sus-
ceptible rabbits developed numerous metastatic
lesions, and such lesions had many bacilli and
large amounts of caseation (2, 7).The disease in
the susceptible rabbits resembled the childhood-
type tuberculosis found in humans (15–17) (see
chapter 1).
In summary, attributes of both resistance and
susceptibility contributed to the dissemination
NATURAL AIRBORNE INFECTION 䡵 221
FIGURE 3 Lungs and other
organs of resistant rabbit A2-6. It
converted its tuberculin reaction at
9 months and died of tuberculosis 9
months later.The primary lesion was
the single (well-encapsulated) cavity
in the right lung.There was tuber-
culosis of the larynx and intestines,
as well as a tuberculous ulcer of the
colon—all from the spread of large
numbers of virulent bovine-type
tubercle bacilli up the bronchial tree
and then into the alimentary canal.
The hilar and mesenteric lymph
nodes were grossly normal, as were
the kidneys. Several nonprogressive
tubercles of hematogenous origin
were present in both lungs. Re-
produced with permission from ref-
erence 2.
of bacilli from the primary pulmonary lesion. In
the resistant rabbits during the first weeks, the
greater interstitial inflammation increased the
spread of bacilli to the hilar lymph nodes, but
the bacilli were inhibited once there. In the
susceptible rabbits, the greater caseation
increased the lymphogenous and hematoge-
nous spread of the bacilli, and the bacilli grew
rather well in the metastatic sites.The decisive
factor in the development of secondary lesions
222 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 Lungs and other organs of susceptible rabbit F4-33. It converted its
tuberculin reaction at 6.2 months and died of tuberculosis 3.0 months later
(Table 1).The primary lesion is the large, completely caseous lesion in the middle
of the left lung.The caseum in this lesion did not liquefy, so no cavity formed.The
homolateral draining hilar lymph nodes show extensive enlargement and massive
caseation. Numerous large progressive caseous tubercles of hematogenous origin are
present in both lungs.The kidneys, pleura, and knee joint show rapidly progressive
caseous lesions. Reproduced with permission from reference 2.
 12.
was the fate of the bacilli in their new locations:
In metastatic sites, the resistant rabbits could
inhibit bacillary growth much more effectively
than could the susceptible rabbits.
CONVERSION OF THE TUBERCULIN
SKIN TEST WITH NO GROSSLY
VISIBLE PRIMARY PULMONARY
LESION AT NECROPSY
In Lurie’s natural airborne infection of rabbits
with virulent bovine-type bacilli, some resis-
tant and susceptible animals became tuber-
culin positive but showed no grossly visible
disease at necropsy (Table 2) (2, 3).This occur-
rence was more common in the natively resis-
tant rabbits and more common in the experi-
ment with the lowest concentrations of
tubercle bacilli in the room air (Table 2) (3). In
the tuberculin-positive rabbits without pul-
monary tuberculous lesions, no grossly visible
tuberculous lesions were found in the hilar
lymph nodes. Such lesions probably had devel-
oped (at least microscopically) and then healed
(see chapter 13).
In Riley’s natural airborne experiments (see
below), guinea pigs inhaled (over many months)
air containing virulent human-type tubercle
bacilli from sputum-positive tuberculous patients
in a hospital ward. In Riley’s Experiment I, 34%
(26 of 77) of the animals became tuberculin
positive without any grossly visible pulmonary
tuberculous lesions (18, 19). In Riley’s Experi-
ment II, 16% (10 of 63) did the same. However,
in contrast to rabbits, most of these guinea pigs
had grossly visible tuberculous lesions in their
hilar lymph nodes, and over half of these guinea
pigs also had lesions in their spleens.
One inhaled unit of 1 to 3 fully virulent
bovine-type (Ravenel) tubercle bacilli in the
alveolar spaces causes progressive disease in rab-
bits (1), and one such unit of fully virulent
human-type or bovine-type bacilli causes pro-
gressive disease in guinea pigs. If this is true, then
how does a rabbit or guinea pig become tuber-
culin positive from inhaled tubercle bacilli with-
out developing grossly visible pulmonary dis-
ease? There are two likely possibilities. (i) The
inhaled bacillary unit may have been weak and
not fully virulent, so that the primary pul-
NATURAL AIRBORNE INFECTION 䡵 223
monary lesion remained small and healed by the
time of necropsy; and (ii) (as discussed above) the
inhaled bacilli may not have multiplied in the
alveoli, but were carried in the lymph (proba-
bly within macrophages) to the hilar lymph
nodes, where in Lurie’s resistant rabbits bacillary
multiplication was inhibited (2, 3), and in Riley’s
guinea pigs bacillary multiplication usually
caused visible tuberculous lesions (18, 19).
ONLY A SINGLE PRIMARY
TUBERCULOUS LESION DEVELOPS
UNDER NATURAL CONDITIONS
In Lurie’s experiments, the rabbits inhaled rel-
atively few tubercle bacilli over many months,
so that the initial primary pulmonary lesion
had time to produce sufficient immunity to
prevent subsequently inhaled tubercle bacilli
from producing grossly visible lesions (1, 2).
Such immunity cannot, however, prevent the
development of microscopic lesions from the
subsequently inhaled bacilli, because immu-
nization does not increase the microbicidal
power of pulmonary alveolar macrophages (see
chapters 11 and 22).
In Riley’s experiments (see below), the guinea
pigs also inhaled relatively few tubercle bacilli
over many months, and also developed only
one primary pulmonary tubercle (18, 19). How-
ever, these guinea pigs were removed from the
infectious air and necropsied soon after they
converted their tuberculin skin test, so there
was little opportunity for exogenous reinfection
to occur. In contrast, Lurie’s rabbits were allowed
to remain in the infectious air for many months,
and usually for the course of the disease.
RESISTANCE TO THE
ESTABLISHMENT OF TUBERCULOUS
LESIONS PRODUCED BY VIRULENT
HUMAN-TYPE BACILLI IN LURIE’S
RABBITS
Human-type tubercle bacilli are never fully vir-
ulent in rabbits (or humans) (1, 3, 5). Rabbits
inactivate many inhaled units of virulent human-
type bacilli for every unit that produces a grossly
visible pulmonary lesion (7, 10, 20). In con-
trast, every inhaled unit of fully virulent bovine-
type tubercle bacilli reaching the pulmonary
alveoli of rabbits produces a progressive disease.
224 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
From lung culture data (10), we know that a
high proportion of inhaled human-type bacilli
are destroyed by rabbit alveolar macrophages
during the first 7 days. From studies in BCG-
immunized rabbits (21, 22), we know that
acquired immunity is also able to decrease the
number of grossly visible pulmonary tubercles.
However, we do not know what proportion of
the grossly visible tubercles are prevented initially
by alveolar macrophages and what proportion
are prevented subsequently by stopping the pro-
gression of microscopic tubercles.
A much greater number of human-type
bacilli are destroyed or inhibited by the alveo-
lar macrophages of Lurie’s resistant rabbits than
by the alveolar macrophages of his susceptible
rabbits (7, 10, 23, 24). Therefore, although the
alveolar macrophages of the resistant group may
trap these bacilli more readily, fewer micro-
scopic lesions would form, because these alve-
olar macrophages are highly activated. In other
words, following the inhalation of human-type
bacilli, the resistant rabbits would prevent the
establishment of a microscopic lesion more
effectively than would the susceptible rabbits—
which is not true for inhalation of virulent
bovine-type bacilli (described above).
RESISTANCE TO THE PROGRESS
OF PULMONARY TUBERCULOSIS
PRODUCED BY VIRULENT HUMAN-
TYPE BACILLI IN LURIE’S RABBITS
The progress of tuberculosis produced by viru-
lent human-type tubercle bacilli is described in
chapter 14 and in references 10 and 20. In brief,
primary pulmonary lesions in Lurie’s resistant
rabbits usually healed within a few months, but
if they liquefied and cavitated, such healing would
probably take over a year.We would expect such
primary lesions to have a similar course in com-
mercial New Zealand White rabbits.
Primary pulmonary lesions in Lurie’s sus-
ceptible rabbits did not heal as quickly, and usu-
ally secondary pulmonary lesions occurred.The
bacilli causing these secondary lesions probably
came from the hilar nodes via efferent lym-
phatics draining into the great veins (see chap-
ter 1). Nevertheless, the secondary lesions caused
by human-type bacilli in Lurie’s susceptible rab-
bits did not progress, but showed fibrosis, which
indicated that they would eventually heal.
In brief, resistance to the establishment of
tuberculosis and resistance to its progress would
not be separable phenomena in rabbits or
humans exposed to virulent human-type tuber-
cle bacilli. In both of these hosts, most inhaled
human-type tubercle bacilli seem to be imme-
diately destroyed by alveolar macrophages (pre-
venting establishment), but once established,
many of these lesions do not progress.
ESTABLISHMENT AND PROGRESS
OF TUBERCULOSIS IN MICE AND
GUINEA PIGS
Mice and guinea pigs are so susceptible to viru-
lent human-type and bovine-type tubercle bacilli
that their alveolar macrophages kill few, if any, of
these bacilli. Also, tuberculosis once established
usually progresses in mice and in guinea pigs and
kills these hosts. It is possible that some mice or
guinea pigs have alveolar macrophages that can
trap occasional airborne human-type tubercle
bacilli (when inhaled over many months) more
effectively than do other mice or guinea pigs. If
so, then these laboratory animals would show
the same separation of establishment and progress
of tuberculosis that was found with Lurie’s rab-
bits inhaling occasional bovine-type bacilli.
Of course, in all hosts, immunization slows the
progress of the disease and has no effect on its
establishment: alveolar macrophage functions
have no immunologic specificity (see chapters
5 and 22).
RILEY’S EXPERIMENTS WITH
NATURAL AIRBORNE INFECTION
Riley performed two major experiments (each
lasting 2 years) with outbred guinea pigs (18, 19,
25–27).The source of infection was air from six
single-bed hospital rooms, each housing a patient
producing sputum that contained virulent
human-type tubercle bacilli. The guinea pigs
were housed on the floor above that of the
patients in a vertical cylindrical chamber aero-
dynamically engineered by Wells (28). This
chamber had six levels that held cages contain-
 12.
ing 4 to 5 guinea pigs per cage, so that an
average of about 150 guinea pigs were con-
tinuously exposed at one time. The animals
were skin-tested with tuberculin every month,
at which time each tuberculin-positive guinea
pig was removed from the chamber, necrop-
sied, and replaced by an uninfected guinea
pig. This procedure eliminated the possibility
of the guinea pigs’ infecting each other,
because early lesions would not shed any bacilli
into the environment. Individual patients were
identified by the antimicrobial susceptibility
of the tubercle bacilli in their sputum (18,
19). Also, various sputum samples were aero-
solized, and their infectivity for guinea pigs was
correlated with the infectivity of the air from
the ward (18).
These experiments clearly established that
tuberculous patients can transmit the disease by
the airborne route and that patients with numer-
ous bacilli in their sputum are more infectious
than those with fewer bacilli. Only one pri-
mary pulmonary lesion was found in each
guinea pig. However, as mentioned above, the
guinea pigs were removed from the source of
contagion as soon as they converted their tuber-
culin reaction, so few if any would have had the
opportunity to develop another lesion from
inhaling a second infectious particle.
Guided by Edward A. Nardell (Harvard
School of Public Health), who was Riley’s col-
league, and by Paul Jensen (Centers for Disease
Control and Prevention), Bernard Fourie and
Karen Weyer of the South African Medical
Research Council (Witbank, South Africa) have
built an experimental tuberculosis ward similar
to the one built by Riley about 50 years ago.The
patients in their study have multidrug-resistant
tuberculosis, and about 50% of them are HIV
infected. The long-term goals of this project
are to (i) investigate the principal determinants
of transmission of tuberculosis and (ii) test infec-
tion control interventions suitable for use in
countries with a high burden of the disease,
such as upper room germicidal irradiation.The
short-term goals are to (i) compare the infec-
tiousness of HIV-positive and HIV-negative
patients, (ii) study the transmissibility of
mycobacterial strain types (including those with
NATURAL AIRBORNE INFECTION 䡵 225
mutations), and (iii) test the efficacy of novel
interventions (such as aerosolized drugs) on
transmissibility.
Each month the tuberculin-positive guinea
pigs will be removed from the source of expo-
sure. Some of these animals will be euthanized
immediately, but many will be allowed to
develop extensive disease over the next 6
months. In every animal, the virulence of the
infecting tubercle bacilli will be assessed at
necropsy for both the extent of the disease and
the type of pathology present.
These natural airborne experiments will pro-
vide information on the infectivity of tubercu-
lous patients that is not obtainable in any other
manner.The bacilli aerosolized by tuberculous
patients are surrounded by bronchial secretions
and usually by components of the liquefied
caseum. Bacilli grown in culture medium (and
subsequently aerosolized) are not surrounded by
these components and spend less time in the air
before they are inhaled by animals in the expo-
sure chamber.Therefore, the infectivity of bacilli
in natural airborne experiments is probably dif-
ferent from the infectivity of bacilli in aerosol
experiments.
Also, the infectivity of human-type tubercle
bacilli for guinea pigs is probably greater than
their infectivity for humans. Guinea pigs must
inhale 3 to 6 units of 1 to 3 bacilli to convert
their tuberculin skin test, whereas humans must
inhale an estimate of 20 to 200 bacillary units
to do so. (Rabbits must inhale 600 to 1,200
units.) Therefore, small changes in bacillary
infectivity for humans might not be detected by
guinea pigs for reasons presented in chapter 11.
However, if rabbits were substituted for guinea
pigs in these experiments, almost none of the
animals would contract the disease or convert
their tuberculin skin tests, because too large an
inhaled dose of human-type tubercle bacilli
would be required to do so.
A similar experiment with different logistics
is being performed in Lima, Peru, by Roderick
Escombe with Carlton Evans of the Johns Hop-
kins Bloomberg School of Public Health in
Baltimore, Md. To date, they have found that
opening doors and windows is the most efficient
method of reducing the concentration of the
226 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
infectious airborne particles in the rooms of
these tuberculous patients.
OTHER METHODS OF
ROOM AIR SAMPLING
Another experiment that detected tubercle
bacilli in hospital room air was published in 1999
(29).Air near the patient’s bed was forced through
a micropore membrane filter, and PCR (poly-
merase chain reaction) was used to detect the
DNA of tubercle bacilli collected on the filter.
This experiment does not, however, distinguish
between live and dead tubercle bacilli or between
large and small particle sizes (30).When inhaled,
bacillary particles of large size do not reach the
alveolar spaces to cause pulmonary disease.
Nevertheless, this method can aid the diagnosis
of tuberculosis by sometimes detecting tubercle
bacilli when they are not detected in sputum.
COMPARISONS OF TUBERCULOSIS
IN RABBITS AND HUMANS
Human tuberculosis usually resembles the disease
produced in rabbits following the inhalation of
human-type bacilli in that resistance to its estab-
lishment and resistance to its progress are not sep-
arable. Persons evidently must inhale numerous
virulent tubercle bacilli before they develop a
microscopic tubercle that converts their tuber-
culin skin test (see chapter 3), because they do
not convert their tuberculin reaction for many
months, even though they live in households
with infectious tuberculous patients. This fact
indicates that most human alveolar macrophages
are able to destroy many inhaled human-type
tubercle bacilli and thereby prevent the estab-
lishment of a primary lesion. Rabbit alveolar
macrophages destroy human-type tubercle bacilli
even more effectively.
Unlike rabbits, humans evidently cannot pre-
vent an already established microscopic pul-
monary lesion from reaching 0.5 to 1.0 mm in
size (31–33): humans are highly sensitive to the
tuberculin-like products of the bacillus, so that
early lesions rapidly develop a small caseous
center (see chapter 3). Nevertheless, in 90% of
humans these 0.5- to 1.0-mm lesions usually
become arrested and remain inactive for the
person’s lifetime. An estimated 5 to 10% of
humans with positive tuberculin skin tests
develop progressive tuberculosis (see chapter 1
and see reference 34 for details).
Humans are more susceptible to human-type
tubercle bacilli than are rabbits. Rabbits usually
recover from active lesions caused by human-
type bacilli, whereas many humans with clinically
active tuberculosis will die of the disease, unless
effective antimicrobial agents are given. On the
other hand, humans are somewhat more resistant
than rabbits to fully virulent bovine-type bacilli,
because tuberculous lesions produced by such
bacilli are fatal in rabbits, but not always fatal in
humans. Except for these slight differences, how-
ever, the disease in rabbits and the disease in
humans resemble each other quite well.
EFFECT OF IMMUNIZATION ON
RESISTANCE TO THE
ESTABLISHMENT AND RESISTANCE
TO THE PROGRESS OF AN ACUTE
SEPTICEMIC DISEASE (MELIOIDOSIS)
AND A CHRONIC, SLOWLY
PROGRESSING DISEASE
(TUBERCULOSIS)
Melioidosis is caused by Burkholderia pseudomal-
lei, a gram-negative bacillus that was formerly
called Malleomyces pseudomallei and Pseudomonas
pseudomallei. When inhaled by mice, virulent
B. pseudomallei produce pulmonary lesions that
usually progress to an acute septicemic form of
the disease followed by death (35–37). These
bacilli grow both intracellularly in phagocytes
and extracellularly in both viable and damaged
host tissues (35, 38).Therefore, once a lesion was
established, the host was usually unable to stop
its progression, because the endotoxins and exo-
toxins of the bacillus kill host defense cells.
Nevertheless, an average of 17 to 75 inhaled
virulent B. pseudomallei bacilli were required to
produce one grossly visible lesion (39; also see
reference 37).This finding suggests that before
the bacilli could establish a microscopic pul-
monary lesion, most of the bacilli were destroyed
by the highly activated alveolar macrophage
population normally existing in the lungs.
In mouse melioidosis, after an aerosol infec-
tion with B. pseudomallei, immunized mice had
fewer primary pulmonary lesions than did the
nonimmunized controls: their resistance was
 12.
increased 4- to 17-fold (37). In other words,
immunization could prevent the establishment
of many microscopic lesions.Apparently, immu-
nization was beneficial, because it produced
antibodies that opsonized B. pseudomallei, thereby
facilitating their ingestion by the alveolar
macrophages. Since most alveolar macrophages
are activated, they evidently killed inhaled B.
pseudomallei before these bacilli could multiply
to cause an uncontrollable progressive lesion.
In rabbit tuberculosis, immunization has no
effect on the establishment of primary micro-
scopic lesions that convert the tuberculin skin
test, because the pulmonary alveolar macrophage
population has no immunologic “memory.”
Memory resides in an expanded antigen-specific
lymphocyte population (see chapters 5 and 22).
Antibodies have little or no effect on the growth
of tubercle bacilli within any macrophage.
Tubercle bacilli are readily phagocytized by
alveolar macrophages without opsonization by
antibodies (40), and the passive transfer of anti-
bodies does not increase the resistance of non-
immunized hosts (41). Therefore, immuniza-
tion against tuberculosis has no effect on the
establishment of a microscopic pulmonary lesion
produced by tubercle bacilli, but can prevent the
progress of such lesions into clinically active
tuberculosis.
In brief, resistance to the establishment of
pulmonary melioidosis in mice is determined by
the alveolar macrophages and is increased by
immunization because of opsonizing antibodies.
Resistance to the progress of melioidosis is only
slightly reduced by immunization, and the dis-
ease continues rapidly to death.
Resistance to the establishment of pulmonary
tuberculosis caused by virulent human-type bacilli
in rabbits is also determined by the alveolar
macrophages, but is not increased by immu-
nization, because alveolar macrophages readily
phagocytize tubercle bacilli without opsoniza-
tion by antibodies. However, resistance to the
progress of tuberculosis is greatly reduced by the
delayed-type hypersensitivity and cell-mediated
immunity that develop during the infection,
and is also reduced by prior vaccination. In fact,
most tuberculous lesions (caused by human-
type tubercle bacilli) are usually arrested in rab-
NATURAL AIRBORNE INFECTION 䡵 227
bits while they are still microscopic, and even
grossly apparent lesions eventually heal.
Although antibodies have no effect on the
establishment of pulmonary tuberculous lesions,
they apparently reduce the progress of such
lesions by accelerating the local accumulation of
macrophages and antigen-specific lymphocytes
(see chapter 5).
A comparison of the characteristics of acute
and chronic bacterial infections is presented in
chapter 24.
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and W. F. Wells. 1959. Aerial dissemination of
pulmonary tuberculosis.A two-year study of con-
tagion in a tuberculosis ward. Am. J. Epidemiol.
142:3–14.
19. Riley, R. L., C. C. Mills, F. O’Grady, L. U. Sul-
tan, F. Wittstadt, and D. N. Shivpuri. 1962.
Infectiousness of air from a tuberculosis ward. Ultra-
violet irradiation of infected air: comparative infec-
tiousness of different patients. Am. Rev. Respir. Dis.
85:511–525.
20. Lurie, M. B., S. Abramson, and A. G. Hep-
pleston. 1952. On the response of genetically re-
sistant and susceptible rabbits to the quantitative
inhalation of human-type tubercle bacilli and the
nature of resistance to tuberculosis. J. Exp. Med.
95:119–134.
21. Lurie, M. B., P. Zappasodi, E. Cardona-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
to the intracutaneous inoculation of BCG as an
index of native resistance to tuberculosis. J. Immunol.
68:369–387.
22. Dannenberg, A. M., Jr., W. R. Bishai, N. Par-
rish, R. Ruiz, W. Johnson, B. C. Zook, J. W.
Boles, and L. M. Pitt. 2001. Efficacies of BCG
and vole bacillus (Mycobacterium microti) vaccines in
preventing clinically apparent pulmonary tubercu-
losis in rabbits: a preliminary report. Vaccine 19:796–
800.
23. Dannenberg, A. M., Jr. 1991. Delayed-type
hypersensitivity and cell-mediated immunity in the
pathogenesis of tuberculosis. Immunol.Today 12:228–
233.
24. Dannenberg, A. M., Jr. 1993. Immunopatho-
genesis of pulmonary tuberculosis. Hosp. Pract.
28:51–58.
25. Riley, R. L., W. F. Wells, C. C. Mills, W. Nyka,
and R. L. McLean. 1957. Air hygiene in tuber-
culosis: quantitative studies of infectivity and con-
trol in a pilot ward.A cooperative study between the
Veterans Administration,The Johns Hopkins Uni-
versity School of Hygiene and Public Health, and
the Maryland Tuberculosis Association. Am. Rev.
Respir. Dis. 75:420–431.
26. Riley, R. L. 2001. How it really happened.What
nobody needs to know about airborne infection.
Am. J. Respir. Crit. Care Med. 163:7–8.
27. Permutt, S. 2002. Richard Lord Riley, 1911–
2001.An appreciation. Am. J. Respir. Crit. Care Med.
166:257.
28. Wells, W. F. 1955. Airborne Contagion and Air
Hygiene. Harvard University Press, Cambridge,
Mass.
29. Mastorides, S. M., R. L. Oehler, J. N. Greene,
J.T. Sinnott IV, M. Kranik, and R. L. Sandin.
1999.The detection of airborne Mycobacterium tuber-
culosis using micropore membrane air sampling and
polymerase chain reaction. Chest 115:19–25.
30. Nardell, E. A. 1999. Air sampling for tuberculo-
sis—homage to the lowly guinea pig. Chest
116:1143–1145.
31. Sutherland, I., and I. Lindgren. 1979.The pro-
tective effect of BCG vaccination as indicated by
autopsy studies. Tubercle 60:225–231.
32. Lindgren, I. 1961. Anatomical and roentgeno-
logic studies of tuberculosis infection in BCG-
vaccinated and non-vaccinated subjects, with bio-
physical investigations of calcified foci. Acta Radiol.
Suppl. 209:1–101.
33. Lindgren, I. 1965.The pathology of tuberculous
infection in BCG—vaccinated humans. Adv.Tuberc.
Res. 14:202–234.
34. Comstock, G. W. 1982. Epidemiology of tuber-
culosis. Am. Rev. Respir. Dis. 125(Suppl.):8–16.
35. Dannenberg,A. M., Jr., and E. M. Scott. 1958.
Melioidosis: pathogenesis and immunity in mice and
hamsters. I. Studies with virulent strains of
Malleomyces pseudomallei. J. Exp. Med. 107:153–166.
36. Dannenberg,A. M., Jr., and E. M. Scott. 1958.
Melioidosis: pathogenesis and immunity in mice
and hamsters. II. Studies with avirulent strains of
Malleomyces pseudomallei. Am. J. Pathol. 34:1099–
1121.
 12.
37. Dannenberg,A. M., Jr., and E. M. Scott. 1960.
Melioidosis: pathogenesis and immunity in mice and
hamsters. III.The effect of vaccination with aviru-
lent strains of Pseudomonas pseudomallei on the re-
sistance to the establishment and the resistance to
the progress of respiratory melioidosis caused by
virulent strains: all-or-none aspects of this disease.
J. Immunol. 84:233–246.
38. Jones, A. L.,T. J. Beveridge, and D. E.Woods.
1996. Intracellular survival of Burkholderia pseudo-
mallei. Infect. Immun. 64:782–790.
NATURAL AIRBORNE INFECTION 䡵 229
39. Dannenberg,A. M., Jr., and E. M. Scott. 1956.
Determination of respiratory LD50 from a number
of primary lesions as illustrated by melioidosis. Proc.
Soc. Exp. Biol. Med. 92:571–575.
40. Schlesinger, L. S. 1996. Role of mononuclear
phagocytes in M. tuberculosis pathogenesis. J. Inves-
tig. Med. 44:312–323.
41. Reggiardo, Z., and G. Middlebrook. 1974.
Failure of passive serum transfer of immunity against
aerogenic tuberculosis in rabbits. Proc. Soc. Exp. Biol.
Med. 145:173–175.
 13
RESPONSE OF RABBITS TO
INHALED TUBERCLE BACILLI

Tuberculosis in rabbits caused by inhaled virulent bovine-type tubercle

bacilli [231]
Tuberculosis in rabbits caused by inhaled virulent human-type tubercle
bacilli [233]
Tubercles in rabbits caused by inhaled BCG [233]

Abstract.Virulent bovine-type and human-type tubercle bacilli and BCG are of decreas-
ing virulence for rabbits, in that order.The host uses the same type of immune response
to control each of these infections, but the response is more effective with bacillary strains
of reduced virulence.
With fully virulent bovine-type tubercle bacilli, only 3 bacillary units of 1 to 3 bacilli
must be inhaled to cause one grossly visible tubercle at 5 weeks. In Lurie’s inbred susceptible
rabbits, bovine-type bacilli produced the childhood form of tuberculosis with hemato-
genous dissemination. In Lurie’s resistant rabbits, bovine-type bacilli produced the adult
form of tuberculosis with pulmonary cavities and bronchial dissemination.
Human-type tubercle bacilli are not fully virulent in rabbits. In Lurie’s inbred resistant
rabbits and in commercially available New Zealand White rabbits, 300 to 1,900 bacillary
units must be inhaled to produce one grossly visible primary pulmonary tubercle at 5 weeks,
depending on the virulence of the infecting strain and on variations among the individ-
ual rabbits. In Lurie’s susceptible rabbits, such tubercles often gave rise to secondary
tubercles. In Lurie’s resistant rabbits and commercial New Zealand White rabbits, human-
type tubercle bacilli usually healed in a few months, except when they formed cavities,
which persisted much longer. However, in all rabbits, lesions produced by human-type bacilli
eventually heal and are never fatal.
BCG is avirulent in all common laboratory species. In commercial rabbits, a large inhaled
dose of aerosolized BCG produces few, if any, tiny nonprogressive tubercles. Most of the
inhaled BCG bacilli are apparently destroyed by the alveolar macrophages before they can
multiply appreciably.Without such multiplication, the degree of immunization would be
negligible. Similar to rabbits, humans should be less immunized by the inhalation of BCG
than by parenteral administration, in which higher doses of BCG can be injected and greater
bacillary multiplication can occur (because the alveolar macrophages are bypassed). In mice
and guinea pigs (which are more susceptible to Mycobacterium tuberculosis), inhaled BCG
would immunize more effectively, because their alveolar macrophages do not destroy inhaled
BCG as readily (see chapter 22).

The purpose of this chapter is to assemble in one
place the characteristics of the disease produced
in rabbits by the inhalation of virulent bovine
and human types of tubercle bacilli and by the
inhalation of BCG. Other chapters in this book
contain the pertinent graphs and photographs.
Tuberculosis in humans usually resembles
that produced by virulent human-type tubercle
bacilli in rabbits in that the primary lesion heals
in most cases. However, human-type bacilli are
somewhat more virulent for humans than they
are for rabbits, because in 5 to 10% of human
cases, the primary lesion progresses or reacti-
vates. When it does, the disease resembles that
produced in rabbits by virulent bovine-type
bacilli. Cavity formation with bronchial spread
of the disease followed by death is produced
in rabbits only by virulent bovine-type bacilli.

230
 13. RESPONSE TO INHALED TUBERCLE BACILLI INCLUDING BCG
(Cavities occasionally form in rabbits with
human-type bacilli, but they are nonprogressive.)
In brief, nonprogressive human tuberculosis
has many of the characteristics of rabbit tuber-
culosis caused by virulent human-type tubercle
bacilli, and clinically progressive human tuber-
culosis has many of the characteristics of rabbit
tuberculosis caused by virulent bovine-type
bacilli.*
TUBERCULOSIS IN RABBITS CAUSED
BY INHALED VIRULENT BOVINE-
TYPE TUBERCLE BACILLI
Overview
Fully virulent bovine-type bacilli (1, 2) usually
produced one grossly visible pulmonary tuber-
cle for each inhaled unit of 1 to 3 bacilli that
reached the alveolar spaces in both susceptible
and resistant inbred Lurie rabbits (3–5). This
conclusion was derived from quantitative air-
borne infection of rabbits, in which the number
of bacilli inhaled was divided by the number of
grossly visible primary pulmonary tubercles gen-
erated (3–5).
This conclusion, however, needs some qual-
ification. During natural airborne infection
(when a single unit of 1 to 3 virulent bovine-
type tubercle bacilli was inhaled over a period
of many weeks), some resistant rabbits became
tuberculin positive and had no visible pulmonary
lesions at necropsy (6, 7). In these rabbits, the sin-
gle inhaled bacillary unit was probably carried
by the lymphatics from the lungs to the hilar
lymph nodes, where bacillary growth could be
more easily controlled (see chapter 12).
Virulent bovine-type bacilli multiplied to
higher titers in susceptible rabbits than they did
in resistant rabbits (Fig. 1) (4).The logarithmic
*Note that Lurie’s rabbit strains are now extinct (see
chapter 14).Therefore, some immunosuppression may be
needed to reproduce the childhood form of tuberculo-
sis in commercial adult rabbits. To our knowledge, no
study of the susceptibility of infant rabbits to aerosolized
virulent tubercle bacilli has been done. Adult guinea
pigs are natively very susceptible to tuberculosis and
would be the animal of choice for reproducing the
childhood form of tuberculosis.
䡵 231
bacillary growth stopped when tissue-damaging
delayed-type hypersensitivity (DTH) to the
tuberculin-like products of the bacillus devel-
oped (8, 9) (see chapters 2 and 5). DTH killed
the nonactivated macrophages in which the
bacillus was proliferating and, in doing so,
destroyed the intracellular environment that was
so favorable to bacillary growth. In the solid
caseous tissue that resulted from DTH killing,
tubercle bacilli did not multiply.
Tuberculosis in Lurie’s Susceptible
Rabbits
In Lurie’s susceptible rabbits, virulent bovine-
type tubercle bacilli grew in the poorly activated
macrophages that surrounded the caseum.Again,
DTH killed these parasitized host cells and
extended the area of caseous necrosis. This
sequence continued. The bacilli entered lym-
phatics and spread to the hilar lymph nodes,
entered blood vessels, and spread throughout
the body. In the lungs, secondary tubercles devel-
oped and were enlarged by the same process
until the host died.
Large caseous hilar lymph nodes developed in
these susceptible rabbits, and tubercles formed
in the kidneys, spleen, bone marrow, and other
sites (see chapters 1, 2, 12, and 14). Systemic
DTH, an endotoxin-like syndrome, probably
contributed to the cause of death. Cavity for-
mation was rare.
Tuberculosis in Lurie’s Resistant
Rabbits
In Lurie’s resistant rabbits, virulent bovine-type
tubercle bacilli did not multiply to as high a titer
as they did in Lurie’s susceptible rabbits (Fig. 1)
(4). The logarithmic bacillary growth was
stopped by the same immune mechanism as
that employed by susceptible rabbits: tissue-
damaging DTH to the tuberculin-like prod-
ucts of the bacillus (8, 9) (see chapters 2 and 5).
The bacillary titer at the end of the logarithmic
stage was lower than that in susceptible rabbits,
because bacillary growth had initially been
inhibited to a greater degree by the alveolar
macrophages of the resistant host (Fig. 1) (8, 9)
(see chapter 2), and these resistant rabbits more
232 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 Number of virulent human-type and bovine-type tubercle bacilli in the lungs of
inbred resistant and susceptible rabbits on various days after airborne infection. Note that bovine-
type bacilli multiplied to higher titers than did human-type bacilli, and that both types multi-
plied to higher titers in susceptible strain C rabbits than they did in resistant strain III rabbits. Repro-
duced with permission from reference 4.

quickly developed effective cell-mediated
immunity (CMI).
The good CMI of the resistant rabbits acti-
vated the macrophages surrounding the solid
caseous centers of the tubercles (8–10) (see
chapter 2). Therefore, when tubercle bacilli
escaped from the edge of the caseum, they were
often engulfed by activated macrophages that
inhibited their intracellular growth (in contrast
to the situation with susceptible rabbits).These
tubercles contained mature epithelioid cells,
which are activated macrophages capable of
inhibiting tubercle bacilli. In the resistant rabbits,
caseation progressed slowly and was less exten-
sive than that in the susceptible rabbits.
Histologically, the lesions usually contained a
caseous center, surrounded by tuberculous gran-
ulation tissue composed of macrophages, lym-
phocytes, plasma cells, fibroblasts, capillaries, and
lymphatics (1, 6, 11; see also references 12 and
13). In time, the lesions became encapsulated by
fibrous tissue (1; see also references 12 and 13).
Because of the good CMI, the hilar lymph
nodes showed minimal disease. Secondary lym-
phogenous and hematogenous lesions in the
lungs and other organs occurred but were usu-
ally nonprogressive.
Frequently, however, the caseous centers in the
resistant host underwent liquefaction, a process
in which fluid was absorbed and the caseous
material softened. In the liquefied menstruum,
the bacilli sometimes multiplied extracellularly to
tremendous numbers (1, 6). The host’s DTH
reaction to the tuberculin-like products of such
bacilli was able to cause much tissue destruction
(see chapters 2 and 12). When this occurred,
the walls of the bronchial tree were frequently
eroded; the bacilli entered the air passages and
spread to other parts of the lungs, where they
sometimes produced areas of caseous pneumo-
nia, as well as new lesions that underwent liq-
uefaction (see chapter 4).The large number of
virulent bovine-type bacilli in the airways over-
whelmed the resistant rabbit’s ability to control
 13. RESPONSE TO INHALED TUBERCLE BACILLI INCLUDING BCG
the disease, so it progressed until the host suc-
cumbed. But for liquefaction and cavity forma-
tion, Lurie’s inbred resistant rabbits would prob-
ably have arrested the infection.
Tuberculosis in Commercial New
Zealand White Rabbits
In commercial New Zealand White rabbits,
aerosolized virulent bovine-type tubercle bacilli
produce a disease that resembles that found in
Lurie’s resistant rabbits (12, 13) (see chapter 4).
TUBERCULOSIS IN RABBITS CAUSED
BY INHALED VIRULENT HUMAN-
TYPE TUBERCLE BACILLI
See references 5, 11, 14, and 15. Human-type
tubercle bacilli are never fully virulent in rabbits.
After an aerosol infection with strain H37Rv (a
standard laboratory strain of virulent human-
type tubercle bacillus), the lesions produced
even in Lurie’s genetically susceptible rabbits
eventually regressed (1).
The use of such aerosolized human-type bacilli
enabled Lurie to develop his tubercle-count
method, which is the most quantitative method
for assaying innate and acquired resistance to
tuberculosis in rabbits as well as the virulence of
the infecting tubercle bacillus (see chapter 11). In
brief, rabbits were exposed to a known quantity
of aerosolized bacilli, and the number of pri-
mary tubercles in the lungs was counted 5 weeks
later.Then, the number of inhaled human-type
tubercle bacilli required to produce one visible
primary pulmonary tubercle (called “the ratio”)
was calculated. In Lurie’s inbred rabbits, 50 to
1,500 inhaled virulent human-type bacilli
(H37Rv) had to be inhaled to produce such a
tubercle,depending on the innate resistance of the
host.Therefore, a wide range of ratios was pro-
vided by using human-type bacilli in rabbits.
With fully virulent bovine-type bacilli, a ratio of
about 3 always occurred, regardless of the host’s
innate resistance. Therefore, the tubercle count
method with bovine-type bacilli is not recom-
mended for assaying the immunity produced by
new vaccines (see chapter 11).
Following the inhalation of virulent human-
type bacilli (H37Rv), Lurie’s resistant rabbits
developed fewer tubercles than did his suscep-
䡵 233
tible rabbits (5).The tubercles in the resistant ani-
mals were usually smaller and contained fewer
bacilli, and their caseous centers were more
mature; i.e., the nuclear debris present was more
completely disintegrated. The cells present in
these tubercles were located more interstitially,
i.e., within the alveolar walls (5). In time, some
of the caseous centers liquefied and even formed
cavities (5), but healing was relatively rapid (16),
calcification sometimes occurred (14), and sec-
ondary lesions were rare (16). (See Table 2 in
chapter 14.)
When compared with tubercles in the resis-
tant rabbits, tubercles in the susceptible rabbits
were usually larger and contained more bacilli.
Their caseous centers were less mature; i.e., the
nuclear debris present was incompletely disin-
tegrated.The cells present in the tubercles of the
susceptible rabbits were located more intra-
alveolarly; i.e., many were within the air spaces
(5). None of the caseous centers liquefied, and
cavities never formed (5). Healing was slow,
and secondary lesions of lymphogenous and
hematogenous origin were common (1, 16).
(See Table 2 in chapter 14.)
Commercial New Zealand White rabbits
responded to the inhalation of virulent human-
type tubercle bacilli in a manner similar to that
of Lurie’s inbred resistant rabbits (15).The Erd-
man strain was more virulent than H37Rv (15),
but not nearly as virulent as the virulent bovine-
type Ravenel strain. The Thorbecke strain of
inbred rabbits (Covance Inc., Denver, Pa.) was
more susceptible than standard New Zealand
White rabbits (17),but not as susceptible as Lurie’s
inbred susceptible rabbits (see chapter 14).
TUBERCLES IN RABBITS
CAUSED BY INHALED BCG
BCG is much less virulent for rabbits than
even human-type tubercle bacilli (1, 2). The
inhalation of several million BCG bacilli was
required to produce a single primary pul-
monary tubercle, which was quite small and
should have healed rapidly (S. Abramson,
unpublished data; quoted on page 223 of ref-
erence 1). In mice and guinea pigs, the inhala-
tion of BCG produces tubercles more readily
(see chapters 15 and 22).
234 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
The lungs were not cultured for tubercle
bacilli. Therefore, we do not know whether
BCG would multiply (in nonactivated
macrophages) during the logarithmic phase at
the same rate as did the virulent bovine and
human types shown in Fig. 1. However, we
would predict from Fig. 1 that the logarithmic
phase of BCG growth would be shorter than the
logarithmic phase of the two more virulent
strains, and that BCG would not reach as high
a titer.The characteristics of the disease produced
in rabbits by intravenous BCG are presented in
references 1 and 18.
REFERENCES
1. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive Mech-
anisms. Harvard University Press, Cambridge, Mass.
2. Lurie, M. B., and A. M. Dannenberg, Jr. 1965.
Macrophage function in infectious disease with
inbred rabbits. Bacteriol. Rev. 29:466–476.
3. Lurie, M. B., A. G. Heppleston, S. Abramson,
and I. B. Swartz. 1950. An evaluation of the
method of quantitative airborne infection and its use
in the study of the pathogenesis of tuberculosis. Am.
Rev.Tuberc. 61:765–797.
4. Allison, M. J., P. Zappasodi, and M. B. Lurie.
1962. Host-parasite relationships in natively resistant
and susceptible rabbits on quantitative inhalation
of tubercle bacilli. Am. Rev. Respir. Dis. 85:553–569.
5. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
culosis in the light of the host-parasite relationships
in natively resistant and susceptible rabbits. Am.
Rev.Tuberc. 72:297–323.
6. Lurie, M. B. 1941. Heredity, constitution and
tuberculosis: an experimental study. Am. Rev.Tuberc.
44(Suppl. to no. 3):1–125.
7. Lurie, M. B. 1944. Experimental epidemiology of
tuberculosis. Hereditary resistance to attack by
tuberculosis and to the ensuing disease and the
effect of the concentration of tubercle bacilli upon
these two phases of resistance. J. Exp. Med. 79:573–
589.
8. Dannenberg,A. M., Jr. 1991. Delayed-type hyper-
sensitivity and cell-mediated immunity in the patho-
genesis of tuberculosis. Immunol.Today 12:228–233.
9. Dannenberg, A. M., Jr. 1993. Immunopatho-
genesis of pulmonary tuberculosis. Hosp. Pract.
28:33–40 (Off. ed. 51–58).
10. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
genesis of tuberculosis: specificity, systemic and local
nature, and associated macrophage enzymes. Bacte-
riol. Rev. 32:85–102.
11. Lurie, M. B. 1932. The correlation between the
histological changes and the fate of living tubercle
bacilli in the organs of tuberculous rabbits. J. Exp.
Med. 55:31–54.
12. Converse, P. J., A. M. Dannenberg, Jr., J. E.
Estep, K. Sugisaki,Y.Abe, B. H. Schofield, and
M. L. M. Pitt. 1996. Cavitary tuberculosis pro-
duced in rabbits by aerosolized virulent tubercle
bacilli. Infect. Immun. 64:4776–4787.
13. Converse, P. J., A. M. Dannenberg, Jr.,
T. Shigenaga, D. N. McMurray, S. W. Phalen,
J. L. Stanford, G. A. W. Rook, T. Koru-
Sengul, H. Abbey, J. E. Estep, and M. L. M.
Pitt. 1998. Pulmonary bovine-type tuberculosis
in rabbits: bacillary virulence, inhaled dose effects,
tuberculin sensitivity, and Mycobacterium vaccae
immunotherapy. Clin. Diagn. Lab. Immunol. 5:871–
881.
14. Heppleston, A. G. 1949. Quantitative air-borne
tuberculosis in the rabbit.The course of human-type
infection. J. Exp. Med. 89:597–610.
15. Manabe, Y. C., A. M. Dannenberg, Jr., S. K.
Tyagi, C. L. Hatem, M.Yoder, S. C.Woolwine,
B. C. Zook, M. L. M. Pitt, and W. R. Bishai.
2003. Different strains of Mycobacterium tuberculosis
cause various spectrums of disease in the rabbit
model of tuberculosis. Infect. Immun. 71:6004–6011.
16. Lurie, M. B., S. Abramson, and A. G. Hep-
pleston. 1952. On the response of genetically re-
sistant and susceptible rabbits to the quantitative
inhalation of human-type tubercle bacilli and the
nature of resistance to tuberculosis. J. Exp. Med.
95:119–134.
17. Dorman, S., C. L. Hatem, S. Tyagi, K. Aird,
J. Lopez-Molina, M. L. M. Pitt, B. C. Zook,
A. M. Dannenberg, Jr.,W. R. Bishai, and Y. C.
Manabe. 2004. Susceptibility to tuberculosis: clues
from studies with inbred and outbred New Zealand
White rabbits. Infect. Immun. 72:1700–1705.
18. Lurie, M. B. 1934.The fate of BCG and associ-
ated changes in organs of rabbits. J. Exp. Med.
60:163–178.
 14
CHARACTERISTICS OF RESISTANCE AND
SUSCEPTIBILITY TO TUBERCULOSIS
IN LURIE’S INBRED RABBITS

History and description of Lurie’s inbred rabbits [235]

Resistance and susceptibility to tuberculosis in Lurie’s inbred
rabbits [237]
Genetic experiments with Lurie’s inbred rabbits [238]
Rate of healing of dermal BCG lesions as a method of determining the
native resistance of inbred rabbit strains [241]
Inbred rabbits today [241]
Use of rabbits for studies other than tuberculosis [244]

Abstract. Lurie’s rabbits were inbred for either susceptibility or resistance to the progress
of tuberculosis.When infected with virulent bovine-type tubercle bacilli, the susceptible
rabbits developed a rapidly progressing, hematogenously spreading “childhood type” of
tuberculosis, and the resistant rabbits developed a slowly progressing, cavitary, bronchial-
spreading “adult type” of tuberculosis.
In lesions produced by virulent bovine-type bacilli, by human-type bacilli, and by BCG,
the same manifestations of genetic resistance to tuberculosis were evident histologically:
mature epithelioid cells (now known as highly activated macrophages) were always more
numerous in the lesions of resistant rabbits than in the lesions of susceptible rabbits, irre-
spective of the differences in virulence of the infecting bacillary strain.
The genetic resistance of these rabbits resides in their ability to activate macrophages
to control the growth of tubercle bacilli, both nonspecifically and immune-specifically.
Crossbreeding showed that the genetic resistance to tuberculosis is multifactorial, with genes
associated with resistance being dominant over susceptibility genes.
The commercially available outbred New Zealand White rabbits seem almost as resistant
as Lurie’s inbred resistant strain III rabbits.Thorbecke inbred rabbits were distinctly more sus-
ceptible than commercial outbred rabbits, but apparently not as susceptible as Lurie’s inbred
C and FC rabbits. van Zutphen’s inbred rabbits (which are hypo- and hyperresponsive to
dietary cholesterol,respectively) have not been adequately studied for resistance to tuberculosis.

HISTORY AND DESCRIPTION OF the specifics of the inbreeding, are presented in

LURIE’S INBRED RABBITS
The rabbit is the only available laboratory ani-
mal species in which tuberculous lesions readily
liquefy, form cavities, and spread bacilli through
the bronchial tree (1). In fact, depending on the
genetic resistance of the host and the virulence
of the infecting bacilli, tuberculosis in rabbits
can mimic all forms of the human disease.
For this reason, in the 1930s, Lurie began
inbreeding different rabbit strains for either re-
sistance or susceptibility to tuberculosis, and he
continued this inbreeding throughout the rest of
his professional career (1, 2). How he began, and
his book (1) and his monograph (2).Table 1 lists
the origins and the degree of resistance to tuber-
culosis of each of Lurie’s rabbit strains.
Between 1948 and 1962, the most important
strains in Lurie’s rabbit colony were the resistant
strain III and the susceptible strains C and FC.
Strain III needed to inhale about 1,070 virulent
human-type bacilli (H37Rv) to generate one
grossly visible primary pulmonary tubercle 5
weeks after infection; strain C needed about
70; strain FC needed about 80; and strain AD (of
intermediate resistance) needed about 360
(Table 1) (3). The inbreeding of these rabbit

235
236 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Relative resistance of Lurie’s inbred rabbit strains, determined by their survival after a standard
intracutaneous inoculation of virulent bovine-type tubercle bacilli (Ravenel), by their response to the quantita-
tive inhalation of human-type tubercle bacilli (H37Rv), or by both
Average survival No. of inhaled human References for
after infection bacilli required to resistance to
Derivation with bovine form one tubercle tuberculosis
Strain bacilli (the “ratio”)
and reference(s)
No. of No. of Bovine Human
Days rabbits Ratios rabbits bacilli bacilli
Ca Carworth farms (26) — — 49 ⫾ 19 9 — (26)
Ca Swift stock (2) 121 5 70 ⫾ 14 30 (2) (3)
CaC Ca ⫻ C (3, 27) — — 97 ⫾ 12 7 — (1)
F Swift stock (2) 141 4 — (2) —
FCa F ⫻ C (26) 132 8 79 ⫾ 9 45 (26) (3)
FCCab FC ⫻ CaC (28) — — 44 ⫾ 3 6 — —
Ac Stubbs (2) 539 6 — (2) —
ADc,d A ⫻ D (29) 197 10 362 ⫾ 103 12 (3) (3)
III (T)a Sawin (26) 422 8 1,065 ⫾ 138 71 (26) (3)
TAc T ⫻ A (30) — — 154 ⫾ 27 9 — (30)
TTC TC ⫻ T (14) — — 315 ⫾ 67 12 — (30)
NZWe Covance, Inc. 500–1,500 26 (31)
Thorbeckef,g Covance, Inc. 100–500 6 (24)
a
The majority of Lurie’s experiments were done with these strains. Strain III was formerly called strain T. The mean ratios
(by Lurie’s method; see chapter 11) and their standard errors are listed. Data from reference 4.
b
See references 9 and 4 for other information on this strain.
c
The resistance of strains A and AD has declined since the ninth and seventh generations, respectively (1).
d
The D strain of Swift stock was of intermediate resistance (2).
e
NZW are commercially available New Zealand White rabbits.
f
Thorbecke rabbits were inbred by the late G. Jeanette Thorbecke, Professor of Pathology, New York University, School of Med-
icine.They were available from Covance, Inc., Denver, PA.
g
Estimated ratios.

strains was not complete, when evaluated by
rejection of skin grafts, blood types, and other
criteria (see reference 4).
The susceptible strain C and resistant strain III
(formerly called T) were albino rabbits.The sus-
ceptible strains Ca and FC and the strain AD of
intermediate resistance were Dutch-belted rab-
bits. Strain A was English in its coat markings;
it was initially resistant, but had become inter-
mediate over the years.
Whether any correlation exists between coat
markings and native resistance to tuberculosis
remains to be determined but seems worthy of
investigation, because more than 50 registered
rabbits strains are available today.These strains are
described in a publication by the American
Rabbit Breeders Association (5).They were bred
for size and pelt characteristics. Upon speaking
with a member of this association, I was left with
the impression that these strains were far from
inbred, and that some were even crossbred with
other strains to maintain the desired size and coat
characteristics. No one, to date, has measured the
overall resistance to tuberculosis of rabbit strains
certified by the American Rabbit Breeders Asso-
ciation, or even determined whether their
response to the inhalation of virulent human-
type tubercle bacilli is more uniform than that
found with commercially available New Zealand
White rabbits. Increased uniformity in resis-
tance to tuberculosis would simplify the evalu-
ation of new tuberculosis vaccines in the rabbit
species, because fewer animals would be required
to obtain significant results.
 14. CHARACTERISTICS OF RESISTANCE AND SUSCEPTIBILITY
RESISTANCE AND SUSCEPTIBILITY
TO TUBERCULOSIS IN LURIE’S
INBRED RABBITS
Characteristics of resistance and susceptibility
to tuberculosis in Lurie’s inbred rabbits are listed
in Table 2, and the details are presented in chap-
ters 2, 11, 12, and 13.The basic tissue responses
of Lurie’s resistant and susceptible rabbits to
tubercle bacilli were independent of the viru-
lence of the infecting bacilli, because tuberculous
lesions produced by highly virulent bovine-type
bacilli, semivirulent human-type bacilli, and
BCG vaccines showed similar characteristics.
In brief, the main inbred characteristic of
Lurie’s resistant rabbits was their ability to activate
macrophages to such a high degree that these
macrophages could inhibit and/or destroy tuber-
cle bacilli. His resistant rabbits nonspecifically
activated their pulmonary alveolar macrophages
to a greater degree than did his susceptible rab-
bits (1, 6–8).Within tuberculous lesions, his resis-
tant rabbits also immunologically activated their
blood-borne macrophages to a greater degree
than did his susceptible rabbits (1, 6–8). (Blood-
borne macrophages enter tuberculous lesions
and become activated by cytokines, mainly from
antigen-specific lymphocytes.)
In Table 2, note (as detailed in the following
paragraphs) how many characteristics of the re-
sistant rabbits were due to the greater activation
developed by their macrophages.
The increased trapping of inhaled bacilli in
the lungs by resistant rabbits (see chapter 12) is
possibly due to their alveolar macrophages’ being
more actively phagocytic for inhaled bacilli (9).
The increased inhibition of bacillary multi-
plication seems to be directly related to the
degree of activation of alveolar macrophages, and
the subsequent inhibition in tuberculous lesions
seems to be directly related to the degree of acti-
vation of blood-borne macrophages. However,
tubercle bacilli grow at equal rates in nonacti-
vated macrophages of both resistant and sus-
ceptible rabbits (see chapters 2 and 15). See
below for drainage of bacilli to lymph nodes.
Mononuclear cells accumulate (and activate)
more rapidly in the early pulmonary lesions of
resistant rabbits. Activated macrophages (and
other cells) produce chemokines that attract
䡵 237
dendritic cells, macrophages, lymphocytes, and
other leukocytes (see chapters 19 and 20).
The primary pulmonary lesions of the resis-
tant rabbits show more interstitial inflammation
(3), which is apparently the cause of the increased
drainage of tubercle bacilli to their hilar lymph
nodes.The susceptible rabbits show less intersti-
tial inflammation and more pneumonic inflam-
mation, i.e., cells in the alveolar spaces.
Mature epithelioid cells are highly activated
macrophages (1, 6–8).They are more numerous
in the lesions of the resistant host. Lurie recog-
nized mature epithelioid cells in tissue sections
by their rounded appearance and homogeneous
cytoplasm (1, 10). Our -galactosidase histo-
chemical procedure to identify mature epithe-
lioid cells (11, 12) was not available when Lurie
was alive.
Maturation of the caseous process seems to
be due to greater hydrolysis of the necrotic
material—probably due to increased levels of
hydrolases in the macrophages of the resistant
hosts.
Increased numbers of lymphocytes and plasma
cells are in the lesions of the resistant rabbits (13).
The plasma cells there and in the lymph nodes are
the source of the greater antibody titers in the re-
sistant rabbits (13).These rabbits may have more
efficient antigen presentation by dendritic cells
and macrophages (see chapters 5 and 6).
Fewer and smaller primary tubercles, fewer
secondary tubercles, and more rapid healing are
due to the decrease in the number of bacilli
caused by the highly activated macrophages of
the resistant group.
Liquefaction of solid caseous tissues and cav-
ity formation in the resistant rabbits seem related
to high levels of hydrolytic enzymes in dead and
live activated macrophages surrounding the
lesion’s caseous center.
The faster rate of healing of the lesions, the
stronger acquired immunity, and the increased
longevity (after infection with virulent bovine-
type bacilli) in the resistant host are mostly due
to many highly activated macrophages capable
of destroying or inhibiting tubercle bacilli.
The increased antibody titers in the resistant
rabbits hasten the local acquired cell-mediated
immune response (see chapter 5).
238 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 2 Characteristics of resistance and susceptibility to tuberculosis in Lurie’s inbred rabbitsa

Resistance Susceptibility
Characteristic Rabbit strain c
Rabbit strain
Degreeb and reference(s) Degreeb and reference(s)
Bacilli
Trapping of tubercle bacilli in lung ⫹⫹⫹⫹ A (33); III (3, 9, 32) ⫹⫹ F(7); C(3, 9, 32, 33); FCCa (9)
Initial inhibition and/or destruction ⫹⫹⫹ III (3, 32) ⫹ C (3, 32)
of inhaled bacilli
Subsequent inhibition of bacillary ⫹⫹⫹⫹ III (3, 32) ⫹⫹ C (3, 32)
multiplication
Drainage of bacilli to ⫹⫹⫹ III (3, 32) ⫹ C (3, 32)
tracheobronchial lymph nodes
Histopathology
Rate of accumulation of ⫹⫹⫹ III (3) ⫹ C (3)
mononuclear cells in early
pulmonary lesions
Interstitial inflammation ⫹⫹⫹ III (3) ⫹ C (3)
Pneumonic inflammation ⫹ III (3) ⫹⫹⫹ C (3)
Rate of epithelioid
cell maturation ⫹⫹⫹⫹ A (2); III (3, 13, 26) ⫹ C (2, 3, 13, 26); F (2)
Maturation of caseous process ⫹⫹⫹⫹ III (3) ⫹⫹ C (3)
Number of lymphocytes and ⫹⫹⫹ III (3, 13) ⫹ C (3, 13)
plasma cells
Gross pathology
Number of gross tubercles 5 weeks ⫹ III (3, 13, 14, 26) ⫹⫹⫹⫹ C (3, 14); Ca (26); FC (3, 13, 26)
after inhalation of human bacilli
Size of tubercles and their caseous ⫹⫹ III (3) ⫹⫹⫹⫹ C (3); FC (3)
centers
Spread of disease to kidneys and ⫹ A (2); III (1, 32) ⫹⫹⫹ F (2); C (2)
other organs
Liquefaction and cavity formation ⫹⫹ A (2); III (3) ⫾ F (2); C (2,3)
Rate of healing of lesionsd ⫹⫹⫹⫹ A (2); III (26) ⫹ F (2); C (2); FC (26)
Absorption of tubercles and ⫹⫹⫹⫹ III (3) ⫹ C (3)
fibroplasiasd
Other factors
Amount of acquired immunity ⫹⫹⫹⫹ A (2); III (13) ⫹ F (2); C (2); FC (13)
Longevity after infection with ⫹⫹⫹⫹ A (2, 33); III (26) ⫹ F (2, 33); C (2, 33); FC (1, 32)
virulent bovine-type bacilli
Antibody titer ⫹⫹⫹⫹ III (13) ⫹⫹ FC (13)
a
Adapted from reference 4, where other characteristics of Lurie’s inbred rabbits are described.
b
⫹, low degree; ⫹⫹ and ⫹⫹⫹, intermediate degrees; ⫹⫹⫹⫹, high degree.
c
Strain III was formerly called strain T.
d
Virulent human-type tubercle bacilli are never fully virulent for rabbits, because pulmonary lesions caused by these bacilli
eventually heal. However, pulmonary lesions caused by fully virulent bovine-type tubercle bacilli never heal.

GENETIC EXPERIMENTS WITH Backcrossing the F1 generation (III ⫻ C) to

LURIE’S INBRED RABBITS resistant strain III produced rabbits (IIIC ⫻ III)
Crossbreeding resistant strain III with suscepti- that were just as resistant as the original strain III
ble strain C produced an F1 generation (III ⫻ rabbits (14) (Fig. 1 and 2). However, backcross-
C) of intermediate resistance (14) (Fig. 1 and 2). ing the F1 generation (III ⫻ C) to susceptible
 14. CHARACTERISTICS OF RESISTANCE AND SUSCEPTIBILITY
strain C did not regain appreciable susceptibil-
ity: this backcross (IIIC ⫻ C) had intermediate
resistance to tuberculosis (14) (Fig. 1 and 2).
Lurie also crossed two susceptible strains (C
with Ca) to produce a first-generation hybrid.
䡵 239
FIGURE 1 Lungs of (top) an F1 hybrid (IIIC ⫽
III ⫻ C), (middle) its backcross to III (IIIC ⫻ III),
and (bottom) its backcross to C (IIIC ⫻ C) 5
weeks after the inhalation of 75,000, 79,000, and
64,000 human-type tubercle bacilli (H37Rv).The
lungs of IIIC 1-31 (top), IIIC III 1-16 (middle),
and IIIC C 1-4 (bottom) contained 497, 142, and
772 tubercles, respectively; the ratios for these rab-
bits were 131, 557, and 82, respectively. See Fig. 2
for the means for each of these combinations.
Reproduced with permission from reference 14.
This hybrid was more resistant than either par-
ent: C12⫽3 (with a ratio of 41) crossed with
Ca5⫽41 (with a ratio of 37) produced CaC
hybrids with a mean ratio of 97 ⫾ 12 (P ⫽
0.001) (see reference 1, p. 240–241).
240 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 Ratios of individual rabbits from resistant strain III, susceptible strain C, and
hybrids III ⫻ C, IIIC ⫻ C, and IIIC ⫻ III.The ratio is the number of inhaled human-type bacilli
(H37Rv) required to generate one grossly visible primary tubercle; i.e., the ratio is the number
of bacilli inhaled by each rabbit divided by the number of primary tubercles found in its lungs
5 weeks later.The mean ratios (by the Lurie method; see chapter 11) with their standard errors
(and log10) are listed on the right.
The original resistant and susceptible rabbit strains were III and C, respectively.The F1 hybrids
were III ⫻ C, and the backcrosses were IIIC ⫻ C and IIIC ⫻ III, respectively.The resistance of
the F1 generation backcrossed to resistant III rabbits was equal to that of the original III group
(ratios of 620 ⫾ 110 and 640 ⫾ 170, respectively). However, the resistance of the F1 generation
backcrossed to susceptible C rabbits was far greater than that of the original C group (ratios of
243 ⫾ 51 and 48 ⫾ 10, respectively). In other words, many of the factors controlling good re-
sistance appeared to be dominant.
This figure also shows the ratio as a log10 (and its arithmetic mean) for the rabbits in this exper-
iment; each subsection represents one rabbit. For example, the IIIC ⫻ C contained 21 rabbits
with ratios of 101.6 to 103.4, and the IIIC ⫻ III group contained 26 rabbits with ratios of 101.9
to 103.7. Note that the resistance of the F1 generation seemed to be less variable than that of the
other groups; i.e., it ranged over five rather than six 0.3 log10 increments (but this result may have
been a chance phenomenon).The shaded boxes represent the rabbits shown in Fig. 1.
Adapted from reference 14.
Note the variation in tubercle counts among the individual rabbits.
 14. CHARACTERISTICS OF RESISTANCE AND SUSCEPTIBILITY
From these crossbreeding experiments, Lurie
concluded that resistance to tuberculosis is mul-
tifactorial or polygenic, with the resistance genes
dominating the susceptibility genes (1, 14).
Alternatively, susceptibility may be an absence of
certain resistance factors (1, 14).Therefore, re-
sistance to tuberculosis is “multiple, complex
and additive in nature” (14).
A similar dominance of resistance over sus-
ceptibility may exist in humans and would partly
explain why immunocompetent humans have
apparently become more resistant to tuberculosis
over the centuries.
RATE OF HEALING OF DERMAL
BCG LESIONS AS A METHOD OF
DETERMINING THE NATIVE RESIS-
TANCE OF INBRED RABBIT STRAINS
About 1950, Lurie used the time of healing of
dermal BCG lesions, instead of the tubercle-
count method, to select for breeding the most
resistant and the most susceptible rabbits and
thereby maintain these characteristics in the
offspring (1, 13).When BCG was used for such
selection in Lurie’s rabbit colony, there was no
apparent spread of the BCG infection to other
rabbits in the colony, because every unvaccinated
rabbit tested remained tuberculin negative. In
other words, even though the BCG lesions
would ulcerate and spread occasional BCG
bacilli into the air of the room, the other rab-
bits in the breeding colony did not inhale a
sufficient quantity of these bacilli to establish a
primary lesion in which the BCG multiplied
(see chapter 13). (Lurie’s rabbits were housed
individually in cages with grates that allowed
urine and feces to pass through.)
Because the time of healing of BCG lesions
cannot be precisely determined, it provides only
a rough measure of host resistance. Healing is too
gradual a process and is in part dependent on
whether or not the lesion ulcerates and dis-
charges much of its liquefied contents contain-
ing bacilli.Therefore, the tubercle-count method
(see chapter 11) is a more precise way of mea-
suring the genetic resistance of different strains
of rabbits.Tubercle counting, however, requires
euthanizing the animal, because not all grossly
visible pulmonary tubercles can be identified
䡵 241
by standard radiographs, or even by computer-
analyzed tomography.
INBRED RABBITS TODAY
Fate of Lurie’s Inbred Rabbits
Unfortunately, Lurie’s rabbit strains became
infertile with time. Strain III rabbits readily
became pregnant, but the fetuses failed to com-
plete development and usually were absorbed.
The last viable strain III (white) rabbits were
produced in commercial black rabbits by trans-
planting early-stage strain III embryos (unpub-
lished data).Two- to 8-cell embryos were col-
lected from the fallopian tubes of a female strain
III rabbit within a few days after mating with a
male strain III rabbit. The embryos were then
placed in the fallopian tubes of a chorionic
gonadotropin-treated female black rabbit. The
black surrogate mother gave birth to white
strain III rabbits. Since the black color is dom-
inant, the origin of the offspring could not be
questioned.
van Zutphen Rabbits
L. F. M. van Zutphen, at the Faculty of Veteri-
nary Medicine of the University of Utrecht in
The Netherlands, has inbred a relative
(IIIVO/JU) of Lurie’s resistant strain III rabbits
(8, 15, 16). It is hyporesponsive to dietary choles-
terol (16). van Zutphen also maintains another
inbred rabbit strain (AX/JU) that is hyperre-
sponsive to dietary cholesterol (16). Neither of
these inbred rabbit strains has been evaluated for
resistance to tuberculosis by Lurie’s tubercle-
count method with aerosolized virulent human-
type tubercle bacilli (17) (see chapter 11). Strain
IIIVO/JU resembled Lurie’s inbred strain III
in its resistance to tuberculosis produced by vir-
ulent bovine-type tubercle bacilli (18, 19), but
the susceptibility of strain AX/JU to tuberculosis
with bovine-type bacilli was never tested.
Bar Harbor Rabbits
The Jackson Laboratories (Bar Harbor, Me.) had
several inbred rabbit strains of unknown resistance
to tuberculosis (20–22) but has stopped inbreed-
ing rabbits. Richard R. Fox of these laboratories
242 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

preserved a few of these inbred strains in a frozen
state as early embryos in the 4- to 8-cell stage (8).
These embryos can be made to continue their
development by placing them into the fallopian
tubes of living female rabbits primed with chori-
onic gonadotropin (see above).This frozen stock
contains embryos of the original resistant strain
III rabbits from which both Lurie and van
Zutphen developed their inbred colonies.
Commercial Rabbits
With the tubercle-count method (23, 24) (see
chapter 11), we evaluated the resistance of out-
bred commercial New Zealand White rabbits
(from Covance Research Products, Inc., Denver,
Pa.) (Tables 1 and 3).They seem to be highly re-
sistant to tuberculosis, perhaps just as resistant as
Lurie’s strain III rabbits (18, 19). However, the
H37Rv strain of human-type tubercle bacilli
used by Lurie and by us was never evaluated for
virulence in the same experiment.Therefore, we
do not know whether Lurie’s strain III rabbits
were more resistant than the commercial out-
bred rabbits available today.We do know, how-
ever, that, when exposed to bovine-type tuber-
cle bacilli, commercial outbred rabbits readily
produce the resistant form of tuberculosis with
cavity formation (18, 19).
Thorbecke Rabbits
Another New Zealand White inbred rabbit
strain was developed by the late G. Jeanette
Thorbecke at New York University (unpub-
lished) and maintained by Covance Research
Products, Inc. These Thorbecke rabbits had
stunted facies and groomed themselves poorly,
and they tended to be more excitable (similar to
Lurie’s susceptible FC and CaC rabbit strains).
The group headed by Yukari C. Manabe
tested the resistance of these Thorbecke inbred
rabbits by the tubercle-count method with
human-type tubercle bacilli (24).When com-
pared with commercial outbred New Zealand
White rabbits, the Thorbecke inbred rabbits
were found to be more susceptible to tubercu-
losis (24); i.e., they had more numerous primary
pulmonary tubercles 5 weeks after aerosol infec-
tion with human-type tubercle bacilli (H37Rv)
(Table 3) (24). These tubercles were larger,
showed more caseous necrosis and surrounding
pneumonitis, and contained more bacilli (Tables
3 and 4).Their hilar lymph nodes were larger

TABLE 3 Tuberculosis in inbred Thorbecke rabbits and outbred commercial rabbits 5 weeks after aerosol
infection with Mycobacterium tuberculosis H37Rv (luciferase)a
Inbred Outbred P values
No. of rabbits 12 6
No. of tubercles 98 ⫾ 12 33 ⫾ 13 0.007
Average diameter of tubercles (mm) 2.73 ⫾ 0.13 1.43 ⫾ 0.09 0.001
No. of bacilli inhaled divided by the number of 128b 323b
tubercles produced (the ratio)
Dermal tuberculin reactions (mm3) 289 ⫾ 35 1,152 ⫾ 348 0.009
No. of viable bacilli cultured from hilar lymph nodes 697 ⫾ 192 26 ⫾ 20 0.004
No. of viable bacilli cultured from each representative 809 ⫾ 210 215 ⫾ 115 0.027
tubercle
No. of viable bacilli cultured from entire right upper 11,475 ⫾ 9,448 1,154 ⫾ 986 0.003
lobe of lungs
a
Two-day dermal reactions to Old Tuberculin were measured at 5 weeks after the aerosol infection. Data from reference 24.
b
Calculated from the means.
H37Rv (luciferase) is a genetically engineered strain of H37Rv. It seemed to have the same virulence (for rabbits) as the par-
ent H37Rv strain by the tubercle count method (24), but it and the parent strain were not compared in the same aerosol exper-
iment.The means and their standard errors are listed.
 14. CHARACTERISTICS OF RESISTANCE AND SUSCEPTIBILITY 䡵 243

TABLE 4 Histologic characteristics of tubercles from inbred Thorbecke rabbits and outbred commercial rab-
bits 5 weeks after aerosol infection with M. tuberculosis (Erdman)a
Inbred rabbits Outbred rabbits P values
Size of tubercles, mm 2b
2.8 ⫾ 0.2 1.8 ⫾ 0.2 0.020
Percentage of tubercles showing caseous necrosis 89 ⫾ 3 60 ⫾ 11 0.045
Area of necrosis in tubercles, mm2 2.6 ⫾ 0.5 0.6 ⫾ 0.2 0.004
No. of neutrophils in tubercles (1⫹ to 4⫹) 2.2 ⫾ 0.3 1.7 ⫾ 0.4 NS
Percentage of mature epithelioid macrophages 58 ⫾ 5 72 ⫾ 3 0.066
No. of lymphocytes and plasma cells in tubercles (1⫹ to 4⫹) 1.3 ⫾ 0.2 3.7 ⫾ 0.2 0.004
Peripheral fibrosis in tubercles (1⫹ to 4⫹) 2.8 ⫾ 0.5 1.8 ⫾ 0.4 NS
Perigranuloma pneumonitis (1⫹ to 4⫹) 3.2 ⫾ 0.3 1.2 ⫾ 0.2 0.005
No. of acid-fast bacilli/mm2 of caseumc 1.04 ⫾ 0.40 0.43 ⫾ 0.15 0.010
a
The means and their standard errors are listed.The P values were determined by Mann-Whitney U nonparametric comparison.
NS, not significant.Adapted from reference 24.
b
The average size of the tubercles listed here is only from those in the tissue sections.This size differs from the average size in
Table 5, which was determined at necropsy from all lesions in the lungs.
c
The number of acid-fast bacilli per mm2 of caseous lung tissue was counted in 3 representative tubercles from each rabbit.

and also contained more bacilli (Table 3), but
their dermal reactions to tuberculin were
decreased (Table 3).The tubercle counts among
these inbred rabbits were less variable than
those found in the outbred rabbits (see the
standard errors in Tables 3 and 5). With the
Erdman strain of human-type bacilli, similar
differences between the inbred and outbred
rabbits were found. However, with this more
virulent strain, more rabbits would be required
to obtain tubercle counts that were statistically
significant (Table 5) (24).
The increased number of tubercles and the
decreased size of the dermal tuberculin reactions
suggest that these inbred rabbits developed less
acquired immunity than did the outbred rab-
bits.The innate immunity of the inbred rabbits
also may be reduced, because in vitro peritoneal
macrophages from uninfected inbred rabbits pro-
duced less tumor necrosis factor alpha than did
uninfected outbred rabbits when both groups
were stimulated by endotoxin or tubercle bacilli
(24). Unfortunately, a fire destroyed all of the
Thorbecke rabbits that Covance Laboratories

TABLE 5 Tuberculosis in inbred Thorbecke rabbits and outbred commercial rabbits 5 weeks after aerosol
infection with M. tuberculosis (Erdman)a
Rabbit group Inbred Outbred P values
No. of rabbits 6 6
No. of tubercles 351 ⫾ 41 305 ⫾ 107 0.170
Average diameter (mm) 3.80 ⫾ 0.30 2.13 ⫾ 0.22 0.004
No. of bacilli inhaled divided by the number of tubercles
produced (the “ratio”) 296b 684b
Dermal tuberculin reactions (mm3) 471 ⫾ 68 1,342 ⫾ 488 0.055
a
Two-day dermal reactions to Old Tuberculin were measured at 5 weeks.The means and their standard errors are listed.Adapted
from reference 24.
b
Calculated from the means.
244 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
was breeding, so they are no longer commercially
available.
USE OF RABBITS FOR STUDIES
OTHER THAN TUBERCULOSIS
Rabbits have been used for studies of arte-
riosclerosis, various eye diseases, arthritis, micro-
bial diseases (including syphilis, tularemia, can-
didiasis, cryptococcosis, coccidioidomycosis,
anthrax, meningitis, and endocarditis), cardiac
allograph rejection, and cardiomyopathy. In addi-
tion, rabbits are often used for testing various
vaccines, for antibody production, and for var-
ious toxicity tests (including those for pyro-
gens) (25).
REFERENCES
1. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive Mech-
anisms. Harvard University Press, Cambridge, Mass.
2. Lurie, M. B. 1941. Heredity, constitution and
tuberculosis: an experimental study. Am. Rev.Tuberc.
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3. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
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in natively resistant and susceptible rabbits. Am.
Rev.Tuberc. Pulm. Dis. 72:297–329.
4. Lurie, M. B., and A. M. Dannenberg, Jr. 1965.
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1996. Standard Bred Rabbits and Cavies, 1996 through
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7. Dannenberg, A. M., Jr. 1993. Immunopatho-
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8. Dannenberg, A. M., Jr. 1994. Rabbit model of
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9. Henderson, H. J., A. M. Dannenberg, Jr., and
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11. Dannenberg, A. M., Jr., O. T. Meyer, J. R.
Esterly, and T. Kambara. 1968.The local nature
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12. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
genesis of tuberculosis: specificity, systemic and local
nature, and associated macrophage enzymes. Bacte-
riol. Rev. 32:85–102.
13. Lurie, M. B., P. Zappasodi, E. Cardone-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
to the intracutaneous inoculation of BCG as an
index of native resistance to tuberculosis. J. Immunol.
68:369–387.
14. Lurie, M. B., P. Zappasodi,A M. Dannenberg,
Jr., and G. H.Weiss. 1952. On the mechanism of
genetic resistance to tuberculosis and its mode of
inheritance. Am. J. Hum. Genet. 4:302–314.
15. van Zutphen, L. F. M., and R. R. Fox. 1977.
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by JAX rabbits: correlation with esterase patterns.
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16. Beynen, A. C., G.W. Meijer, A. G. Lemmens,
J. F. C. Glatz, A. Versluis, M. B. Katan, and
L. F. M. van Zutphen. 1989. Sterol balance and
cholesterol absorption in inbred strains of rabbits
hypo- or hyperresponsive to dietary cholesterol.
Atherosclerosis 77:151–157.
17. Dannenberg, A. M., Jr. 1998. Lurie’s tubercle-
count method to test TB vaccine efficiency in rab-
bits. Front. Biosci. 3:c27–33. Available at
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18. Converse, P. J., A. M. Dannenberg, Jr., J. E.
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H.Abbey, J. E. Estep, and M. L. .M. Pitt. 1998.
Pulmonary bovine-type tuberculosis in rabbits:
bacillary virulence, inhaled dose effects, tuberculin
sensitivity, and Mycobacterium vaccae immunotherapy.
Clin. Diagn. Lab. Immunol. 5:871–881.
20. Fox, R. R. 1974.Taxonomy and genetics, p. 1–22.
In S. H.Weisbroth, R. E. Flatt, and A. L. Kraus (ed.),
The Biology of the Laboratory Rabbit.Academic Press,
Inc., New York, N.Y.
21. Fox, R. R. (ed.). 1975. Handbook on Genetically
Standardized JAX Rabbits.The Jackson Laboratory,
Bar Harbor, Me.
22. Altman, P. L., and D. D. Katz. 1979. Rabbit, p.
565–606. In Inbred and Genetically Defined Strains of
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Rabbit, and Chicken. Federation of American Soci-
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23. Manabe, Y. C., A. M. Dannenberg, Jr., S. K.
Tyagi, C. L. Hatem, M.Yoder, S. C.Woolwine,
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24. Dorman, S., C. L. Hatem, S. Tyagi,
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comer (ed.). 1994. The Biology of the Laboratory
Rabbit, 2nd ed., p. 367. Academic Press, Inc., New
York, N.Y.
26. Lurie, M. B., S. Abramson, and A. G. Hep-
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sistant and susceptible rabbits to the quantitative
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nature of resistance to tuberculosis. J. Exp. Med.
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27. Allison, M. J., P. Zappasodi, and M. B. Lurie.
1961. Metabolic studies of mononuclear cells from
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mals. Am. Rev. Respir. Dis. 84:364–370.
28. Dannenberg,A. M., Jr., K. Mizunoe, M. Peace,
and P. Zappasodi. 1965. Dermal response to the
䡵 245
lipopolysaccharide PmKo from tubercle bacilli as an
index of resistance to tuberculosis. Bull. Johns Hop-
kins Hosp. 117:174–194.
29. Lurie, M. B., A. G. Heppleston, S. Abramson,
and I. B. Swartz. 1950. An evaluation of the
method of quantitative airborne infection and its use
in the study of the pathogenesis of tuberculosis.
Am. Rev.Tuberc. 61:765–797.
30. Lurie, M. B., P. Zappasodi, R. S. Levy, and
R. G. Blaker. 1959. On the role of the thyroid
in native resistance to tuberculosis. I.The effect of
hyperthyroidism. Am. Rev. Respir. Dis. 79:152–
179.
31. Dannenberg, A. M., Jr., W. R. Bishai, N. Par-
rish, R. Ruiz, W. Johnson, B. C. Zook, J. W.
Boles, and M. L. M. Pitt. 2000. Efficacies of
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cines in preventing clinically apparent pulmonary
tuberculosis in rabbits: a preliminary report. Vaccine
19:796–800.
32. Allison, M. J., P. Zappasodi, and M. B. Lurie.
1962. Host-parasite relationships in natively resis-
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85:553–569.
33. Lurie, M. B. 1944. Experimental epidemiology of
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phases of resistance. J. Exp. Med. 79:573–589.
 15
COMPARISONS OF TUBERCULOSIS
IN RABBITS, MICE, AND GUINEA PIGS

Overview [247]
Tuberculosis in rabbits [247]
Tuberculosis in mice [247]
Tuberculosis in guinea pigs [254]
Tuberculosis in monkeys [257]
Comparison of tuberculosis in mice with that in rabbits, humans, and guinea
pigs [259]
Virulence of inhaled tubercle bacilli in rabbits, mice, and guinea
pigs [260]
Bacillary titers in the stationary stage after aerosol infection of rabbits, mice,
and guinea pigs [261]
Fate of aerosolized BCG in rabbits, mice, and guinea pigs [262]
Tuberculin sensitivity among the three common laboratory species [262]
Organ resistance in rabbits and guinea pigs [262]
Early tuberculous lesions in humans [263]
Cavity formation in various species [263]
Role of DTH and CMI in mouse, guinea pig, and rabbit tuberculosis [263]

Abstract. In recent times, mice have been by far the most frequently used animal for the
study of tuberculosis. Guinea pigs and rabbits are used less often, and monkeys are used
only occasionally.
Rabbits are highly susceptible to bovine-type tubercle bacilli and are the only common
laboratory species in which chronic cavitary tuberculosis with bronchial spread is readily
produced. Rabbits develop grossly visible pulmonary tubercles following the inhalation of
virulent human-type tubercle bacilli, but these tubercles usually regress, as they do in most
humans. Rabbit pulmonary lesions caused by virulent human-type tubercle bacilli some-
times form nonprogressive cavities.Rabbits die after an infection with virulent bovine-type
tubercle bacilli, but eventually heal an infection with virulent human-type tubercle bacilli.
Mice develop slowly progressing pulmonary tubercles with both bovine and human
strains of tubercle bacilli, but the disease progresses more rapidly with the bovine strain.
With virulent human-type bacilli, the tubercles of mice contain a larger number of viable
bacilli than do the tubercles of rabbits and guinea pigs. Apparently, the low levels of
delayed-type hypersensitivity (DTH) in mice and the rarity of caseous necrosis allow the
bacillus to grow to higher titers in the logarithmic stage. After these titers are reached, the
good cell-mediated immunity (CMI) developed by mice reduces the intracellular multi-
plication of the majority of the bacilli to almost a dormant state. However, the low per-
centage of nondormant bacilli causes the disease to slowly progress until the animal dies.
Guinea pigs are highly susceptible to both human and bovine strains of tubercle bacilli.
They usually develop a rapidly progressing, hematogenously spread form of tuberculosis,
similar to that developed by infants and immunosuppressed individuals. Because guinea
pigs develop relatively high sensitivity to tuberculin, which causes considerable caseous
necrosis, their lesions contain a rather low number of viable bacilli. In other words, despite
the rather poor CMI developed by guinea pigs, their good DTH effectively reduces the
number of viable bacilli in their lesions. However, because of the extensive lung destruc-
tion caused by tissue-damaging DTH, guinea pigs often die in less time than do rabbits
and mice.
Rhesus monkeys are very susceptible to tuberculosis, but cynomolgus monkeys are more
resistant. Some cynomolgus monkeys can even stop the progression of the disease.

246
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS
OVERVIEW
In all three species of common laboratory ani-
mals, if the tubercle bacillus is fully virulent it
will cause progressive disease terminating in
death. However, after aerosol infection, despite
the progression of the disease, the number of
viable bacilli in the lungs of all three species stops
increasing at 3 to 4 weeks (Fig. 1, 2, and 3). At
this time, the host has developed both delayed-
type hypersensitivity (DTH) to the tuberculin-
like products of the bacillus and cell-mediated
immunity (CMI), which together keep the
number of viable bacilli rather constant (see
chapter 2).
The number of viable bacilli present in the
lungs does not necessarily parallel increases in
antigen levels. Antigen levels can increase with
no change in the number of viable bacilli if
their rate of multiplication and their rate of
death are equal. In this case, bacillary secretory
products, carcasses (1, 2), and other components
(3) can increase if they are not easily destroyed.
In fact, viable bacilli persisting at constant levels often
cause progressive disease (see Fig. 1, 2, and 3) (4).
Only after the number of viable bacilli has
been substantially reduced do the antigen con-
centrations actually decrease and the lesions
begin to heal. However, when tubercle bacilli are
fully virulent for the host, the number of viable
bacilli in the lungs never decreases, although it
may remain constant for months.
In mice, which are animals with little or no
tissue-damaging DTH, the majority of tubercle
bacilli persist intracellularly in macrophages
without multiplying or dying (1, 2), but some
bacilli multiply and are killed, adding to the
antigenic load (discussed more fully below.)
Table 1 presents the main characteristics of
tuberculosis in humans and in laboratory ani-
mals. Reference 5 reviews the literature on pul-
monary tuberculous cavities in rabbits, mice,
guinea pigs, humans, and nonhuman primates.
TUBERCULOSIS IN RABBITS
The disease in rabbits is unique, because in this
species virulent human-type tubercle bacilli and
virulent bovine-type bacilli produce disease of
markedly different severity. Tuberculosis pro-
䡵 247
duced by the virulent bovine type progresses
until the host dies, whereas tuberculosis pro-
duced by the virulent human type uniformly
heals, although it may take over a year to do so.
The pathogenesis of tuberculosis in rabbits
with both bovine and human bacillary types is
described in chapter 13. In brief, 5 weeks after
the inhalation of either type of virulent tuber-
cle bacillus, typical tubercles are usually present,
often with small caseous centers.These tubercles
progress or regress, depending on the amount of
immunity (acquired resistance) developed by the
host and the virulence of the infecting strain. In
hosts that develop good acquired resistance, liq-
uefaction and cavity formation may occur (6),
even with human-type bacilli (7). However, only
the bovine type produces progressive tuberculosis
with spread through the bronchial airways.
TUBERCULOSIS IN MICE
Bacillary Growth Curves and Type of
Disease
Similar to rabbits and guinea pigs, the number
of virulent bovine- or human-type bacilli in
the lungs of mice stops increasing at 3 to 4
weeks after an aerosol infection (Fig. 2).Then,
the bacillary titers remain more or less constant
until near death (8–11).The pulmonary gran-
ulomas enlarge progressively (often with nearby
satellite lesions [12]) until the mouse succumbs
(4, 13, 14).The lesions show little or no necro-
sis (Table 1) before the final stages of the disease.
Ending the Logarithmic (Symbiotic)
Stage of Bacillary Growth
The logarithmic stage ends in 3 to 4 weeks
when CMI and DTH develop (Fig. 2). Mice
develop only low levels of tuberculin sensitivity,
which explains why there is little or no necro-
sis. They probably use apoptosis to kill bacilli-
laden macrophages and stop the logarithmic
stage of bacillary growth. Cells killed by apop-
tosis are rapidly ingested and destroyed by
macrophages without any nearby tissue reac-
tion (15–17). Apoptosis is apparently caused at
least in part by cytotoxic T lymphocytes, because
the multiplication of Mycobacterium tuberculosis
is uncontrolled in knockout mice (see glossary)
248 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 The number of viable human- and bovine-type tubercle bacilli in the lungs of
natively resistant and susceptible rabbits at different intervals following quantitative airborne infec-
tion (6, 7, 75).This graph shows the increase in the number of viable bacilli relative to the ini-
tial number deposited into the pulmonary alveoli (see explanation below). All human-type
tubercle bacilli are of reduced virulence for rabbits.The means are shown (see references 7 and
75 for the standard errors).
By 1 week after infection, the resistant rabbits had inhibited the growth of the human-type
bacilli in their lungs about 25 times more effectively than did the susceptible rabbits, and the re-
sistant rabbits had inhibited the growth of bovine-type bacilli about 5 times more effectively. From
then on, the bacillary growth curves in both strains of rabbit were parallel. At 4 weeks, similar
comparisons of the number of bacilli in the susceptible and resistant rabbits were about 20 times
for the human type and about 2.5 times for the bovine type.
These findings indicate that bovine-type bacilli are harder to inhibit than human-type bacilli—
both by native resistance (pulmonary alveolar macrophages) and by acquired resistance (T lym-
phocytes activating macrophages).These findings also indicate that both native and acquired re-
sistance are much more effective in the genetically resistant group. Commercially available New
Zealand White rabbits resemble Lurie’s resistant strain of rabbits (68, 69, 94–97).
The number of bacilli in the lungs of the resistant group failed to decrease during the period
illustrated, because liquefaction with extracellular multiplication of the bacillus readily occurs in
these rabbits (6). Liquefaction usually does not occur in the susceptible rabbits (6), because their
macrophages probably develop only low levels of hydrolytic enzymes. Reproduced with permission
from reference 75.
Note that this figure was derived from the results of many experiments, each using a some-
what different inhaled dosage. For this reason, the y axis represents the number of viable tuber-
cle bacilli in the entire lung divided by one-third the number of bacilli inhaled.This ratio shows
increases (or decreases) in the number of viable pulmonary bacilli at various times after infec-
tion.At 8 weeks, the lungs of the resistant and susceptible rabbits contained, respectively, an aver-
age of 2.2 ⫻ 108 and 3.4 ⫻ 108 viable bovine-type bacilli, and 2.2 ⫻ 106 and 25.5 ⫻ 106 human-
type bacilli (see references 7 and 75).
The number of bacilli was estimated by culturing lung homogenates.The number of inhaled
bacilli was calculated by sampling and culturing the aerosols with an impinger and determining
the amount of air breathed by each rabbit calculated from its weight (6, 7, 75).Two-thirds of the
small bacillary particles breathed by each rabbit impinge on the mucosa of the bronchial tree and
are eventually swallowed. Only one-third reach the alveoli where the infection begins (6). In the
graph, the number of tubercle bacilli inhaled into the alveoli is therefore one-third the number
of bacilli that the rabbit breathed.
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS
that lack such lymphocytes (18–21).The killing
of bacilli-laden macrophages is necessary to stop
the logarithmic stage of bacillary multiplication,
because during this stage many macrophages
contain too many actively growing bacilli to be
controlled by CMI alone (see chapter 2).*
Maintaining the Stationary Stage of
Bacillary Growth
In maintaining the stationary stage, mice appar-
ently differ from humans, rabbits, and guinea
*The logarithmic stage of bacillary growth apparently
ends sooner after an intravenous infection with tuber-
cle bacilli than after an aerosol infection (10) for the fol-
lowing reasons. (i) A major portion of the intravenous
dose directly enters the spleen, which is a lymphoid
organ that can rapidly produce acquired immunity (10).
(ii) The dose of bacilli given intravenously is usually
higher than that given by aerosol, thereby providing a
greater immunological stimulus. And (iii) the dose of
bacilli given by aerosol is lowered even further, because
the pulmonary alveolar macrophage population is highly
activated and immediately inhibits or slows the multi-
plication of many inhaled bacilli. Bacilli entering the
lungs following an intravenous injection bypass the
alveolar macrophage population and are ingested by
nonactivated macrophages. Following an aerosol infec-
tion, some dissemination of bacilli from the lungs to the
lymph nodes, liver, and spleen occurs in mice, but the
disease in other organs is more readily produced by an
intravenous infection. Since the inhalation of tubercle
bacilli is the most common way that humans contract
tuberculosis, we have limited our comparisons of tuber-
culosis in rabbits, mice, and guinea pigs to this method
of infection.
䡵 249
FIGURE 2 The number of viable viru-
lent human-type (H37Rv) and bovine-
type (Ravenel) tubercle bacilli in the lungs
of C57BL/6 mice at different intervals fol-
lowing quantitative airborne infection (8).
Note that the in vivo bacillary growth
curves in mice resembled those found in
rabbits (see Fig. 1), but in contrast to rab-
bits, H37Rv and Ravenel tubercle bacilli
multiplied to the same levels in C57BL/6
mice (a relatively resistant strain). Repro-
duced with permission from reference 8.
pigs. After immunity develops in mice, many
macrophages in the lesions are probably activated
and do not allow the ingested bacilli to multi-
ply appreciably.The good CMI evidently keeps
tubercle bacilli in a rather dormant state in
which their multiplication is inhibited (Fig. 4)
(1, 2). In other words, during the stationary
stage relatively few tubercle bacilli multiply
within the macrophages, so the number of dead
bacilli does not increase. Dead bacilli are only
slowly destroyed over many weeks (Fig. 4).
Nevertheless, a small amount of bacillary
replication probably occurs in mouse macro-
phages during the stationary stage, followed by
apoptosis of these macrophages (1, 2).This small
amount of bacillary multiplication causes the
disease to progress slowly until the mouse suc-
cumbs.
In brief, the CMI evidently activates sufficient
macrophages in the tuberculous lesions of mice
to keep most intracellular tubercle bacilli from
multiplying appreciably. Unfortunately, this inhi-
bition of intracellular bacillary growth is incom-
plete and is insufficient to stop the progression
of the disease, which eventually kills the host.
Tissue Necrosis in Mice
Tissue necrosis will occur in mice when large
numbers of bacilli grow within macrophages
(22). The exact cause of such necrosis has not
been identified. Some of the necrosis in mice
may be due to their low level of DTH that
kills macrophages when the tuberculin-like
250 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 3 The number of viable virulent human-type tubercle bacilli (H37Rv) in the lungs
of BCG-vaccinated and control guinea pigs at different intervals following quantitative airborne
infection (40). Note that the in vivo bacillary growth curves in guinea pigs resemble those found
in rabbits (Fig. 1) and in mice (Fig. 2). Other experiments showed that the stationary phase in
guinea pigs continues for at least 18 weeks (41). Note also that in the BCG-vaccinated guinea
pigs, the logarithmic growth stage ends soon and that the number of viable tubercle bacilli in the
stationary stage is markedly reduced (see text).The guinea pigs were immunized intradermally
with live BCG 6 weeks before the aerosol challenge. Reproduced with permission from refer-
ence 40.
The number of tubercle bacilli in the stationary stage in guinea pigs is lower than the num-
ber found in mice (compare Fig. 2 and 3), because guinea pigs probably end the logarithmic stage
sooner (although insufficient time points were plotted in these two figures to show this differ-
ence).Therefore, the good DTH of guinea pigs is probably more effective than the good CMI
of mice in ending the logarithmic stage, which supports the principle that tissue-damaging DTH
is an important host defense mechanism in controlling intracellular bacillary multiplication.

products of the bacilli reach a high local con-
centration. Some of the necrosis may be merely
degeneration of macrophages when they con-
tain numerous bacilli (11, 18, 22–26). Some of
the necrosis may be a local Shwartzman phe-
nomenon (see glossary) (27, 28) (characterized
by intravascular fibrin deposits that impair the
local blood supply [29]) and/or an antigen-
antibody reaction causing an Arthus reaction
(see glossary). In mice, such large numbers of
tubercle bacilli often occur in various types of
knockout mice (22, 27, 30, 31) and may occur
terminally in wild-type mice (22, 27).
The intragranulomatous necrosis found in
the more chronic stages of pulmonary tuber-
culosis in mice is somewhat different from the
typical caseous necrosis found in rabbits, guinea
pigs, and humans. Mice show little damage to
the lung tissue surrounding the granulomas,
and they show little vascular thrombosis, which
is a major factor in producing caseous necro-
sis in the other three species (see below).
Mouse intragranulomatous necrosis (22, 27)
does not seem as “mature” as the typical
caseous necrosis found in other species. Mature
caseum is a homogeneous necrosis with the
nuclear debris of the dead cells finely dispersed
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS 䡵 251

TABLE 1 Characteristics of tuberculosis in humans and in laboratory animalsa,b

Species Tuberculin-type Caseous necrosis Cell-mediated Cavity

allergy (DTH) (due to DTH)c immunity (CMI) formation
Isolated human populationsd ⫹⫹⫹⫹⫹ ⫹⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹
Rhesus monkeyse ⫹⫹ ⫹⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹
Guinea pigs ⫹⫹⫹ ⫹⫹⫹⫹⫹ ⫹⫹ ⫹
Modern humans
Immunocompetent ⫹⫹⫹⫹⫹ ⫹⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹⫹
Immunosuppressed ⫹ ⫹⫹⫹⫹⫹ ⫹ ⫹
Rabbits
Resistant ⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
Susceptible ⫹⫹ ⫹⫹⫹⫹⫹ ⫹⫹ 0
Mice ⫹ ⫹ ⫹⫹⫹ 0
a
Reproduced with permission from reference 4 and adapted from a table by Francis (43). References 60 and 93 reproduce
the entire Francis table, which contains many animal species, including elephants.
b
The assignment of ⫹ to ⫹⫹⫹⫹⫹ is a generalization of a given species as a whole, because much variation exists within a
species. Good CMI and DTH are both needed to arrest the disease. Guinea pigs have good DTH but relatively poor CMI, and
mice have good CMI but poor DTH. Both species show progressive disease leading to their death. Most humans and rabbits (infected
with virulent human-type tubercle bacilli) have adequate amounts of DTH and CMI to arrest the disease.
c
Tissue-damaging DTH is the cause of caseous necrosis.Tubercle bacilli are dormant in solid caseum, and many do not sur-
vive there. In arrested tuberculous lesions, solid caseum is often encapsulated by fibrous tissue.
d
Isolated human populations are those that have never before been exposed to tuberculosis, such as the Senegalese troops brought
from Africa to Europe during World War I (65).
e
Cynomolgus monkeys are more resistant to tuberculosis than rhesus monkeys and may even arrest the disease (see text).

or absent (probably due to proteases and nucle-
ases) (6, 7).
Tuberculosis Produced in Mice by
Virulent Bovine-Type and Virulent
Human-Type Tubercle Bacilli
In some mouse strains, the virulent bovine-type
and the virulent human-type bacilli multiplied
in the lungs to the same titers (Fig. 2) (8). How-
ever, the bovine type (Ravenel) killed mice
faster than did the human type (H37Rv): by 160
days, all the mice inhaling Ravenel were dead,
whereas all the mice inhaling H37Rv were alive
(8). By 200 days, only 20% of the mice inhaling
H37Rv had died (8). In this experiment, since
the number of live pulmonary bacilli of each
strain was the same, components of the dead
bovine-type bacilli evidently persisted longer
in the lungs than components of the dead
human type, causing the granulomas to increase
in size more rapidly (8, 26, 32) and kill the host
sooner (see references 8, 26, and 33). Perhaps, live
bovine-type bacilli produce and/or secrete larger
amounts of various antigens (or more reactive
antigens) than do live virulent human-type
bacilli. Bovine-type bacilli may also stimulate a
greater chemokine response in the host. An
increase in chemokines would cause a more
rapid growth of the pulmonary lesions and
therefore cause earlier death.
In other mouse strains, virulent bovine-type
bacilli multiply in the lung to higher titers dur-
ing the logarithmic stage than do virulent
human-type bacilli (33; K. Bosio, personal com-
munication). Similarly, the virulent H37Rv
strain multiplies in mouse lungs to higher titers
than does the relatively avirulent R1Rv strain
(33, 34).
Genetic Differences in Resistance to
Tuberculosis among Mouse Strains
As in rabbits, genetic differences among various
inbred mouse strains influence the course of
the disease. During the stationary phase after an
intravenous injection, the number of viable
H37Rv bacilli in the lungs of BALB/c mice was
252 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 Viable counts and total counts of virulent tubercle bacilli (H37Rv)
in the lungs of mice from 9 weeks to 25 weeks after an intravenous infection.
After the lungs were homogenized, the viable counts were calculated from the
CFU developing on plates containing solid culture medium.The total counts
were calculated from the bacilli observed microscopically after acid-fast stain-
ing of spread smears made from the homogenates. During this time period, one
of the four groups of mice received isoniazid-pyrazinamide (PZA/INH) daily
to kill the bacilli.
Note that for untreated mice the average total counts were 0.3 to 0.4 logs
higher (2.0 to 2.5 times) than the average viable counts, and that PZA/INH treat-
ment markedly reduced the viable counts but had relatively little effect on the
total counts.These findings indicate that (i) most of the dead tubercle bacilli per-
sisted in mouse lungs for many weeks, (ii) most of the live bacilli in the untreated
mice were in a dormant state, because if they had been multiplying and then
had been killed, the total counts (including the dead bacilli) would have
increased, and (iii) the good CMI developed by mice activated macrophages suf-
ficiently to prevent the intracellular multiplication of most of the tubercle
bacilli.
Note also that the 2.0- to 2.5-fold mean difference in total and viable counts
suggests that at least some bacillary growth and destruction were occurring. In
other words, at least some of the bacilli were not inhibited by the good CMI,
which is probably the reason for the progression of the disease in mice until they
succumb.
These results were confirmed in reference 2 using quantitative real-time PCR.
Reproduced with permission from reference 1.The same figure appears as
Fig. 1 in chapter 6 of this volume.
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS 䡵 253

FIGURE 5 A primary pulmonary tuberculous lesion in a C57BL/6

mouse 8 weeks after an aerosol inhalation of virulent human-type tubercle
bacilli. On the right is the lesion’s center containing large viable epithelioid
macrophages, some of which probably inhibited (or destroyed) the tubercle
bacilli that they had ingested during the earlier stages of the infection.A few
polymorphonuclear leukocytes have accumulated where some of these
macrophages have disintegrated (far right). In the middle of the photograph
is part of the mantle of compact lymphocytes, which surrounds the center
of epithelioid macrophages. On the far left are a few foamy macrophages
within the surrounding alveolar spaces. Many of these foamy macrophages
are pulmonary alveolar macrophages that tend to accumulate around pul-
monary tuberculous lesions (see Fig. 5 of chapter 9). Pulmonary alveolar
macrophages often ingest surfactant, which gives them the foamy appearance.
Stained with hematoxylin and eosin. Magnification, ⫻275. Photograph pro-
vided by P.-J. Cardona.

about two logs lower than the number in the
lungs of DBA/2 mice (35).Also, BALB/c mice
and C57BL/6 mice lived about twice as long as
the DBA/2 and C3H/HeJ mice: their mean
survival times were about 8 and 4 months,
respectively (14).
Among the mouse strains tested, aerosol
infection produced results similar to the intra-
venous infection: C57BL/6 and BALB/c mice
are more resistant than DBA/2, 129/SvJ, CBA,
C3H/HeN, and C3H/HeJ mice (12, 14, 27).
Also, knockout mice (with one or more com-
ponents of innate or acquired immune response
removed from their genome by molecular biol-
ogy techniques) show higher bacillary titers
during the stationary stage (22, 24, 31; see also
reference 25). SCID mice are so susceptible that
virtually no stationary phase occurs before the
animal dies (36).
Histopathology of Mouse Pulmonary
Tuberculous Lesions
Lesion progression in mice occurs locally (12, 37).
Macrophages in which tubercle bacilli are grow-
ing evidently migrate from the primary lesions
into neighboring alveoli and cause secondary
lesions (Fig.5 and 6).Therefore,the secondary and
tertiary lesions are often nearby, and confluence
occurs in the terminal stages of the disease.
Bacilli-laden foamy macrophages are present
in bronchoalveolar lavages (12, 37).The vacuoles
causing their foamy appearance are probably
254 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 The outer layer of the 8-week lesion shown in Fig. 5. Note
how these foamy cells fill the alveolar spaces.Tubercle bacilli (not shown) are
occasionally present in some of these foamy macrophages.The bacilli were
probably carried to the lesion’s periphery within macrophages that had
migrated at an earlier time from the center. Such bacilli sometimes cause sec-
ondary lesions in nearby alveoli. Stained with hematoxylin and eosin. Mag-
nification, ⫻275. Photograph provided by P.-J. Cardona.
In mice, few, if any, tubercle bacilli remain free in viable tissues or even
in lymphatics, because bacilli released from disintegrating macrophages are
soon ingested by other (still viable) macrophages. In animal species where
cavities form, many free tubercle bacilli enter the airspaces, and some of these
bacilli may be ingested by the alveolar macrophages. Mouse lesions do not
liquefy and form cavities, so extracellular tubercle bacilli are rarely present.

filled with the lipids from both dead tubercle
bacilli and dead apoptotic host cells, as well as
the surfactants that these cells ingest.
It is not known how the bacilli get from the
necrotic center to the periphery. They could
travel in lymphatics in a free state or within
macrophages. In mouse pulmonary granulomas,
lymphatics may or may not develop along with
the vasculature. (The walls of the alveoli have no
lymphatics. Lymphatics first appear in the lungs
at the level of the respiratory bronchioles.)
caseous necrosis (6, 38; see also reference 39).
Such thrombosis does not usually occur in the
tubercles of mice, and functional capillaries and
venules, as well as viable lymphocytes, dendritic
cells, and macrophages, are usually present
throughout the tubercles.
See references 12, 22, 27, and 35 for other
photomicrographs, and see references 23, 30, and
31 for reviews of factors regulating tubercu-
lous granulomas in mice, including CD4 and
CD8 lymphocytes and various cytokines.

Microvascular Patency in Mouse

Pulmonary Lesions
The absence of caseous necrosis in the pul-
monary tubercles of mice is partly due to intact
microvasculature (Fig. 7). In the tubercles of
other laboratory animal species, microvascular
thrombosis is a major factor contributing to the
TUBERCULOSIS IN GUINEA PIGS
Bacillary Growth Curves
In guinea pigs, as in rabbits and mice, inhaled
virulent tubercle bacilli show a logarithmic
growth phase followed by a stationary phase in
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS 䡵 255

FIGURE 7 A tuberculous lesion in a mouse similar to those shown in Fig. 5 and

6. Little or no necrosis occurs in mouse lesions, mainly because there is no throm-
bosis of the microvasculature. This photograph shows patent microvessels (white
arrows) within the dense lymphocyte layer that surrounds the center, containing
numerous viable epithelioid macrophages.The microvessels are easily identified by
the intact erythrocytes that they contain. Stained with hematoxylin and eosin. Mag-
nification, ⫻295. Photograph provided by P.-J. Cardona.

which multiplication and killing of bacilli appar-
ently occur at equal rates (Fig. 3) (40–42).
Gross Pathology
Tuberculosis in guinea pigs, produced by both
virulent human-type bacilli and virulent bovine-
type bacilli, has many of the characteristics of
tuberculosis caused by virulent bovine-type
bacilli in Lurie’s susceptible strains of rabbits
(Table 1) (also see chapters 2, 13, and 14).
1. One grossly visible primary pulmonary
tubercle is usually produced by every
inhaled virulent tubercle bacillary unit
that reaches the alveolar spaces.
2. The “childhood type” of tuberculosis with
large caseous hilar lymph nodes usually
develops.
3. Both Lurie’s susceptible rabbits and guinea
pigs develop relatively weak CMI.Tuber-
cle bacilli multiply within the poorly acti-
vated macrophages that surround the
caseous center. Killing or inhibition of
these bacilli seems to be largely due to tis-
sue-damaging DTH, but in each case a
proportion of this inhibition (or killing) is
due to activation of macrophages by CMI.
4. The considerable caseous necrosis (43) is
apparently due to thrombosed microvas-
culature (see reference 44)—in contrast to
mouse tuberculous lesions, in which such
thrombosis is usually absent.
5. Acquired (adaptive) immunity, i.e., DTH
and CMI, slows the progression of the
disease, but not enough to arrest it.
6. Fatal progressive destruction of the lungs
and hematogenous spread of this disease
occur, mainly due to the tissue-damaging
DTH. In guinea pigs, the hilar nodes and
spleen are major sites of the metastatic dis-
ease. (The lymphogenous and hematoge-
nous spread of tubercle bacilli from the ini-
tial site of infection occurs throughout the
course of the disease. Initially, the bacilli are
probably in macrophages that transport
them to the hilar nodes and then [via the
efferent lymph and great veins] back to the
lungs. Later in the disease, the caseous
256 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
necrotic process destroys the adjacent
microvasculature, and free bacilli in the
caseum can directly enter the bloodstream
[see reference 6]).
Two factors may change the typical suscep-
tible type of tuberculosis just described for
guinea pigs into a more resistant type (see ref-
erence 45). The first is the virulence of the
infecting strain: Isoniazid-resistant catalase-
negative tubercle bacilli are often, but not always,
of reduced virulence for guinea pigs (46–48). In
fact, guinea pigs infected with such resistant
strains may even arrest the disease, depending on
the degree of attenuation (46, 48).The second
is the number of tubercle bacilli inhaled: a very
low inhaled dose sometimes converted the
tuberculin skin test with no gross evidence of
disease at necropsy.This sometimes occurred in
Riley’s experiments in which guinea pigs
inhaled an occasional bacillus over periods of
many weeks (see chapter 12).
Note also that fully virulent tubercle bacilli
may be present in the sputum of isoniazid-
resistant patients, because not all bacilli in spu-
tum are identical (46–48).
Histopathology
Figures 8 and 9 show typical guinea pig lesions
4 weeks after an aerosol infection with virulent
human-type tubercle bacilli. Each has a caseous
necrotic center surrounded by tuberculous gran-
ulation tissue.
Reference 49 presents a detailed histological
description of guinea pig tuberculous granulo-
mas during various stages of their development,
including the identification of CD4 and CD8
cells and the cells undergoing apoptosis. Refer-
ence 50 presents the effects of BCG on these
granulomas.
Immunization
In effectively immunized guinea pigs (Fig. 3), the
growth of tubercle bacilli in both primary and
metastatic lesions is decreased, and fewer bacilli
spread to other organs (40, 44, 51, 52). In other
words, guinea pigs are able to develop some
effective CMI, i.e., activated macrophages that
prevent the growth of ingested tubercle bacilli.
Unfortunately, this acquired immunity is rarely
strong enough to stop the eventually fatal
progress of the disease.
Cavity Formation
When guinea pigs inhale such low numbers of
bacilli that only 1 to 3 primary pulmonary
lesions are produced, cavities may form after
several months (53–55). However, as stated
above, tuberculosis in guinea pigs is primarily a
hematogenously spread disease, similar to that
described in Lurie’s susceptible strains of rabbits.
Inbred Guinea Pigs
Two inbred guinea pig strains exist: strain 2 and
strain 13 (56).These strains did not significantly
differ in their response to aerosol challenge with
virulent human-type tubercle bacilli (H37Rv)
(57).When a protein deficit existed in their diet,
BCG immunization no longer reduced the
growth of inhaled H37Rv bacilli in the lungs of
strain 13, and reduced the growth of inhaled
H37Rv bacilli in strain 2 only slightly (57).
Fibrin Meshworks
After DTH develops, fibrin readily forms in
guinea pig tuberculous lesions (6, 44).The asso-
ciated thrombosis not only increases the amount
of necrosis, but also partly obstructs the lym-
phatics draining the lesions (6, 44). Therefore,
tuberculin positivity in guinea pigs reduces the
spread of tubercle bacilli from tuberculous
lesions to the draining lymph nodes, whereas
tuberculin positivity in rabbits enhances the
spread of bacilli from lesions to the draining
nodes. Rabbits produce relatively little fibrin, and
the inflammatory processes increase the flow
of lymph (6, 44).
After an aerosol infection, the number of
tubercle bacilli reaching the hilar lymph nodes
has relatively little effect on the amount of
tuberculosis produced there, which depends on
whether the host inhibits the growth of the
bacilli in these nodes (6). Guinea pigs (and
Lurie’s susceptible rabbits) usually inhibit bacil-
lary growth in the nodes poorly, whereas
Lurie’s resistant rabbits (and commercial rab-
bits) usually inhibit such bacillary growth
effectively.
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS 䡵 257

FIGURE 8 A guinea pig tuberculous lesion 4 weeks after an aerosol infection with virulent
human-type tubercle bacilli. Note (from left to right) the pronounced caseous center, the typi-
cal tuberculous granulation tissue with many lymphocytes, and the nearby alveoli with slightly
thickened walls. Stained with hematoxylin and eosin. Magnification, ⫻80. Photograph provided
by P.-J. Cardona.

Koch Phenomenon in rabbits (44) (and possibly in humans) may not

The Koch phenomenon is classically defined as
the local necrotic reaction caused by an intra-
dermal injection of tubercle bacilli (or their
culture filtrate) into a tuberculous guinea pig.
Because such guinea pigs have strong tuber-
culin sensitivity, the developing dermal lesion
soon caseates, ulcerates, and discharges much
of its necrotic contents.An intradermal challenge
may also increase the amount of necrosis in the
existing (pulmonary) lesions.
The Koch phenomenon is thought to be
protective, because many bacilli are discharged
from the ulcer, and, in guinea pigs, few bacilli
reach the draining lymph nodes due to the fib-
rin meshworks in the draining lymphatics. How-
ever, as mentioned above, the fibrin meshworks
be as effective as those in guinea pigs in reduc-
ing the spread of bacilli to the nodes. Details and
possible mediators of the Koch phenomenon are
presented in reference 58.
TUBERCULOSIS IN MONKEYS
Rhesus monkeys (Macaca mulatta) are highly
susceptible to virulent M. tuberculosis and viru-
lent Mycobacterium bovis (43, 59–64).They resem-
ble isolated human populations that had never
been previously exposed to the tubercle bacil-
lus (Table 1) (6), such as the Senegalese troops
taken from Africa to Europe to fight in World
War I (65).
Tuberculosis in rhesus monkeys is usually a
rapidly progressive pulmonary disease, frequently
258 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 9 Another guinea pig tuberculous lesion 4 weeks after an aerosol infection with vir-
ulent human-type tubercle bacilli. Note (from left to right) the pronounced caseous center, the
typical tuberculous granulation tissue, the surrounding fibroblasts, and the intact pleura. Stained
with hematoxylin and eosin. Magnification, ⫻80. Photograph provided by P.-J. Cardona.

with both bronchial and hematogenous spread
of the bacilli. Extensive areas of caseous necro-
sis occur, as well as liquefaction of the caseum
and cavities (43, 59, 61, 63, 64, 66).The cavities
often have ragged linings harboring numerous
tubercle bacilli (63) that spread through the air-
ways.Areas of tuberculous bronchopneumonia
are common (43, 61, 63, 66), as are enlarged
caseous hilar nodes (61, 63, 66).
BCG immunization of rhesus monkeys
reduces the severity of the disease (59, 61, 66)
and can prevent grossly visible pulmonary lesions
in some animals if only a few virulent M. tuber-
culosis cells are initially inhaled (66).
Since monkeys cough, they can readily infect
handlers and other monkeys, if appropriate pre-
cautions are not taken. Once a rhesus monkey
becomes tuberculin positive from infection with
virulent tubercle bacilli, it usually dies within 6
months (67). Because of such a rapid course,
tuberculosis in rhesus monkeys is usually a poor
model for the slowly progressive fibrotic cavi-
tary disease found in modern adult humans
and in commercially available rabbits infected
with virulent bovine-type bacilli (68, 69).The
disease in cynomolgus monkeys is a better
model.
Cynomolgus monkeys (Macaca fascicularis)
appear to be more resistant than rhesus monkeys
to infection with virulent human-type tubercle
bacilli (70, 71). When infected intratracheally
with 10 to 100 bacilli (Erdman strain), cynomol-
gus monkeys often develop a chronic, slowly
progressive granulomatous pulmonary disease
with hilar lymph node involvement and variable
degrees of tuberculin skin test positivity (70). In
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS
addition, a significant proportion of cynomol-
gus monkeys contain their infection in a non-
progressive subclinical state (70).
When 17 cynomolgus monkeys were infected
bronchoscopically with about 25 CFU of viable
tubercle bacilli (Erdman strain) (71), no clinically
apparent disease was evident in about 40% of the
animals, even though they were radiographed
periodically for 15 to 20 months. They were,
however, clearly infected, as most were tuber-
culin positive. One of these animals with arrested
tuberculosis was necropsied at 17 weeks. It con-
tained a few 1-mm fibrotic granulomas with
caseous centers and also a hilar caseous lymph
node with calcification and peripheral fibrosis.
Among the remaining 60% of the cynomol-
gus monkeys with active tuberculosis, one had
a rapidly progressive disease lasting about 2.5
months, and one had reactivated tuberculosis
with cavity formation and bronchial spread (71).
The majority of these monkeys with active dis-
ease, however, showed slowly progressive chronic
tuberculosis, characterized by granulomas with
caseous centers and outer fibrosis, accompanied
by caseous hilar lymph nodes.
In brief, cynomolgus monkeys varied con-
siderably in the type of tuberculosis produced by
infection with virulent human-type bacilli. A
substantial number (40%) of the infected animals
developed no clinically apparent disease, and a
substantial number (60%) developed a chronic
fibrotic granulomatous type similar to that found
in human populations. Cavities occurred, but
apparently not as frequently as cavities occur in
humans.
When both cynomolgus and rhesus mon-
keys were infected intratracheally with 3,000
virulent human-type bacilli (Erdman strain)
(72), the disease was less severe in the cynomol-
gus group.With this large infecting dose, prior
BCG vaccination substantially reduced the
amount of disease in the cynomolgus group, but
had little effect in the rhesus group (72).There-
fore, cynomolgus monkeys, rather than rhesus
monkeys, would be the preferred nonhuman
primate for the evaluation of newly developed
tuberculosis vaccines.These findings were sim-
ilar to those found with Lurie’s susceptible and
resistant inbred rabbits, namely, that the suscep-
䡵 259
tible rabbits benefited little from BCG vaccina-
tion (73) (see chapter 23).
See references 71, 72, and 74 for reviews of the
tuberculosis literature in nonhuman primates.
COMPARISON OF TUBERCULOSIS IN
MICE WITH THAT IN RABBITS,
HUMANS, AND GUINEA PIGS
Mouse tuberculosis is quite different from that
found in rabbits, guinea pigs, and humans.Table
2 lists the various characteristics of pulmonary
tuberculous lesions in the three laboratory ani-
mal species. Not all of these characteristics have
been directly observed, but they are listed in
Table 2 because they seem to be consistent with
the histopathology.
Mice have poor DTH, good CMI, and no
true caseous necrosis.
Mouse lesions contain a greater number of
viable virulent human-type tubercle bacilli than
do noncavitary lesions of the other species.
In mouse lesions, cycles of bacillary growth
and destruction (bacillary turnover) seem to be
reduced, because (i) most of these bacilli are in
a semidormant state within (probably activated)
macrophages (Fig. 4) (1, 2), and (ii) macrophage
turnover is probably reduced; i.e., fewer new
macrophages apparently enter from the blood-
stream and fewer are killed in the lesions (see
chapter 10). New macrophages are nonacti-
vated and provide an opportunity for renewed
intracellular bacillary multiplication. DTH
increases macrophage turnover, but mice have
only low levels of DTH.
Mouse lesions show little or no thrombosis of
the microvasculature, whereas in lesions of the
other species, microvascular thrombosis proba-
bly causes most of the caseous necrosis.
Mouse lesions never undergo liquefaction
and cavity formation, whereas the tuberculous
lesions of rabbits and humans frequently do so.
Extracellular multiplication of the bacillus often
occurs in liquefied caseum and cavities.
Despite having a much higher number of
viable tubercle bacilli in their lesions (see Table
3), mice are actually more resistant than guinea
pigs. The progression of the disease is deter-
mined by the total number of live-plus-dead
bacilli, not just the number of live bacilli (see
260 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 2 Characteristics of pulmonary tuberculous lesions in rabbits, mice, and guinea pigs
Lurie’s
resistant rabbits Lurie’s Mice Guinea
Characteristica (including susceptible (resistant pigs
commercial rabbits strains)
rabbits)
Delayed-type hypersensitivity ⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹
(DTH)
Cell-mediated immunity (CMI) ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹
Caseous necrosis (solid, not liquefied)* ⫹⫹ ⫹⫹⫹ ± ⫹⫹⫹
Number of tubercle bacilli in noncavitary ⫹ ⫹⫹ ⫹⫹⫹ ⫹
lesions*
Turnover (growth and destruction) of ⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹⫹⫹
tubercle bacilli in lesions*
Dormancy of tubercle bacilli in ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹
macrophages of lesions
Dormancy of tubercle bacilli in solid ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
caseum of lesions
Turnover of macrophages in lesions ⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹⫹⫹
Thrombosis of the microvasculature ⫹⫹⫹ ⫹⫹⫹ 0 ⫹⫹⫹
causing caseous necrosis*
Cavity formation and extracellular growth ⫹⫹ 0 0 ⫾
of tubercle bacilli*
a
This table is derived from our knowledge of tuberculous lesions produced by virulent human tubercle bacilli. Bovine-type
tubercle bacilli are more virulent for rabbits than the human type.The categories marked with an asterisk (*) would be increased
in rabbit lesions caused by the bovine type, especially the amount of caseation and number of cavities.

reference 1). Dead tubercle bacilli and live tuber-
cle bacilli produce almost identical tissue reac-
tions (6). Mouse lesions probably contain a
lower number of live-plus-dead tubercle bacilli
than do guinea pig lesions.Therefore, the disease
progresses more slowly in mice than in guinea
pigs.
Mice never arrest pulmonary lesions caused
by virulent human-type tubercle bacilli, but
rabbits and humans readily do so. The CMI
and apoptotic mechanisms (used to kill mac-
rophages overloaded with bacilli) found in mice
are probably not as effective in stopping the
progression of the disease as the CMI and
tissue-damaging DTH found in rabbits and
humans.
Mouse lesions are never encapsulated, i.e.,
completely surrounded by a fibrous tissue. In
fact, in mouse lesions, collagen fibers are actu-
ally scattered centrally among bacilli-laden
macrophages (22, 27, 28). In contrast, capsules
readily form around arrested tuberculous lesions
of rabbits and humans.
VIRULENCE OF INHALED TUBERCLE
BACILLI IN RABBITS, MICE, AND
GUINEA PIGS
In immunocompetent rabbits, fully virulent
Ravenel strains of M. bovis produce one grossly
visible tubercle for each 3 units (of 1 to 3 bacilli)
inhaled (6), whereas fully virulent H37Rv strains
of M. tuberculosis produce one such tubercle for
each 300 to 3,000 bacillary units inhaled (Table
3) (6,7,75).The Ravenel strain eventually kills the
rabbit,but the H37Rv strain rarely does so (6,75).
In both mice and guinea pigs, 3 to 6 inhaled
bacillary units of either fully virulent human- or
bovine-type tubercle bacilli apparently produces
one grossly visible tubercle (Table 3) (see refer-
ences 32, 76, and 77). In these animals, the
bovine type produces a more rapidly progressive
disease than the human type (8, 26, 32). Rabbits
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS 䡵 261

are unique in that they uniformly survive infec-
tion with the virulent human type (6, 78).
Unless the infecting bacillary strain is
markedly attenuated, the doubling times of the
infecting bacilli should not vary greatly during
their logarithmic stage of growth, whether this
growth directly follows an intravenous infection
(79) or whether it follows an aerosol infection
after the blood-borne nonactivated macrophages
prevail in the lesion. However, markedly atten-
uated bacilli do not multiply very well, even in
nonactivated macrophages (79).
After the inhalation of attenuated tubercle
bacilli, the bacillary titers would be lower in the
stationary stage, because many of these bacilli
would be killed by the alveolar macrophages, and
DTH and CMI would develop sooner. Molec-
ular biologists are producing genetically altered
tubercle bacilli of reduced virulence (see refer-
ences 80 and 81). Such bacilli should multiply to
substantially lower levels in vivo, similar to the
semivirulent M. tuberculosis R1Rv (33, 34).
In rabbits, virulent bovine-type bacilli reach
higher titers in the logarithmic stage of growth
than do human-type bacilli (Fig. 1). The loga-
rithmic stage ends when the host kills
macrophages that harbor numerous intracellular
bacilli (see chapters 2 and 5).Why the killing of
virulent bovine-type bacilli is delayed in rabbits
is not known.The secreted and constitutive anti-
gens of the bovine type may be different from
those of the human type, and/or they may be
released more slowly.When discovered, the prop-
erty of bovine-type bacilli that causes the delay
in host acquired immunity will add yet another
virulence characteristic to those already known.
BACILLARY TITERS IN THE
STATIONARY STAGE AFTER
AEROSOL INFECTION OF RABBITS,
MICE, AND GUINEA PIGS
As stated above, after inhalation, the number of
viable virulent tubercle bacilli increases loga-
rithmically in the lungs of these three laboratory
animal species for about 3 weeks and then fails
to increase further until the host approaches
the terminal stages of the disease.The bacillary

TABLE 3 Number of inhaled tubercle bacilli required to produce one primary pulmonary tubercle and the
amount of multiplication during the logarithmic growth phase in rabbits, mice, and guinea pigsa
Lurie’s
resistant rabbitsb
(including Mice Guinea pigs
commercial (C57BL/6)c
rabbits)
Average no. of inhaled virulent bovine-type 3 3–6 3–6
bacilli (Ravenel) required to produce one
pulmonary tubercle
Average no. of inhaled virulent human-type 300–3,000 3–6 3–6
bacilli (H37Rv) required to produce one
pulmonary tubercle
Increase in the number of virulent bovine-type 1,000,000 ⫻ 10,000 ⫻ No data
bacilli (Ravenel) before the stationary phase (from Fig. 1) (from Fig. 2)
Increase in the number of virulent human-type 1,000 ⫻ 10,000 ⫻ 1,000 ⫻
bacilli (H37Rv) before the stationary phase (from Fig. 1) (from Fig. 2) (from Fig. 3)
a
Adapted from reference 4. Note that, although many factors determine the amount of bacillary multiplication in the lung (see
text), species differences and genetic differences within species play important roles. This table clearly shows the importance of
tissue-damaging DTH in limiting bacillary titers: guinea pigs (with strong DTH) have lower titers than mice (with weak DTH).
Yet, despite having fewer viable bacilli, guinea pigs seem to have a more rapid progression of the disease.
b
Lurie’s resistant rabbits (e.g., strain III) (6, 7, 75). Commercially available New Zealand White rabbits seem to be similar (68, 69).
c
Data from reference 8. Bacillary multiplication in other mouse strains may be different from those listed here for C57BL/6
(see references 14 and 35). C57BL/6 is a relatively resistant strain of mice.
262 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
titer during the stationary stage is determined by
several factors: (i) the species of laboratory ani-
mal that inhaled the bacilli, (ii) the native
(genetic) resistance of the host (6, 35), (iii) the
virulence of the infecting tubercle bacilli, (iv) the
degree of immunization provided by a vaccine,
and (v) perhaps the number of bacilli initially
inhaled (69) (see chapter 11).
Bacillary titers in the stationary stage often
differ among rabbits, mice, and guinea pigs
(Table 3 and Fig. 1, 2, and 3) (4). In rabbits,
bovine-type tubercle bacilli (Ravenel) multi-
plied the most of any strain of tubercle bacillus
(1,000,000-fold), and human-type tubercle
bacilli (H37Rv) multiplied the least (1,000-
fold). In C57BL/6 mice, both types of tubercle
bacilli multiplied 10,000-fold, and such multi-
plication may be even higher in more suscep-
tible strains of mice (14, 35). In guinea pigs,
H37Rv multiplied 1,000-fold, similar to H37Rv
in rabbits. Therefore, guinea pigs (which are
generally considered the most susceptible of
these three laboratory species) stop the bacillary
increase in the logarithmic (symbiotic) stage
quite effectively (Fig. 3)—probably because
guinea pigs develop rather high DTH to the
tuberculin-like products of the bacillus.
Vaccination with BCG produces both DTH
and CMI. The stopping of the logarithmic
stage requires some DTH, because tubercle
bacilli in nonactivated macrophages are often
too numerous for the developing CMI to con-
trol. In immunized guinea pigs, the logarithmic
stage ends sooner (Fig. 3), initially due to pre-
existing DTH. However, once the stationary
stage is reached, both CMI and DTH would
play more equal roles: the CMI would prevent
renewed intracellular bacillary growth, and the
DTH would be effective whenever CMI failed
to do so.
FATE OF AEROSOLIZED BCG IN
RABBITS, MICE, AND GUINEA PIGS
It takes 300 to 3,000 inhaled virulent human-
type bacilli to produce one grossly visible pri-
mary pulmonary tubercle in commercial rab-
bits, and BCG is even less virulent (6). After
commercial rabbits inhaled several million BCG
bacilli by aerosol, only a rare primary tubercle
(1 mm or less) was visible in their lungs 5 weeks
later (S. Abramson, unpublished data, 1964 [as
cited in reference 6]). Evidently, the alveolar
macrophage population of rabbits and their
acquired immunity readily destroy this attenu-
ated bacillus before it multiplies extensively.
In contrast, BCG inhaled by mice multiplies
readily (82) and produces many pulmonary
tubercles that heal only after a period of many
weeks (83).Therefore, aerosol immunization of
mice should be quite effective, whereas aerosol
immunization of rabbits, and probably humans,
might not be (discussed further in chapter 22).
In guinea pigs, aerosolized BCG provided
substantial immunization against aerosol chal-
lenge with virulent tubercle bacilli (40, 77).
TUBERCULIN SENSITIVITY
AMONG THE THREE COMMON
LABORATORY SPECIES
The amount of DTH and CMI developed dur-
ing infection with tubercle bacilli differs among
these laboratory species (Table 1). Humans are
by far the most sensitive to tuberculin, followed
in descending order by guinea pigs, rabbits, and
mice (see DTH and PPD in the glossary).
The positive skin test is a DTH response to
antigens present in Old Tuberculin, or PPD (a
purified tuberculin). Caseous necrosis is a DTH
response to these and other antigens of the
tubercle bacillus. Usually, no necrosis is pro-
duced by the small amount of tuberculin
injected into the skin. However, in pulmonary
lesions of humans, guinea pigs, and rabbits, the
local concentrations of bacillary antigens may
reach such high levels that considerable necro-
sis occurs. Therefore, the size of the dermal
tuberculin reaction does not necessarily reflect
the amount of caseation within the pulmonary
lesions (see chapter 5).
ORGAN RESISTANCE IN
RABBITS AND GUINEA PIGS
The livers of rabbits and guinea pigs show oppo-
site susceptibilities to mycobacteria. In rabbits,
the liver is resistant and the kidneys are suscep-
tible. In guinea pigs, the liver is susceptible and
the kidneys are resistant (see chapters 7 and 25).
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS
EARLY TUBERCULOUS LESIONS
IN HUMANS
Humans are more resistant than mice and guinea
pigs to virulent human-type tubercle bacilli,
because only about 5% of recently infected
humans continue to progress to clinically active
disease (see chapter 3). In humans, each estab-
lished microscopic pulmonary tubercle develops
a small caseous center and reaches grossly visi-
ble size. However, its progression is usually
stopped while it is only 0.5 to 3 mm in diam-
eter (84–86) (reviewed in chapter 3).
CAVITY FORMATION IN
VARIOUS SPECIES
Rabbits are the main laboratory animal species
that readily forms chronic fibrotic cavities simi-
lar to those found today in adult humans (87–89)
(Table 1). Guinea pigs do not readily form cavi-
ties, probably because they die rather soon of a
hematogenously disseminated (often miliary-
type) tuberculosis.When infected by aerosol with
only 1 to 10 bacillary units, however, guinea pigs
may develop cavities (53–55). Mice never develop
true cavities, apparently because of their low sen-
sitivity to the tuberculin-like products of the
bacillus (see references 90 and 91 and chapter 4).
In both mice and guinea pigs, some local
bronchial spread of virulent human-type
(H37Rv) and bovine-type (Ravenel) tubercle
bacilli does occur 7 to 12 weeks after an aerosol
infection (12, 26). Such spread is apparently due
to a discharge of semisolid caseous necrotic ma-
terial into bronchioles and smaller bronchi. How-
ever, in both mice and guinea pigs, more distant
spread of the disease via the bronchial tree is rare.
Rhesus monkeys form cavities but rarely
develop a slowly progressive fibrotic version of the
disease. Cynomolgus monkeys do develop such
a chronic disease, but cavities apparently do not
occur as frequently as they do in humans and in
rabbits infected with virulent bovine-type bacilli.
In the human population, cavities are the
major reason why the tubercle bacillus spreads
from person to person. Drugs to prevent the liq-
uefaction of solid caseum and the formation
and progression of cavities are greatly needed,
both for therapy and for reducing the number
of new cases of this disease (92) (see chapter 25).
䡵 263
ROLE OF DTH AND CMI IN MOUSE,
GUINEA PIG, AND RABBIT
TUBERCULOSIS
Definitions: Similarities and
Differences between CMI and DTH
In brief, CMI and DTH are both Th1 lympho-
cyte responses to antigens of the tubercle bacil-
lus, and both can activate macrophages to kill or
inhibit ingested mycobacteria at the appropriate
concentration. The difference between DTH
and CMI resides in the concentration of the
antigen that produces each reaction: with DTH,
the tuberculin-like products of the bacillus acti-
vate macrophages at an extremely low concen-
tration, and at a slightly higher concentration
kill bacilli-laden macrophages and cause caseous
necrosis. With CMI, other antigenic products
of the bacillus activate macrophages to kill or
inhibit ingested mycobacteria at higher con-
centrations and do not cause caseous necrosis at
concentrations usually present in the host.There-
fore, the difference between DTH and CMI is
merely the host’s sensitivity to the antigen that
elicits these reactions. More details are presented
in chapter 5.
Species Differences
Mice, guinea pigs, rabbits, and humans control
the progress of tuberculosis by using DTH and
CMI in differing proportions (Table 1 and Fig.
10).Yet, each DTH/CMI proportion seems to
be effective in preventing the number of viable
tubercle bacilli in the lungs from increasing or
decreasing for many months.
Apoptosis and Tissue Necrosis
Both apoptosis and tissue necrosis probably play
a role in all species. However, mice seem to
have a high proportion of apoptosis; guinea pigs
and humans (with progressing disease) seem to
have a high proportion of necrosis; and rabbits
seem to have intermediate levels of necrosis.
The role of DTH in apoptosis has never been
investigated.
Intracellular and Extracellular
Bacillary Dormancy
Mice apparently develop so much CMI that
tubercle bacilli can remain dormant within
264 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 10 Overview of the characteristics of tuberculosis in laboratory animals and humans.

Note that these groups overlap because of variations among individual members. Liquefaction, cav-
ity formation, extracellular bacillary multiplication, and bronchial dissemination can overwhelm the
hosts in the middle group. CMI, cell-mediated immunity (highly activated macrophages). Caseation,
caseous tissue necrosis.Adapted from reference 4. See Table 1 for additional information.

presumably activated macrophages for months. Tuberculosis in Guinea Pigs

This type of intracellular dormancy must occur
in all species, but whether it occurs to the same
extent as in mice remains to be investigated. In
other words, the relative amounts of intracellular
dormancy in macrophages and extracellular
dormancy in solid caseum among the various
laboratory species are still unknown.
Tuberculosis in Mice
Mice develop a progressive granulomatous dis-
ease with generalized thickening and eventual
fibrosis of the lung parenchyma (11, 33).They
develop only low sensitivity to tuberculin, and
their lesions usually show relatively little necro-
sis. Since the vasculature remains patent, lym-
phocytes, macrophages, and antigen-presenting
cells are functional throughout each lesion. Mice
evidently develop good CMI and seem to kill
bacilli-laden macrophages by apoptosis, rather
than by tissue-damaging DTH.
When infected with moderate or large numbers
of virulent tubercle bacilli, guinea pigs develop
a progressive tissue-destructive type of disease,
associated with strong tissue-damaging DTH.
They develop appreciable CMI, but not enough
to arrest the disease. As in Lurie’s susceptible
strains of rabbits, bacillary multiplication in
guinea pigs seems to be mostly controlled by
DTH, which produces extensive caseous necro-
sis. Bacilli are spread by the hematogenous route,
secondary lesions form, and the disease pro-
gresses until the animal dies.
Tuberculosis in Rabbits
Rabbits (and present-day humans) develop a
spectrum of tuberculosis that varies from that
found in mice to that found in guinea pigs,
depending on the virulence of the bacillus and
the native and acquired resistance expressed by
the host. In commercially available New Zealand
 15. TUBERCULOSIS IN RABBITS, MICE,AND GUINEA PIGS
White rabbits (and in immunocompetent adult
humans), the disease produced by human-type
tubercle bacilli is usually controlled rather well,
due to an effective balance of tissue-damaging
DTH and CMI. However, with bovine-type
bacilli in rabbits (and with both types in
humans), a solid caseous focus may liquefy and
form a cavity. Then, the bacilli may multiply
extracellularly (for the first time during the
course of the disease) and may reach such large
numbers that even the strong CMI and DTH
developed by these hosts cannot prevent a fatal
progression of the disease.
Because of the differences in the pathogenesis
of tuberculosis in rabbits, mice, and guinea pigs,
all three species should be used for testing new
vaccines prior to their trials in humans. The
aerosol route of challenge is preferable to other
routes, because it measures both the microbici-
dal efficiency of the alveolar macrophage pop-
ulation (a component of innate resistance) and
the acquired immunity (DTH and CMI) pro-
duced by the vaccine.
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and A. W. Thomas. 2001. Divergent effect of
Bacillus Calmette-Guérin (BCG) vaccination on
Mycobacterium tuberculosis infection in highly related
macaque species: implications for primate models
in tuberculosis vaccine research. Proc. Natl.Acad. Sci.
USA 98:11497–11502.
73. Lurie, M. B., P. Zappasodi, E. Cardona-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
to the intracutaneous inoculation of BCG as an
index of native resistance to tuberculosis. J. Immunol.
68:369–387.
74. McMurray, D. N. 2000. A nonhuman primate
model for preclinical testing of new tuberculosis
vaccines. Clin. Infect. Dis. 30(Suppl. 3):S210–S212.
75. Allison, M. J., P. Zappasodi, and M. B. Lurie.
1962. Host-parasite relationships in natively resis-
tant and susceptible rabbits on quantitative inhala-
tion of tubercle bacilli. Their significance for the
nature of genetic resistance. Am. Rev. Respir. Dis.
85:553–569.
76. Ho, R. S., J. S. Fok, G. E. Harding, and D. W.
Smith. 1978. Host-parasite relationships in exper-
imental airborne tuberculosis. VII. Fate of Myco-
bacterium tuberculosis in primary lung lesions and in
primary lesion-free lung tissue infected as a result
of bacillemia. J. Infect. Dis. 138:237–241.
77. Middlebrook, G. 1961. Immunological aspects
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Bacteriol. Rev. 25:331–346.
78. Dannenberg, A. M., Jr. 1994. Rabbit model of
tuberculosis, p. 149–156. In B. R. Bloom (ed.),
Tuberculosis: Pathogenesis, Protection, and Control.
ASM Press,Washington, D.C.
79. North, R. J., and A. A. Izzo. 1993. Mycobacte-
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ter equipped to resist growth-inhibiting functions
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1733.
80. Jacobs, W. R. 1992. Advances in mycobacterial
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81. Bloom, B. R. 1989. New approaches to vaccine
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S466.
82. Morrison, N. E., and F. M. Collins. 1975.
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83. Nuermberger, E. L., T.Yoshimatsu, S. Tyagi,
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cibacillary tuberculosis in mice after prior aerosol
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84. Lindgren, I. 1961. Anatomical and roentgeno-
logic studies of tuberculous infections in BCG-
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85. Sutherland, I., and I. Lindgren. 1979.The pro-
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86. Lindgren, I. 1965.The pathology of tuberculous
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87. Rich, A. R. 1951. The Pathogenesis of Tuberculosis,
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88. Canetti, G. 1955. The Tubercle Bacillus in the Pul-
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89. Dannenberg,A. M., Jr., and J. F.Tomashefski,
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91. Yamamura, Y., Y. Ogawa, H. Maeda, and
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93. Dannenberg, A. M., Jr. 1984. Pathogenesis of
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94. Bishai, W. R., A. M. Dannenberg, Jr., N. Par-
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96. Manabe, Y. C., A. M. Dannenberg, Jr., S. K.
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 Section 5.

EFFECTS OF HORMONES
AND X-IRRADIATION
ON TUBERCULOSIS
This page intentionally left blank
 16
EFFECTS OF CORTISONE AND
ADRENOCORTICOTROPIC
HORMONE ON TUBERCULOSIS

Effects of pharmacological concentrations of cortisone on the development

of tuberculosis [273]
Effects of cortisone withdrawal [275]
Reactivation by glucocorticoids of healing pulmonary tubercles [279]
Effects of physiological concentrations of glucocorticoids, illustrated by
studies with adrenocorticotropic hormone (ACTH) in inbred rabbits
with small adrenals [280]
Effects of dehydroepiandrosterone (DHEA) and its derivative
3,17-androstenediol (AED) [282]
Addendum [283]

Abstract. Pharmacological amounts of glucocorticoids are frequently given as therapy for

a variety of allergic, autoimmune, and inflammatory conditions, such as asthma and
rheumatoid arthritis.When such drugs are continued for long periods of time, latent tuber-
culosis may reactivate.
In tuberculous rabbits infected with human-type tubercle bacilli, pharmacological
amounts of glucocorticoids decreased cell-mediated immunity and delayed-type hyper-
sensitivity. Macrophages were poorly activated, and tubercle bacilli grew to large numbers
within these phagocytes.After glucocorticoid administration was stopped, the tuberculin
sensitivity returned, and (because of the large numbers of bacilli) liquefaction, cavity for-
mation, tuberculous bronchopneumonia, and hematogenous dissemination occurred in
some of the rabbits.
One of Lurie’s inbred rabbit strains was evidently deficient in glucocorticoid produc-
tion. In these rabbits, the administration of physiological doses of adrenocorticotropic hor-
mone increased their resistance to tuberculosis.

Glucocorticoids are hormones that physiolog-
ically enable the host to adapt to stress (1). In
fact, rabbits with tuberculosis show hyperplasia
of the adrenal cortices (2), which helps them
control the progress of this disease. Only phar-
macological concentrations of glucocorticoids
have an adverse effect on tuberculosis (see
below). References 3 through 5 review their
effects on inflammatory processes.
EFFECTS OF PHARMACOLOGICAL
CONCENTRATIONS OF CORTISONE
ON THE DEVELOPMENT OF
TUBERCULOSIS
In a series of experiments in the 1950s, Lurie (2,
6–9) injected rabbits intramuscularly with cor-
tisone acetate (2 mg/kg) every 2 days for 38
days. This dosage produced pharmacological
glucocorticoid effects, many of which are listed
in Table 1: (i) a decrease in the number of cir-
culating lymphocytes and in the size of the
spleen (from both bone marrow suppression
and from lympholysis) (3, 4), (ii) an increase in
fasting blood sugar and a decrease in blood
ascorbic acid, (iii) an increase in liver size (from
an increase in glycogen and fat content), (iv) a
decrease in the size of the adrenals, thyroid, and
testicles (from suppression of various pituitary
hormones), (v) an increase in liver and spleen
phagocytosis of carbon particles (India ink)
injected intravenously, and (vi) a decrease in the
dermal reaction to nonspecific irritants (2, 9).

273
274 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Effects of cortisone on tuberculosis produced by the inhalation of human-type tubercle bacilli
(H37Rv) 5 weeks previouslya
Rabbits receiving cortisone Controls P values
No. of inhaled tubercle bacilli required to 35 ⫾ 2.9 120 ⫾ 22 0.002
produce one primary pulmonary tubercle
(“the ratio”)b
Average diameter of tubercles (mm) 2.5 ⫾ 0.33 5.6 ⫾ 0.24 0.000
Adrenal weight (mg/kg body weight) 66 ⫾ 6 128 ⫾ 10 0.000
Liver weight (g/kg body weight) 48 ⫾ 2.9 20 ⫾ 0.9 0.000
Spleen weight (mg/kg body weight) 310 ⫾ 20 530 ⫾ 30 0.000
Thyroid weight (mg/kg body weight) 40 ⫾ 0.5 47 ⫾ 1.3 0.000
Testes weight (mg/kg body weight) 714 ⫾ 96 1480 ⫾ 60 0.000
a
The pharmacological effects of cortisone are shown by the reduction in weight of the adrenals, spleen, thyroid, and testicles,
and by the increase in weight of the liver (see text).Adapted from reference 2.
b
“The ratio” is the number of inhaled human-type tubercle bacilli (H37Rv) divided by the number of grossly visible primary
pulmonary tubercles, usually counted at 5 weeks after infection, i.e., the number of inhaled bacilli required to produce one such
tubercle.The ratios in this table were calculated by the Lurie method (see chapter 11). In the cortisone-treated rabbits, the num-
ber of such primary tubercles was increased, i.e., fewer inhaled tubercle bacilli were needed to produce one such tubercle, but the
size of these tubercles was decreased (an anti-inflammatory effect).

On the third day after the second cortisone
injection, the rabbits were exposed to an aerosol
of virulent human-type tubercle bacilli (H37Rv)
(6). Five weeks later, the rabbits were euthanized,
and the number of primary pulmonary tuber-
cles was counted.The cortisone-treated rabbits
had 3 to 4 times the number of tubercles in their
lungs when compared with the untreated rab-
bits (Table 1 and Fig. 1). Although they were
more numerous, the tubercles in the cortisone-
treated group were much smaller (due to the
anti-inflammatory effects of the steroid) (Table
1 and Fig. 1) and contained many more tuber-
cle bacilli within live and dead macrophages,
which formed intra-alveolar plugs (Fig. 2) (2, 9).
In the cortisone-treated rabbits, the alveolar
walls remained more or less normal, whereas
those in the control rabbits were thickened by
cell infiltration and often destroyed by the
inflammatory process (Fig. 2). In the steroid-
treated group, fewer bacilli drained (via lym-
phatics) to the hilar lymph nodes (because of
reduced perifocal inflammation) (2, 9). The
steroid also reduced the size of the tuberculin
skin test reaction (2, 9).
In an experiment similar to Lurie’s cortisone
experiment, commercial rabbits were treated
with dexamethasone (10). Dexamethasone
treatment had the expected pharmacological
effects, because at 5 weeks the number of cir-
culating white blood cells as well as the weights
of the spleens and adrenal glands were decreased.
The dexamethasone-treated rabbits also showed
a decreased percentage of lung CD4 cells and
splenic CD4 and CD8 cells (10). However,
unlike Lurie’s rabbits treated with cortisone,
the number of grossly visible pulmonary tuber-
cles in the dexamethasone-treated group was not
significantly increased over the number in the
controls, and the size of these tubercles was not
significantly smaller. The difference in results
from those found by Lurie was probably because
commercial rabbits are more resistant to tuber-
culosis than Lurie’s susceptible rabbits and
because a somewhat less virulent strain of
human-type tubercle bacillus (CDC1551) was
used to infect the commercial rabbits.
In 1978, our laboratory studied the effects of
cortisone acetate (2 mg/kg every 2 days) on rab-
bit dermal BCG lesions (11), because we had
recently developed the -galactosidase histo-
chemical test for macrophage activation (12–14).
This steroid greatly reduced the size of the BCG
lesions (Fig.3),the amount of cell infiltration (Fig.
4), the number of activated macrophages present
(Fig. 4), and the size of their caseous centers
 16. CORTISONE AND ANDRENOCORTICOTROPIC HORMONE 䡵 275

FIGURE 1 Lungs of control rabbit FC 2-46 (left) and cortisone-treated rabbit FC 2⫽1
(right) 5 weeks after the inhalation of aerosolized virulent human-type tubercle bacilli (H37Rv).
The lungs are black, because shortly before the rabbits were euthanized, an intravenous injection
of carbon particles (India ink) was made to assess the phagocytic abilities in the liver and spleen.
Note that (i) fewer grossly visible primary pulmonary tubercles are present in the corticosteroid-
treated animal, (ii) their size is smaller, (iii) their caseous centers are whiter, and (iv) little perifocal
inflammation exists around the caseous centers. Reproduced with permission from reference 6.

(Fig. 5).Their dermal tuberculin reactions were EFFECTS OF CORTISONE

also reduced (Fig. 3).
In brief, pharmacological amounts of gluco-
corticosteroids decreased the ability of the
macrophages to destroy tubercle bacilli by
decreasing both the inflammatory and immune
responses of the host (3–5).
Despite these adverse effects of glucocorti-
coids on host resistance to tuberculosis, they
may be lifesaving in patients with either tuber-
culous meningitis or caseous pneumonia when
combined with effective antimicrobial therapy
(15–18; see also reference 19).
WITHDRAWAL
Lurie’s susceptible Ca rabbits were infected with
aerosolized human-type tubercle bacilli
(H37Rv) and similarly treated with cortisone for
6.5 weeks (2). Then, the glucocorticoid treat-
ment was stopped. Five weeks later, these ani-
mals died of massive caseous pneumonia, liq-
uefaction, and spread of bacilli by both bronchial
and hematogenous routes (Fig. 6). Such large
numbers of bacilli had accumulated in the lungs
of the cortisone-treated host that the disease
progressed to death. As expected, the control
276 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 A tissue section of a

primary tubercle from the lungs of
a cortisone-treated rabbit (A) and
a control rabbit (B), 5 weeks after
the inhalation of an aerosol of viru-
lent human-type tubercle bacilli
(H37Rv). These specimens were
from the same experiment as that
depicted in Fig. 1. (A) The tubercle
of the cortisone-treated rabbit shows
intra-alveolar plugs of necrotic
macrophages containing very large
numbers of tubercle bacilli.The alve-
olar walls are thin with little or no
cell infiltration, because the gluco-
corticoids reduced the inflamma-
tory response. (B) The tubercle of the
control rabbit (which received no
glucocorticoids) shows extensive
perifocal inflammation and more
mature (i.e., more homogeneous)
caseation. Relatively few bacilli were
present (not visible at this magnifi-
cation). Reproduced with permis-
sion from reference 6.

(untreated) susceptible rabbits in this experi-
ment showed regressive pulmonary tubercles
with evidence of healing (Fig. 6).All untreated
rabbits, including inbred susceptible rabbits, usu-
ally recover from infection with virulent human-
type tubercle bacilli.
The liquefaction of tuberculous lesions and
bronchial spread of the disease never occurred
before in Lurie’s susceptible rabbits.These rab-
bits apparently do not form cavities.That these
phenomena occurred in susceptible rabbits after
withdrawal of corticosteroids suggests that the
 16. CORTISONE AND ANDRENOCORTICOTROPIC HORMONE 䡵 277

FIGURE 3 Size of BCG lesions and size of dermal tuberculin reactions in cortisone-treated
and control rabbits at various times after the BCG lesions were produced. Cortisone treatment
markedly reduced the size of both the BCG lesions and the tuberculin reactions, because there
was less cell infiltration in each (see Fig. 4). Reproduced with permission from reference 11.

local presence of large numbers of tubercle
bacilli plays some role in the liquefaction process
(see chapter 4).
In a similar experiment, after corticosteroid
withdrawal, Lurie’s resistant rabbits showed a
chronic cavitary form of the disease (Fig. 7) (2).
The high resistance of these rabbits enabled them
to survive during the period of study, despite the
fact that glucocorticoid treatment had caused
large numbers of human-type tubercle bacilli
to be present in their lungs. In this experiment,
the control (untreated) resistant rabbits showed
 FIGURE 4 Tissue sections of 12-day BCG lesions from a cortisone-treated rabbit (A) and a
control rabbit (B) stained for -galactosidase, our marker enzyme for macrophage activation (12–
14). Near the necrotic center of the control lesion are large -galactosidase-positive epithelioid
cells (activated macrophages). Such mature epithelioid cells are almost absent in the lesion of the
cortisone-treated rabbit (P ⫽ 0.001). Stained with 5-bromo-4-chloro-3-indolyl--D-galactoside,
hematoxylin, and carbol-fuchsin.
Both photographs are at the same magnification (⫻155).When macrophages become activated,
they greatly increase in size. Reproduced with permission from reference 11.

278
 16. CORTISONE AND ANDRENOCORTICOTROPIC HORMONE 䡵 279

FIGURE 5 Tissue sections of 18-day BCG lesions from a cortisone-treated rabbit (A) and a
control rabbit (B), showing the size of their necrotic liquefied centers. Note that cortisone treat-
ment reduced the lesion size, the amount of cell infiltration, the amount of necrosis, and the -
galactosidase activity (recognized here by the dark-staining cells in the infiltrate). Stained with
5-bromo-4-chloro-3-indolyl--D-galactoside, hematoxylin, and carbol-fuchsin. Magnification,
⫻10. Reproduced with permission from reference 11.
The volumes of the necrotic centers illustrated were 16 mm3 in the cortisone-treated rabbit
and 46 mm3 in the control rabbit.The average volume of these centers for the six cortisone-treated
rabbits (in the 18-day BCG group of this experiment) was 5.0 ⫾ 2.5 mm3, and that for the six
control rabbits was 28 ⫾ 7 mm3 (P ⫽ 0.01) (11).

only a few minute regressive pulmonary tuber- Commercial rabbits were infected by aerosol
cles (Fig. 7). with human-type tubercle bacilli (H37Rv) and
divided into experimental and control groups.
REACTIVATION BY Ten weeks after infection, when the pulmonary
GLUCOCORTICOIDS OF HEALING lesions were healing, the rabbits in the experi-
PULMONARY TUBERCLES mental group were injected intramuscularly with
The following data are as yet unpublished (Y. C. dexamethasone (0.15 mg/kg on 6 days per
Manabe et al., in preparation). week) for the next 5 weeks.Twenty weeks after
280 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Lungs of a control rabbit of the inbred susceptible Ca strain (Ca 5-23) (left)
and a cortisone-treated rabbit of the same strain (Ca 4-7) (right).These rabbits had inhaled
aerosols of virulent human-type tubercle bacilli (H37Rv) 11 weeks previously.The corti-
sone treatment was started 4 days before infection and continued for 6 weeks during infec-
tion. Five weeks after the cortisone treatment was stopped, the rabbit died of massive
caseous pneumonia with liquefaction (shown on the right). It had both bronchial and
hematogenous spread of the disease.At this time, the control rabbit (which had received dilu-
ent alone) had regressive pulmonary tubercles with evidence of healing (shown on the left).
These studies clearly demonstrate the dangers of pharmacological glucocorticoid ther-
apy in tuberculous hosts, i.e., a marked decrease in the host’s ability to control bacillary growth
and, after hormone withdrawal, an extensive delayed-type hypersensitivity reaction to accu-
mulated bacillary products (see text).
Reproduced with permission from reference 2.

infection (5 weeks after the glucocorticoid treat- EFFECTS OF PHYSIOLOGICAL

ment was stopped), the rabbits were euthanized.
In four of the six rabbits in the experimen-
tal group, dexamethasone had allowed many
more bacilli to persist in the lesions and then
multiply after the glucocorticoid was stopped.
The host’s reaction to these numerous bacilli
made these lesions much larger than the lesions
in the controls.
Real-time PCR identified several genes that
seem to be involved in mycobacterial survival in
vivo in rabbit tuberculous lesions.
CONCENTRATIONS OF
GLUCOCORTICOIDS,
ILLUSTRATED BY STUDIES
WITH ADRENOCORTICOTROPIC
HORMONE (ACTH) IN INBRED
RABBITS WITH SMALL ADRENALS
Lurie’s susceptible FC rabbits had relatively small
adrenals, which apparently produced suboptimal
amounts of glucocorticoids (6).To stimulate the
adrenals of these FC rabbits, sheep ACTH in
gelatin (0.5 mg/kg intramuscularly) was given
daily (Table 2).The rabbits were then infected
 16. CORTISONE AND ANDRENOCORTICOTROPIC HORMONE 䡵 281

FIGURE 7 Lungs of control rabbits of the inbred resistant strains (IIIFIII 1-16 and III IIIC
1-5) on the left, and lungs of cortisone-treated rabbits of the same strains (IIIFIII 1-6 and III IIIC
1-4) on the right. These rabbits had inhaled aerosols of virulent human-type tubercle bacilli
(H37Rv) 13 weeks previously.The cortisone treatment was started 9 days before infection and
continued for 8 weeks during infection. Four and one-half weeks after the treatment was
stopped, the rabbits were euthanized.
The cortisone-treated rabbit IIIFIII 1-6 (right) had two large thick-walled cavities in the right
lung, one of which is shown in the photograph.The cortisone-treated rabbit III IIIC 1-4 (right)
had numerous active tuberculous lesions in both lungs.The two control rabbits, however, had only
a few minute regressing tubercles.
Although this experiment was similar to the one portrayed in Fig. 6, the inbred IIIFIII and
III IIIC rabbits, which were genetically much more resistant than the inbred Ca rabbits in
Fig. 6, developed fewer primary tubercles.These two factors, especially the native resistance of
the host, evidently made the adverse effects of the glucocorticoids less severe.
Reproduced with permission from reference 2.

by aerosol with virulent human-type bacilli
(H37Rv), and the ACTH injections continued
for 5 weeks, at which time the rabbits were
euthanized.The number of primary pulmonary
tubercles found in the lungs of the ACTH-
treated rabbits was about half that found in the
untreated controls (Table 2). In other words,
the ACTH reduced the number of tubercles
rather than increased them. The diameters of
these tubercles were not recorded, but apparently
they were similar to those of the controls,
because the ACTH produced no pharmaco-
logical glucocorticoid effects (Table 2) (6).
This ACTH regimen had no appreciable
effect on another inbred susceptible strain of rab-
bits (strain C), which innately had normal-size
282 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 2 Effects of adrenocorticotropic hormone (ACTH) on tuberculosis produced by the inhalation of

human-type tubercle bacilli (H37Rv)a
Sheep ACTH in Porcine ACTH
FC rabbits of low Sheep ACTH in C in FC rabbits of
resistance (with small rabbits of low resistance low resistance (with
adrenals) (with normal adrenals) small adrenals)
Treated Control Treated Control Treated Control
Number of inhaled 110 ⫾ 11* 62 ⫾ 14 50 ⫾ 7 47 ⫾ 4 70 ⫾ 19 82 ⫾ 14
tubercle bacilli
required to
produce one
primary
pulmonary
tubercle
(the ratio)b
Average diameter 2.9 ⫾ 0.2** 3.7 ⫾ 0.2
of tubercles (mm)
Adrenal weight 147 ⫾ 11 120 ⫾ 19 259 ⫾ 13 245 ⫾ 14 160 ⫾ 23 133 ⫾ 12
(mg/kg body
weight)
Liver weight 23 ⫾ 1 21 ⫾ 1 33 ⫾ 2† 25 ⫾ 1 25 ⫾ 2‡ 19 ⫾ 1
(g/kg body
weight)
Adapted from reference 6.Values significantly different from control: *P ⫽ 0.01; **P ⫽ 0.007; †P ⫽ 0.004; ‡P ⫽ 0.008.
a
b
The ratio is explained in Table 1.The larger the number of inhaled bacilli required to produce one tubercle, the fewer the
number of tubercles produced in the lung by a given inhaled dose.

adrenals (Table 2). It seems, therefore, that
additional glucocorticoids will only be benefi-
cial in hosts that are deficient in glucocorticoid
production.
To determine the physiological amount of
glucocorticoids is difficult. A porcine ACTH
in gelatin that was given to another set of FC
rabbits at the same dosage as the sheep ACTH
was not beneficial (Table 2).This porcine ACTH
preparation caused pharmacological glucocor-
ticoid effects, such as an increase in liver weight
from glycogen and fat, and a decrease in the size
of the primary pulmonary tubercles—an anti-
inflammatory effect (Table 2).
In brief, a slight increase in glucocorticoids in
a corticosteroid-deficient host increased its re-
sistance to tuberculosis, but additional gluco-
corticoids had no effect, or even an adverse
effect, on hosts with normal adrenal function.A
physiological concentration of glucocorticoids
probably increases rather than decreases the local
cytokine production that activates macrophages
to destroy or inhibit ingested tubercle bacilli,
whereas a pharmacological concentration only
decreases cytokine production.This dose-effect
principle has been established for systemic hor-
mones (19, 20) as well as for cytokines (21)
(which are local hormones). See the addendum
below.
EFFECTS OF
DEHYDROEPIANDROSTERONE
(DHEA) AND ITS DERIVATIVE
3,17-ANDROSTENEDIOL (AED)
This chapter would not be complete without
mention of DHEA and AED. These cortico-
steroids counteract the adverse effects of gluco-
corticoids on tuberculosis (22). Unfortunately,
DHEA and AED have only been evaluated in
the mouse model (22). Briefly, these antigluco-
corticoids reduced the number of mycobacte-
rial titers in the lungs of BALB/c mice, reduced
the extent of the disease, and shifted the
Th1/Th2 lymphocyte ratio toward Th1 (22).
 16. CORTISONE AND ANDRENOCORTICOTROPIC HORMONE
ADDENDUM
The physiological and pharmacological effects of
glucocorticoids are well understood (reviewed in
references 19 and 20). Physiologically, they keep
normal inflammatory and immune responses
from becoming excessive. In acute septicemic
infections, the host produces such high levels of
endogenous glucocorticoids that shock and death
may sometimes be prevented.The high physio-
logical levels of glucocorticoids evidently reduce
the production of proinflammatory and fever-
producing cytokines (e.g., interleukin-1, tumor
necrosis factor , and interleukin-6) that enter the
circulation (18, 19). Pharmacologically, gluco-
corticoids reduce both local and systemic inflam-
matory responses (again by reducing cytokine
production) and are helpful in diseases such as
asthma, arthritis, and dermatoses whenever other
therapies are ineffectual.
The adrenals of different animal species
secrete different proportions of glucocorticoids:
e.g., rabbits and rats have low ratios of 17-
hydroxycorticosterone (hydrocortisone) to cor-
ticosterone, whereas monkeys and humans have
high ratios (23). Five weeks after infection with
human-type tubercle bacilli, Lurie’s resistant
strain III rabbits apparently produced higher
levels of both hormones than did his suscepti-
ble strain FC rabbits (23).
REFERENCES
1. Seyle, H. 1946.The general adaptation syndrome
and the diseases of adaptation. J. Clin. Endocrinol.
6:117–230.
2. Lurie, M. B., P. Zappasodi,A. M. Dannenberg,
Jr., and E. Cardona-Lynch. 1953. Constitu-
tional factors in resistance to infection: the effect of
cortisone on the pathogenesis of tuberculosis, p. 84–
99. In G. Shwartzman (ed.), The Effect of ACTH and
Cortisone upon Infection and Resistance. Columbia
University Press, New York, N.Y.
3. Claman, H. N. 1983. Glucocorticosteroids I: anti-
inflammatory mechanisms. Hosp. Pract. 18:123–126,
131–134.
4. Claman, H. N. 1983. Glucocorticosteroids II: the
clinical responses. Hosp. Pract. 18:143–146, 149–
151.
5. Dannenberg, A. M., Jr. 1979. The anti-
inflammatory effects of glucocorticosteroids: a brief
review of the literature. Inflammation 3:329–343.
6. Lurie, M. B., P. Zappasodi,A. M. Dannenberg,
Jr., and E. Cardone-Lynch. 1953.The effect of
䡵 283
cortisone and ACTH on the pathogenesis of tuber-
culosis. Ann. N.Y.Acad. Sci. 56:779–792.
7. Lurie, M. B., P. Zappasodi,A. M. Dannenberg,
Jr., and I. B. Swartz. 1951. Constitutional factors
in resistance to infection: the effect of cortisone on
the pathogenesis of tuberculosis. Science 113:234–
237.
8. Lurie, M. B., and P. Zappasodi. 1955. On the
mode of action of cortisone on the pathogenesis of
tuberculosis and its implications for the nature of
genetic resistance to the disease, p. 246–258. In
Ciba Foundation Symposium on Experimental Tuber-
culosis. Churchill, London, United Kingdom.
9. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive
Mechanisms, p. 244–264. Harvard University Press,
Cambridge, Mass.
10. Kesavan, A. K., S. Mendez, C. L. Hatem,
J. Lopez-Molina, M. Brooks, R. Fujiwara,
K. Aird, M. L. M. Pitt, A. M. Dannenberg, Jr.,
and Y. C. Manabe. 2005. Effects of dexamethasone
and transient malnutrition on rabbits infected with
aerosolized Mycobacterium tuberculosis CDC1551.
Infect. Immun. 73:7056–7060.
11. McCue, R. E., A. M. Dannenberg, Jr.,
S. Higuchi, and M. Sugimoto. 1978.The effect
of cortisone on the accumulation, activation, and
necrosis of macrophages in tuberculous lesions.
Inflammation 3:159–176.
12. Yarborough, D. J., O. T. Meyer, A. M. Dan-
nenberg, Jr., and B. Pearson. 1967. Histo-
chemistry of macrophage hydrolases. III. Studies
on -galactosidase, -glucuronidase and aminopep-
tidase with indolyl and naphthyl substrates. J. Retic-
uloendothel. Soc. 4:390–408.
13. Dannenberg, A. M., Jr., O. T. Meyer, J. R.
Esterly, and T. Kambara. 1968.The local nature
of immunity in tuberculosis, illustrated histochem-
ically in dermal BCG lesions. J. Immunol. 100:931–
941.
14. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
genesis of tuberculosis: specificity, systemic and local
nature, and associated macrophage enzymes. Bacte-
riol. Rev. 32:85–102.
15. Johnson, J. R., and W. N. Davey. 1954. Corti-
sone, corticotropin, and antimicrobial therapy in
tuberculosis in animals and man: a review. Am. Rev.
Tuberc. 70:623–636.
16. Spink, W. W. 1957. ACTH and adrenocortico-
steroids as therapeutic adjuncts in infectious diseases.
N. Engl. J. Med. 257:979–983 (continued).
17. Spink, W. W. 1957. ACTH and adrenocortico-
steroids as therapeutic adjuncts in infectious diseases.
N. Engl. J. Med. 257:1031–1035 (concluded).
18. Silverstein, R., and D. C. Johnson. 2003.
Endogenous vs exogenous glucocorticoid responses
to experimental bacterial sepsis. J. Leukoc. Biol.
73:417–427.
284 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
19. Webster, J. I., and E. M. Sternberg. 2004. Role
of the hypothalamic-pituitary-adrenal axis, gluco-
corticoids and glucocorticoid receptors in the toxic
sequelae of exposure to bacterial and viral products.
J. Endocrinol. 181:207–221.
20. Webster, J. I., L.Tonelli, and E. M. Sternberg.
2002. Neuroendocrine regulation of immunity.
Annu. Rev. Immunol. 20:125–163.
21. Nathan, C. 2002. Points of control in inflamma-
tion. Nature 420:846–852.
22. Hernandez-Pando, R., M. de la Luz Streber,
H. Orozco, K. Arriaga, L. Pavon, S. A.
Al-Nakhli, and G.A.W. Rook. 1998.The effects
of androstenediol and dehydroepiandrosterone on
the course and cytokine profile of tuberculosis in
BALB/c mice. Immunology 95:234–241.
23. Kass, E. H., O. Hechter,T.W. Mou, and M. B.
Lurie. 1955. Effects of adrenal steroids on resistance
to infection. Differences in the relative amounts of
corticosterone and hydrocortisone secreted and their
biologic effects. Arch. Intern. Med. 96:397–402.
 17
EFFECTS OF ESTROGEN, CHORIONIC
GONADOTROPIN, AND THYROID
HORMONES ON TUBERCULOSIS

Effects of estrogen and chorionic gonadotropin [285]

Effects of thyroid hormones [286]

Abstract. Since estrogen increased the hyaluronic acid and water content of the skin, it
decreased the spread of intradermally injected virulent bovine-type tubercle bacilli in rab-
bits. Chorionic gonadotropin had the reverse effect.These two sex hormones had no appre-
ciable effect on the innate or acquired ability of the host to control the progression of tuber-
culosis, because neither hormone appreciably changed the number of primary pulmonary
tubercles generated by aerosols of virulent human-type tubercle bacilli. However, estro-
gen markedly suppressed the development of amyloid in the spleens of rabbits dying of
the more chronic form of tuberculosis caused by bovine-type bacilli.
In rabbits, triiodothyronine or thyroxine increased host resistance in that they decreased
the number of grossly visible primary pulmonary tubercles produced by the inhalation of
virulent human-type tubercle bacilli, whereas thyroidectomy or propylthiouracil treatment
increased the number of such primary tubercles.Thyroid hormones were most beneficial
in inbred rabbits of intermediate resistance.The resistance of the most susceptible inbred
C rabbits and that of the most resistant III(r) rabbits were not appreciably increased by thy-
roid hormones.

EFFECTS OF ESTROGEN AND extracellular matrix (reviewed in references 3

CHORIONIC GONADOTROPIN
The effects of estrogen and chorionic go-
nadotropin on tuberculosis in rabbits were stud-
ied by Lurie (1–4) because of the inferred effects
of these hormones on the disease in humans.
Latent tuberculosis tends to reactivate during
adolescence (especially in females) and also dur-
ing the first 4 months of pregnancy, when the
levels of chorionic gonadotropin in the blood
are high (4). During the last 5 months of preg-
nancy, blood estrogen levels are high, and reac-
tivation of tuberculosis rarely occurs.
Lurie injected -estradiol dipropionate (0.5
mg in sesame oil) subcutaneously into inbred
rabbits (usually his resistant strain A and his sus-
ceptible strain C) once a week throughout the
experiment (1, 4). By the second week, this
estrogen had increased the turgidity of the skin,
apparently by enhancing its hyaluronic acid
content and the amount of water bound to the
and 4). Estrogen also enlarged the nipples,
increased the size and congestion of the vulva,
and increased the weight of the uterus and
vagina (observed at necropsy) (1).
Soon after the effects of estrogen became
apparent, the rabbits were injected intrader-
mally with virulent bovine-type tubercle bacilli
(Ravenel).When compared with control rabbits
receiving only the diluent, estrogen markedly
reduced the size of the resulting dermal tuber-
culous lesion and usually reduced the spread of
disease to other sites in the body (1–3). Specif-
ically, the lungs and kidneys often showed less
extensive tuberculosis, and the other organs
often showed fewer metastatic lesions.
Estrogen did not affect the power of the host
to inhibit the growth of tubercle bacilli (3, 4),
nor did it affect the basic immunity that the host
developed (3, 4) (Table 1). After the inhalation
of virulent human-type bacilli (H37Rv), the

285
286 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Effects of estrogen on tuberculosis produced by the inhalation of human-type tubercle bacilli
(H37Rv) in female susceptible CaC rabbitsa
Estrogen-treated Controls P value
Weight of uterus 29.9 ⫾ 1.4 g 18.0 ⫾ 0.8 g 0.001
Weight of ovaries 380 ⫾ 24 mg/kg 500 ⫾ 57 mg/kg 0.038
4-h spread of India ink 687 ⫾ 44 mm2 846 ⫾ 80 mm2 0.045
4-h spread of Evans Blue dye 728 ⫾ 44 mm2 1,290 ⫾ 251 mm2 0.012
Maximum tuberculin sensitivity 78 ⫾ 13 mm3 264 ⫾ 35 mm3 0.001
Number of inhaled tubercle bacilli
required to produce one primary
pulmonary tubercle (“the ratio”)b 27 ⫾ 3.6 28 ⫾ 2.2 No difference
Average diameter of tubercles 2.6 ⫾ 1.4 mm 3.8 ⫾ 0.1 mm 0.01
a
Estrogen was given weekly, starting 2 weeks before the airborne infection, and was continued throughout the experiment.
Note that estrogen increased the weight of the uterus, decreased the weight of the ovaries, and reduced the spread in the skin of
both India ink particles and a solution of Evans Blue dye.Therefore, the hormone was active in the dosage given.The reduced
spread of these markers was apparently due to increased dermal turgidity (produced by increased hyaluronic acid and the water
it binds).Adapted from reference 4.
b
“The ratio,” a measure of both the native and acquired resistance of the host, was calculated by the Lurie method (see chap-
ter 11).The absence of a significant change in the ratio indicates that estrogen had no appreciable effect on the resistance of these
rabbits to tuberculosis.

same number of primary pulmonary tubercles
was produced in the estrogen-treated group as
in the control group (Table 1) (4).
Estrogen’s main effect on tuberculosis seemed
to be a reduction in the spread of the bacilli
through the connective tissue and a reduction
in the inflammatory response of the skin to
irritants, such as heat-killed tubercle bacilli and
pertussis endotoxin. In tuberculous hosts, estro-
gen reduced the size of the tuberculin reaction
(Table 1) (2, 4).
Unexpectedly, the estrogen treatment
almost eliminated the amount of amy-
loid in the spleens of rabbits dying of
chronic tuberculosis caused by the bovine-
type bacilli (3, 4).
The effects of chorionic gonadotropin were
the reverse of those produced by estrogen. Lurie
injected chorionic gonadotropin (0.02 to 0.2
mg) intravenously (usually into his resistant
strain A rabbits) every 10th day throughout the
experiment.This pituitary hormone causes the
ovaries to form corpora lutea, which produce
progesterone, a hormone that depolymerizes
hyaluronic acid and thereby reduces the turgid-
ity of the skin (3, 4).
In the chorionic gonadotropin-treated rab-
bits, lesions caused by the intradermal injection
of virulent bovine-type tubercle bacilli were
larger than those in control rabbits, and the
spread of the tubercle bacilli from an intrader-
mal site of injection was increased (1).
Reference 5 reviews the older literature on
the effects of these and several other hormones
on tuberculosis.
EFFECTS OF THYROID HORMONES
The effects of thyroid hormones on tuberculo-
sis in rabbits are presented in references 6, 7, and
8. The thyroid hormones L-triiodothyronine
(40 to 50 g) or L-thyroxine (100 g) were
injected intramuscularly daily (or on alternate
days) into several strains of inbred rabbits
throughout the experiment (5). After about 4
weeks, the rabbits were exposed to aerosols of
virulent human-type tubercle bacilli (H37Rv).
The rabbits were euthanized 5 weeks later, and
the grossly visible primary pulmonary tubercles
were counted.
In inbred rabbits of intermediate resistance, a
significant reduction in the number of grossly
visible tubercles was produced by treatment with
thyroid hormones (Table 2) (7, 8). These hor-
mones had much less effect on the resistance of
Lurie’s susceptible rabbits (strain C) (7) and on
the resistance of his resistant rabbits (strain III) (8).
The thyroid hormones apparently increased
both the native and the acquired resistance of the
 17. ESTROGEN, CHORIONIC GONADOTROPIN,THYROID HORMONES 䡵 287

TABLE 2 Effects of L-triiodothyronine, dinitrophenol (DNP), and L-thyroxine on tuberculosis produced by

the inhalation of human-type tubercle bacilli (H37Rv) in inbred rabbitsa
L-Triiodothyronine- DNP-treated Thyroxine-treated
treated AD rabbits AD rabbits (of IIIA rabbits
(of intermediate intermediate (of intermediate
resistance) resistance) resistance)
Treated Control Treated Control Treated Control
Change in basal 41% 11% 28% 11% 65% 1%
metabolic
rate during
infection
Maximal 206 ⫾ 50 mm3 809 ⫾ 62 mm3
tuberculin P ⬍ 0.001
sensitivity
Number of 273 ⫾ 55 155 ⫾ 22 89 ⫾ 27 155 ⫾ 22 659 ⫾ 99 154 ⫾ 27
inhaled P ⫽ 0.03 P ⫽ 0.038 P ⬍ 0.001
tubercle
bacilli
required to
produce
one primary
pulmonary
tubercle
(the ratio)b
Average 2.5 ⫾ 0.2 3.3 ⫾ 0.3 3.2 ⫾ 0.2 3.3 ⫾ 0.3 2.7 ⫾ 0.1 3.7 ⫾ 0.2
diameter of P ⫽ 0.02 P ⬍ 0.001
tubercles
(mm)
Number of Few Moderate Many Moderate Few Moderate
bacilli
Type of Interstitial More More Interstitial Interstitial More
inflammation pneumonic pneumonic pneumonic
Amount of None to Moderate Extensive Moderate None to Moderate to
caseation in slight slight extensive
the tubercles
a
Note that both thyroid hormones and DNP increased the basal metabolism of these rabbits. However, only the hormones
increased their resistance to tuberculosis. In fact, DNP decreased their resistance. Data from Table 4 in reference 8 and Table 7 in
reference 7.
b
The higher the ratio, the fewer the number of grossly visible tubercles (see footnote b in Table 1).

responding rabbits.The hormone-treated rabbits reduced in size in the hormone-treated group

had smaller tubercles that showed greater inter-
stitial inflammation, fewer bacilli, and less
caseation than tubercles in the control rabbits
(Table 2) (7). The thyroid hormones decreased
the number of tubercle bacilli present in the
lungs (Fig. 1) but increased the number of tuber-
cle bacilli reaching the hilar nodes.These find-
ings were similar to those observed in the tuber-
culosis of Lurie’s resistant rabbits (9) (see chapter
14). The tuberculin skin test reactions were
(Table 2) (7).
In another experiment, with triiodothyro-
nine treatment beginning 3 weeks after the
inhalation of virulent human-type tubercle
bacilli, the number of grossly visible primary
tubercles found at necropsy 7 weeks later (10
weeks after infection) was decreased to 36%
that of the control rabbits (Table 3) (7). This
result suggests that thyroid hormones may have
a therapeutic effect on tuberculosis.
288 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 Effect of thyroxine on the number of human-type tubercle bacilli

(H37Rv) cultured from the lungs of inbred III IIIC rabbits (which are of rather high
resistance) at various times after aerosol infection.The points represent the mean
number of viable bacilli at weekly intervals divided by the number on the first day.
Reproduced from reference 7.
Note that thyroxine treatment of these rabbits inhibited bacillary growth, espe-
cially during the first 7 days. Evidently, the pulmonary alveolar macrophages of these
hyperthyroid rabbits had an increased ability to destroy or inhibit the inhaled
bacilli.

TABLE 3 Effect on tuberculosis of triiodothyronine treatment begun 3 weeks after the inhalation of human-
type tubercle bacilli (H37Rv) and continued for 7 subsequent weeks in III IIIC rabbits of rather high
resistancea
Triiodothyronine-treated Controls P value
Number of inhaled tubercle bacilli required to
produce one primary pulmonary tubercle
(the ratio)b 1,004 ⫾ 371 359 ⫾ 64 0.05
Average diameter of tubercles (mm) 2.7 ⫾ 0.2 3.3 ⫾ 0.3 0.05
a
Note that treatment with triiodothyronine that began after pulmonary tubercles were well established at 3 weeks still reduced
the number of grossly visible tubercles found 7 weeks later to 36% of those found in controls (calculated from ratios).These find-
ings suggest that thyroid hormones increased the acquired (adaptive) resistance of the host. Data from Table 5 of reference 7.
b
The higher the ratio, the fewer the number of grossly visible tubercles (see footnote b of Table 1).
TABLE 4 Effects of thyroidectomy (Thyx) and propylthiouracil treatment (PtuRx) on tuberculosis produced by the inhalation of human-type tubercle
bacilli (H37Rv)a
Thyroidectomy
Propylthiouracil-treated
IIIA rabbits III rabbits CaC rabbits CaC rabbits

17.
(of intermediate (of high (of low (of low
resistance) resistance) resistance) resistance)

ESTROGEN, CHORIONIC GONADOTROPIN,THYROID HORMONES

Thyx Control Thyx Control Thyx Control PtuRx Control
Change in basal ⫺20% 0% ⫺20% 0% ⫺22% 0% ⫺19% 0%
metabolic P ⫽ 0.001
rate before
infection
Intradermal 1,551 ⫾ 3,050 ⫾ 2,152 ⫾ 3,359 ⫾
turpentine- 157 mm3 312 mm3 381 mm3 267 mm3
induced P ⫽ 0.001 P ⫽ 0.009
inflammation
Maximal 54 ⫾ 9 mm3 306 ⫾ 103 mm3 86 ⫾ 23 mm3 303 ⫾ 48 mm3 167 ⫾ 23 mm3 303 ⫾ 48 mm3
tuberculin P ⫽ 0.01 P ⫽ 0.001 P ⫽ 0.01
sensitivity
Weight of spleen 305 ⫾ 39 mg 461 ⫾ 35 mg 185 ⫾ 10 mg 300 ⫾ 20 mg 119 ⫾ 11 mg 339 ⫾ 24 mg 321 ⫾ 11 mg 339 ⫾ 24 mg
(mg/kg) P ⫽ 0.004 P ⫽ 0.001 P ⫽ 0.001
Weight of 12 ⫾ 0.8 mg 9 ⫾ 0.2 mg
pituitary P ⫽ 0.001
(mg/kg)
Weight of 184 ⫾ 23 mg 64 ⫾ 3 mg
thyroid P ⫽ 0.001
(mg/kg)
No. of inhaled 16 ⫾ 3.4 26 ⫾ 2.2 1,500 ⫾ 260 5,100 ⫾ 1,200 9⫾1 18 ⫾ 2 9⫾1 18 ⫾ 2
tubercle P ⫽ 0.01 P ⫽ 0.004 P ⫽ 0.001 P ⫽ 0.001
bacilli
required to
produce one

䡵
primary
pulmonary
tubercle

289
(the ratio)b (continued next page)
 290
䡵
PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
TABLE 4 Effects of thyroidectomy (Thyx) and propylthiouracil treatment (PtuRx) on tuberculosis produced by the inhalation of human-type tubercle
bacilli (H37Rv)a (continued)
Thyroidectomy
Propylthiouracil-treated
IIIA rabbits III rabbits CaC rabbits CaC rabbits
(of intermediate (of high (of low (of low
resistance) resistance) resistance) resistance)
Thyx Control Thyx Control Thyx Control PtuRx Control
Average 3.4 ⫾ 0.1 mm 3.0 ⫾ 0.3 mm 3.1 ⫾ 0.3 2.5 ⫾ 0.3 2.7 ⫾ 0.2 3.4 ⫾ 0.1 2.9 ⫾ 0.1 3.4 ⫾ 0.1
diameter of P ⫽ 0.11 P ⫽ 0.07 P ⫽ 0.001 P ⫽ 0.001
tubercles
Percent of 29 ⫾ 6 7⫾2 61.1 ⫾ 1.8 11.3 ⫾ 2.7 51.2 ⫾ 8.2 11.3 ⫾ 2.7
tubercles with P ⫽ 0.002 P ⫽ 0.001 P ⫽ 0.001
white caseous
centers
Percent of 24 ⫾ 7.8 6 ⫾ 2.4
tubercles P ⫽ 0.02
liquefied, with
or without a
cavity
a
Note that thyroidectomy had a glucocorticoid-like effect on turpentine-induced inflammation, tuberculin sensitivity, and the lymphoid tissues of the spleen. Note also that in resis-
tant strain III rabbits, a large percentage of the pulmonary lesions liquefied and/or formed cavities in the thyroidectomized group, probably because the lesions contained so many more
bacilli.This experiment lasted 15 weeks (see reference 6 for details).Adapted from Tables 1, 2, and 3 of reference 8.
b
The higher the ratio, the fewer the number of grossly visible tubercles (also see Table 1).
 17. ESTROGEN, CHORIONIC GONADOTROPIN,THYROID HORMONES
Decreasing thyroid function by thyroidec-
tomy or the administration of propylthiouracil
(averaging 100 mg subcutaneously each day
throughout the experiment) markedly decreased
host resistance to tuberculosis produced by the
inhalation of H37Rv (Table 4) (8).The effects
of the lowered metabolic rate resembled those
produced by pharmacological doses of gluco-
corticoids, i.e., increased numbers of bacilli,
decreased perifocal inflammation, decreased
drainage of bacilli to the hilar lymph nodes,
decreased size of dermal tuberculin reactions,
and decreased weight of the spleen (except in
the propylthiouracil-treated rabbits) (Table 4)
(8).These hypothyroid effects were also similar
to those found innately in rabbits with low re-
sistance to tuberculosis (8).
2,4-Dinitrophenol (roughly 10 mg/kg in
peanut oil subcutaneously each day throughout
the experiment) increased the host’s metabolic
rate but reduced (rather than increased) host
resistance (Table 2) (8).Therefore, the beneficial
effects of thyroid hormones were due not
merely to increased oxygen consumption by
the host, but also to other metabolic effects.
REFERENCES
1. Lurie, M. B., S. Abramson, and M. J. Allison.
1949. Constitutional factors in resistance to infec-
tion. I. The effect of estrogen and chorionic
䡵 291
gonadotropin on the course of tuberculosis in
highly inbred rabbits. Am. Rev.Tuberc. 59:168–185.
2. Lurie, M. B., T. N. Harris, S. Abramson, and
M. J. Allison. 1949. Constitutional factors in re-
sistance to infection. II. The effect of estrogen on
tuberculin skin sensitivity and on the allergy of the
internal tissues. Am. Rev.Tuberc. 59:186–197.
3. Lurie, M. B., S. Abramson, A. G. Heppleston,
and M. J. Allison. 1949. Constitutional factors in
resistance to infection. III. On the mode of action
of estrogen and gonadotropin on the progress of
tuberculosis. Am. Rev.Tuberc. 59:198–218.
4. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive
Mechanisms, p. 181–191. Harvard University Press,
Cambridge, Mass.
5. Lurie, M. B. 1955. On the role of hormones in
experimental tuberculosis. Adv.Tuberc. Res. 6:18–48.
6. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive
Mechanisms, p. 265–301. Harvard University Press,
Cambridge, Mass.
7. Lurie, M. B., P. Zappasodi, R. S. Levy, and
R. G. Blaker. 1959. On the role of the thyroid in
native resistance to tuberculosis. I. The effect of
hyperthyroidism. Am. Rev.Tuberc. 79:152–179.
8. Lurie, M. B., P. Zappasodi, R. G. Blaker, and
R. S. Levy. 1959. On the role of the thyroid in
native resistance to tuberculosis. II. The effect of
hypothyroidism: the mode of action of thyroid hor-
mones. Am. Rev.Tuberc. 79:180–203.
9. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
culosis in the light of the host-parasite relationships
in natively resistant and susceptible rabbits. Am.
Rev.Tuberc. 72:297–329.
 18
EFFECTS OF WHOLE-BODY
X-IRRADIATION ON
TUBERCULOSIS

Effects of X-irradiation on dermal BCG lesions [293]

Effects of X-irradiation on pulmonary alveolar macrophage
populations [293]
Effects of X-irradiation on the number of AM from granulomatous
lungs [294]
Recovery from the effects of X-irradiation [296]
Effects of X-irradiation on tuberculosis produced by the inhalation of
virulent human-type bacilli (H37Rv) [296]
Conclusions concerning the effects of 400 rads of whole-body X-irradiation
on tuberculosis in rabbits [297]
Comment on the public health consequences of medical irradiation [298]

Abstract. Commercial rabbits were irradiated with 400 rads of whole-body X-irradia-
tion—a sublethal dose. At 2 or 10 days thereafter, they were injected intradermally with
BCG. Between 2 and 4 weeks after irradiation, the BCG lesions and 48-h tuberculin reac-
tions in the irradiated group were smaller than those of the nonirradiated controls.The
BCG lesions in the irradiated group also contained more bacilli.
This dose of whole-body X-irradiation evidently decreased the supply of macrophages
and lymphocytes from “cell factories” in the bone marrow and lymphoid tissues, so that
fewer cells were available to infiltrate the BCG lesions.These cell factories had apparently
recovered 4 to 5 weeks after irradiation, because BCG lesions starting at this time were
the same size as those in the nonirradiated controls and contained the same number of
bacilli.
Pulmonary alveolar macrophages (AM) recovered by bronchoalveolar lavage (BAL) from
irradiated rabbits contained higher levels of hydrolytic enzymes than did AM from non-
irradiated controls.The AM from the irradiated group were apparently an older (more acti-
vated) cell population, because they had ingested inhaled particles for a longer period of
time. The irradiation evidently had reduced the young macrophage population that
replenishes the AM population.
When heat-killed tubercle bacilli in oil were injected intravenously 1 day after irradi-
ation, the BAL specimens obtained 9 and 10 days later (from the resulting granulomatous
lungs) contained about half as many macrophages as did those from nonirradiated con-
trols.This finding indicated that the irradiation reduced the supply of macrophages from
the bone marrow.When the heat-killed tubercle bacilli were injected 4 weeks after irra-
diation, BAL specimens from the granulomatous lungs of the irradiated and the nonirra-
diated animals contained similar numbers of macrophages, which indicated that the sup-
ply of these macrophages had recovered.
Rabbits were infected by aerosol with virulent human-type tubercle bacilli (H37Rv)
at 12 or 30 days after irradiation. In each case, 5 weeks after infection, the number of pri-
mary pulmonary tubercles in their lungs was the same in both the irradiated and the non-
irradiated groups. Also, the number of viable bacilli in these tubercles was the same.
Therefore, this sublethal dose of irradiation had no appreciable effect on the development
and progress of primary pulmonary tubercles in rabbits.
In brief,X-irradiation reduces the bone marrow’s capacity to provide defense cells to pro-
tect the host against infection.When the host is challenged by inhaled virulent human-type
tubercle bacilli,an adequate supply of defense cells is available.However,with acute infections
(requiring many more defense cells), the irradiated host would have an inadequate supply.

292
 18.
After a single dose of whole-body X-irradi-
ation in the 50% lethal dose (LD50) range (i.e.,
600 to 700 rads for rabbits and probably
humans), the recipient may live relatively well for
7 to 10 days and then develop a progressive
systemic infection that ends in death (reviewed
in reference 1). In most cases, this infection is
caused by microorganisms that normally inhabit
the gastrointestinal or respiratory tract.
In unirradiated hosts, these microorganisms
frequently gain access to host tissues, but they are
rapidly destroyed by mononuclear phagocytes
(primarily those in the liver and spleen) and
also by granulocytes that are circulating and
sequestered (mainly in the lung).
In hosts irradiated in the LD50 range, serum
and leukocyte microbicidins decline after 7 to 10
days, and the bone marrow’s supply of granulo-
cytes, macrophages, and lymphocytes becomes
low.At this time,endogenous microorganisms are
no longer destroyed, and septicemia results.Also,
at this time,or shortly afterward,the macrophages
of the host seem to be deficient in microbicidal
and other abilities.These effects are partly due to
the direct effects of irradiation and partly due to
indirect effects, such as increased intestinal per-
meability to bacterial endotoxins, partial starva-
tion, decreased microbicidal factors in the serum
(including antibodies), increased adrenal gluco-
corticoid production, and decreased granulo-
cytes, which, live or dead, may synergistically aid
macrophage function (reviewed in reference 1).
We studied the effects of sublethal whole-
body irradiation in rabbits injected with tuber-
cle bacilli, and reported our findings in a series
of four papers (1–4). They are summarized in
this chapter.
EFFECTS OF X-IRRADIATION
ON DERMAL BCG LESIONS
Rabbits were irradiated with 400 rads of whole-
body X-irradiation and injected intradermally
with BCG 2 or 10 days thereafter (1, 3). This
amount of irradiation is about 60% of the LD50.
Therefore, all of the rabbits in these experi-
ments survived.
The effect of such irradiation was clearly
demonstrated by the peripheral blood counts
(Fig. 1). During the first 2 weeks, the number of
WHOLE-BODY X-IRRADIATION 䡵 293
circulating leukocytes per mm3 of blood was
markedly reduced, mainly due to decreases in
lymphocytes and granulocytes (1, 3).The BCG
lesions and tuberculin reactions were smaller in
the irradiated group than in the controls (Fig.
2), because evidently fewer cells were available
to infiltrate them (4). In the irradiated group, the
total number of bacilli present in the entire
BCG lesion, as well as the number within each
parasitized macrophage therein, was increased.
This observation suggests that the ability of the
macrophages to destroy the bacillus was impaired
(1).The bone marrow and lymphoid tissues had
apparently recovered in 4 to 5 weeks, because
the blood counts had returned to normal (Fig.
1), and the BCG lesions and tuberculin reac-
tions, begun at this late time, showed no appre-
ciable difference in size from those in the non-
irradiated controls (3).
EFFECTS OF X-IRRADIATION
ON PULMONARY ALVEOLAR
MACROPHAGE POPULATIONS
Alveolar macrophages (AM) are the host’s ini-
tial defense against inhaled microorganisms.
They are highly activated cells (5, 6) because
they continuously ingest and digest a variety of
airborne particles (6). (The AM population eval-
uated were roughly 90% macrophages with
some lymphocytes, a few granulocytes, and pos-
sibly a few dendritic cells.)
Ten to 11 days after whole-body irradiation
with 400 rads, the rabbits were euthanized and
bronchoalveolar lavage (BAL) specimens were
obtained (2).Although a comparable number of
pulmonary AM were found in the unirradiated
and irradiated animals (75 and 67 million, respec-
tively) (Table 1), the pulmonary AM obtained
from the irradiated hosts showed increased lev-
els of a variety of hydrolytic enzymes, namely,
lysozyme, -glucuronidase, -galactosidase, acid
phosphatase, RNase, DNase, and a benzoyl-
phenylalanine--napththol esterase.
The reason for this effect on enzyme content
is not known. It was probably due to the presence
of older, more mature macrophages in the AM
population. Irradiation reduces the number of
young unstimulated macrophages from the bone
marrow that migrate via the bloodstream into the
294 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 Blood counts (cells

per mm3) in control rabbits (shaded
areas) and rabbits irradiated with 400
rads on day 0 (line graphs). The
means and their standard errors are
shown for each group.
Note the differences in the total
leukocyte count, the granulocyte
(PMN) count, and the lymphocyte
count 2 days after irradiation. PMN
were high at 2 days, probably because
the sequestered PMN (mainly in the
lungs) were released into the circula-
tion. Lymphocytes are very radiosen-
sitive, so their number markedly
decreased soon after irradiation.
During the first 2 weeks, the bone
marrow could not maintain normal
circulating leukocyte levels, but then
it gradually recovered so that the
leukocyte counts were nearly normal
at 4 to 5 weeks.
Reproduced with permission
from reference 3.

alveolar spaces (see reference 4). Therefore, the
AM population, although reduced in size 10 to
11 days after whole-body irradiation, should con-
tain a high proportion of older AM with a high
enzyme content, and this is what we found (2).
Other stimuli contributing to these increased
enzyme levels following irradiation would include
lymphocytic debris, and endotoxin and bacteria
from the gut (see above).
AM are continually ingesting dust particles,
inhaled microorganisms, and extravasated eryth-
rocytes. Such stimuli activate these cells and
increase their content of hydrolytic (and meta-
bolic) enzymes (5–7).The longer macrophages
reside in the alveolar spaces, the longer they are
exposed to such stimuli and the higher these
enzyme levels should become.The percentage
of older (highly activated) AM in the population
is increased if the entry of new young (poorly
activated) AM is reduced by irradiation (see ref-
erence 4).
EFFECTS OF X-IRRADIATION
ON THE NUMBER OF AM FROM
GRANULOMATOUS LUNGS
Commercial rabbits were irradiated with 400
rads (see Table 1). On the next day, they were
injected intravenously with 0.2 mg of heat-killed
18. WHOLE-BODY X-IRRADIATION 䡵 295

FIGURE 2 Size of the BCG

lesions and 2-day tuberculin reac-
tions in control rabbits and in rabbits
that received 400 rads of whole-body
radiation 2 days before 0.1 ⫻ 106
viable BCG were injected intrader-
mally in each of several sites. The
means and their standard errors are
shown.
Note that between 2 and 3
weeks the size of the BCG lesions
and the size of the tuberculin reac-
tions were reduced in the irradiated
rabbits, apparently because the bone
marrow could not supply the lesions
with the usual number of leuko-
cytes (mainly macrophages and lym-
phocytes).
Reproduced with permission
from reference 1.
296 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Effect of whole-body irradiation on the number of rabbit pulmonary alveolar macrophages (AM)
obtained by bronchoalveolar lavage (BAL) after an intravenous injection of heat-killed tubercle bacilli in oila
Experiment Rabbit group No. of AM obtained (106)
Effect of irradiation Normal noninjected 75 ⫾ 13
Irradiated noninjected 67 ⫾ 21 (P ⫽ 0.375)
Normal injected 317 ⫾ 54
Irradiated injected 161 ⫾ 16 (P ⫽ 0.007)
Effect of bacillary injection Normal noninjected 75 ⫾ 13
Normal injected 317 ⫾ 54 (P ⬍ 0.001)
Irradiated noninjected 67 ⫾ 21
Irradiated injected 161 ⫾ 16 (P ⫽ 0.001)
a
Eight rabbits in each group received 400 rads of whole-body X-irradiation on day 0.They were injected intravenously with
lyophilized heat-killed tubercle bacilli (BCG) in mineral oil on day 1 and killed for BAL on day 10 or 11.The means and their
standard errors are listed.Adapted from reference 2.

lyophilized tubercle bacilli (BCG) in mineral oil.
They were euthanized 10 or 11 days after radi-
ation and then bronchoalveolar lavaged (usually
three times) (2).At 10 and 11 days the lungs con-
tained many small granulomatous lesions.
Irradiation had no effect on the number of
AM obtained by BAL from rabbits that did not
receive the intravenous bacillary injection (Table
1) (2). However, irradiation reduced to half the
larger number of macrophages obtained after the
bacillary injection (Table 1) (2).
In the unirradiated rabbits, the intravenous
bacillary injection increased 4.2-fold the num-
ber of macrophages in the lavages (Table 1) (2).
In contrast, in the irradiated rabbits, the bacil-
lary injection increased only 2.4-fold the num-
ber of macrophages in the lavages (Table 1) (2).
Therefore, the number of macrophages available
to migrate (from the bone marrow) into the
lungs in response to this bacterial stimulus was
markedly reduced by irradiation.
In the unirradiated group, the intravenous
injection of heat-killed tubercle bacilli increased
the levels of several enzymes per million AM, but
in the irradiated group, the intravenous bacillary
injection caused no further enzyme increase (2).
Apparently, the AM enzymes in the irradiated
group were at maximal levels before the bacillary
injection.
RECOVERY FROM THE EFFECTS
OF X-IRRADIATION
By 4 to 7 weeks, the rabbits had apparently
recovered from the effects of whole-body radi-
ation (1–3). (i) Their peripheral blood counts had
returned to normal (Fig. 1) (1, 3). (ii) Their
BCG lesions, begun 4 to 5 weeks after the irra-
diation, were similar in size to the controls and
histologically contained comparable numbers
of bacilli (3). And (iii) the number of their
macrophages obtained by BAL from the gran-
ulomatous lungs was also similar to that of con-
trols (2).
Recovery from the effects of X-irradiation in
the LD50 range in other laboratory animals is
reviewed in reference 3. The recovery times
varied from 3 weeks to more than 8 weeks.
EFFECTS OF X-IRRADIATION ON
TUBERCULOSIS PRODUCED BY THE
INHALATION OF VIRULENT HUMAN-
TYPE BACILLI (H37Rv)
Three experiments were performed. In each, the
rabbits were irradiated with 400 rads. In Exper-
iment I, the rabbits were infected by aerosol
with virulent human-type tubercle bacilli
(H37Rv) 8 days after irradiation. Six to 8 days
after infection, the lungs were cultured for viable
tubercle bacilli. No appreciable difference was
 18.
found in the number of tubercle bacilli cul-
tured from the lungs of the control and the
irradiated rabbits (3).
In Experiment II, the rabbits were infected by
aerosol with H37Rv 12 days after irradiation (3).
Five weeks later, the number of grossly visible
tubercles in the lungs was the same in both the
control and irradiated groups, as were the size of
these tubercles and the number of culturable
bacilli in them.
In Experiment III, the rabbits were infected
by aerosol 30 days after irradiation (3). Five
weeks later, the tubercle count, tubercle size, and
number of culturable bacilli per tubercle were
also the same in both the control and irradiated
groups.
These findings suggest that resident AM are
rather resistant to X-radiation (2, 3).This pop-
ulation is composed of highly activated (differ-
entiated) macrophages (5, 6), which in rabbits
can destroy many inhaled virulent human-type
tubercle bacilli (3).
In our inhalation experiments, the highly
activated AM population ingested the bacilli in
both the irradiated and control rabbits. In our
intravenous experiments with heat-killed tuber-
cle bacilli, the AM population was bypassed,
because the lesions began in the pulmonary
capillaries where only nonactivated macrophages
exist. Also, in our intravenous experiments,
numerous pulmonary granulomas were pro-
duced, whereas in the inhalation experiments,
relatively few pulmonary lesions were produced.
Therefore, the absence of any recognizable effect
of 400 rads of X-irradiation on tuberculosis
(produced in rabbits by aerosols of virulent
human-type tubercle bacilli) could be explained
(i) by the high activation of the AM population
initially ingesting the inhaled bacilli, and (ii) by
the relatively small number of pulmonary lesions
in such rabbits.
In mice, 2 weeks after irradiation, most of the
radioresistant AM population had apparently died
off and was not appreciably replaced by new
macrophages from the bone marrow (reviewed in
reference 3). At this time, but not before, the
mice had a reduced ability to control the growth
of inhaled (rapidly growing) virulent strepto-
WHOLE-BODY X-IRRADIATION 䡵 297
cocci (8).The delayed detrimental effect of irra-
diation on the AM population of mice was not,
however,evident in rabbits inhaling (slowly grow-
ing) virulent human-type tubercle bacilli (3).
CONCLUSIONS CONCERNING THE
EFFECTS OF 400 RADS OF WHOLE-
BODY X-IRRADIATION ON
TUBERCULOSIS IN RABBITS
1. Whole-body irradiation with 400 rads
decreases the production of macrophages,
lymphocytes, and other cells by the bone
marrow and lymphoid tissues, so that
fewer defense cells are available to infiltrate
early dermal BCG lesions (4).
2. By 30 days after this whole-body irradi-
ation, the production of macrophages,
lymphocytes, and other cells had recovered
almost completely, with few, if any, resid-
ual effects.
3. X-irradiation with 400 rads on 8, 12, or 30
days before aerosol infection with virulent
human-type bacilli (H37Rv) had little or
no effect on the establishment and progress
of the resulting pulmonary lesions. (i) The
AM population was rather radioresistant.
(ii) Early pulmonary lesions contained rel-
atively few bacilli, compared with dermal
BCG lesions. And (iii) after this dose of
sublethal X-irradiation, macrophages and
lymphocytes were probably still produced
in sufficient numbers by the bone marrow
and lymphoid tissues.
4. In rabbits, one of the most sensitive indi-
cators of radiation injury was the bone
marrow’s capacity to provide large
quantities of macrophages.These large
quantities were required to produce the
multiple pulmonary granulomas that fol-
lowed an intravenous injection of numer-
ous heat-killed tubercle bacilli. One to 2
weeks after such an intravenous injection,
the number of macrophages obtained by
BAL was reduced 50% in the irradiated
rabbits (when compared with the number
in the nonirradiated controls).When lungs
containing no granulomas were lavaged,
similar numbers of macrophages were
298 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
obtained from both the control and the
irradiated groups. In other words, after
400 rads of whole-body radiation, the
bone marrow function should be quite
adequate if the host is not challenged by
numerous microorganisms.
COMMENT ON THE PUBLIC HEALTH
CONSEQUENCES OF MEDICAL
IRRADIATION
Cells differ in their sensitivity to irradiation.
Sperm production and egg production in the
reproductive organs are affected by a few rads.
Lymphocytes in the lymphoid tissues are injured
by 50 rads. Myeloblasts (producing macrophages
and granulocytes in the bone marrow) are injured
by 500 rads.
Because of these differing sensitivities, a lower
GI series, i.e., gastrointestinal X-irradiation (usu-
ally 5 rads) following barium installation, should
be avoided if possible in young people hoping
eventually to have children. However, the
adverse effects of such irradiation on dormant
ova (in contrast to the adverse effects on devel-
oping ova [9] and fetuses) have not been eval-
uated in human populations.
REFERENCES
1. Kambara, T., S. Chandrasekhar, A. M. Dan-
nenberg, Jr., and O.T. Meyer. 1970. Radiation,
infection and macrophage function. I. Effects of
whole body radiation on dermal tuberculous lesions
in rabbits: development, histology and histochem-
istry. J. Reticuloendothel. Soc. 7:53–78.
2. Meyer, O.T., and A. M. Dannenberg, Jr. 1970.
Radiation, infection and macrophage function. II.
Effect of whole body radiation on the number of
pulmonary alveolar macrophages and their levels of
hydrolytic enzymes. J. Reticuloendothel. Soc. 7:79–90.
3. Dannenberg, A. M., Jr., W. G. Roessler, O. T.
Meyer, S. Chandrasekhar, and T. Kambara.
1970. Radiation, infection and macrophage func-
tion. III. Recovery from the effects of radiation
illustrated by dermal BCG lesions: resistance of
pulmonary alveolar macrophages to radiation illus-
trated by tuberculosis produced by the airborne
route. J. Reticuloendothel. Soc. 7:91–108.
4. Chandrasekhar, S., K. Shima, A. M. Dannen-
berg, Jr., T. Kambara, J. I. Fabrikant, and
W. G. Roessler. 1971. Radiation, infection and
macrophage function. IV.The effect of radiation on
the proliferative abilities of mononuclear phagocytes
in tuberculous lesions of rabbits. Infect. Immun.
3:254–259.
5. Dannenberg, A. M., Jr., M. S. Burstone, P. C.
Walter, and J.W. Kinsley. 1963.A histochemical
study of phagocytic and enzymatic functions of
rabbit mononuclear and polymorphonuclear exu-
date cells and alveolar macrophages. I. Survey and
quantitation of enzymes, and states of cellular acti-
vation. J. Cell Biol. 17:465–486.
6. Mizunoe, K., and A. M. Dannenberg, Jr. 1965.
Hydrolases of rabbit macrophages. III. Effect of
BCG vaccination, tissue culture, and ingested tuber-
cle bacilli. Proc. Soc. Exp. Biol. Med. 120:284–290.
7. Dannenberg, A. M., Jr., P. C.Walter, and F. A.
Kapral. 1963. A histochemical study of phago-
cytic and enzymatic functions of rabbit mononu-
clear and polymorphonuclear exudate cells and
alveolar macrophages. II. The effect of particle
ingestion on enzymes activity; two phases of in vi-
tro activation. J. Immunol. 90:448–465.
8. Shechmeister, I. L., V. P. Bond, and M. N.
Swift. 1952.The susceptibility of irradiated mice
to infection as a function of post-irradiation time.
J. Immunol. 68:87–95.
9. Pearson, R. 1989. Radiography in women of
childbearing ability. Br. Med. J. 299:1175–1176.
 Section 6.

CYTOKINES AND VASCULAR ADHESION

MOLECULES IN TUBERCULOUS LESIONS
This page intentionally left blank
 19
CYTOKINE PRODUCTION IN
PRIMARY BCG LESIONS

Cytokine mRNA in cells within tissue sections of BCG lesions [304]

Cell types containing MCP-1, IL-1, IL-8, and TNF- mRNAs [304]
Immunohistochemical studies for cytokine proteins [305]
IFN- mRNA identified by RT-PCR [305]
Comparisons among the five cytokines evaluated [306]
Studies on cytokines produced by tuberculosis in humans and
in mice [307]
Conclusions: nonspecific and antigen-specific cytokine production [309]

Abstract. A sequential histochemical study of cytokines in developing and healing rab-

bit tuberculous (BCG) lesions is described. In tissue sections, interleukin-1 (IL-1), tumor
necrosis factor alpha (TNF-), macrophage chemoattractant (activating) protein 1 (MCP-
1), and IL-8 were evaluated for cytokine mRNA by in situ hybridization techniques and
for cytokine protein by immunohistochemical techniques. In tissue homogenates, gamma
interferon (IFN-) mRNA was evaluated by reverse transcription-PCR.
In the BCG lesions, the percentage of mononuclear cells that contained the mRNAs
of these cytokines showed a biphasic pattern.At 1 to 3 days, a peak occurred, which was
apparently a nonspecific inflammatory response caused by the tubercle bacilli in the BCG
vaccine.At 5 days, the percentage of mononuclear cells containing the cytokine mRNAs
was significantly reduced, but by 9 days, the percentage had again increased, and the rab-
bits had become tuberculin positive.This second peak was apparently antigen specific.With
IFN-, the two mRNA peaks were delayed by 2 days.
Mononuclear cells containing IL-1 and IL-8 mRNAs were more numerous sur-
rounding the caseous center.These cytokines evidently recruited the polymorphonuclear
leukocytes that were common in this location. Mononuclear cells containing MCP-1
mRNA were more numerous in the outer third of the lesion where new macrophages and
lymphocytes were being recruited.
Both the nonspecific and antigen-specific cytokine responses of BCG vaccines are evi-
dently synergistic.The early nonspecific cytokine (chemokine) response causes a local accu-
mulation of antigen-presenting cells and lymphocytes, which explains, at least in part, why
tubercle bacilli are good immunological adjuvants.This adjuvant effect should be consid-
ered in developing improved vaccines for the prevention of tuberculosis, because vaccines
producing a strong early nonspecific cytokine (chemokine) response should be more
immunogenic than vaccines with similar antigens producing a weak response.

Cytokines from macrophages, lymphocytes, and
other cells (including vascular endothelial cells
and local fibroblasts) play major roles in the
pathogenesis of tuberculosis.A large number of
cytokines exist (listed in references 1 to 3), and
more are being discovered every year.This chap-
ter describes sequential changes in the mRNAs
and/or proteins of five major cytokines during
the 5-week period when rabbit dermal BCG
lesions developed and healed.
Interleukin 1 (IL-1) and tumor necrosis factor
alpha (TNF-) are primary cytokines that up-
regulate the production of other cytokines (4–6).
The chemokine macrophage chemoattractant
(activating) protein 1 (MCP-1) (7–13) attracts
mononuclear cells (macrophages and lympho-
cytes) into tuberculous lesions, and the chemo-
kine interleukin 8 (IL-8) attracts granulocytes.
Gamma interferon (IFN-) (14–18) and TNF-
(4, 5, 19–21) play major roles in activating

301
302 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 1 The percentage of mononuclear cells labeled for IL-1 (A),TNF- (B), MCP-1
(C), and IL-8 (D) mRNAs in BCG lesions at various times during their development and regres-
sion. Note the biphasic pattern of these cytokine mRNAs.The first peak is due to nonspecific
irritation by the injected BCG.The second peak is probably due to the development of antigen-
specific immunity.The means and their standard errors are shown. Reproduced with permission
from reference 11.
 19. CYTOKINE PRODUCTION IN PRIMARY BCG LESIONS 䡵 303

FIGURE 1 Continued.

macrophages (22), so that the macrophages can
inhibit and/or kill tubercle bacilli (16, 18, 24). See
glossary for additional information on various
cytokines.
Tuberculosis is a locally controlled disease, i.e.,
the host arrests the disease locally at the sites
where bacilli are present (see chapter 5) (25).
Our findings are presented as a percentage of
mononuclear cells (MN) (mainly macrophages
with some lymphocytes and probably some
dendritic cells) that are labeled for cytokine
mRNA (or for cytokine protein) in the densely
cell-infiltrated areas of the BCG lesions.A local
increase in the percentage of cells labeled for
304 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
chemokines indicates that more MN will be
attracted to the area and that the lesion will
enlarge. Conversely, a local decrease in the per-
centage of cells labeled for chemokines indicates
that fewer MN will be attracted to the area and
that the lesion will enlarge more slowly or even
regress, depending on the rate of turnover of the
MN (see chapter 10).
Cytokines are local hormones that primar-
ily act on nearby cells. In tuberculous lesions,
two adjacent macrophages may even produce
different cytokines (see reference 26 and chap-
ter 6). In tissue sections, in situ hybridization
and immunohistochemical methods for identi-
fying cytokine mRNA and protein allow us to
evaluate the distribution of cytokines to dif-
ferent parts of tuberculous lesions as they
develop and regress. In other words, the cells
producing certain cytokines can be identified
along with the nearby cells they have recruited
and activated. In homogenates of tuberculous
lesions, such cell-cell interaction would not be
recognized.
CYTOKINE mRNA IN CELLS WITHIN
TISSUE SECTIONS OF BCG LESIONS
Methods
In tissue sections of rabbit BCG lesions of vari-
ous ages, cells were identified with cytokine 35S-
labeled antisense mRNAs by in situ hybridization
techniques followed by autoradiography (6, 11).
Cytokine 35S-labeled sense RNAs were used as
(negative) controls. Cells were considered labeled
when they contained three or more silver grains
above the number found in the background.
Cytokine proteins were identified by immuno-
histochemical techniques (6, 11).
The Biphasic Pattern
The percentage of MN labeled for IL-1,TNF-
, MCP-1, and IL-8 mRNAs showed a bipha-
sic pattern as these lesions developed (Fig. 1A to
D), i.e., a large initial peak at 1 to 3 days, fol-
lowed by a trough at 5 days, and then another,
smaller peak.The initial MCP-1 mRNA peak
indicated that this MN-attracting chemokine
was a major mediator of the early nonspecific
cell infiltration caused by BCG. The smaller
second peak was apparently caused by the devel-
opment of cell-mediated immunity and delayed-
type hypersensitivity.
The Distribution of Labeled Cells
At 3 days, all of the MN labeled for four cytokine
mRNAs were located in the exudate between the
collagen fibers.However,from 5 days on,after dis-
tinct areas of necrosis were established, the MN
labeled for each of the four cytokine mRNAs had
their own distribution patterns. (i) Cells con-
taining IL-1 and IL-8 mRNAs were found
near the necrotic centers and sometimes even
within such centers (Fig. 2). Therefore, IL-1
and IL-8 were probably involved in the recruit-
ment of the polymorphonuclear leukocytes
(PMN) that accumulated near the caseous cen-
ter. (ii) MN containing TNF- mRNAs were
more widely distributed than those containing
IL-1 and IL-8 mRNAs, and were most numer-
ous among the infiltrating cells of 3-day BCG
lesions. (iii) The majority of the MN containing
MCP-1 mRNA were found in the outer third of
the areas densely infiltrated with cells. These
labeled MN were closely associated with the
microvasculature, a propitious location for cells
producing chemoattractants. Cells containing
MCP-1 mRNA were also present more periph-
erally in the highly vascularized areas located
between the hair follicles (see chapter 8).
CELL TYPES CONTAINING MCP-1,
IL-1, IL-8, AND TNF- mRNAs
In BCG lesions, MCP-1 mRNA was frequently
found in MN and occasionally in vascular
endothelial cells, but never in PMN (6, 11). IL-
1 and IL-8 mRNAs were found mainly in
MN and occasionally in PMN.TNF- mRNA
was found only in MN (6, 11).
In healing (37-day) lesions, large MN were
often labeled for MCP-1 mRNA. However, in
the 37-day lesions, mature epithelioid cells were
not labeled for MCP-1 (or IL-1, TNF-, or
IL-8) mRNA. In other words, fully activated
macrophages were apparently programmed to
destroy or inhibit tubercle bacilli rather than to
produce cytokines.
The keratinocytes of the epidermis and hair
follicles were occasionally labeled for IL-8
mRNA, but not for the other three cytokines.
 19. CYTOKINE PRODUCTION IN PRIMARY BCG LESIONS
IMMUNOHISTOCHEMICAL STUDIES
FOR CYTOKINE PROTEINS
BCG lesions of various ages were stained by
immunohistochemical techniques for MCP-1
and TNF- proteins (11) (Table 1). The per-
centage of cells containing the proteins of these
two cytokines was highest at 1, 3, and 5 days and
reduced at 9 days. No decrease occurred in the
percentage of MN staining positive for these
cytokine proteins at 5 days, even though the per-
centage of MN containing the mRNAs was
decreased at that time (see Fig. 1). Evidently,
cytokine proteins have a slower turnover rate
than the mRNAs that produce them. Perhaps,
TABLE 1 Percentage of mononuclear cells stained
for MCP-1 and TNF-a
Age of BCG MCP-1
lesions (days) protein
1 ⫹⫹⫹
3 ⫹⫹⫹
5 ⫹⫹⫹
9 ⫹⫹
23 ⫾ ⫺
37 ⫹ ⫺
a
Symbols: ⫺ ⫽ 0–0.5%; ⫾ ⫽ 0.5–1%; ⫹ ⫽ 1–2%; ⫹⫹ ⫽
2–5%; ⫹⫹⫹ ⫽ ⬎5%.Adapted from reference 11.The absence
of a biphasic response suggests that these cytokine proteins have
a slower turnover rate than their mRNAs.
䡵 305
FIGURE 2 A 16-day dermal BCG lesion
in situ hybridized for IL-8 mRNA. Lique-
fied caseum is in the left lower part of the
photograph. Solid caseum is shown at the far
right of the photograph. Large macrophages
in the liquefied caseum adjacent to the solid
caseum are heavily labeled for IL-8 mRNA.
IL-8 is a major chemokine that attracts
PMN, and numerous PMN are present
throughout the liquefied material. Fixed-
frozen tissue section hybridized with anti-
sense IL-8 35S-labeled RNA, autoradi-
ographed, and counterstained with Giemsa.
Magnification, ⫻350. Reproduced with
permission from reference 11.
the cytokine proteins are stabilized by compo-
nents of the extracellular matrix (27).
The cells stained for MCP-1 protein were
mainly large macrophages (Fig. 3A and B).At 23
and 37 days, some microvascular endothelial
cells stained for MCP-1 protein, but none
stained for TNF- protein.
In tissue sections of BCG lesions, the MN
staining positive for MCP-1 and TNF- pro-
teins showed the same distribution pattern as the
MN containing MCP-1 and TNF- mRNAs.
This finding further supports the conclusion
that cells containing cytokine mRNA were
actively producing cytokine protein.
IFN- mRNA IDENTIFIED BY RT-PCR
We were unable to demonstrate rabbit IFN- in
tissue sections of dermal BCG lesions by in situ
hybridization techniques.Therefore, the reverse
transcription-polymerase chain reaction (RT-
TNF- PCR) (see glossary) was used to measure IFN-
protein mRNA in BCG lesions of various ages (11).
⫹
The ratio of the IFN- mRNA to the “house-
⫹⫹
keeping” GAPDH (glyceraldehyde-3-phosphate
⫹⫹
dehydrogenase) mRNA was highest at 5 days,was
⫾
decreased at 7 days, and was increased again at
9 days (Fig. 4).The 9-day level was maintained at
23 days, which is when the BCG lesions were
largest in size (11).Thus, similar to the mRNA
levels of the four cytokines represented in Fig. 1,
the IFN- mRNAs showed a biphasic pattern,
but it was somewhat delayed.
306 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 3 (A) An unfixed-frozen

tissue section of a 3-day dermal
BCG lesion stained immunohisto-
chemically for MCP-1 protein.
Large mononuclear cells in the
partly necrotic exudate between
the remaining collagen fibers are
darkly stained for MCP-1 protein.
Anti-rabbit MCP-1 polyclonal anti-
body, biotinylated anti-guinea pig
IgG, and the avidin-biotin-peroxi-
dase procedure, counterstained with
Giemsa. Magnification, ⫻200.
Reproduced with permission from
reference 11. (B) A lightly fixed
tissue section of a 3-day dermal
BCG lesion embedded in glycol
methacrylate and stained with
Giemsa. This photograph shows
some of the histologic detail that is
absent in frozen sections. In the cen-
ter and to the left are a few large
mononuclear cells that are similar
to those stained histochemically in
panel A. Note the frequent occur-
rence of PMN in the 3-day BCG
lesions. Magnification, ⫻460. Repro-
duced with permission from refer-
ence 11.

COMPARISONS AMONG THE FIVE However,the changes in the mRNA levels of

CYTOKINES EVALUATED
With in situ hybridization, the antisense RNA
probes for one type of cytokine do not necessarily
hybridize with the same affinity as do the anti-
sense probes for another type of cytokine.There-
fore, in Fig. 1, the percentages of MN containing
IL-1,TNF-, MCP-1, and IL-8 mRNAs can-
not be precisely compared with one another,
nor can the in situ hybridization technique be
precisely compared with the RT-PCR technique
used to measure lesion IFN- mRNA.
each cytokine can be compared as the BCG
lesions developed and healed (Fig. 1, 4, and 5).
With all five cytokines, the highest levels of
mRNA seemed to occur early.Subsequently,the
mRNAs decreased, but, during the immune
phase,the number of MN containing IFN- and
IL-8 mRNAs seemed to remain higher (relative
to the early peak) than the number containing IL-
1,TNF-,and MCP-1 mRNAs (Fig.1 and 4).
As stated above, IFN- and TNF- activate
macrophages to destroy or inhibit the tubercle
 19. CYTOKINE PRODUCTION IN PRIMARY BCG LESIONS
bacilli that they phagocytize. IL-8 is produced by
macrophages, but its major function is to attract
and activate granulocytes (mainly PMN) (7).
PMN are known to accumulate in areas of bac-
terial invasion and around areas of necrosis (28).
Therefore, the high levels of IL-8 mRNA on day
3, when the BCG bacilli are numerous, and on
days 9 and 23, when necrosis is present, would
be expected (Fig. 1D). At 37 days, when tissue
necrosis was much reduced and the ulcer (in
the center of the lesion) was becoming epithe-
lialized, the levels of IL-8 seemed to be decreased.
STUDIES ON CYTOKINES
PRODUCED BY TUBERCULOSIS
IN HUMANS AND IN MICE
The role of cytokines in tuberculosis is a very
active field of investigation. Here, we list a few
of these studies with comments on their appli-
cation to the pathogenesis of the human disease.
Humans
The human monocytic-leukemia cell line THP1
was infected with Mycobacterium tuberculosis
(H37Rv) at an average of 10 bacilli per
macrophage (29). In these cells, the mRNAs of
numerous chemokines were increased several-
fold during the first 6 to 12 h after infection.
These in vitro results with human macrophages
䡵 307
FIGURE 4 Changes in IFN- mRNA
in rabbit BCG lesions, relative to those of
GAPDH mRNA (see text). (The amount of
GAPDH mRNA in cells is known to
remain rather constant without wide fluc-
tuations, as the cells up- and downregulate
their various activities.) Note that the bipha-
sic response was somewhat delayed when
compared with that of the other cytokine
mRNAs shown in Fig. 1.This delay suggests
that the initial mononuclear cells produced
cytokines (including chemokines) that sub-
sequently upregulated the IFN- produc-
tion in other cells (mostly in nearby lym-
phocytes). ***P ⬍ 0.01 for day 5 vs. day 7.
The means and their standard errors are
shown. Reproduced with permission from
reference 11.
are consistent with the early nonspecific in vivo
cytokine response found in the macrophages
of rabbit BCG lesions described above.
Several investigators evaluated the cytokines
at the sites of active tuberculosis in humans
(reviewed in references 30, 31, and 32). In tuber-
culous pleuritis (a self-healing form of human
tuberculosis), cytokine proteins and mRNAs
were found in the pleural exudates and/or in
pleural tissue biopsies. The cytokines present
were TNF (33), IFN- (33, 34), IL-10 (34), IL-
12 (35), and others. In tuberculous granulomas
within human lungs, IL-1 protein (36) and
TGF- (37) were present. None of these stud-
ies, however, evaluated changes in cytokine
mRNA or protein as the lesions progressed and
regressed.
Other investigators evaluated the cytokines in
peripheral blood mononuclear cells (PBMC)
from tuberculous patients. With the RT-PCR
technique, IL-1, IL-8, TNF-, and IFN-
mRNAs were found in PBMC of these patients
but were absent or reduced in PBMC from
healthy tuberculin-negative persons (38).When
cultured 24 h in the presence of tubercle bacilli,
the PBMC of tuberculous patients produced less
IFN- mRNA and IFN- protein than did the
PBMC of healthy tuberculin-positive controls
(39). Also, more TGF-, an immunosuppressive
308 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 5 Summary of the histologic characteristics and cytokines in early and peak rabbit
dermal BCG lesions. Note that the cytokines upregulated in the early lesions were downregulated
by the time the lesions peaked at 14 to 23 days. Reproduced with permission from reference 11.

cytokine, was produced by the PBMC of patients Mice

with tuberculosis than by the PBMC of healthy
tuberculin-positive controls (32, 37, 40).
Although analyses of human PBMC may be
prognostically useful, they do not always reflect
the situation in the tuberculous lesions them-
selves. For example, in hosts with poor resistance,
an increase in suppressor cytokines may reflect
(and be the cause of) the progression of the
local disease. On the other hand, in hosts with
good resistance, an increase in such suppressors
may reflect the regression or control of the local
disease, because the further accumulation of
host defense cells may no longer be needed and
may even be detrimental.
Cytokines participating in mouse tuberculosis
are described in references 41 through 49.
Mouse lungs, infected by aerosol with virulent
M. tuberculosis, contained the mRNAs of IL-1,
TNF-, IL-2, IFN- and the chemokines
RANTES, MCP-1, MCP-3, MIP-1, MIP-2,
and IP-10. To measure the mRNAs of these
cytokines, lung homogenates were prepared,
the RT-PCR was used.The amount of cytokine
mRNA was compared with the amount of a
“housekeeping” gene.The highest levels of most
cytokine mRNAs in the mouse tuberculous
lungs occurred between 3 and 9 weeks after
onset of the infection (47). However, the exact
 19. CYTOKINE PRODUCTION IN PRIMARY BCG LESIONS
role of each cytokine and its effect on neigh-
boring cells in tuberculous lesions remain to be
elucidated.
CONCLUSIONS: NONSPECIFIC AND
ANTIGEN-SPECIFIC CYTOKINE
PRODUCTION
Cytokines (including chemokines) play a major
role in the development, progression, and regres-
sion of tuberculous lesions. They cause host
defense cells to enter and become activated, so
that those cells can destroy or inhibit the tuber-
cle bacillus. Early cytokine production is non-
specific, caused by the irritant and adjuvant
effects of the bacillus, but subsequent cytokine
production is mostly antigen specific and caused
by the host’s immune response.
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 19. CYTOKINE PRODUCTION IN PRIMARY BCG LESIONS 䡵 311

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 20
CYTOKINE PRODUCTION IN
REINFECTION BCG LESIONS
AND IN TUBERCULIN REACTIONS

Production of primary and reinfection BCG lesions and tuberculin

reactions [313]
Local cell infiltration [313]
Lesion size, ulceration, and healing [313]
Tuberculin reactions [313]
Mononuclear cells (MN) and granulocytes (PMN) in primary and reinfection
BCG lesions [315]
Number of tubercle bacilli in early primary and reinfection BCG
lesions [319]
Cytokines in primary and reinfection BCG lesions and in tuberculin
reactions [319]
IL-1 and IL-8 mRNAs in PMN [321]
Causes of cytokine downregulation [321]
Cytokine networks [323]
Conclusions [323]

Abstract. Reinfection BCG lesions provide a simple model of how tuberculosis vaccines
would affect an exogenous infection with virulent tubercle bacilli.Therefore, rabbit der-
mal primary and reinfection BCG lesions were produced and evaluated during the first
5 days of their existence.Tissue sections of the lesions were prepared, and the types of cells
and their cytokine mRNAs and proteins were analyzed by histochemical methods.The
cytokines studied were interleukin-1, macrophage chemoattractant (activating) protein
(MCP-1), interleukin-8, and tumor necrosis factor alpha (see chapter 19).
Our most informative findings were with MCP-1, one of the main chemokines attract-
ing mononuclear cells (MN). (In tuberculous lesions, MN are mostly macrophages but also
contain dendritic cells and lymphocytes.) At 3 h, both the reinfection lesions and the pri-
mary lesions contained the same percentage of MN labeled for MCP-1 mRNA. How-
ever, the reinfection lesions were 400 to 500 times larger and therefore contained many
more of these MN.This high cell number alone would cause the total chemokine pro-
duction to exceed by far that occurring in the primary lesions.
By 1 day, the percentage of MN containing MCP-1 mRNA (and protein) had markedly
decreased in the reinfection lesions, but remained high for at least 2 days in the primary
lesions, which were beginning to increase in size.This finding suggests that chemokine pro-
duction is turned off when sufficient MN have accumulated. In other words, the local accu-
mulation of MN is carefully regulated, so that excessive cell infiltration into the lesions is
prevented.
The rapid local accumulation of MN (macrophages, dendritic cells, and antigen-
specific lymphocytes) in the early reinfection BCG lesions seemed to be due to the
presence of antibodies that developed during the first BCG infection (see chapter 5).
The antigen-antibody complexes formed at the site of reinfection evidently produced
chemotactic factors that markedly hastened the cell infiltration.
In general, cytokine production in tuberculin reactions showed the same pattern as that
found in the early reinfection BCG lesions.

312
 20. CYTOKINE PRODUCTION IN REINFECTION BCG LESIONS
Rabbits were immunized with BCG.Then, the
effects of this immunization on the cell compo-
sition and the cytokines of BCG lesions of rein-
fection (made 24 days later) were evaluated.
These studies provide insight into how vaccines
would affect the development and progress of a
challenging infection with virulent bacilli.
Chapter 19 describes briefly the functions
of the cytokines that we evaluated and the
methodology used. More details are presented
in references 1, 2, and 3.
PRODUCTION OF PRIMARY AND
REINFECTION BCG LESIONS AND
TUBERCULIN REACTIONS
New Zealand White rabbits (about 2.6 kg) were
immunized by the intradermal injection of
about 5 ⫻ 106 (log-phase) Tice BCG bacilli on
the right and left flanks (2, 3).Twenty-four days
later, they were reinfected with 5 ⫻ 106 (log-
phase) Tice BCG bacilli in two similar sites (2,
3).At that time, for comparison, primary BCG
lesions were also produced in nonimmunized
rabbits. Two experiments were performed. In
Experiment I, the size of the BCG lesions was
followed for 42 days (Fig. 1). In Experiment II,
tissue sections of the lesions were made and
evaluated for only 5 days, when the difference
in the size of primary and reinfection BCG
lesions was most striking (see Fig. 1).
LOCAL CELL INFILTRATION
Figures 1A and C show the size of primary and
reinfection BCG lesions at various times in
Experiments I and II, respectively. Within the
first 3 h, the reinfection lesions had substantially
increased in size, and peaked at 1 or 2 days. At
that time, the primary lesions were still very
small; in fact, the primary lesions and reinfection
lesions showed a 400- to 500-fold difference in
size.The cell infiltration, which caused this dif-
ference in size, was antigen specific. It could
only have been initiated by an antigen-
antibody reaction, because few, if any, memory
T cells are present in skin. (See chapter 5 for a
discussion of the role of antibodies in acceler-
ating local cell-mediated immunity [CMI] and
delayed-type hypersensitivity [DTH]).
䡵 313
By 4 and 5 days, the size of the reinfection
BCG lesions had markedly decreased, and the
size of the primary lesions had moderately
increased, so that the two types of lesion became
almost the same size (Fig. 1A and C). From 5
days on, the sizes of the primary and reinfection
BCG lesions followed a similar pattern. How-
ever, a second (immune-mediated) peak in size
occurred in the reinfection lesions at 8 days
(Fig. 1A) and in the primary lesions at 12 days.
The second peak occurred earlier in the rein-
fection group, because the second BCG injec-
tion boosted the already existing CMI and
DTH. In both groups, a gradual decline in lesion
size followed the second peak.
LESION SIZE, ULCERATION,
AND HEALING
No apparent difference was found in the times
when the primary and reinfection BCG lesions
ulcerated and when they healed—probably
because of the variations present among these
outbred rabbits.The ulceration was followed by
discharge of the lesion’s necrotic contents, which
was a major contributor to the decrease in lesion
size associated with healing. Sometimes, however,
the discharge of necrotic material was incomplete,
which slowed the healing process.At 6 weeks, the
sites of the BCG injections still could be read-
ily discerned, but at 14 weeks, grossly visible
evidence of the lesions could not usually be
found, either on the skin surface or, at necropsy,
on the underside of the skin.
TUBERCULIN REACTIONS
In Experiment I, the rabbits were injected intra-
dermally at various times with a 1:30 dilution of
4⫻ Old Tuberculin, and the induration of the
resulting tuberculin reaction was measured with
calipers 2 days later (Fig. 1B) (3). In the primary
BCG group, tuberculin sensitivity was not pre-
sent initially, but became strong between 9 and 23
days, and then slowly declined. In the reinfection
group, tuberculin sensitivity was rather strong
before the second injection of BCG and declined
thereafter. If a booster effect of BCG on the 2-
day tuberculin reactions occurred in these rabbits,
it was no longer apparent 10 days later (3).
 FIGURE 1 (A) Size of primary and reinfection BCG lesions from 3 h to 42 days in rabbits of
Experiment I (3).The reinfected rabbits had been sensitized intradermally by BCG 24 days pre-
viously. Note that the reinfection BCG lesions were many times larger than the primary BCG
lesions at 3, 12, 24, and 48 h, a fact that was apparently initiated by an antigen-antibody reaction
(see chapter 5). Note also that the size of the reinfection BCG lesions reached a second peak at

314
 20. CYTOKINE PRODUCTION IN REINFECTION BCG LESIONS 䡵 315

In Experiment II (which is the main one pro-
viding the data for this chapter), we injected Old
Tuberculin at the same time that we injected
BCG to produce lesions in the primary and rein-
fection groups of rabbits.The resulting skin lesions
were then measured over the next 5 days (Fig.1C)
(3). In the reinfected host, the size of lesions pro-
duced by both BCG and tuberculin increased and
decreased in parallel (Fig. 1C). By 5 days, how-
ever, the local amount of tuberculin had proba-
bly decreased substantially and no longer provided
the stimulus that it had provided earlier. Note that
in this experiment, no pre-reinfection tuberculin
tests were given, so we could not determine
whether BCG and tuberculin had a booster effect
on tuberculin sensitivity.
During the same 5-day period of study, var-
ious cell types and their cytokines were evalu-
ated (Fig. 2 through 6). The cell types and
cytokines in the tuberculin reactions and those
in the reinfection BCG lesions increased and
decreased in a rather similar manner (3), despite
the fact that the antigenic compositions of Old
Tuberculin and intact tubercle bacilli are some-
what different (see reference 4).
MONONUCLEAR CELLS (MN) AND
GRANULOCYTES (PMN) IN PRIMARY
AND REINFECTION BCG LESIONS
Figure 2 shows the number of MN per mm2 of
tissue section of BCG lesions. MN include
macrophages, dendritic cells, medium and large
lymphocytes, and some activated fibroblasts.
Figure 2 also shows the number of PMN
(including some eosinophils) per mm2. In the
early BCG lesions, small areas of necrosis
occurred, probably at sites containing the
most bacilli. PMN were present near these
necroses, but PMN were rare in more periph-
eral areas. The cell counts were made in the
large intermediate area between the more
centrally located necroses and the relatively
cell-poor areas in the lesion periphery. This
intermediate area was densely infiltrated with
MN along with some PMN.
Many more MN per mm2 were present in the
reinfection BCG lesions than in primary BCG
lesions until day 5, at which time both types of
lesion contained the same number of MN per
mm2 (Fig. 2A).The number of MN per mm2 was
usually much greater than the number of PMN
per mm2 (Fig. 2 and Fig. 13 of chapter 6) (2, 3).
The early PMN response depicted in Fig. 2B
was probably due to the nonspecific irritants in
the vaccine (see chapter 19) (2). PMN are
thought to play only a minimal role in the devel-
opment and healing of tuberculous lesions (5).
Eosinophils were included in the PMN
group, because PMN are eosinophilic in the
rabbit (see chapter 6). Rabbit eosinophils can
sometimes be differentiated from PMN by their
large oval granules (6). By this criterion,

8 days, whereas the primary lesions reached a similar peak at 12 days.These second peaks were
apparently caused by an antigen-specific CMI/DTH reaction (see chapter 5). After the second
peaks, the lesions slowly regressed. Each point represents the mean size of the lesions and its stan-
dard error. (B) Size of 2-day tuberculin reactions in rabbits of Experiment 1 (3). In the reinfected
host, tuberculin sensitivity was highest before challenge.This sensitivity declined thereafter, and
no booster effect from the second BCG injection was apparent. In contrast, hosts with primary
BCG infections had strong tuberculin sensitivity by 9 days, which tended to remain higher than
that present in the reinfected hosts, possibly because the infecting bacilli were not destroyed as
readily. Each point represents the mean size of the tuberculin reactions and its standard error.
(C) Size of primary and reinfection BCG lesions and tuberculin reactions, each measured from
3 h to 5 days in Experiment II (3).As in Experiment I, the reinfected rabbits were sensitized intra-
dermally by BCG 24 days previously. Note that the reinfection BCG lesions and the tuberculin
reactions showed the same pattern, and that the primary BCG lesions remained very small until
DTH and CMI started to develop at 4 or 5 days.The tuberculin reactions in the rabbits that had
only primary BCG lesions are not shown, because they were small or absent (see panel B). Each
point represents the mean size of the lesions and its standard error.
In panels A and C, reinfection BCG lesions versus primary BCG lesions: *P ⬍ 0.05 and **P
⬍ 0.01. Reproduced with permission from reference 3. Note that this figure also appears as Fig.
5 in chapter 5.
316 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 Number of mononuclear cells (MN) (A) and PMN (B) per mm2 of tissue section
at various times in reinfection BCG lesions, tuberculin reactions, and primary BCG lesions (3).
The reinfection BCG lesions and tuberculin reactions show no differences in the density of either
cell type, but the primary lesions contain fewer MN per mm2 than the other two at 12 h and
at 1 and 2 days.The total number of MN and PMN in each type of lesion can be estimated by
combining the data per mm2 (this figure) with the lesion size (Fig. 1). Each point represents the
mean and its standard error (see reference 3 for P values). Reproduced with permission from ref-
erence 3.
The number of MN and PMN per mm2 in primary BCG lesions at later time periods is shown
in Fig. 13 in chapter 6.
 20. CYTOKINE PRODUCTION IN REINFECTION BCG LESIONS 䡵 317

FIGURE 3 Percentage of MN immunostained for CD4 (A) and CD8 (B) in the three types
of lesion (3).At 2 days, the reinfection BCG lesions and tuberculin reactions contained a higher
percentage of CD8 cells per mm2 than did the primary lesions, suggesting that tuberculin sen-
sitivity favors the production of cytotoxic T lymphocytes. Note that CD4 cells are much more
numerous than CD8 cells (compare the scales on the y axes).The means and their standard errors
are shown (see reference 3 for P values).The CD4/CD8 ratios in primary BCG lesions at later
times are shown in Table 4 of chapter 6. Reproduced with permission from reference 3.This fig-
ure also appears as Fig. 12 in chapter 6.
 FIGURE 4 Percentage of mononuclear cells (MN) labeled for IL-1 mRNA (A), MCP-1
mRNA (B), and IL-8 mRNA (C) in the three types of lesion (3).The percentage of MN con-
taining the three cytokine mRNAs shows early peak levels at 3 h.Then, this percentage of MN
rapidly declines in BCG lesions of reinfection and in tuberculin reactions, but remains relatively

318
 20. CYTOKINE PRODUCTION IN REINFECTION BCG LESIONS
eosinophils seemed to compose less than 10% of
the granulocyte population in BCG lesions.
CD4 MN (mainly lymphocytes) were more
numerous than CD8 MN in both primary and
reinfection BCG lesions (Fig. 3). Both CD4
and CD8 cell types increased more rapidly in the
reinfection lesions. See chapter 6 for other stud-
ies and a more complete discussion of CD4
and CD8 lymphocytes in the rabbit model of
tuberculosis.
In brief, the cells mainly responsible for the
fate of rabbit tuberculous lesions are macro-
phages and lymphocytes. (The percentage of
dendritic cells in such lesions has not been
determined.) Within the lymphocyte popula-
tion, CD4 cells predominate.
NUMBER OF TUBERCLE BACILLI IN
EARLY PRIMARY AND REINFECTION
BCG LESIONS
In tissue sections, we were unable to accurately
assess the total number of tubercle bacilli in the
primary and reinfection BCG lesions by count-
ing the number of acid-fast bacilli in the central
areas of the lesions (3). Before day 5, the lesions
of the two groups differed so markedly in size
(Fig. 1) that differences in the number of bacilli
per mm2 were rather meaningless:the same num-
ber of bacilli would be distributed over markedly
different volumes. On day 5, however, when the
primary and reinfection BCG lesions were
approximately the same size (Fig.1),similar num-
bers of tubercle bacilli seemed to be present.
Homogenizing BCG skin lesions is difficult
because of the collagen present.Therefore,no cul-
tures were made to determine the effect of immu-
nization on the number of viable bacilli present.
CYTOKINES IN PRIMARY AND
REINFECTION BCG LESIONS AND IN
TUBERCULIN REACTIONS
In tissue sections of primary and reinfection
BCG lesions and tuberculin reactions, we
determined the percentage of cells containing
high in primary BCG lesions at 2 days, especially the percentage containing MCP-1 mRNA.At
2 days, the primary lesions are growing in size, whereas the reinfection lesions and tuberculin reac-
tions are regressing (see Fig. 1C).The means and their standard errors are shown (see reference
3 for P values). Reproduced with permission from reference 3.
䡵 319
the mRNAs and/or the proteins of the cyto-
kines interleukin-1 (IL-1), tumor necrosis
factor alpha (TNF-), macrophage chemoat-
tractant-activating protein-1 (MCP-1), and
IL-8 (Fig. 4 and 5) as described in chapter 19
and reference 3.
Cytokine mRNAs
At 3 h, the percentage of MN labeled for IL-1
mRNA, MCP-1 mRNA, and IL-8 mRNA
showed peak levels in all three types of lesion:
primary BCG lesions, reinfection BCG lesions,
and tuberculin reactions (Fig. 4). Since the rab-
bits with primary lesions were not immunized,
their 3-h reaction was nonspecific (2), whereas
that in the reinfection lesions was probably a
combination of a nonspecific response and an
antigen-antibody response (see chapter 5). In
reinfection lesions, the percentage of MN con-
taining the MCP-1 mRNAs rapidly declined
between 3 and 12 h (Fig. 4), but in the primary
lesions this decline was delayed (Fig. 4).
At 2 days, the percentages of MN labeled for
IL-1, MCP-1, and IL-8 mRNAs in primary
lesions were higher than those in the reinfection
lesions (Fig. 4).This finding does not mean that
the primary BCG lesions contained a greater
number of MN labeled for cytokine mRNAs
than did the reinfection lesions, because the
primary lesions were so much smaller than the
reinfection lesions (see Fig. 1C).
Changes in the percentage of cells labeled for
cytokine mRNA foretell the course of the
lesion. For example, an increase in the per-
centage of cells labeled for MCP-1 mRNA
indicates that more MCP-1 protein will be
produced and that more MN will infiltrate the
lesion because of being attracted there by this
chemokine. Conversely, a decrease in the per-
centage of cells labeled for MCP-1 mRNA
indicates that MN infiltration will be down-
regulated. Similarly, increases or decreases in
the percentage of cells containing a cytokine
320 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 5 Percentage of mononuclear cells (MN) labeled for MCP-1 protein (A) and
TNF- protein (B) in the three types of lesion (3).The percentage of MN containing these
two cytokine proteins shows the same pattern as the percentage of MN containing MCP-
1 mRNA in Fig. 4, but the early peak at 3 h in the tuberculin-sensitive hosts is much less
pronounced. (We did not label TNF- mRNA in these lesions.) The means and their stan-
dard errors are shown (see reference 3 for P values). Reproduced with permission from
reference 3.
 20. CYTOKINE PRODUCTION IN REINFECTION BCG LESIONS
protein indicate that the lesion is progressing or
regressing, respectively.
Cytokine Proteins
The MCP-1 and TNF- proteins were visual-
ized immunohistochemically with specific anti-
bodies (Fig. 5) (2, 3). In primary and reinfection
BCG lesions, the percentage of MN stained for
MCP-1 and TNF- proteins followed roughly
the same pattern as the percentage of MN
stained for MCP-1 mRNA (compare Fig. 4
with Fig. 5).This finding indicates that the cells
containing MCP-1 mRNA were actively pro-
ducing MCP-1 protein. (TNF mRNA was not
evaluated in these experiments.)
During the first 5 days, the cytokines in tuber-
culin reactions resembled those in reinfection
BCG lesions (Fig. 4 and 5). By 5 days, however,
most of the tuberculin at the site had probably
been removed by hydrolysis or by diffusion into
lymphatics.
IL-1 AND IL-8 mRNAs IN PMN
IL-1 is a primary cytokine that tends to upreg-
ulate other cytokines (7–9), and IL-8 is a
chemokine that attracts PMN into the local
site and activates them (10). (We did not find
MCP-1 mRNA in rabbit PMN [1, 2]).
PMN were evaluated in the densely infiltrated
areas of BCG lesions that were not adjacent to
areas of necrosis (see chapter 19). In reinfection
BCG lesions,the percentage of PMN labeled for
IL-1 mRNA and IL-8 mRNA showed a peak
at 3 h and then remained at lower levels (Fig. 6).
In primary BCG lesions,the 3-h peak was smaller,
and less regression occurred (Fig.6).In tuberculin
reactions, the PMN labeled for IL-1 and IL-8
mRNAs peaked at 3 h and were low at 1, 2, and
5 days (Fig. 6).
In all lesions, the intensity of the PMN stain-
ing for the IL-1 and IL-8 mRNAs indicates
the amounts present.The intensity was strongest
at 3 h.Thereafter, few PMN stained, and those
that did stained only faintly.
A higher percentage of PMN than MN were
labeled for IL-1 and IL-8 mRNAs (compare
Fig. 4 and 6). At the 3-h peak of reinfection
BCG lesions, 36% of the PMN and 5% of the
MN were labeled for IL-1 mRNA, and 48%
䡵 321
of the PMN and 7% of the MN were labeled for
IL-8 mRNA.This difference in the percentages
of cytokine-labeled PMN and MN was also
present at a later time. In other words, a high
proportion of PMN were engaged in IL-1
and IL-8 production, and only a relatively small
proportion of the MN were so engaged. Most
of the MN were probably involved in numer-
ous other activities (11, 12).
CAUSES OF CYTOKINE
DOWNREGULATION
The literature identifies many factors that seem
to be involved in the downregulation of cyto-
kines.
1. High local concentrations of both anti-
gens and certain cytokines no longer
stimulate nearby cells and can even induce
their apoptosis (13)—called active apop-
tosis (14, 15). Fas ligand and TNF are
involved (14, 16).
2. Very low antigen concentrations (caused
by a decrease in bacilli and their products)
stop the production of certain cytokines
and result in apoptosis of nearby cells—
called passive apoptosis (14). The with-
drawal of IL-2 induces this type of apop-
tosis (14).
3. Regulatory T cells (17–25) (including T
suppressor cells [23, 24, 26]) and regulatory
dendritic cells (25, 27–29) play distinct
roles (also see chapter 6).
4. The TIM gene family (30–32) stops both
excessive T-cell proliferation and excessive
cytokine production.
5. Decreases in the number and type of
chemokine receptors also play a role (33–
35), as do increases in soluble cytokine
receptors (36–38) and increases in soluble
inhibitors, such as IL-1 receptor antago-
nist (39).
6. In addition, the production of various
cytokines is downregulated by transform-
ing growth factor  (17, 40), IL-10 (17, 28,
41), IL-4 (17), IL-6 (41), prostaglandin E2
(40),lipoxygenase-derived eicosanoids (e.g.,
15-hydroperoxy-eicosatetraenoic acid)
(42a, 43), inducible NO (44–46), platelet-
activating factor (40), adenosine (47),
322 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 6 Percentage of PMN labeled for IL-1 mRNA (A) and IL-8 mRNA (B) in
the three types of lesion (3). Rabbit PMN do not contain MCP-1 mRNA (1–3). Similar
to the MN in Fig. 4, the percentage of PMN containing these cytokine mRNAs showed
a 3-h peak.Then, the percentage of labeled PMN declined in the reinfection BCG lesions
and tuberculin reactions, but remained somewhat elevated in the primary BCG lesions, again
similar to the MN in Fig. 4.
 20. CYTOKINE PRODUCTION IN REINFECTION BCG LESIONS 䡵 323

adenosine triphosphate (48), catecho-
lamines (49), immune complexes (50),
annexin I (lipocortin 1) (51), suppressors of
cytokine signaling (52, 53), and, at times,
even gamma interferon (IFN-) (54).
The exact role of each of these factors in the
early downregulation of chemokines in rein-
fection BCG lesions is not known. It is clear,
however, that when sufficient numbers of MN
are present, the chemokine production causing
further MN infiltration is turned off.
CYTOKINE NETWORKS
Our studies describe the rise and fall of various
cytokines within primary and reinfection rab-
bit BCG lesions during the first 5 days. Multi-
ple mechanisms are probably involved, because
cytokines are enhanced and suppressed by host
factors, as well as by factors from the microor-
ganisms themselves (55). We may never fully
understand the in vivo cytokine network in
tuberculous lesions or, in fact, in any inflamma-
tory lesion (see reference 56).We can, however,
visualize (as we have done here) the overall
cytokine pattern and thereby gain some insight
into which cytokines are participating.
Chemokine production is influenced in vivo
by cell-cell, cell-adhesion molecule, and cell-
extracellular matrix interactions, and by other
cytokines (57). For example, cocultures of
monocytes with endothelial cells produce more
IL-8, MCP-1, and macrophage inflammatory
protein-1 than do cultures of either cell type
alone (57). Similarly, monocyte-fibroblast cocul-
tures produce more MCP-1 and macrophage
inflammatory protein-1 than do cultures of
either cell type alone (57). Such cell-cell inter-
actions can also downregulate the production of
certain chemokines (57), probably depending on
the type and stage of the inflammatory lesions
involved.
The initial exposure to one cytokine evi-
dently determines the type of macrophage acti-
vation and renders the macrophage temporar-
ily unresponsive to activation of another type by
another cytokine (58). Such studies may explain
the heterogeneity of macrophage functions
within lesions caused by tubercle bacilli (11,
12). Of interest is that IL-12, IFN-, and IL-2
bind to heparin and heparan sulfate, which keep
these cytokines localized near the cells that pro-
duce them (reviewed in reference 59).
An immunohistochemical study of cytokines
in tuberculous mice is described in reference 60,
and the effects of BCG vaccination of guinea
pigs on their cytokines are described in refer-
ences 61 and 62.
CONCLUSIONS
Our studies suggest that BCG vaccination pro-
tects in the following manner. After the host
inhales virulent tubercle bacilli, chemotaxins
would initially be produced when bacillary anti-
gens combine with circulating and cytophilic
antibodies (see chapter 5). These chemotaxins
would cause a rapid accumulation of dendritic
cells, macrophages, and antigen-specific (mem-
ory) T lymphocytes at the sites of bacillary lodge-
ment.The infiltrating cells would then produce
cytokines, such as MCP-1, IL-1,TNF-, and
IFN-, that enhance cell infiltration and activate
the accumulating macrophages (and lympho-
cytes).After activation, the macrophages would
kill or inhibit the tubercle bacillus and thereby
prevent many microscopic tubercles from
becoming clinically apparent (see chapter 11)
(63; http://www.bioscience.org/1998/v3/c/
dannenbe/list.htm).

Note that at 3 h in reinfection BCG lesions, a much higher percentage of PMN (this figure) than
MN (Fig. 4) contained IL-1 mRNA and IL-8 mRNA. At their peaks, IL-1 mRNA was 36%
for PMN and 5% for MN, and IL-8 mRNA was 48% for PMN and 8% for MN. Evidently, many
PMN produce cytokines as soon as they enter sites of inflammation.
The means and their standard errors are shown (see reference 3 for P values). Reproduced with
permission from reference 3.
324 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
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 21
VASCULAR ADHESION MOLECULES
IN TUBERCULOUS LESIONS

Identification of microvessels in tissue sections of BCG lesions [329]

Quantitation of vascular adhesion molecules in tissue sections [329]
Vascular adhesion molecules in primary BCG lesions [329]
Vascular ICAM, ELAM, and VCAM in reinfection BCG lesions [331]
Vascular adhesion molecules in acute inflammatory lesions [331]
Leukocyte ligands for adhesion molecules in BCG lesions [333]
Adhesion molecules in epithelioid cells [333]
Overview of ICAM, ELAM, and VCAM functions [333]
How activation of microvascular endothelium may contribute to caseous
necrosis [334]
Questions to be answered [335]

Abstract.Vascular adhesion molecules enable host defense cells to leave the bloodstream
and enter tuberculous lesions.After the inhalation of tubercle bacilli, Lurie’s resistant rab-
bits had a larger number of mononuclear cells within developing lesions than did his sus-
ceptible rabbits.Therefore, the rapid local accumulation of mononuclear cells seems to be
one of the factors associated with resistance to the progress of this disease.Vascular adhe-
sion molecules enable such an accumulation to occur.
With immunohistochemical techniques, we evaluated the rise and fall of three major
vascular adhesion molecules as rabbit dermal BCG lesions developed and healed. ICAM-
1 (intercellular adhesion molecule 1) is important for the adherence of polymorphonu-
clear leukocytes (PMN), monocytes, and lymphocytes to activated vascular endothelium
before they emigrate from the bloodstream into sites of inflammation and infection.
VCAM-1 (vascular cell adhesion molecule 1) is a major factor in monocyte, lymphocyte,
and eosinophil emigration. ELAM-1 (endothelial-leukocyte adhesion molecule 1, now
called E-selectin) aids the emigration of granulocytes (and some monocytes and T lym-
phocytes).
In primary BCG lesions, ICAM and VCAM peaked at 1 to 2 weeks and decreased as
the lesions healed. In reinfection BCG lesions, ICAM and VCAM were upregulated
much sooner, beginning at 3 to 12 h and peaking at 1 to 2 days.The upregulation of these
two adhesion molecules apparently caused the rapid infiltration of mononuclear cells into
sites of BCG reinfection. ELAM-1 seemed to be less involved.
In tuberculosis, epithelioid cells are macrophages that adhere to one another in an epithe-
lial-like pattern.This adherence seems to be due in part to the ICAM-1 of one macrophage’s
binding to its ligand LFA-1 (lymphocyte function-associated antigen 1) (CD11a/CD18)
on a neighboring macrophage.Whether or not this epithelial-like pattern benefits the host
remains to be determined.
From these studies of vascular adhesion molecules, we developed a theory of why so much
tissue destruction occurs in tuberculosis: ICAM,VCAM, and ELAM are markers for acti-
vated vascular endothelial cells. In tuberculous lesions, such activated endothelial cells can
capture and present local mycobacterial antigens and therefore may be killed by antigen-
specific cytotoxic T lymphocytes.When the vascular endothelium is no longer intact, throm-
bosis occurs, and the local tissues (now lacking a blood supply) undergo caseous necrosis.

327
328 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

Vascular adhesion molecules, such as ICAM-1,
VCAM-1, and ELAM-1 (1–4), enable the dia-
pedesis of leukocytes. These adhesion mole-
cules are upregulated in the postcapillary venules
of all inflammatory reactions (5, 6), including
those produced in rabbits by BCG (1). The
upregulation is produced by the primary
cytokines interleukin-1 (IL-1) and tumor
necrosis factor alpha (TNF-) (7–10) as well as
by chemokines (11, 12).The leukocytes in these
venules upregulate their ligands for these vascular
adhesion molecules (13, 14) and then diapedese
into the tissues (5).
The local vascular endothelium is a major
participant in the inflammatory process (2–4, 13,
14). It produces cytokines, eicosanoids, nitric
oxide, vasoactive peptides, procoagulants and
anticoagulants, and fibrinolytic factors, all of
which probably play important roles in devel-
opment (and regression) of tuberculous lesions.
This chapter describes the upregulation and
downregulation of three major vascular adhesion

FIGURE 1 Microvessels in a 9-
day rabbit BCG lesion, stained
immunohistochemically for ICAM-
1 (A) and for VCAM-1 (B).Vessels
immunostained for ELAM were
similar in appearance. Mouse mon-
oclonal antibodies to rabbit ICAM-
1 and VCAM-1, rabbit anti-mouse
IgG, counterstained with Giemsa.
Magnification, ⫻475. Reproduced
with permission from reference 1.
 21. VASCULAR ADHESION MOLECULES IN TUBERCULOUS LESIONS
molecules in rabbit dermal BCG lesions as they
developed and healed.
IDENTIFICATION OF MICROVESSELS
IN TISSUE SECTIONS OF BCG
LESIONS
The size of the BCG lesions as they developed
and regressed is shown in Fig. 1A. In general, the
larger the lesion, the greater the number of
microvessels it will contain.
The total number of microvessels in the lesions
is best measured by the gelatin–colloidal car-
bon perfusion method in thick (250-m) tissue
sections (15, 16) (see chapter 8). However, the
sections prepared by this method are too thick
for the evaluation of individual cells, including
vascular endothelial cells.We therefore immuno-
stained the microvessels for von Willebrand (vW)
factor in cryostat-prepared tissue sections 5 m
thick. (In standard tissue sections, the microvas-
culature is rarely sectioned lengthwise. The
microvessels are usually cut crosswise [Fig. 2].)
vW factor is present in almost all functional
vascular endothelial cells (17, 18) and therefore
is an excellent marker for the total number of
these microvessels in a tissue section. vW factor
is both stored and secreted by endothelial cells
(17, 18). When secreted, it binds to vascular
basement membrane, where it helps platelets
initiate the clotting cascade if overlying endothe-
lium is no longer intact (17, 18). vW factor cir-
culates bound to Factor VIII (19, 20) and also in
free form (20a).
QUANTITATION OF VASCULAR
ADHESION MOLECULES IN TISSUE
SECTIONS
Vascular endothelial cells in tissue sections of
BCG lesions were stained for vW factor and for
ICAM, ELAM, and VCAM by immunohisto-
chemical methods (Fig. 1) (1).The areas occu-
pied by the stained endothelial cells in the walls
of the microvessels were measured using the
probe of a computerized image analyzer. (The
inflammatory process activates microvascular
endothelial cells causing them to increase in
size, so that their area in cross section is easily
measured with a computerized image analyzer.)
The lumens of the microvessels were not
䡵 329
included in these measurements. Stained areas
the size of a single cell were also not included,
because such cells may or may not have been
part of the microvasculature.
From these measurements, we calculated the
area of functional (vW-staining) microvascula-
ture per mm2 of lesion in the tissue section, and
we calculated the area per mm2 that stained for
each of the three vascular adhesion molecules
(1).The percentage of the vW-positive microvas-
culature that stained for each vascular adhesion
molecule could then be determined (see below).
VASCULAR ADHESION MOLECULES
IN PRIMARY BCG LESIONS
The size of the BCG lesions as they developed
and healed is shown in Fig. 2A.They peaked in
size at 2 to 3 weeks, when the host became
tuberculin positive. Such lesions showed much
cellular infiltration, but little edema. The cells
present apparently hydrolyzed the ground sub-
stance and collagen, so that the microvessels
were not spread apart as they would have been
if frank edema had been present.
The microvessels that stained for both vW
factor and adhesion molecules reached an early
peak and then declined as the lesions healed
(Fig. 2B, C, and D). The peaks for ICAM and
VCAM seemed to be earlier than the peak for
ELAM (Fig. 2C) (21).This increase in microvas-
culature was due partly to an increase in the
number of vessels per unit area (see chapter 8)
and partly to thickening (activation) of the ves-
sel endothelium.
A similar pattern was found in the percentage
of vW-positive microvessels that stained for these
three adhesion molecules (Fig. 2D).Therefore,
the increase in the adhesion molecules per unit
area was due not only to greater vascular density
but also to the upregulation of ICAM, ELAM,
and VCAM within the functional microvessels.
A comparison of Fig. 2A with Fig. 2B, C, and
D shows that the vascular adhesion molecules
peaked before the number of cells in the BCG
lesions peaked, and they started to decline before
the number of cells in the BCG lesions declined.
In other words, the upregulation of vascular
adhesion molecules increases BCG lesion size,
and their downregulation decreases lesion size.
330 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
 21. VASCULAR ADHESION MOLECULES IN TUBERCULOUS LESIONS 䡵 331

VASCULAR ICAM, ELAM, AND VCAM
IN REINFECTION BCG LESIONS
The percentages of vascular adhesion molecules
in primary BCG lesions and those in reinfection
BCG lesions and in tuberculin reactions during
the first 5 days are plotted in Fig. 3 (21, 22).
Reinfection BCG lesions showed an ELAM
peak at 3 h, an ICAM peak at about 2 days, and
a possible VCAM peak at 5 days.Therefore, in
reinfection lesions, the upregulation of ELAM
(which is associated with granulocyte emigra-
tion) was rapid, and the upregulation of ICAM
and VCAM (which are associated with a
mononuclear cell response) was slower and more
prolonged. In primary BCG lesions, the upreg-
ulation of all these adhesion molecules was more
delayed (Fig. 3).
In tuberculin reactions produced in the rein-
fected rabbits, the vascular adhesion molecules
rose and fell in a pattern similar to that found in
their BCG lesions (Fig. 3). The slight differ-
ences were probably due to the antigens in the
tuberculin reactions and the BCG lesions leav-
ing at different rates.Vascular VCAM was upreg-
ulated in tuberculin reactions of both humans
(23) and rhesus monkeys (24).
VASCULAR ADHESION MOLECULES
IN ACUTE INFLAMMATORY LESIONS
We also studied these vascular adhesion molecules
in acute inflammatory lesions produced in rab-
bits by placing a drop of 1% sulfur mustard on the
skin (1).These acute lesions reached their peak
size in 1 day and were almost healed in 6 days.
The sulfur mustard lesions showed higher levels
of ELAM-1 than VCAM-1, whereas the more
chronic BCG lesions showed the reverse (data in
reference 1).These findings are consistent with the
high numbers of granulocytes found in the crust
of sulfur mustard lesions and the high numbers
of mononuclear cells (macrophages and lym-
phocytes) found in the tuberculous granulation
tissue of BCG lesions. Of interest is that dilated
lymphatics are easily seen in the acute edematous
sulfur mustard lesions (Fig.4).In the more chronic
BCG lesions, the lymphatics are not nearly as
dilated and are hard to recognize (see Fig. 18 in
chapter 4).

FIGURE 2 (A) Size (in mm3) of rabbit dermal BCG lesions at various times after their onset.
(B) Area of stained microvasculature in such lesions as a percentage of a 1-mm2 area of tissue sec-
tion.The tissue sections were stained immunohistochemically for von Willebrand (vW) factor (a
measure of the total functional microvasculature) and for the adhesion molecules ICAM-1, ELAM-
1, and VCAM-1 (1) (see text).The microvasculature was evaluated only in the intact areas of the
BCG lesions that were densely infiltrated with inflammatory cells. Normal skin values are shown
at zero time.The means and their standard errors are shown. Note that peak levels of adhesion
molecules preceded the peak size of the BCG lesions, because the upregulation of these mole-
cules enabled the cell infiltration that caused the BCG lesions to grow in size.To produce this
panel, each stained microvessel was circled with the probe of a computerized image analyzer, and
its lumen was also circled.The two circled areas were subtracted to provide the area (in mm2) of
the vessel wall.Then, the areas occupied by vessel walls in 1 mm2 of tissue section were com-
puted. Reproduced with permission from reference 1. (C) Microvasculature stained for ICAM-
1, ELAM-1, and VCAM-1 in BCG lesions as a percentage of a 1-mm2 area of tissue section.This
panel confirms the data in panel B, with important 3-day measurements added. Note that ICAM
and VCAM peaked at 3 days, suggesting that these adhesion molecules enabled the initial cell infil-
tration of mononuclear cells (macrophages and lymphocytes) into the BCG lesions. ELAM upreg-
ulation was more delayed.The means and their standard errors are shown. Reproduced with per-
mission from reference 21. (D) Vasculature in BCG lesions stained for adhesion molecules as a
percentage of the total functional vasculature (vW factor-stained vessels). Presenting the data in
this way identifies the upregulation and downregulation of the adhesion molecules more precisely,
because changes in the total functional vasculature have been factored out.The means and their
standard errors are shown. Reproduced with permission from reference 1.
332 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
 21. VASCULAR ADHESION MOLECULES IN TUBERCULOUS LESIONS 䡵 333

LEUKOCYTE LIGANDS FOR fibroblasts, and epithelial cells, including ker-

ADHESION MOLECULES IN BCG
LESIONS
For leukocytes to emigrate from the bloodstream,
they must contain ligands on their surface for the
adhesion molecules of the vascular endothelium.
The leukocyte ligands for ICAM-1 are LFA-1
(CD11a/CD18) and Mac-1 (CD11b/CD18)
(25).We therefore immunostained the cells in the
BCG lesions with monoclonal antibodies to these
ligands.Throughout these lesions, mononuclear
cells (mostly macrophages) stained for LFA-1.
Close to areas of caseous necrosis, granulocytes
(PMN) stained for Mac-1.
VCAM is monocyte/lymphocyte selective
(26–31) because its ligand,VLA-4, is present on
monocytes and lymphocytes, but not on PMN.
VCAM and VLA-4 are especially important
factors involved in the accumulation of lym-
phocytes in tissues (28, 32–34). Unfortunately,
we were able to immunostain only a few
mononuclear cells for VLA-4 in BCG lesions,
apparently because the only available antibody
was for human VLA-4, rather than for rabbit
VLA-4, and did not appreciably cross-react.
ADHESION MOLECULES IN
EPITHELIOID CELLS
Both ICAM-1 and its ligand, LFA-1, were
strongly expressed by epithelioid cells of the
BCG granuloma (Fig. 5).This fact suggests that
the ICAM–LFA-1 pair plays a major role in
the binding of these mature macrophages to
one another. Such binding is the cause of their
epithelial-like appearance (1).
OVERVIEW OF ICAM, ELAM, AND
VCAM FUNCTIONS
ICAM-1 is produced by many cells in the body:
lymphocytes, dendritic cells, endothelial cells,
atinocytes (4). ICAM makes these cells adhere
to their surroundings and plays an important role
in the adherence of PMN, monocytes, and lym-
phocytes to activated vascular endothelium.This
adherence is followed by the emigration of these
leukocytes from the circulation into the tissues.
ELAM plays a similar role in the emigration of
granulocytes, monocytes, and a subpopulation of
T cells (4, 9, 23, 25, 35–41), and VCAM plays a
similar role in monocyte, lymphocyte, and
eosinophil emigration (4, 42).
Details of the mechanisms involved—rolling,
adherence, penetration, and migration toward
chemoattractants—are reviewed in references
25, 35, 40, 43, 44, and 45, and the various lym-
phocyte adhesion molecules are reviewed in
references 13 and 46.
In most chronic inflammatory lesions,
mononuclear cells predominate over PMN. Our
studies suggest that vascular VCAM is a major
player in this selection (43, 47–49). However, in
inflammatory processes, interactions between
leukocytes, platelets, and endothelial cells are
complex and even reciprocal (47), involving (i)
vascular ICAM, ELAM, VCAM, PADGEM
(GMP-140, P-selectin) (50), and others; (ii)
leukocyte ligands for each of these adhesion
molecules; (iii) various cytokines, including IL-
1 and tumor necrosis factor alpha (51, 52); (iv)
chemokines, such as macrophage chemoattrac-
tant (activating) protein-1 and IL-8 (44); (v) his-
tamine (53) and platelet-activating factor (53);
(vi) thrombin (53, 54), fibrinogen (47), and
fibrin (55); and, finally, (vii) elements of extra-
cellular matrix (56), e.g., fibronectin (57, 57a),
laminin, and collagen. Details on many of these
interactions are reviewed in references 35, 43,
and 56.

FIGURE 3 Areas of microvasculature stained immunohistochemically for von Willebrand (vW)

factor, ICAM-1,VCAM-1, and ELAM-1, as a percentage of 1-mm2 areas of tissue sections of der-
mal BCG lesions during the first 5 days.The functional microvasculature (per mm2) (recognized
by vW staining) and the three adhesion molecules increased much more rapidly in reinfection
lesions (and in tuberculin reactions) than in primary lesions. Most of the functional microvascu-
lature stained for ICAM, but only about half as much stained for VCAM and ELAM (compare
the scales on the y axes). In the primary BCG lesions, ELAM-1 was highest at 5 days, but in the
reinfection BCG lesions, ELAM was highest at 3 h.The means and their standard errors are shown.
Reproduced with permission from reference 22 (where P values can be found).
334 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 A dilated lymphatic vessel in a 1-day acute dermal inflammatory lesion produced
in a rabbit by the topical application of 1% sulfur mustard (1).These lesions are markedly ede-
matous at 1 and 2 days, but they are healed in 6 to 10 days. Note the valve in the center of the
lymphatic, the nerve on the upper left of the photograph, and the capillary with three erythro-
cytes below. Numerous leukocytes, especially PMN, are present in the connective tissue. Lym-
phatics remain patent in rabbit inflammatory lesions (74). In fact, extravasated serum proteins in
1-day sulfur mustard lesions are replaced every 8 h (75). Glycol methacrylate-embedded tissue
section (1 to 2 m thick) stained with Giemsa. Magnification, ⫻475.

Overviews of the various roles of adhesion delayed-type hypersensitivity reactions to the

molecules in inflammatory processes have been
published (1, 2, 4, 58–61).The roles of rabbit vas-
cular adhesion molecules in arteriosclerosis and
in cardiac allograph rejection are presented in
references 62, 63, 64, and 65.
A rather complete list of animal models in
which anti-adhesion therapy in vivo decreased
granulocyte or mononuclear cell infiltration
into inflammatory sites appears in reference 35.
HOW ACTIVATION OF
MICROVASCULAR ENDOTHELIUM
MAY CONTRIBUTE TO CASEOUS
NECROSIS
Vascular endothelium is activated in both acute
and chronic inflammatory reactions. Such acti-
vation upregulates various adhesion molecules
and causes the endothelial cells to produce var-
ious cytokines and express major histocompat-
ibility complex class I (MHC-I) and MHC-II
molecules (2, 66–69).
In humans, rabbits, and guinea pigs, well-
developed tuberculous lesions have solid caseous
or liquefied necrotic centers.Tissue-damaging
tuberculin-like products of the bacilli are a
major cause of such necrosis (70, 71). If this
immune reaction is directed only against
macrophages containing bacilli (see references 70
71), why is there so much damage to adjacent
tissue? The answer seems to be injury to nearby
vascular endothelial cells, followed by clot for-
mation (see reference 15), ischemia, and local
necrosis.
The sequence of events would be as follows.
Vascular endothelial cells are activated by the
inflammatory process. Since activated endothe-
lial cells upregulate MHC class I and MHC
class II (2, 66–69), they could present the tuber-
culin-like antigens released by nearby tubercle
bacilli (1, 72). Cytotoxic T lymphocytes could
then kill these endothelial cells, initiating the
clotting cascade. Local thrombosis would follow,
causing ischemia and death of the surrounding
tissues (see reference 15).
The dead endothelial cells cause blood clot-
ting and subsequent thrombosis by two mech-
anisms (73): (i) they no longer produce heparin-
like glycoproteins that prevent platelet adherence,
 21. VASCULAR ADHESION MOLECULES IN TUBERCULOUS LESIONS
and (ii) the collagen in the vascular basement
membrane is no longer covered by endothe-
lium, so platelets become activated, and throm-
bin is formed from prothrombin.
In tuberculous lesions, it has always been puz-
zling why cytotoxic reactions directed against
bacilli-laden macrophages cause so much necro-
sis of nearby tissues (see references 70 and 71).
The killing of activated endothelial cells (con-
taining MHC-associated bacillary antigens) by
cytotoxic T cells and the resulting thrombosis
could explain much of the tissue necrosis.
䡵 335
FIGURE 5 (A and B) Both pho-
tographs show a group of rather
mature epithelioid cells (macro-
phages) surrounding a small necrotic
area in a 37-day BCG lesion. The
cells in panel A were immunostained
for ICAM-1, whereas those in panel
B were immunostained for CD11a,
which is a component of LFA-1, the
ligand on macrophages that binds to
ICAM-1.These findings suggest that
the ICAM-1–LFA-1 pair is a major
contributor to the rather firm cell-cell
adherence that characterizes epithe-
lioid cells. Mouse monoclonal anti-
body to rabbit ICAM and CD11a,
biotinylated rabbit anti-mouse IgG,
counterstained with methyl green.
Magnification, ⫻200. Reproduced
with permission from reference 1.
QUESTIONS TO BE ANSWERED
This theory on the role of vascular endothelial
cells in the tissue necrosis found in tuberculo-
sis raises several questions that need to be
answered experimentally. (i) Do activated vas-
cular endothelial cells capture and present local
mycobacterial antigens? (ii) Are the concentra-
tions of tuberculin-like antigens on vascular
endothelial cells high enough to be recognized
by cytotoxic T cells? (iii) Does the death of
these cells result in a change in the endothelial
lining so that thrombosis occurs? Endothelial
336 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
cells may just undergo apoptosis, and the gap in
the endothelial lining may be quickly repaired.
(iv) Is antigen-induced endothelial damage the
main cause of the extensive tissue necrosis found
in many cases of progressive human tuberculo-
sis, or are other mechanisms operating?
The study of vascular endothelial cell activa-
tion must be more extensively pursued before
we can fully understand the role of endothelial
cells in the pathogenesis of tuberculosis.
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 Section 7.

TUBERCULOSIS VACCINES
This page intentionally left blank
 22
PRINCIPLES AND GUIDELINES
FOR DEVELOPING BETTER
TUBERCULOSIS VACCINES

Effects of vaccines on the establishment of a microscopic pulmonary

tubercle [342]
Effects of vaccines on the progress of a microscopic pulmonary
tubercle [342]
Effective routes of vaccine administration [343]
Immunization of mice and guinea pigs [343]
Benefits of vaccines containing few, if any, tuberculin-like antigens [344]
Composition of improved vaccines against tuberculosis [345]
Hosts do not need high levels of tuberculin sensitivity to control
the disease [346]
Live tuberculosis vaccines should be standardized before comparing their
relative efficacies [346]
Immunotherapy [347]
Maximal effectiveness of tuberculosis vaccines in humans [347]
Maximal effectiveness of tuberculosis vaccines in rabbits [348]
Other vaccine possibilities [349]
BCG must multiply in the host to be most effective [350]
BCG as a carrier of antigens from other pathogens [350]

Abstract. Tuberculosis vaccines have little or no effect on the establishment of a micro-

scopic pulmonary lesion produced by the inhalation of a virulent tubercle bacillus. Such
a lesion is established only when the pulmonary alveolar macrophages fail to destroy the
inhaled bacillus.Alveolar macrophages do not expand their population in response to spe-
cific antigens. Therefore, the establishment of a microscopic pulmonary tubercle is not
affected by vaccination. Effective tuberculosis vaccines may, however, stop the progression
of a tiny established lesion, because the vaccination has expanded antigen-specific lym-
phocyte populations.These lymphocytes enter the early lesion, where they cause a rapid
local delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI) response
that often prevents progression of the disease.
When comparing their relative efficacies, two or more live vaccines should be stan-
dardized for equal numbers of live and dead bacilli, equal numbers of log-phase and dor-
mant bacilli, and equal numbers of clumps and isolated bacilli.
Vaccines will probably never be 100% effective in preventing active tuberculosis in
humans, because humans with arrested tuberculous lesions are able to be reinfected with
exogenous tubercle bacilli. However, the efficacy of BCG could be improved by recom-
binant constructs that contain major protective antigens.Also, the immunity provided by
BCG could be increased by booster injections of such antigens.
If vaccines more effective than BCG are ever developed, they would probably produce
in the host a higher CMI/DTH ratio, i.e., an expanded antigen-specific lymphocyte pop-
ulation capable of producing increased numbers of activated macrophages and decreased
amounts of tissue necrosis.To do this, the improved vaccine would probably contain increased
bacillary glycolipid-protein components and decreased tissue-damaging tuberculin-like pro-
tein components.The vaccine should also contain components that increase the Th1/Th2
ratio.

341
342 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
EFFECTS OF VACCINES ON
THE ESTABLISHMENT OF A
MICROSCOPIC PULMONARY
TUBERCLE
Role of Pulmonary Alveolar
Macrophages
When first inhaled, 1 bacillary unit of 1 to 3
bacilli (which is the only size that can stay sus-
pended in the airstream long enough to reach
the alveolar spaces) does not contain enough
antigen to stimulate the immune system. In
both humans and rabbits, pulmonary alveolar
macrophages apparently ingest and destroy most
inhaled virulent human-type tubercle bacilli,
and the tuberculin skin test remains negative.A
microscopic primary tubercle begins in the lung
only if a phenotypically more virulent bacillary
unit is ingested by a relatively weak alveolar
macrophage (1, 2) (see chapters 2 and 11).
In humans and rabbits, the alveolar macro-
phage population is highly activated (non-
specifically) and therefore is capable of destroy-
ing many inhaled human-type tubercle bacilli
before they multiply (see chapter 11).This con-
clusion is supported in rabbits by quantitative
airborne infection (3) and in humans by the
absence of conversion of the tuberculin skin
test in many persons (including nurses) living in
an environment containing low numbers of air-
borne tubercle bacilli. However, the average
number of inhaled virulent tubercle bacilli
required to convert the tuberculin skin test in
humans is not known.This number could not
be determined in Riley’s studies of a hospital
ward containing tuberculous patients (4) (where
the concentration of tubercle bacilli in the air
was calculated), because the exposure times of
personnel entering and leaving the ward was not
known (see chapter 12).
Tuberculosis vaccines cannot prevent the
establishment of an infection with the tubercle
bacillus, because alveolar macrophages (a highly
activated population) show no immunologic
specificity. Alveolar macrophages readily ingest
tubercle bacilli nonspecifically (via mannose
and other receptors [5–7]) and do not require
opsonization of these bacilli by antibodies.
In rabbits and humans, the alveolar macro-
phage population shows a bell-shaped activation
distribution—weakly activated ones at the left,
extremely activated ones at the right, and the
majority (highly activated) in the middle. Immu-
nization does not change this distribution after
the nonspecific effects of the vaccine have sub-
sided.
EFFECTS OF VACCINES ON THE
PROGRESS OF A MICROSCOPIC
PULMONARY TUBERCLE
Progress of Early Pulmonary Tubercles
in Nonvaccinated Rabbits
After an inhaled bacillary unit multiplies in a
weak alveolar macrophage, chemotactic factors
are produced that attract dendritic cells,
macrophages, and antigen-specific lymphocytes
to the site (see chapters 5 and 6). In nonvacci-
nated rabbits challenged with virulent human-
type tubercle bacilli, these host defense cells
prevent most of the microscopic lesions from
reaching grossly visible size.Those lesions that
do reach visible size form the basis of Lurie’s
tubercle count method for assessing vaccine
efficacy (see chapter 11).
Progress of Early Pulmonary Tubercles
in Vaccinated Rabbits
The sequence just described for nonvaccinated
rabbits also occurs in vaccinated rabbits, but in
the vaccinated group the number of recirculat-
ing antigen-specific lymphocytes has been
highly expanded.Therefore, the local immune
response is greatly accelerated, i.e., macrophages
and antigen-specific lymphocytes rapidly accu-
mulate (see chapter 20).The macrophages soon
activate and keep many microscopic lesions
from reaching grossly visible size (8–10).A good
vaccine will prevent over 80% of the primary
tubercles from becoming visible at necropsy 5
weeks after aerosol challenge (9, 11).
Progress of Early Pulmonary Tubercles
in Humans
Humans are much more sensitive than rabbits to
the tuberculin-like products of tubercle bacilli.
Therefore, each microscopic pulmonary tuber-
cle that converts the tuberculin reaction in non-
vaccinated persons soon forms a tiny caseous
center. However, in 95% of cases, these early
 22. DEVELOPING BETTER TUBERCULOSIS VACCINES
lesions are arrested when they are 0.5 to 2.0
mm in size, and the person has no clinical evi-
dence of the disease, except for the conversion
of the tuberculin skin test (12–14). These tiny
inapparent lesions often calcify, so that they are
detectable at necropsy many years later (12–14).
In BCG-vaccinated individuals, such lesions are
arrested sooner and therefore are smaller in size
than those in unvaccinated individuals (12–14)
(see chapter 3).
EFFECTIVE ROUTES OF VACCINE
ADMINISTRATION
The most effective route of immunization
remains to be determined. No comparisons of
oral, intradermal, subcutaneous, intramuscular,
intranasal, and aerosol routes of immunization of
rabbits—followed by aerosol challenge and
tubercle counts—have yet been made.Therefore,
we can only mention several theoretical possi-
bilities.
The aerosol route can deliver only a relatively
small number of live BCG into the lungs,whereas
intradermal and parenteral routes can deliver
thousands.If the live BCG multiplies extensively
in the lungs and throughout the body (as in mice
[15, 16] and evidently in guinea pigs [17, 18]),
then the route of vaccine administration would
matter little. However, if BCG multiplies rela-
tively little (as in rabbits [S.Abramson, discussed
by Lurie in 1964; see reference 35, below] and
possibly in humans), then the intradermal and
parenteral routes would be preferable,because the
dose administered could be so much higher.
The intranasal and oral routes deliver antigens
in the vaccine to bronchial-associated and gut-
associated lymphoid tissues (BALT and GALT,
respectively). Lymphocytes from BALT and
GALT home mainly to mucosal surfaces (19),
and these surfaces are not easily infected by
inhaled tubercle bacilli. However, some anti-
gen-specific lymphocytes from BALT and
GALT do not home to mucosal surfaces (19)
and should be able to enter early tubercles in the
peripheral lung.
In the 1930s, infants were immunized with
BCG by the oral route during the first 10 days
of life (20).The oral route was chosen because
bovine-type tubercle bacilli were sometimes
䡵 343
present in (unpasteurized) milk. In neonates,
the gut is more permeable to antigens, so they
are absorbed intact more readily than in older
individuals. Also, in all neonates (because of
their low resistance to mycobacteria), BCG can
multiply to higher titers. For both reasons, oral
immunization should be more effective in
neonates than in older hosts. However, in recent
times, infants are immunized with BCG only by
the intradermal route. In children and adults,
antigens administered by the oral route may
elicit tolerance to these antigens (reviewed in
reference 19).
The intradermal, subcutaneous, and intra-
muscular routes deliver a large number of viable
BCG (and their antigens) to the draining lymph
nodes, where they clonally expand the antigen-
specific lymphocyte population that recircu-
lates through these nodes.
The intravenous route delivers the BCG
throughout the body—especially to the spleen,
which contains much lymphoid tissue.The intra-
venous route, therefore, should be quite effective
in laboratory animals, but should not be used in
human populations because of the greater pos-
sibility that live BCG will cause minute lesions
in critical sites, such as the eye and brain.
IMMUNIZATION OF MICE
AND GUINEA PIGS
The multiplication of a live vaccine and the
degree of its persistence have marked effects on
its efficacy (reviewed in reference 21). BCG
multiplies extensively in mice (15, 16) and
apparently in guinea pigs (18), but less so in
rabbits and probably humans, which are more
resistant species.Yet, to our knowledge, live vac-
cine multiplication has not been carefully com-
pared among these species.
In mice and guinea pigs, the aerosol route of
BCG immunization was equally effective as
other routes (and sometimes more effective) in
reducing the multiplication of inhaled virulent
human-type bacilli in the lungs (reviewed in ref-
erences 15 and 18). In mice (15, 16) and prob-
ably in guinea pigs (18), inhaled BCG multiplies
readily, apparently because their alveolar
macrophages cannot inhibit inhaled BCG as
easily as can rabbit alveolar macrophages.
344 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
In mice, aerosolized BCG persisted in the
lungs for at least 12 weeks in relatively high
titers (15, 16). Such aerosol-immunized mice
reduced the growth of subsequently inhaled vir-
ulent tubercle bacilli (H37Rv) by about 2 logs
(15, 18).
In guinea pigs, the aerosol, subcutaneous, and
intradermal routes of BCG immunization were
equally effective in reducing tuberculosis after
challenge by aerosol with virulent human-type
tubercle bacilli (H37Rv)—even though the
BCG in the subcutaneous and intradermal
routes was at much higher dose levels (18). Evi-
dently, aerosolized BCG multiplies extensively
in the lungs of guinea pigs (see Table 16 in ref-
erence 18).
In rabbits, aerosolized BCG only rarely pro-
duced any visible pulmonary lesion (S.Abram-
son, discussed by Lurie, 1964), apparently because
aerosolized BCG did not grow or persist in
titers as high as those in mice and guinea pigs.
In human beings, the efficacy of the aerosol
route of immunization has never been evaluated.
However, humans were immunized by aero-
solized BCG (22, 23), and they sometimes, but
not always, converted their tuberculin skin tests
(22, 23).
In brief, vaccine efficacy varies greatly among
laboratory animal species because of differences
in (i) their ability to allow the live attenuated
tubercle bacilli in the vaccine to multiply and
TABLE 1 Benefits of tuberculosis vaccines produc-
ing strong CMI and weak DTH, especially those pro-
ducing little or no tuberculin sensitivitya
1. Continued usefulness of the tuberculin skin test
for diagnosing infection with virulent tubercle
bacilli
2. Prevention of clinical tuberculosis (prophylaxis)
3. Treatment of clinical tuberculosis
(immunotherapy)
4. Vaccination of tuberculin-positive individuals
without harm
5. Preventive therapy, as a replacement for
chemoprophylaxis with isoniazid or rifampin
6. Further enhancement of existing acquired
immunity by repeated vaccinations (which are not
advised with current BCG vaccines)
a
Adapted from reference 2.
persist, and (ii) the susceptibility of each species
to the virulent tubercle bacilli used to assess
the degree of immunization: the more virulent
the challenge strain of tubercle bacillus used for
such assessment, the less effect the immunization
would appear to have on the disease. There-
fore, the tubercle-count method (see chapter 11)
would measure vaccine efficacy in rabbits more
precisely than it would in mice and guinea pigs.
(Human-type tubercle bacilli are much less vir-
ulent for rabbits.) On the other hand, if mice and
guinea pigs were challenged with tubercle bacilli
of reduced virulence, a precision comparable to
that found in rabbits could be achieved.
BENEFITS OF VACCINES
CONTAINING FEW, IF ANY,
TUBERCULIN-LIKE ANTIGENS
There are many benefits in reducing or elimi-
nating tuberculin-like antigens from BCG vac-
cine (Table 1) (2).
1. Vaccinated individuals with little or no
sensitivity to tuberculin would not
become appreciably tuberculin positive.
Therefore, tuberculin testing of such indi-
viduals would still be useful for detecting
infection with virulent tubercle bacilli.
2. As with all effective vaccines, such a vac-
cine would prevent the development of
clinically apparent tuberculosis.
3. Such a vaccine could be used as
immunotherapy for active tuberculosis,
because it would expand the T-cell pop-
ulation responding to protective antigens
of the tubercle bacillus and not expand
the T-cell population responding to the
tuberculin-like antigens that are involved
in caseous necrosis, liquefaction, and cav-
ity formation.
4. Such a vaccine could be given without
harm to tuberculin-positive individuals
with inapparent Mycobacterium tuberculosis
infection to reduce the likelihood of reac-
tivation. Reference 24 emphasizes the
need for a vaccine to increase the immu-
nity of individuals who are already tuber-
culin positive and harbor virulent tuber-
cle bacilli. Such individuals constitute a
large proportion of the world’s population.
 22. DEVELOPING BETTER TUBERCULOSIS VACCINES
5. Such a vaccine could replace isoniazid
(INH) as preventive therapy in persons
who had recently become tuberculin pos-
itive. Substituting such a vaccine for INH
would eliminate the danger of hepato-
toxicity and the development of drug re-
sistance to INH.
6. Because it produces negligible levels of
delayed-type hypersensitivity (DTH), such
a vaccine could be given more than once,
to create a higher level of immunity.
Boosting immunity with a better BCG or
another live vaccine in individuals who are
tuberculin positive from an arrested virulent
infection may temporarily lower resistance to
that endogenous infection (25), because a pro-
portion of the recirculating antigen-specific
lymphocytes now enter the sites of BCG (or live
vaccine) multiplication.Therefore, fewer defense
cells would be available to keep the virulent
infection arrested. (See comments after reference
58 of Appendix C.) However, the negative
effects of the boosting with important antigens
would not be long lasting, and the subsequent
positive effects should far outweigh any tem-
porarily harmful effects that may have occurred.
For reasons discussed in chapters 2 and 5,
DTH to some of the antigens of tubercle bacilli
is required to effectively control this disease.
However, the amount of tuberculin sensitivity
had no relation to the amount of protection
against clinical tuberculosis that was produced by
vaccination with BCG or vole bacilli (Mycobac-
terium microti) (26, 27).Therefore, antigens caus-
ing low levels of tuberculin sensitivity could be
just as effective as vaccines producing high lev-
els (discussed further below).
COMPOSITION OF IMPROVED
VACCINES AGAINST TUBERCULOSIS
Antigen Composition
The antigens in vaccines causing the most
favorable cell-mediated immunity (CMI)/DTH
ratio will produce an expanded (memory) T-
lymphocyte population with the same favorable
CMI/DTH ratio. Then, when the vaccinated
individual inhales virulent tubercle bacilli, the
memory lymphocytes infiltrating the resulting
䡵 345
lesion will also have this favorable CMI/DTH
ratio, and the lesion would probably have less
caseation and undergo liquefaction less readily.
Some antigenic fractions of the tubercle bacil-
lus, i.e., certain tuberculoproteins (or peptides)
complexed with certain carbohydrates and lipids
(28–34), seem to stimulate CMI with minimal
tissue-damaging DTH (28, 34).To improve cur-
rently available strains of BCG, their content of
such antigens should probably be increased.
Other antigenic fractions (i.e., tuberculopro-
teins that produce the tuberculin reaction) seem
to stimulate tissue-damaging DTH with less
CMI (28, 35, 36). These tuberculoproteins are
not protective as a vaccine (28, 35, 36). In fact,
tuberculoproteins can produce severe necrosis in
the tuberculin-positive host if their concentra-
tion exceeds that which is safe for that host
(37). To improve currently available strains of
BCG, their content of tuberculoproteins should
probably be decreased.
The engineering of vaccines to produce an
increase in the CMI/DTH ratio seems logical,
but still remains to be established. Also, the
immunogenic role of proteins secreted by live
tubercle bacilli (32, 38, 39) and the immuno-
genic role of carbohydrate fractions of the bacil-
lus (28, 34) need further investigation.
Adjuvant Composition
The intradermal injection of BCG apparently
initiates an early, pronounced nonspecific
cytokine response, both in the resident cells and
in the mononuclear cells that soon infiltrate the
site (40). Mononuclear cells at sites of BCG
injection have upregulated their IL-1,TNF-,
MCP-1, and IL-8 mRNAs by the third day,
which is long before the time when the immune
response occurs (40) (see chapter 19). These
cytokines, as well as the accompanying upreg-
ulation of vascular adhesion molecules (41) (see
chapter 21), play important roles in the accu-
mulation of dendritic cells, macrophages, lym-
phocytes, and granulocytes at the site of BCG
injection and in the draining lymph nodes.
Incorporation into live vaccines of genes that
produce cytokines and/or costimulatory mole-
cules for antigen presentation is a field of active
investigation (42–46).
346 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
Lurie showed that acquired resistance to
tuberculosis was superimposed on, and deter-
mined by, the native (innate) resistance of his
inbred rabbits (9, 35). The early tuberculous
lesions (and probably their draining lymph
nodes) of Lurie’s resistant rabbits contained
greater numbers of lymphocytes and macro-
phages (and probably dendritic cells) than did
the early lesions of his susceptible rabbits (3).
Such large numbers of local mononuclear cells
contributed to the high levels of acquired
immunity to tuberculosis that developed in his
resistant rabbits. By analogy, a vaccine that
recruited a large number of these cells into sites
where antigens are located should produce more
immunity than a vaccine that recruited a small
number of these cells.
Therefore, a more effective vaccine than the
currently available BCG requires not only a
more appropriate mycobacterial antigen com-
position, but also an adjuvant composition that
recruits the same or a larger number of dendritic
cells, macrophages, and lymphocytes to sites
where the vaccine is deposited.
Additional aspects of designing an improved
vaccine for tuberculosis have been reviewed (24,
44,47–54).Of considerable importance is increas-
ing the number of memory lymphocytes that
remain in the host for many years following
immunization (see chapter 6). During the first
year after the initial immune response, up to 90%
of the antigen-specific T lymphocytes are elim-
inated by apoptosis—a phenomenon that seems
to prevent autoimmunity (42).The remaining T
cells become memory cells (42). New (recom-
binant) vaccines that reduce cell death in the
initial expanded antigen-specific T-cell population
should have greatly increased efficacy (42).
HOSTS DO NOT NEED HIGH LEVELS
OF TUBERCULIN SENSITIVITY TO
CONTROL THE DISEASE
In humans and rabbits, tissue-damaging DTH
seems to be required to stop the initial logarith-
mic growth of tubercle bacilli within nonacti-
vated macrophages (see chapters 2 and 15), as
well as the subsequent logarithmic growth within
nonactivated macrophages that continuously
enter established lesions from the bloodstream
(55) (see chapter 10).Therefore, would vaccines
that produce little or no sensitivity to tuber-
culin still be able to stop such logarithmic intra-
cellular growth? The answer seems to be “yes.”
In the British Medical Research Council tri-
als on human populations, one lot of vole bacil-
lus vaccine produced a high incidence of tuber-
culin positivity and another lot did not. Yet,
both lots showed equally high protective
potency against tuberculosis (27).This and other
studies with different strains of BCG (51) clearly
demonstrate that the antigens causing appre-
ciable tuberculin positivity were not required to
protect human beings.
When applied to the pathogenesis of tuber-
culosis, these findings indicate that the killing of
nonactivated macrophages, in which the bacilli
are growing intracellularly in a logarithmic fash-
ion, is not uniquely due to the antigens in tuber-
culin preparations. It can apparently be pro-
duced by a DTH response to other antigens of
the bacillus, especially when these antigens are
in sufficiently high concentrations. In fact, in the
mouse model, lymphocytes that transfer pro-
tective immunity can be dissociated from those
that transfer tuberculin sensitivity (56).
LIVE TUBERCULOSIS VACCINES
SHOULD BE STANDARDIZED
BEFORE COMPARING THEIR
RELATIVE EFFICACIES
Two types of live vaccines should be standard-
ized before we can conclude that the antigens
in one are more effective than the antigens in the
other.
1. They should be administered in the same
manner. Intradermal administration is
more accurate than the percutaneous
(multiple puncture) method. Oral and
intranasal administration may perhaps be
the least accurate because of variations in
antigen absorption.
2. The two vaccines should contain the same
number of live bacilli and also the same
number of dead bacilli. Some antigens are
only secreted by live bacilli (57). Immu-
nization of mice by live, but not dead,
tubercle bacilli produces antigen-specific
lymphocytes (in the spleen) that can adop-
tively transfer immunity to naive mice
(50).An increased percentage of live bacilli
 22. DEVELOPING BETTER TUBERCULOSIS VACCINES
may also increase the Th1/Th2 lympho-
cyte ratio. This ratio seems to play an
important role in host resistance to tuber-
culosis (57, 58).
3. The bacilli in each vaccine should be in the
same stage of their growth cycle. Actively
growing log-phase bacilli may multiply to
higher titers in recipient hosts than dormant
(frozen or lyophilized) bacilli, because dor-
mant bacilli may be slow in starting to
grow in the host and may not reach as
high a titer before acquired (adaptive)
immunity develops (see chapter 11 and
reference 59).
4. The two vaccines should contain the same
number of clumped and isolated bacilli.
Clumps are more common in thawed
frozen bacilli and in reconstituted lyo-
philized bacilli.When injected parenter-
ally, such clumps may resist destruction by
the host better than do isolated bacilli.
We prepared fresh 100% live nonclumped
and 100% viable (log-phase) bacilli (11) for the
rabbit experiments with BCG and M. microti
(the vole bacillus) described in chapter 23. How-
ever, for trials in human populations, reconsti-
tuted lyophilized vaccines, which usually con-
tain many clumps and many dead bacilli (60), are
more practical. Reference 61 describes how to
obtain nonclumped BCG preparations that are
90 to 99% viable after freezing and storing at
⫺70°C, even after a year.
Excellent reviews of the factors just listed (as
well as others) have been published (60–64).
Included were vaccine stability, the growth of
BCG in the organs of mice, and the degree of
protection produced, as well as standardization
of how the vaccine was injected. Because of
such variables, different laboratories were unable
to rank the efficacy of different BCG prepara-
tions in animals in any consistent order (65).
IMMUNOTHERAPY
Mycobacterium vaccae
Heat-killed M. vaccae, a nonpathogenic soil bacil-
lus, is being used as immunotherapy in patients
with active tuberculosis (reviewed in references
58, 66, and 67). Heat-killed M. vaccae does not
produce adverse systemic DTH reactions in
䡵 347
tuberculous hosts, because it elicits minimal
tuberculin sensitivity. Due to the presence of
common mycobacterial antigens, M. vaccae
increases the Th1/Th2 ratio (58), thereby
enhancing the host’s CMI.
Repeated immunization with M. vaccae appar-
ently reduced the required length of antimi-
crobial therapy and helped in the treatment of
multidrug-resistant tuberculosis (67). Since the
host’s immune forces work in synergy with
antimicrobials, studies on the use of this and
other immunotherapeutic agents should be
actively pursued. (In the two experiments that
we did on immunotherapy with M. vaccae in
rabbits with cavitary tuberculosis, the variation
among the rabbits was too large to detect any
significant effect [see chapter 4].)
Dendritic Cells as Vaccine Carriers
The use of dendritic cells as vaccine carriers is
being explored (68). Dendritic cell-vaccine car-
riers may find their best use as immunotherapy
in immunocompetent patients with multidrug-
resistant tuberculosis.
Surgical Removal of a Cavitary Lesion
Most multidrug-resistant tubercle bacilli arise
in cavities where the growth of extracellular
bacilli may be tremendous. If antimicrobial ther-
apy with second-line drugs is unsuccessful,
surgery to remove the cavitary lesion may be
indicated. Doing so would remove the major
source of multidrug-resistant bacilli. In immuno-
competent patients, noncavitary lesions con-
taining multidrug-resistant bacilli (originating
metastatically from cavities) would probably be
controlled by the host’s immune forces, but if
such lesions were not controlled, boosting immu-
nity with immunotherapy could be beneficial.
MAXIMAL EFFECTIVENESS
OF TUBERCULOSIS VACCINES
IN HUMANS
In theory, humans should have maximal immu-
nity after they arrest an infection with virulent
tubercle bacilli. In most of these tuberculin-
positive persons, there is a tiny caseous calcified
lesion (0.5 to 2.0 mm in size) (see chapter 3).
If such persons remain tuberculin positive,
348 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
tubercle bacilli may occasionally escape from
the lesion, enter nonactivated blood-borne
macrophages, multiply, and then be inhibited by
the DTH and CMI response. This sequence
maintains immunity and tuberculin sensitivity.
Nevertheless, (i) careful epidemiological studies
(69), (ii) restriction fragment length polymor-
phism with DNA fingerprinting (70), (iii) phage
typing (71), and (iv) antimicrobial-resistant spec-
tra (71, 72) have shown that tuberculin-positive
individuals are able to develop active reinfection
pulmonary tuberculosis when the exposure to
exogenous virulent tubercle bacilli is high (70,
73a). However, active reinfection tuberculosis is
rare when the exposure is low (73, 73a, 74).
Because such natural reinfection occurs, immu-
nity in humans apparently is never complete.
Therefore, one should not expect immunity
produced by any vaccine to be complete.
Active tuberculosis produced by a different
strain from the one causing the primary infec-
tion is most frequently due to a reinfection with
an exogenously inhaled bacillus. However, dif-
ferent strains of tubercle bacilli may be found in
the same sputum specimen (75), because of bacil-
lary mutations occurring within the cavities pro-
duced by the original bacillary strain (76).
Factors Influencing Vaccine
Effectiveness
Vaccines are less effective in human immuno-
deficiency virus (HIV)-infected persons and
others with decreased immune function.These
individuals develop active tuberculosis more
readily after inhaling exogenous tubercle bacilli.
They also develop active tuberculosis more read-
ily from endogenous tubercle bacilli that had
been latent in primary tuberculous lesions.They
are more susceptible to tuberculosis because
they have a reduced Th1 lymphocyte popula-
tion. Helminth infection and/or environmental
mycobacteria can also shift a Th1 lymphocyte
response toward a Th2 response (reviewed in ref-
erence 77). Tuberculosis vaccines have often
been ineffective in populations in sub-Saharan
Africa because of parasitic infections (77). Every
tuberculosis vaccine trial in developing countries
should attempt to eliminate helminth infection
before the vaccine is administered.
Environmental mycobacteria may also lessen
the difference between vaccinated and control
groups (26, 27). Constant exposure to mycobac-
teria in the soil (and in dust arising from the soil)
can increase host resistance to tuberculosis
(reviewed in reference 26).
MAXIMAL EFFECTIVENESS OF
TUBERCULOSIS VACCINES IN
RABBITS
Lurie’s Experiments with Virulent
Human-Type Tubercle Bacilli
Rabbits should have maximal immunity while
they are arresting an infection with human-
type tubercle bacilli (which are never fully vir-
ulent in this animal species). Lesions produced
by the human type may caseate, liquefy, and
cavitate, but do not progress to death, but lesions
produced by the bovine type almost always
progress to death (35).
Therefore, to test their immunogenicity, Lurie
(78, 79) infected rabbits intravenously with vir-
ulent human-type bacilli (H37Rv), and 6
months later he reinfected them intravenously
with virulent bovine-type bacilli.After the rein-
fection, Lurie euthanized the rabbits at various
intervals (up to 2 months) and cultured the
lungs for tubercle bacilli. He found that the
bacilli were mostly the human type.The mul-
tiplication of the fully virulent bovine type had
been markedly reduced by the presence of the
residual human type. (The human-type bacilli
were identified by their eugonic colonies on
solid culture media, and the bovine-type bacilli
were identified by their dysgonic colonies [78].)
This experiment indicates that residual pri-
mary lesions caused by human-type bacilli
(which are only semivirulent in rabbits) offer
considerable protection against reinfection by
bovine-type bacilli (which are highly virulent in
rabbits).
Proposed Studies in Rabbits with the
Same Strain of Tubercle Bacilli
With modern recombinant technology, virulent
human-type bacilli can be made to produce
green or red fluorescent proteins. Rabbits can
then be exposed to “green” bacilli by aerosol and
challenged with “red” bacilli by aerosol weeks
 22. DEVELOPING BETTER TUBERCULOSIS VACCINES
later (suggested by William R. Bishai). The
tubercles produced can then be counted (see
chapter 11) and cultured to determine whether
they arose from the primary or challenge
aerosol. Controls of rabbits that had not been
previously infected, but were challenged with
“red” bacilli, should be included.Also, the exper-
iment should be repeated with the infection by
“green” and “red” bacilli in reverse order.
OTHER VACCINE POSSIBILITIES
Recombinant Technology To Improve
Vaccines
Protection of rabbits against reinfection by vir-
ulent human-type bacilli assumes that these
bacilli contain a full complement of antigens and
that a host recovering from infection with such
bacilli will have the most effective immune
response. However, certain antigens may be
more protective than others. If such antigens are
increased substantially in BCG by recombinant
technology (see references 44 and 80–84), it is
possible that the resulting BCG vaccine will
immunize more effectively than even virulent
human-type tubercle bacilli. The BCG strain
should be chosen carefully, because differences
among various BCG strains exist (64, 85, 86).
It is also possible that the additional antigens
will increase the virulence of BCG and make it
unsafe to use as a vaccine. Such a vaccine should
be tested in mice, guinea pigs, rabbits, and per-
haps monkeys before human trials are per-
formed (see references 87–89).
DNA Vaccines
DNA vaccines are those in which the DNAs of
one or more mycobacterial antigens are incor-
porated into a plasmid and usually injected
intramuscularly. Such DNA vaccines increased
the immunity of mice and guinea pigs to viru-
lent tubercle bacilli, but no more than BCG did,
and the immunity produced was shorter lived
(reviewed in references 24 and 90).
DNA vaccines or vaccines incorporating
mycobacterial protein, lipid, and/or glycolipid
antigens (in adjuvants) may be useful in HIV
patients, because such vaccines cannot cause
tuberculosis in immunosuppressed individuals.
䡵 349
Postinfection Immunization
A substantial percentage of the world’s human
population is already tuberculin positive from
inhaling virulent tubercle bacilli. Therefore,
postinfection immunization to prevent endoge-
nous (and exogenous) reinfection would be
especially useful. Subunit vaccines (including
DNA vaccines) are probably the safest type to use
for this purpose (reviewed in references 90 and
91).Antigens should be chosen that do not elicit
tuberculin sensitivity, because systemic tuber-
culin can exacerbate existing tuberculous lesions
and even induce cavities to form (see chapter 4).
Combination Vaccines
Combination vaccines will probably provide
the most effective protection against active
tuberculosis (see references 24, 44, and 90–92).
Such an immunization often consists of an ini-
tial BCG vaccination followed by one or more
booster immunizations with important my-
cobacterial antigens (including those produced
by DNA vaccines [93]). The antigens in such
boosters would expand the appropriate T-
lymphocyte population almost as well as if these
antigens had been incorporated into the live
vaccine itself, and would avoid the possibility of
an increase in virulence that a direct incorpo-
ration into the BCG genome might produce.
However, booster immunization would proba-
bly have to be given more than once to provide
the best results.
M. tuberculosis Mutants
as Possible New Vaccines
Reference 94 lists the in vivo growth charac-
teristics in mice of several M. tuberculosis mutants
that lack specific genes. (See reference 95 for a
more detailed discussion of the production of
such vaccines and their use.) These mutants
often do not grow or persist in the host. Some
of these mutants may eventually find use as an
improved vaccine for tuberculosis (especially in
HIV/AIDS individuals) or as a booster for
immunity after BCG vaccination.
Reviews of Possible New Vaccines
References 24, 44, 45, 58, 87, 90, 91, and 94
briefly review some of the newly developed
350 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
potential vaccines for tuberculosis that were
evaluated in mice and/or guinea pigs.
BCG MUST MULTIPLY IN THE HOST
TO BE MOST EFFECTIVE
We favor the use of live vaccines for immuno-
competent persons.The bacilli in live vaccines
multiply in the host and produce (and even
secrete) certain antigens that are not present in
killed preparations. Live bacilli also persist longer
in the host than do killed bacilli. Reference 96
reviews the older literature on nonliving vac-
cines against tuberculosis.
BCG AS A CARRIER OF ANTIGENS
FROM OTHER PATHOGENS
Because BCG tends to persist in the host for
many years, recombinant BCG vaccines con-
taining specific antigens from various viral, bac-
terial, and parasitic microorganisms are currently
being evaluated (reviewed in reference 83).
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1. Lurie, M. B. 1944. Experimental epidemiology of
tuberculosis: hereditary resistance to attack by tuber-
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the concentration of tubercle bacilli upon these
two phases of resistance. J. Exp. Med. 79:573–589.
2. Dannenberg,A. M., Jr. 1990. Controlling tuber-
culosis: the pathologist’s point of view. Res. Micro-
biol. 141:192–196, 262–263.
3. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
culosis in the light of the host-parasite relationships
in natively resistant and susceptible rabbits. Am.
Rev.Tuberc. 72:297–329.
4. Riley, R. L., C. C. Mills, F. O’Grady, L. U. Sul-
tan, F. Wittstadt, and D. N. Shivpuri. 1962.
Infectiousness of air from a tuberculosis ward. Ultra-
violet irradiation of infected air: comparative infec-
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85:511–525.
5. Schlesinger, L. S. 1996. Role of mononuclear
phagocytes in M. tuberculosis pathogenesis. J. Inves-
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6. Fenton, M. J., L.W. Riley, and L. S. Schlesinger.
2005. Receptor-mediated recognition of Mycobac-
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Cole, K. D. Eisenach, D. N. McMurray, and W. R.
Jacobs, Jr. (ed.), Tuberculosis and the Tubercle Bacillus.
ASM Press,Washington, D.C.
7. Beharka, A., C. D. Gaynor, B. K. Kang, D. R.
Voelker, F. X. McCormack, and L. S.
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 23
CHARACTERISTICS OF RABBIT BCG
LESIONS AND EFFICACIES OF BCG AND
MYCOBACTERIUM MICROTI VACCINES

Characteristics of dermal BCG lesions in commercial New Zealand White

rabbits [354]
Comparison of dermal BCG lesions and pulmonary lesions produced by
virulent tubercle bacilli in rabbits [358]
Healing of dermal BCG lesions as a measure of host resistance [359]
Lurie’s intravenous BCG experiments in rabbits [359]
Systemic nature of BCG vaccination in humans [360]
Intradermal BCG vaccination in Lurie’s inbred resistant and susceptible
rabbits [361]
Efficacies of BCG and M. microti vaccines in commercial New Zealand
White rabbits [363]

Abstract. In rabbits (and humans), dermal BCG lesions have the same components as
lesions caused by virulent tubercle bacilli, i.e., a caseous liquefied center surrounded by
tuberculous granulation tissue.Therefore, BCG lesions can be used as a model in which
to study the host response to this disease.
In immunocompetent hosts, BCG lesions do not progress and always heal.The rate of
healing of dermal BCG lesions reflected the native and acquired resistance of Lurie’s sus-
ceptible and resistant inbred rabbit strains. Similarly, the rate of healing of BCG lesions has
been shown to reflect the resistance to tuberculosis of human populations. However, the
healing of BCG lesions in individual rabbits and humans may vary considerably from the
mean.
In Lurie’s natively resistant rabbits, immunization with BCG was quite effective in pre-
venting many grossly visible primary pulmonary tubercles (i.e., clinically apparent tuber-
culosis). In his natively susceptible rabbits, BCG was hardly effective at all. In other words,
the rabbits that needed it the least were helped by BCG vaccination the most, and the
rabbits that needed it the most were helped the least. This principle also applies to
human populations: persons with poor resistance to clinical tuberculosis would develop
less immunity from BCG administration than persons with strong resistance.
The efficacies of various BCG and Mycobacterium microti (the vole bacillus) vaccines were
evaluated in commercial outbred rabbits by the tubercle-count method. Both of these vac-
cine types were effective. However, the variations in resistance among outbred rabbits pre-
vented our rating any one vaccine above another. Recombinant BCG vaccines that con-
tain additional antigenic components may be more promising than currently used BCG
vaccines (see chapter 22).

CHARACTERISTICS OF DERMAL BCG
LESIONS IN COMMERCIAL NEW
ZEALAND WHITE RABBITS
BCG Preparation
In our laboratory, strains of BCG and M. microti
were usually maintained on solid Lowenstein-
Jensen slants with periodic transplantation to
fresh slants. Seven to 10 days before use, the
BCG was again transplanted to fresh Lowenstein-
Jensen slants, so that, when harvested, the BCG
colonies were still in the log phase of growth.
They were then ground in an agate mortar with
10 to 20 µl of Tween 80, suspended in a solution
of Becton-Dickinson’s BBL Oleic Albumin
Complex (diluted 1:10 in 0.9% NaCl), and cen-
trifuged for 10 min at 2,000 rpm to remove
clumps containing more than a few bacilli (1).
Then, the bacilli in the supernatant fluids were

354
 23. RABBIT DERMAL BCG LESIONS 䡵 355

counted microscopically in a hemacytometer were almost healed at 5 to 7 weeks (Fig. 1) (3).

(to obtain the number of live plus dead bacilli in
the preparation) and adjusted to 106 to 107 bacilli
per 0.1 ml. Every bacillary unit was found to be
viable when cultured.This method of prepara-
tion standardized the vaccines to be compared
(discussed in chapter 22). In tissue sections, tuber-
cle bacilli were identified by acid-fast staining
with carbol-fuchsin (2).
Dermal BCG Lesion Development
and Healing
In rabbits, BCG lesions (produced by the Tice
strain) reached peak size in 2 to 3 weeks and
Variations in size among the lesions were often
due to the time of ulceration and the quantity
of necrotic material discharged from the lique-
fied centers. The rabbits were slightly tuber-
culin positive 9 days after the injection of BCG
and strongly tuberculin positive from 14 days on
(Fig. 1). At 3 to 5 days, the transected lesions
showed small foci of necrosis on gross exami-
nation. Between 9 and 23 days, most of these
necrotic foci had fused to form a relatively large
caseous center, which liquefied and usually
ulcerated, discharging some of its contents.At 37
days, the lesions were almost healed: they were

FIGURE 1 Size of BCG lesions in rabbits at various times after receiving 8 intrader-
mal injections of about 5 ⫻ 106 BCG bacilli on each flank (3).The biphasic nature of the
curve is due to a decline in the initial nonspecific inflammatory response to the vaccine
followed by the development of an antigen-specific response.The size of 2-day tuberculin
reactions in these rabbits is shown below the curve: ⫺ ⫽ 0 to 100 mm3, ⫹ ⫽ 100 to 200
mm3, ⫹⫹ ⫽ 200 to 400 mm3, and ⫹⫹⫹ ⫽ greater than 400 mm3.The rabbits had sig-
nificant tuberculin reactions from 9 days on.The means and their standard errors are shown
along with the statistical significance between adjacent points: *P ⬍ 0.05 and ***P ⬍ 0.01.
Reproduced with permission from reference 3.
356 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 2 Mononuclear cells (MN) and granulocytes (PMN) per mm2 of tissue sec-
tion in rabbit BCG lesions at various times during their development and healing. PMN
accumulated around the caseous necrotic areas. Note that, although the number of MN
per mm2 remained high during healing (at 37 days), the volumes of the BCG lesions were
diminished (see Fig. 1). ***P ⬍ 0.01. Reproduced with permission from reference 3.

much reduced in size, the ulcer had usually
epithelialized, and little or no necrotic contents
remained inside.
The lesions that had discharged most of their
liquefied necrotic contents seemed to heal the
fastest. We purposely selected such healing
lesions for our 37-day evaluations. Compar-
isons of primary and reinfection dermal BCG
lesions are presented in chapter 20.
Histopathology
At 1 to 3 days, the BCG lesions showed scattered
areas of intact and fragmented leukocytes among
the collagen fibers.At 9, 23, and 37 days, few, if
any, collagen fibers were present in the necrotic
areas or in the adjoining areas densely infiltrated
with cells. (Both granulocytes and macrophages
contain collagenases [see reference 4].) The
peripheral areas of BCG lesions were not
included in our quantitative evaluations. Such
peripheral areas contained more dispersed cells,
as well as more collagen fibers, including newly
formed collagen as the lesions healed.
The cell infiltrate was mainly composed of
mononuclear cells (mostly macrophages and
lymphocytes, but probably some dendritic cells)
(Fig. 2) (3). Polymorphonuclear leukocytes were
present in the initial infiltrate and near the
caseous and liquefied areas of the older lesions,
but elsewhere they were only a minor compo-
nent of the cell infiltrate (Fig. 2). Eosinophils
were rare, but plasma cells and fibroblasts were
common as the BCG lesions healed.
The ratio of CD4 to CD8 mononuclear cells
(mainly lymphocytes) in primary BCG lesions
did not change appreciably during their devel-
opment and healing (Table 1) (5).A larger per-
centage of CD4 cells than CD8 cells was always
present (5). (In rabbits, 5 weeks after an aerosol
inhalation of Mycobacterium tuberculosis strain
 23. RABBIT DERMAL BCG LESIONS 䡵 357

TABLE 1 CD4 and CD8 cells in mononuclear cell populations within primary BCG lesions during their
development and healinga
Age of BCG lesion (days) % of CD4 cells % of CD8 cells Ratio CD4/CD8
9 13.2 ⫾ 1.2 8.7 ⫾ 2.1 1.7 ⫾ 0.5
23 17.8 ⫾ 1.1 5.4 ⫾ 0.9 3.9 ⫾ 0.5
37 15.7 ⫾ 2.4 6.3 ⫾ 1.6 2.9 ⫾ 0.6
a
Listed are the percentages (mean ⫾ standard error) of CD4 and CD8 cells identified in the tissue sections by immunohis-
tochemical techniques. Note that CD4 cells are more numerous than CD8 cells.Adapted from reference 5.

CDC1551 [6] or strain H37Rv [Y. C. Manabe,
unpublished data], single-cell suspensions of
lung homogenates also contained [by flow
cytometry] a larger percentage of CD4 cells
than CD8 cells.)
In 23-day primary BCG lesions, epithelioid
cells were present in the tuberculous granulation
tissue surrounding the caseous/liquefied center,
and fibroblasts were present in the periphery as
the lesions healed. Epithelioid cells are macro-
phages that adhere to one another, so they
resemble epithelium (see chapter 6).They have
often ingested and digested tubercle bacilli
(7, 8). In addition, they have differentiated for a
variety of functions, e.g., secretion, digestion of
ingested materials, and/or cytokine production
(9, 10) (see chapter 6).
Vasculature-cell interactions in developing
and healing BCG lesions are presented in chap-
ter 21 and in references 1 and 5.A complete his-
tological description of developing and healing
BCG lesions in Lurie’s resistant and susceptible
inbred rabbits is published in reference 11.
Number of Bacilli in BCG Lesions
Acid-fast bacilli were always more numerous
where necrosis was present.At 1 to 3 days, many
bacilli were present in both intact macrophages

FIGURE 3 Number of bacilli in

central areas of rabbit BCG lesions of
various ages, visualized by acid-fast
staining with carbol-fuchsin. As the
lesions progressed, a shift occurred from
bacilli located intracellularly (mostly in
intact and necrotic macrophages) to
bacilli located extracellularly in areas
of (liquefied) caseous necrosis. By 23
days, the number of bacilli was greatly
reduced. Reproduced with permission
from reference 3.
358 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 4 Liquefied caseous

center of a 5-day dermal BCG lesion
stained for tubercle bacilli. Many
bacilli appear as clumps within
darkly stained macrophages. Other
bacilli are located extracellularly in
the liquefied debris. All darkly
stained tiny particles are bacilli. Since
this is necrotic material, the macro-
phages are not distinctly recognizable
in the photograph, but their rounded
outline and size clearly identify their
cell type. Fixed-frozen tissue sec-
tions stained by carbol-fuchsin and
counterstained with methylene blue.
Magnification, ⫻800. Reproduced
with permission from reference 3.

(and polymorphonuclear leukocytes) and in the
necrotic cells (Fig. 3).At 5 to 9 days, the number
of bacilli seen in intact cells was much reduced,
but many bacilli remained in necrotic foci that
were coalescing and liquefying (Fig. 3 and 4).At
23 days, very few bacilli could be seen in intact
tissues, and the number of bacilli in the caseous
necrotic center was greatly reduced (Fig. 3).At 37
days, the BCG lesions were almost healed.
Homogenized dermal BCG lesions were cul-
tured by Lurie for viable bacilli (11). These
bacilli multiplied for 2 weeks in the susceptible
rabbits but did not multiply appreciably in the
resistant rabbits (Table 2). We did not culture
BCG lesions produced in commercial New
Zealand White rabbits, but we would expect the
number of viable bacilli in them to follow the
pattern found in Lurie’s resistant rabbits.
Tuberculin Reactions
For the tuberculin tests, rabbits were injected
intradermally (0.1 ml per site) with an equiva-
lent to a 1:10 dilution of standard Old Tuber-
culin (7; see also reference 3) (see glossary).The
resulting tuberculin reactions were measured
with calipers, as described in reference 12. Old
Tuberculin contains more tuberculin-like anti-
gens than purified protein derivative and was
preferred by Lurie for testing the tuberculin
sensitivity of rabbits (7).
When skin tested, the rabbits were tuber-
culin negative at 3 days after the BCG injection,
but at 9 days, they had become tuberculin pos-
itive (Fig. 1).At 14 to 23 days, tuberculin sensi-
tivity frequently reached a peak and was some-
what reduced as the BCG lesions healed (5)
(see Fig. 5B).
After the rabbits became tuberculin positive,
the BCG lesions developed distinct caseous
centers, which soon liquefied. In hosts possess-
ing delayed-type hypersensitivity, caseous necro-
sis and liquefaction are caused by the high local
concentrations of tuberculin-like products (7)
from the large number of bacilli injected (see
chapter 4).
COMPARISON OF DERMAL BCG
LESIONS AND PULMONARY LESIONS
PRODUCED BY VIRULENT TUBERCLE
BACILLI IN RABBITS
Dermal BCG lesions can serve as a model of
pulmonary lesions caused by fully virulent
tubercle bacilli. In rabbits, both types of lesion
usually develop tuberculous granulation tissue,
caseous necrosis, liquefaction, ulceration or cav-
ities, and encapsulation by fibrous tissue.Also, the
same host factors control their progression and
regression.
The main differences between dermal BCG
lesions and those caused by fully virulent bacilli
 23. RABBIT DERMAL BCG LESIONS 䡵 359

TABLE 2 Relative numbers of tubercle bacilli in dermal BCG lesions of resistant strain III rabbits and suscep-
tible strain C rabbits at various times after infectiona
Avg. no. of BCG bacilli cultured from the
No. rabbits of each Interval after BCG injection same aliquot of the entire BCG lesion
strain tested
Resistant strain III Susceptible strain C
2 1 day 48 30
2 3 days — 54
2 1 week 65 155
2 2 weeks 40 575
2 4 weeks 6 42
a
On “day zero,” 12 ⫻ 106 viable BCG bacilli were injected intradermally into each of the 20 rabbits. On the days and weeks
listed, the entire lesion was surgically removed, ground in a mortar, and cultured for viable bacilli. Note that the BCG bacilli in
the dermal lesions of the resistant strain III rabbits were greater on day 1, did not increase appreciably over time, and declined more
rapidly than the BCG bacilli in the susceptible C rabbits.These findings were apparently due to a greater initial localization of
the bacilli at the dermal site of injection, a more rapid development of delayed-type hypersensitivity and cell-mediated immu-
nity, and a more rapid ulceration (with discharge of liquefied caseum containing bacilli) in the resistant group.Adapted from ref-
erence 11.

in the lungs is that dermal BCG lesions, usually
started by the injection of over a million tuber-
cle bacilli in one site, soon heal, whereas pul-
monary lesions, caused by inhalation of virulent
tubercle bacilli, begin with only 1 unit of 1 to
3 bacilli in a given site.
In addition, in rabbits, virulent human- and
bovine-type tubercle bacilli multiply to a greater
extent than BCG, and the virulent bovine-type
causes progressive disease (see chapter 13).
Mycobacteria are currently being studied by
molecular biology techniques to identify their
virulence factors (13–16).
HEALING OF DERMAL BCG
LESIONS AS A MEASURE
OF HOST RESISTANCE
Lurie (11) used the rapid healing of dermal
BCG lesions to select breeders for his resistant
rabbits (Fig. 5A) and used the delayed healing of
dermal BCG lesions to select breeders for his
susceptible rabbits. Resistance determined by
this method of selection paralleled that deter-
mined by his tubercle-count method (see chap-
ter 11), but did not always match on an indi-
vidual basis (11).
In human populations, people whose BCG
lesions healed quickly should usually be more
resistant to clinical tuberculosis than those whose
BCG lesions healed more slowly. Meyer and
Palmer (17) did a study that supported this
premise.They found that smaller BCG lesions
and the rapid development of tuberculin sensi-
tivity were, in general, associated with a greater
resistance to clinical tuberculosis.
LURIE’S INTRAVENOUS BCG
EXPERIMENTS IN RABBITS
Following the intravenous injection of 1 mg
wet weight of BCG (about 5 ⫻ 106), rabbits
developed small lesions in many organs of the
body, including the lungs, liver, spleen, and
lymph nodes (7, 18, 19). By 4 weeks most of the
BCG had been destroyed, and because of the
development of cell-mediated immunity and
delayed-type hypersensitivity, the BCG lesions
soon healed.
Among the various organs, bacillary multi-
plication varied greatly. In general, similar to
more virulent mycobacteria, the bacilli in the
liver multiplied the least, and those in the lung
(and spleen) multiplied the most (7, 18, 19).
Some caseous necrosis was even present in the
tracheobronchial lymph nodes, and viable BCG
occasionally persisted in these (and other) nodes
for many months.
In one of these studies (7, 18), the BCG
strain injected intravenously was somewhat
more virulent than the strains available today,
but in a subsequent study, the standard BCG was
360 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

FIGURE 5 Size of dermal BCG lesions and tuberculin reactions in Lurie’s inbred re-
sistant and susceptible rabbits at various times after infection. Note that at 1 and 2 weeks,
the BCG lesions (A) were largest in resistant strain III rabbits, because their tuberculin sen-
sitivity (B) developed more quickly and to a greater degree.Then, the BCG lesions in this
resistant strain III declined rapidly and healed sooner than did those in the susceptible strain
C. Strain A rabbits were of intermediate resistance when this experiment was performed.
Reproduced with permission from reference 11.

used (7, 19). The results from each of these
experiments were similar, although, as expected,
the more virulent strain multiplied more exten-
sively in the various organs.
SYSTEMIC NATURE OF BCG
VACCINATION IN HUMANS
In Scandinavia, necropsies were performed on
20 infants, children, and young adults who died
6 to 40 months after intradermal BCG vacci-
nation (usually of trauma) (20). Over half of
these individuals had granulomas, most often in
the liver and/or lungs, and sometimes in the
bronchial lymph nodes, spleen, or kidneys.
Therefore, following BCG vaccination in
humans, the BCG bacilli are apparently widely
disseminated throughout the body and produce
nonprogressing lesions in many organs. These
 23.
studies indicate that BCG vaccination is not
confined to the intradermal site of injection
and the draining lymph nodes, but is systemic in
nature (reviewed in references 20 and 21).
INTRADERMAL BCG VACCINATION
IN LURIE’S INBRED RESISTANT AND
SUSCEPTIBLE RABBITS
The BCG Vaccine
The vaccine used in these rabbit experiments
was Phipps BCG produced by Joseph D.Aron-
son (22, 23). It was freshly prepared each week
(by techniques similar to those described by
Calmette and Guérin [24]) and used within 3
days. Each rabbit received an intradermal injec-
tion of 106 to 107 viable bacilli, the exact dose
depending on the experiment (11).
The BCG Lesions
The lesions of the resistant strain III rabbits
enlarged more rapidly, reached a peak more
quickly, ulcerated more frequently, and healed
sooner than did those of the susceptible strain
C rabbits (Fig. 5A) (11). In resistant rabbits,
tuberculin sensitivity developed earlier (Fig. 5B)
(11), the bacilli multiplied for a shorter period
of time, the bacilli were more rapidly elimi-
nated (Table 2) (11), and antibodies were pro-
duced in higher titers (Fig. 6) (11). Histologically,
in resistant hosts, the tissue responses to BCG
were accelerated, i.e., mature epithelioid cells
(which are highly activated macrophages [9, 25,
26]) and plasma cells appeared sooner and in
greater numbers, as did fibroblasts and collagen
bundles (11).
The Amount of Protection
Ten to 11 weeks after the BCG vaccination,
Lurie’s resistant and susceptible rabbits were
challenged by aerosol with virulent human-
type tubercle bacilli (H37Rv) (11). Five weeks
after this aerosol challenge, the rabbits were
euthanized, and the grossly visible primary
tubercles in the lungs were counted.
In resistant strain III rabbits, the number of
inhaled human-type bacilli (H37Rv) required to
produce one grossly visible pulmonary tubercle
(“the ratio”) was increased by BCG from 642 to
2,948 (by the Lurie method of calculating ratios)
(Table 3) (11). In other words, the average num-
RABBIT DERMAL BCG LESIONS 䡵 361
FIGURE 6 Antibody titers in inbred resistant strain
III rabbits and susceptible strain FC rabbits at various
times after an intradermal injection of BCG.The anti-
bodies were determined by the hemagglutination
method of Middlebrook (31) with Old Tuberculin as the
antigen. Note that such antibodies reached much higher
titers in the resistant group.Adapted from reference 11.
The FC strain rabbits were similar to the C strain rab-
bits in their resistance to tuberculosis (see chapter 14).
ber of tubercles in the BCG-vaccinated rabbits
was 22% of the number of tubercles in the
unvaccinated controls, if no allowance was made
for “ratio inflation” by the Lurie method (see
chaper 11).
In susceptible strain FC rabbits, the ratio was
increased by BCG from 107 to 124. In other
words, the average number of tubercles (similarly
determined) in the BCG-vaccinated rabbits was
86% of the number of tubercles in the unvac-
cinated controls (which was not statistically sig-
nificant) (Table 3) (11).
The difference in the ability of inbred resis-
tant and susceptible rabbits to be immunized
against tuberculosis should have been expected.
Resistant rabbits, which are known to develop
good acquired resistance during an infection
with virulent tubercle bacilli, should also develop
good acquired resistance from immunization
with BCG.This acquired resistance from BCG
would then protect them substantially against an
aerosol challenge with virulent tubercle bacilli.
362 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 3 Number of inhaled human-type tubercle bacilli producing one grossly visible primary pulmonary
tubercle in unvaccinated and BCG-vaccinated inbred resistant and susceptible rabbits (“the ratio” by the Lurie
method)a
A B A B
Unvaccinated Vaccinated Unvaccinated Vaccinated
resistant resistant susceptible susceptible
strain III strain III strain FC strain FC
No. of rabbits 8 8 12 9
No. of inhaled bacilli 642 ⫾ 107 2,948 ⫾ 914 107 ⫾ 200 124 ⫾ 18
producing one visible (536) (1,073) (69) (98)
tubercle (ratios)
Amount of protection: 22% 86%
no. of visible tubercles P ⫽ 0.013 P ⫽ 0.270
in the vaccinated rabbits
compared to the no.
in the controls
a
The rabbits were vaccinated intradermally with 1.6 ⫻ 106 Phipps strain BCG.Ten to 11 weeks later, they were challenged
by aerosol with 4,000 to 12,000 virulent human-type tubercle bacilli (H37Rv) inhaled per rabbit. Five weeks after challenge, the
rabbits were euthanized, and the grossly visible primary pulmonary tubercles were counted.The vaccinated strain III rabbits were
paired with vaccinated strain FC rabbits and infected with H37Rv at the same times. However, the data for the unvaccinated rab-
bits were assembled mostly from experiments in which the resistant and susceptible rabbits were infected with H37Rv at differ-
ent times (11). Note that BCG vaccination was much more effective in resistant strain III rabbits than in the susceptible strain FC
rabbits (see text).The ratios were determined by the Lurie method, which inflates the higher ratios but is the best way to pool
data from experiments differing in inhaled bacillary dosages (see chapter 11).The numbers in parentheses are noninflated ratios
derived from averages (see chapter 11).Adapted from reference 11.

TABLE 4 Vaccine efficacy: number of grossly visible tubercles in vaccinated rabbits and their diameters as per-
centages of those found in controlsa
No. of grossly visible tubercles as a Diameters of tubercles as a percentage
Vaccine percentage of the no. in controls of those in controls
BCG
Danish 54, 69, 64 74, 86, 78
Japanese 48, 61 77, 83
Tice 65 77
Pasteur 13, 52 85, 76
M. microti
OV 254 40, 25, 22, 61 81, 62, 80, 69
NCO 8712 56 65
ATCC 35781 62 77
ATCC 35782 65 79
ATCC 11152 29, 20 96, 81
ATCC 19422 73 72
a
This table summarizes the results of numerous experiments, each containing 6 to 8 commercially available outbred New Zealand
White rabbits in the control and vaccinated groups. Five weeks after these rabbits inhaled virulent human-type tubercle bacilli
(H37Rv), the animals were euthanized, and the grossly visible primary tubercles in their lungs were counted. Note that every vac-
cine evaluated decreased both the tubercle count and the tubercle size. However, because of the variability among outbred rab-
bits, no vaccine proved consistently better than the others.Adapted from reference 29, with some unpublished experiments added
from Yukari C. Manabe.
 23.
In contrast, susceptible rabbits, which are known
to develop poor acquired resistance during an
infection with virulent bacilli, should also
develop poor acquired resistance from BCG,
and therefore, benefit less from the immuniza-
tion. In other words, BCG vaccination helped
the most the hosts that needed it the least; con-
versely, it helped the least the hosts that needed
it the most.
These findings are just another example of
Lurie’s cardinal principle: “Acquired resistance
is superimposed upon and determined by native
resistance” (7). Recent studies, especially those
by the Janeway group (27, 28), provide insight
into some of the mechanisms by which innate
resistance affects the development of acquired re-
sistance (see chapter 5).
If these results are applied to human popula-
tions, tuberculosis vaccines would be most effec-
tive in people who are able to produce a good
immune response,which fortunately is the major-
ity of the immunocompetent persons living today.
Such vaccines would be less effective in people
who are unable to mount a good immune
response, e.g., those treated with immunosup-
pressive agents and those infected with
HIV/AIDS.Such persons need the protection the
most, but vaccination with BCG would proba-
bly help them relatively little. In fact, live vaccines
may be dangerous to give to such persons.
EFFICACIES OF BCG AND M. MICROTI
VACCINES IN COMMERCIAL NEW
ZEALAND WHITE RABBITS
We have recently used the tubercle-count
method (see chapter 11) to evaluate the efficacy
of various strains of BCG and M. microti (the vole
bacillus) in commercial New Zealand White
rabbits (29). All of the vaccines tested so far
reduced the average number of pulmonary
tubercles found 5 weeks after the inhalation of
virulent human-type bacilli (H37Rv) and
decreased the average diameter of these tuber-
cles (Table 4) (29). However, the number of
tubercles produced in these outbred rabbits was
so variable that we could not determine which
vaccine was significantly better than the others.
The late Professor Helen Abbey of our Biosta-
tistics Department calculated that about 18 of
RABBIT DERMAL BCG LESIONS 䡵 363
these rabbits (rather than 6 to 8) in each cate-
gory of a given experiment would be required
to obtain statistical significance, if any exists.
The reduction in tubercle size did not seem to
be correlated with the reduction in the number
of tubercles (29). Also, the number of bacilli
inhaled seemed to have no effect on the size of
the resulting tubercles (29).
In human populations, no one BCG prepa-
ration was found to be more efficacious than any
other (30). However, human trials cannot be
standardized for variables as carefully as those
performed in laboratory animals.
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1. Abe,Y., K. Sugisaki, and A. M. Dannenberg,
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inate in chronic inflammation. J. Leukoc. Biol.
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2. Bartholomew, J.W. 1981. Stains for microorgan-
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3. Sugisaki, K., A. M. Dannenberg, Jr., Y. Abe,
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T.Yoshimura, P. J. Converse, and P. Mounts.
1998. Nonspecific and immune-specific up-
regulation of cytokines in rabbit dermal tubercu-
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4. Woessner, J. F., Jr., A. M. Dannenberg, Jr.,
P. J. Pula, M. G. Selzer, C. L. Ruppert,
K. Higuchi, A. Kajiki, M. Nakamura, N. M.
Dahms, J. S. Kerr, and G.W. Hart. 1990. Extra-
cellular collagenase, proteoglycanase and products
of their activity, released in organ culture by intact
dermal inflammatory lesions produced by sulfur
mustard. J. Investig. Dermatol. 95:717–726.
5. Shigenaga, T., A. M. Dannenberg, Jr., D. B.
Lowrie, W. Said, M. J. Urist, H. Abbey, B. H.
Schofield, P. Mounts, and K. Sugisaki. 2001.
Immune responses in tuberculosis: antibodies and
CD4-CD8 lymphocytes with vascular adhesion
molecules and cytokines (chemokines) cause a rapid
antigen-specific cell infiltration at sites of bacillus
Calmette-Guérin reinfection. Immunology 102:466–
479.
6. Kesavan, A. K., S. Mendez, C. L. Hatem,
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7. Lurie, M. B. 1964. Resistance to Tuberculosis: Exper-
imental Studies in Native and Acquired Defensive Mech-
anisms. Harvard University Press, Cambridge, Mass.
8. Ando, M., A. M. Dannenberg, Jr., M. Sugi-
moto, and B. S. Tepper. 1977. Histochemical
studies relating the activation of macrophages to the
intracellular destruction of tubercle bacilli. Am. J.
Pathol. 86:623–634.
9. Suga, M., A. M. Dannenberg, Jr., and
S. Higuchi. 1980. Macrophage functional hetero-
geneity in vivo: macrolocal and microlocal
macrophage activation, identified by double-staining
tissue sections of BCG granulomas for pairs of
enzymes. Am. J. Pathol. 99:305–324.
10. Nathan, C. F. 1987. Secretory products of
macrophages. J. Clin. Investig. 79:319–326.
11. Lurie, M. B., P. Zappasodi, E. Cardona-Lynch,
and A. M. Dannenberg, Jr. 1952.The response
to the intracutaneous inoculation of BCG as an
index of native resistance to tuberculosis. J. Immunol.
68:369–387.
12. Dannenberg, A. M., Jr., M. Ando, and
K. Shima. 1972. Macrophage accumulation, divi-
sion, maturation and digestive and microbicidal
capacities in tuberculous lesions. III.The turnover
of macrophages and its relation to their activation
and antimicrobial immunity in primary BCG
lesions and those of reinfection. J. Immunol.
109:1109–1121.
13. Manabe, Y. C., A. M. Dannenberg, Jr., and
W. R. Bishai. 2000.What can we learn from the
Mycobacterium tuberculosis genome sequencing pro-
jects? Int. J.Tuberc. Lung Dis. 4:S18–S23.
14. Brodin, P., C. Demangel, and S.T. Cole. 2005.
Introduction to functional genomics of the Mycobac-
terium tuberculosis complex, p. 143–153. In S.T. Cole,
K. D. Eisenach, D. N. McMurray, and W. R. Jacobs,
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Press,Washington, D.C.
15. Brosch, R., and M. A. Behr. 2005. Comparative
genomics and evolution of Mycobacterium bovis
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16. Goulding, C. W., D. Pal, and D. Eisenberg.
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McMurray, and W. R. Jacobs, Jr. (ed.), Tuberculosis and
the Tubercle Bacillus.ASM Press,Washington, D.C.
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to BCG vaccination as an index of familial suscep-
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18. Lurie, M. B. 1932. The correlation between the
histological changes and the fate of living tubercle
bacilli in the organs of tuberculous rabbits. J. Exp.
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19. Lurie, M. B. 1934.The fate of BCG and associ-
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60:163–178.
20. Gormsen, H. 1956. On the occurrence of epithe-
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Scand. Suppl. 111:117–120.
21. Lindgren, I. 1961. Anatomical and roentgeno-
logic studies of tuberculous infections in BCG-
vaccinated and non-vaccinated subjects, with
biophysical investigations of calcified foci. Acta
Radiol. 209(Suppl):1–101.
22. Aronson, J. D., E. I. Parr, and R. M. Saylor.
1940. BCG vaccine. Its preparation and the local
reaction to its injection. Am. Rev.Tuberc. 42:651–666.
23. Aronson, J. D., C. F. Aronson, and H. C.Tay-
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Med. 101:881–893.
24. Calmette, A., and C. Guérin. 1924.Vaccination
des bovides contre la tuberculose et méthode nou-
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Inst. Pasteur 38:371–398.
25. Dannenberg, A. M., Jr., O. T. Meyer, J. R.
Esterly, and T. Kambara. 1968.The local nature
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26. Dannenberg, A. M., Jr. 1968. Cellular hyper-
sensitivity and cellular immunity in the patho-
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27. Janeway, C. A., Jr., P.Travers, M.Walport, and
M. J. Shlomchik. 2001. Immunobiology: the Immune
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28. Medzhitov, R., and C. Janeway, Jr. 2000. Innate
immunity. N. Engl. J. Med. 343:338–344.
29. Dannenberg, A. M., Jr., W. R. Bishai, N. Par-
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Boles, and M. L. M. Pitt. 2000. Efficacies of
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30. Milstien, J. B., and J. J. Gibson. 1990. Quality
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 Section 8.

PAST, PRESENT, AND FUTURE

This page intentionally left blank
 24
SUMMARY AND CONCLUSIONS
Part I. Summary [367]
Host-parasite interactions [367]
Stages in the pathogenesis of tuberculosis
Role of DTH and CMI in the pathogenesis of tuberculosis
Part II. Conclusions [370]
Principles of host-parasite interaction established by this
disease [370]
Application to other infectious diseases
PART I. SUMMARY
HOST-PARASITE INTERACTIONS
The pathogenesis of tuberculosis can be con-
sidered a series of battles between the host and
the tubercle bacillus, each of which has its own
weapons that can be used against the other. In
addition, both the host and the bacillus have sites
of vulnerability that can be exploited by the
adversary (1).
The weapons of the host are:
1. Cell-mediated immunity (CMI), which
activates macrophages so that they can
kill or inhibit tubercle bacilli that they
ingest
2. Delayed-type hypersensitivity (DTH),
which stops the intracellular growth of
bacilli in nonactivated macrophages by
killing these macrophages. DTH thereby
transforms an environment that is favor-
able for the bacillus into an environment
that is inhibitory, i.e., solid caseous tissue.
The weapons of the bacillus are:
1. The ability to multiply intracellularly in
nonactivated macrophages, i.e., within the
monocytes/macrophages that emigrate
from the bloodstream into the tissues at
the sites of the infection
2. The ability to multiply extracellularly in
the liquefied caseum of a cavity wall,
sometimes reaching tremendous numbers
367
[367]
[368]
[372]
The vulnerabilities of the host are:
1. Nonactivated macrophages that provide a
favorable environment for the intracellu-
lar growth of the bacillus
2. Liquefied caseous material, the only men-
struum in the host that can support the
extracellular growth of the bacillus
The vulnerabilities of the bacillus are:
1. The inability to survive within a fully
activated macrophage
2. The inability to multiply in solid caseous
tissue
Table 1 correlates the multiplication of the
bacilli with the type of disease and the immune
response of the host.
STAGES IN THE PATHOGENESIS
OF TUBERCULOSIS
See chapter 2 and reference 2.An inhaled bacil-
lary unit of 1 to 3 bacilli will reach the alveo-
lar spaces and will be ingested by an alveolar
macrophage (Stage I). In humans and rabbits,
most of these macrophages seem to be acti-
vated sufficiently to destroy the bacillary unit
before any lesion is produced. However, if the
alveolar macrophage is not sufficiently activated,
the ingested bacilli multiply and disrupt the
alveolar macrophage.
The released bacilli are then ingested by non-
activated monocytes/macrophages that are
368 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS

TABLE 1 Multiplication of tubercle bacilli, the type of disease, and the host immune responsea
No multiplication of virulent tubercle bacilli occurs in the majority of human and rabbit pulmonary
alveolar macrophages, because they are already highly activated.
Disease: No disease.
Immune response: No cell-mediated immunity (CMI) and no delayed-type hypersensitivity (DTH) or
conversion of the tuberculin skin test.
Bacilli are dormant in the solid caseous center of tuberculous lesions. (In humans the bacilli may remain
dormant in solid caseum for over 30 years.)
Disease: Arrested disease. Bacilli that escape from the edge of the caseum are soon ingested and, in resistant
hosts, are inhibited by nearby highly activated macrophages.
Immune response: Good CMI and (in humans) strong or weak DTH, depending on the extent of residual
disease and the amount of bacillary growth and destruction at the edge of the caseum.
Intracellular multiplication of bacilli occurs in poorly activated macrophages surrounding the solid caseous
center.Tissue-damaging DTH kills these macrophages. (Intracellular bacillary multiplication also occurs in
nonactivated macrophages before DTH and CMI develop.)
Disease: Progressive disease. If only poorly activated macrophages are nearby, the solid caseous center
enlarges, and hematogenous and lymphatic spread of bacilli occurs.
Immune response: Poor CMI, but often good DTH, i.e., tuberculin sensitivity (except terminally).
Extracellular multiplication of bacilli occurs in liquefied caseum, especially in the liquefied caseum lining the
walls of cavities. (Coughing spreads these extracellular bacilli into the environment.)
Disease: Bronchial spread of bacilli, causing new pulmonary lesions and sometimes bronchopneumonia.
Immune response: Good CMI (which may be overwhelmed by a large number of bacilli), and usually good
DTH (except terminally).
a
Revised from reference 1.

chemotactically attracted there from the blood-
stream. In these phagocytes the bacilli grow
logarithmically (Stage II). Lurie called this the
stage of symbiosis, because both macrophages
and tubercle bacilli increase in the developing
lesion, with no apparent injury to either.
When the host becomes tuberculin positive,
a caseous center may develop in the lesion
(Stage III).A lesion with a small caseous center
(up to 2 mm in diameter) may heal or stabilize
before it is detectable by radiography. A larger
caseous lesion may enlarge and shed bacilli into
the blood and lymph (Stage IVa), or it may heal
or stabilize (Stage IVb).
Alternatively, a caseous lesion may liquefy
and form a cavity (Stage V). Now, the bacilli can
grow extracellularly (for the first time), enter the
bronchial tree, and spread to other parts of the
lung and to the environment.
In general, each stage of tuberculosis is won
by the host with increasing difficulty. Further-
more, in the same lung, some lesions may
progress while other lesions may regress.Tuber-
culosis is a locally controlled disease that depends
on the growth of bacilli in nonactivated
macrophages or in liquefied caseum, or on the
inhibition of bacilli in activated macrophages or
in solid caseum.
ROLE OF DTH AND CMI IN THE
PATHOGENESIS OF TUBERCULOSIS
See chapter 5. Tissue-damaging DTH will
directly or indirectly kill nonactivated macro-
phages that have permitted more than a few
tubercle bacilli to grow in their cytoplasm.
(These bacilli apparently release high local con-
centrations of tuberculin-like products.) The
killing of such macrophages eliminates the
intracellular environment that is favorable to
bacillary growth and replaces it with the extra-
cellular environment of solid caseous tissue that
is inhibitory to bacillary growth. The bacillus
 24. SUMMARY AND CONCLUSIONS 䡵 369

itself is nontoxic; it damages host tissue mainly
by means of the host’s own immune response
to its tuberculin-like products.
CMI activates macrophages so that they can
kill or inhibit ingested tubercle bacilli.The bacil-
lus does not multiply within highly activated
macrophages.Therefore, to arrest this disease, the
host must produce many activated macrophages
at sites where the bacilli lodge.
To control bacillary multiplication, both CMI
and tissue-damaging DTH are required. This
statement was proved by correlating bacillary
growth curves (Fig. 1) with the observed gross
pathology and histopathology (see chapter 2).
Lurie produced these growth curves by infect-
ing susceptible and resistant inbred rabbits with
aerosolized virulent tubercle bacilli and cultur-
ing the lungs for viable bacilli at different times
during the course of the resulting disease (3, 4).
The resistant rabbits developed strong immunity
(CMI) that activated macrophages to destroy
or inhibit tubercle bacilli.The susceptible rab-
bits developed only weak CMI that often was
incapable of activating macrophages sufficiently
to control the growth of the bacilli.Therefore,
in Fig. 1, the complete parallelism found in the
bacillary growth curves between the two strains
of rabbits was totally unexpected.
This riddle was solved by the realization that
two methods of controlling bacillary growth
exist: CMI and tissue-damaging DTH. After
tubercle bacilli are inhaled, they are ingested
by alveolar macrophages. The alveolar macro-
phages of resistant rabbits are apparently more
highly (nonspecifically) activated and therefore
destroy or inhibit tubercle bacilli more effec-
tively than those of susceptible rabbits.Therefore,
at 7 days after infection, there is about a 20- to

FIGURE 1 Changes in the number of human-type tubercle bacilli in the lungs of

Lurie’s natively resistant and natively susceptible rabbits at different intervals after aerosol
infection. By 7 days after infection, the resistant rabbits had inhibited the growth of the
bacilli 20 to 30 times more effectively than did the susceptible rabbits, but, from then
on, the two curves were parallel.At 4 to 5 weeks, the lungs of susceptible rabbits con-
tained about 13 times the number of primary pulmonary tubercles that the lungs of re-
sistant rabbits contained.The means and their standard errors are shown. (The y axis of
this figure is on a log scale.) Reproduced with permission from reference 3.
The number of tubercle bacilli in the lungs of the resistant rabbits failed to decrease
during the period illustrated, because liquefaction with extracellular multiplication of
the bacillus readily occurred in these rabbits. Liquefaction did not occur in the susceptible
rabbits, possibly because only low levels of hydrolytic enzymes developed in their
macrophages.
The logarithmic and stationary phases of this bacillary growth curve also occur in mice
and guinea pigs, following the inhalation of virulent tubercle bacilli (see chapter 15).
370 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
30-fold difference in the number of bacilli in the
lungs of the two strains of rabbits (Fig. 1).
From this point on, however, the bacillary
growth curves in resistant and susceptible rabbits
follow a parallel course. In both rabbit strains, dur-
ing the logarithmic stage of symbiosis, the bacilli
apparently multiply in nonactivated macrophages
without any inhibition. Also, the logarithmic
stage stops in both rabbit strains at the same time,
because each rabbit strain develops tuberculin
sensitivity (DTH), which kills the nonactivated
macrophages in which the bacilli are multiplying.
The susceptible rabbits often had tuberculin reac-
tions comparable in size to those of the resistant
rabbits, because the immune system of the sus-
ceptible rabbits (although weaker) had been stim-
ulated by 20 to 30 times the quantity of bacillary
antigens. Such antigens are released by the bacilli
growing within poorly activated macrophages.
Such antigens would also stimulate CMI, but
the bacillary load is too high in the susceptible
rabbits, and their ability to produce CMI (acti-
vated macrophages) is too weak, to effectively
arrest the progress of the lesion.
Subsequently, the two bacillary growth curves
still follow a parallel course (Fig. 1), but for dif-
ferent reasons. In the susceptible rabbits, bacilli
escape from the edge of the caseum and are
ingested by poorly activated macrophages.Again,
intracellular multiplication of the bacilli occurs,
and, again, the poorly activated macrophages
harboring these bacilli are killed by tissue-
damaging DTH. The process is repeated, and
much of the surrounding lung tissue is
destroyed. Nevertheless, this strategy is effec-
tive for months, because the total number of
viable bacilli in the lung does not increase.
In the resistant rabbits, bacilli escaping from
the edge of the caseum are often ingested by
highly activated macrophages that surround the
caseum.These activated macrophages ingest and
inhibit or destroy the bacilli, so that the disease
would be arrested at this time were it not for the
process of liquefaction and cavity formation.
In the liquefied caseum, tubercle bacilli can
grow extracellularly for the first time, and at such
sites they cannot be reached, even by highly
activated macrophages. In the resistant rabbits
(and in commercial New Zealand White rab-
bits), liquefaction may begin 8 weeks after an
aerosol infection with fully virulent bovine-
type tubercle bacilli, whereas in the susceptible
rabbits, liquefaction does not occur.
All of these principles developed in the rab-
bit model seem applicable to the pathogenesis
of tuberculosis in humans. The type of tuber-
culosis described in the resistant rabbits resem-
bles that found in immunocompetent adults.The
type of tuberculosis described in the susceptible
rabbits resembles that found in infants and
immunocompromised adults.
The various chapters of this book provide
more details on the pathogenesis and immunol-
ogy of tuberculosis, on the role of antigen-
presenting cells, macrophages, and lymphocytes,
and on the role of antibodies. Chapters on the
vascular endothelium, cytokines, and hormones
are included, as well as chapters on improving
vaccines. Chapter 15 compares the characteris-
tics of tuberculosis found in rabbits, mice, and
guinea pigs.
For a quick overview of the entire book, the
reader is referred to appendix F, where the
abstracts of these chapters are collected.
PART II. CONCLUSIONS
PRINCIPLES OF HOST-PARASITE
INTERACTION ESTABLISHED BY
THIS DISEASE
The following list succinctly presents some of
the principles described above and adds addi-
tional ones.
1. Tuberculosis is controlled locally at each
site where the bacillus resides in the host.
2. Local DTH and CMI are both needed to
arrest the progress of this disease.
3. Tissue-damaging DTH kills overloaded
macrophages in which tubercle bacilli are
multiplying intracellularly when the
tuberculin-like products locally reach high
concentrations.The bacillus does not mul-
tiply in the resulting solid caseous tissue.
4. CMI (via the cytokines of Th1 lympho-
cytes) activates local macrophages, so that
these macrophages can now inhibit
and/or kill the tubercle bacilli that they
phagocytize.
 24. SUMMARY AND CONCLUSIONS 䡵 371

TABLE 2 General characteristics of acute and chronic bacterial infectionsa

Acute septicemic infections, Chronic granulomatous
Characteristics such as pneumococcal infections, such as
pneumonia tuberculosis
1. Rate of accumulation of ⫹⫹⫹⫹ ⫹
bacteria in tissues
2. Endotoxins (lipopolysaccharides) ⫹⫹⫹⫹ ⫾
from gram-negative bacteria
and similarly acting components
of gram-positive bacteriab that
can cause local necrosis, blood
vessel damage, and systemic
shock, as well as terminal
irreversibility of the
disease process
3. DTH, which can cause local ⫾ ⫹⫹⫹⫹
necrosis and systemic toxicity
4a. Bacterial intracellular None in PMN and macrophages Only in nonactivated macrophages
multiplication
4b. Bacterial extracellular In tissues and blood In the liquefied caseum of
multiplication pulmonary cavities
5. Predominant type of
phagocyte:
granulocytes (PMN) ⫹⫹⫹⫹ ⫾
mononuclear cells ⫹ ⫹⫹⫹
(lymphocytes and macrophages)
6. Importance of circulating ⫹⫹⫹⫹ ⫾
antibodies in recovery of the
host
7. Degree of resistance to Completely resistant to reinfection Incompletely resistant to
exogenous or endogenous with the same strain of reinfection with M. tuberculosis
reinfection pneumococcus
8. Frequency of reactivation of ⫾ ⫹⫹⫹
the primary infection
9. Frequency of symptom-free ⫾ ⫹⫹⫹
inapparent infection that
increases antigen-specific
acquired resistance
a
Exceptions exist.
b
For example, peptidoglycans, teichoicated peptidoglycans, lipoteichoic acid, and phosphorylcholine attached to teichoic acids
(5, 6).

5. Antigen-antibody reactions (by produc- reinfection may be stopped at an earlier

ing chemotactic complement components)
hasten the local accumulation of both
macrophages and antigen-specific Th1 cells
at sites of reinfection. In this manner, anti-
gen-antibody reactions greatly enhance
local cell-mediated immune response (i.e.,
DTH and CMI) wherever reinfecting
tubercle bacilli lodge. Because of circulat-
ing antibodies, the progression of lesions of
time, i.e., while they are still microscopic
in size. However, without an expanded
Th1 cell population (produced by vac-
cines or a primary infection), antibodies
would have little effect on the progress of
the disease.
6. The amount of cell infiltration is under
strict control. Apparently, after sufficient
lymphocytes and macrophages have
372 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
entered the site of bacillary multiplica-
tion, the production of chemokines (and
probably other chemotaxins) is down-
regulated.
7. Liquefaction of solid caseous tissues
enables the bacillus to multiply extracel-
lularly for the first time, often reaching
such large numbers that even strong
acquired resistance may be overwhelmed.
Bronchial walls are eroded by tissue-
damaging DTH, and the bacilli spread
through the airways to other parts of the
lung and into the environment, where
they may be inhaled by other persons.
8. In humans, antimicrobial-resistant tuber-
cle bacilli mostly occur within the bacil-
lary population that grows profusely in the
liquefied caseum of the cavity wall.
9. The prevention of liquefaction and cav-
ity formation should greatly reduce the
progress of tuberculosis in immunocom-
petent hosts and greatly reduce the spread
of tuberculosis to other individuals.
APPLICATION TO OTHER
INFECTIOUS DISEASES
Acute and chronic bacterial infections show a
spectrum of host responses. At one end of the
spectrum is typical pneumococcal pneumonia, a
rapidly progressing acute disease somewhat like
anthrax and plague (Table 2).At the other end of
the spectrum is typical adult-type tuberculosis, a
slowly progressing chronic disease somewhat like
chronic brucellosis and some of the chronic
mycotic infections. Most infectious diseases are
intermediate between these two extremes,
because they possess some of the characteristics
of both the acute and the chronic types.
Of course, disease produced by any given
microorganism is not always typical and may
vary across the spectrum. However, the structure
presented in Table 2 can serve as a format to
which many of the attributes of most infec-
tious diseases can be assigned.
REFERENCES
1. Dannenberg, A. M., Jr. 1999. Pathophysiology:
basic aspects. I. Pathogenesis of tuberculosis. II.
Immunology of tuberculosis, p. 17–47. In D. Schloss-
berg (ed.), Tuberculosis and Nontuberculous Mycobac-
terial Infections, 4th ed. The W. B. Saunders Co.,
Philadelphia, Pa.
2. Dannenberg, A. M., Jr. 1993. Immunopatho-
genesis of pulmonary tuberculosis. Hosp. Pract.
28:33–40 (Off. ed. 51–58).
3. Lurie, M. B., P. Zappasodi, and C. Tickner.
1955. On the nature of genetic resistance to tuber-
culosis in the light of the host-parasite relationships
in natively resistant and susceptible rabbits. Am.
Rev.Tuberc. Pulmon. Dis. 72:297–329.
4. Allison, M. J., P. Zappasodi, and M. B. Lurie.
1962. Host-parasite relationships in natively resistant
and susceptible rabbits on quantitative inhalation
of tubercle bacilli: their significance for the nature
of genetic resistance. Am. Rev. Respir. Dis. 85:553–
569.
5. Weber, J. R., P. Moreillon, and E. I.Tuomanen.
2003. Innate sensors for Gram-positive bacteria.
Curr. Opin. Immunol. 15:408–415.
6. McCullers, J. A., and E. I. Tuomanen. 2001.
Molecular pathogenesis of pneumococcal pneu-
monia. Front. Biosci. 6:d877–d889.
 25
SUGGESTED FUTURE RESEARCH
AND UNANSWERED QUESTIONS

Part I. Suggested research on the host [373]

Local control of the host response [373]
Organ resistance and its implications [374]
Ideal ratio of DTH to CMI [374]
Role of vascular thrombosis in causing caseous necrosis and
inhibition of bacillary growth [375]
Role of granulocytes in tuberculosis [376]
Formation of liquefied caseum and cavities and their
prevention [376]
Prognostic tests that reflect disease progression or
regression [377]
Comparisons of tuberculous lesions produced by live and dead
tubercle bacilli [378]
Role of antibodies in host resistance to reinfection [378]
New resistant and susceptible rabbit strains [378]
Part II. Suggested research on the bacillus [378]
Bacillary survival and virulence [378]
Part III. Suggested research on prophylactic vaccines and
immunotherapy [380]
More effective vaccines [380]
Immunotherapy to supplement antimicrobial therapy and to treat
multidrug-resistant tuberculosis [380]
Other vaccine possibilities [380]
Part IV. Development of new drugs for the treatment and prevention of
tuberculosis [380]
Drugs acting on the host [380]
Drugs acting on the bacillus [380]
Part V. Sources of additional information [382]

PART I. SUGGESTED RESEARCH differentiation? How many macrophages carry

ON THE HOST
LOCAL CONTROL OF THE
HOST RESPONSE
The cells participating in tuberculous lesions
show a heterogeneity of functions, even among
cells of identical appearance (see chapter 6). One
macrophage may differentiate in one direction,
e.g., to produce reactive oxygen intermediates
(ROIs) and reactive nitrogen intermediates
(RNIs) to kill bacilli, while a nearby macrophage
may differentiate in another direction, e.g., to
secrete interleukin-12 that stimulates lymphocytes
to produce Th1-type cytokines. What factors
control each of the many types of macrophage
out multiple functions, and how many are spe-
cialized for only a single function? What cells
direct the local differentiation of macrophages?
How do the parenchymal cells in different organs
and the type of infiltrating cell affect macrophage
differentiation?
What cytokines are produced by each type of
infiltrating cell? How are such cytokines upreg-
ulated and downregulated? How do these
cytokines affect each other? What cells have
receptors for these cytokines, and how are these
receptors upregulated and downregulated?
These questions on macrophage differentia-
tion would apply to the dendritic cells (DCs) in

373
374 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
tuberculous lesions. In addition, we do not know
how many DCs are actually present there, how
they interact with the other cells, and how they
differentiate into their various functional types
that determine the type of immune response. See
chapter 6 for more details on dendritic cells.
The interactions that occur within a tuber-
culous lesion are complex. For example,
macrophages and lymphocytes can be stimula-
tory or inhibitory, depending on their local
environment (discussed in reference 1).We can
get some insights from cell culture experiments,
but such experiments are not equivalent to
those made in vivo. Cell cultures have no con-
stant influx and egress of participating cells, no
continuous fresh blood supply to provide nutri-
ents and removal of waste products, and no nat-
ural cell–extracellular matrix interactions. His-
tochemistry (including immunocytochemical
and in situ hybridization procedures) can pro-
vide some answers, but histochemistry is limited
by the availability of reagents and by what fac-
tors remain localized in the tissue section and
are not destroyed during its preparation. Are
frozen sections necessary? Would cold-embed-
ding in glycol methacrylate (2, 3) preserve fac-
tors not preserved in paraffin sections? Would
lyophilization (4) of tuberculous lesions before
embedding preserve more of these factors? A
variety of fixation and embedding techniques
exist (4). Some of these techniques may preserve
certain enzymes, cytokines, and other tissue
and bacillary components better than other
techniques.
ORGAN RESISTANCE AND
ITS IMPLICATIONS
In rabbits, tubercles in the liver rarely progress to
grossly visible size, but tubercles in the kidneys
do so (5). In guinea pigs, tubercles in the liver
continue to progress, but tubercles in their kid-
neys never do so (5).These facts are puzzling, but
insight into their causes may provide new
approaches to the control of tuberculosis. The
local conditions surrounding the infiltrating den-
dritic cells, macrophages, and lymphocytes deter-
mine how these cells function. Specifically, liver
cells and kidney cells in various species may
produce different cytokines (and different cofac-
tors for antigen presentation) that activate the
infiltrating cells in different ways (see chapter 5).
The cytokines and cytokine receptors that
are involved in the immune response within
different organs should be identified by molec-
ular biology techniques. Liver and kidney cells
from rabbits and guinea pigs could be cultured.
These cells could be stimulated with lipopolysac-
charides, or tuberculin, or live tubercle bacilli
with unstimulated cells as controls. Cytokine
proteins could be assayed by immunological
techniques, and cytokine mRNAs could be
assayed by reverse transcription-PCR techniques.
Immunohistochemical and/or in situ hybridiza-
tion technology could also be applied to tissue
sections of tuberculous lesions from each organ.
Knowledge of the cytokines and the cofactors
for antigen presentation that prevent or enhance
the development of tuberculous lesions in each
organ might lead to new therapeutic agents for
the control of this disease. For example, if the kid-
ney cytokines of the guinea pig can prevent the
progress of tuberculous lesions, then pharmaco-
logical agents to increase the production of such
inhibitory cytokines might be found.
IDEAL RATIO OF DTH TO CMI
(i) Definitions and Goals. As discussed
in chapters 2, 5, and 15, DTH and CMI play
major roles in the pathogenesis of tuberculosis.
With DTH, macrophages containing too many
tubercle bacilli for CMI to control are killed.
With CMI, lymphocytes produce cytokines that
activate macrophages to destroy or inhibit tuber-
cle bacilli. Both DTH and CMI are mediated by
Th1 lymphocytes and their cytokines.
Within tuberculous lesions, the main differ-
ence between DTH and CMI is the amount of
host sensitivity to the bacillary products that
elicit each response.The local tissue-damaging
DTH (which kills macrophages containing
numerous tubercle bacilli) is caused by low con-
centrations of tuberculin-like products, whereas
the local activation of macrophages (CMI) is
apparently caused by higher concentrations of
other bacillary products (see chapter 5).Tuber-
culin in very low concentrations within tuber-
culous lesions can activate macrophages without
 25.
necrosis (6).Therefore, the sensitivity of the host
to each bacillary antigen determines whether
that antigen produces a local CMI or a local tis-
sue-damaging DTH response.
An ideal ratio of DTH to CMI would be one
that produces the least amount of tissue damage
with the greatest destruction (or inhibition) of
the tubercle bacillus. Our goal would be to
identify the antigenic composition that pro-
duces such an ideal ratio and then use this
knowledge to make a more effective vaccine.
Tissue necrosis in clinical tuberculosis could
be reduced by vaccines that cause little or no
sensitivity to tuberculin (see chapter 22). Such
vaccines would expand the Th1 lymphocyte
population that does not react to tuberculin-like
products without expanding the population that
does react to these products. In other words, this
vaccine would create a population of lympho-
cytes that could respond with a high CMI/
DTH ratio. When such a vaccinated person
inhales virulent tubercle bacilli, the expanded
lymphocyte population that infiltrates the result-
ing pulmonary lesions would produce more
acquired cellular resistance (CMI, i.e., activated
macrophages capable of inhibiting the growth
of ingested tubercle bacilli) but show less tissue
damage from DTH.After challenge with inhaled
virulent tubercle bacilli, the recipient of this
low-DTH-producing vaccine should have
reduced lung destruction and even reduced cav-
ity formation if the disease progresses.
(ii) Species Variations in CMI/DTH
Ratios. In mice, exactly what stops the loga-
rithmic stage remains to be evaluated. DTH in
mice (although weak) probably causes apopto-
sis, which kills macrophages containing too many
bacilli. Alternatively, macrophages overloaded
with tubercle bacilli may simply burst. Then,
most of the released bacilli would be ingested by
the highly activated macrophages that CMI in
mice produces. Highly activated macrophages
must play a major role in mice, because in the sta-
tionary stage the majority of bacilli are dormant
for weeks within macrophages (7, 8).
In rabbits and guinea pigs, the CMI that causes
bacillary dormancy within macrophages has never
been studied. In rabbit tuberculous lesions, tuber-
SUGGESTED FUTURE RESEARCH 䡵 375
cle bacilli multiply in nonactivated macrophages
and in weakly activated macrophages, but tuber-
cle bacilli are inhibited (and even killed) both in
strongly activated macrophages and in solid
caseous tissues.However,we do not know the rel-
ative importance of activated macrophages (CMI)
and solid caseum (DTH) in controlling the
progress of tuberculosis in either rabbits or guinea
pigs.
How does solid caseous tissue inhibit bacil-
lary growth and even reduce the number of
live tubercle bacilli? Do anoxia, toxic fatty acids,
high osmolarity, and lower pH all play a role (see
chapters 2 and 7 and reference 9)?
In brief, in the various laboratory animal
species, the roles of apoptosis and necrosis need
to be investigated. Both processes kill macro-
phages that contain too many tubercle bacilli for
CMI to control. In each species, the roles of
intracellular dormancy within activated macro-
phages (produced by CMI) and extracellular
dormancy in solid caseous tissues (produced by
DTH) also need to be investigated.
(iii) DTH and CMI and Turnover of
Macrophages. The turnover of macrophages
in tuberculous lesions provides a constant sup-
ply of nonactivated macrophages in which the
bacillus may multiply. Such turnover has been
documented in rabbit tuberculous lesions (see
chapter 10) but has not been studied in mice or
guinea pig lesions. Species variations probably
exist. In guinea pigs, many lesions show caseous
necrosis, and they should have high macrophage
turnover. In mice, few, if any, lesions have caseous
necrosis, and they should have the least macro-
phage turnover. Comparisons of macrophage
turnover in various laboratory species would
provide insights into what might be occurring
in various types of human tuberculous lesions.
ROLE OF VASCULAR THROMBOSIS
IN CAUSING CASEOUS NECROSIS
AND INHIBITION OF BACILLARY
GROWTH
In rabbits, guinea pigs, and humans, microvas-
cular thrombosis causes much of the tissue dam-
age found in tuberculosis (see chapters 8 and 21).
However, the amount of tissue necrosis pro-
duced by vascular thrombosis and the amount
376 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
produced by other DTH mechanisms still need
to be compared. In rabbits, guinea pigs, and
humans, thrombosis (with subsequent caseous
necrosis) seems to be a major factor in killing
macrophages that contain numerous bacilli, but
killing such macrophages by other mechanisms
(including cytotoxic T cells) may be sufficient
without the additional local tissue damage pro-
duced by vascular thrombosis.
What role does activation of vascular
endothelial cells play in presenting mycobacte-
rial antigens and in the thrombosis present in
caseous necrosis (described in chapter 21)?
Would decreasing the ability of the blood to clot
be beneficial in tuberculous hosts, as it seems to
be in the prevention and treatment of myocar-
dial infarctions and strokes?
ROLE OF GRANULOCYTES
IN TUBERCULOSIS
Polymorphonuclear leukocytes (PMN) (neu-
trophils in humans, mice, and guinea pigs, but
eosinophilic heterophils in rabbits) are com-
mon near solid and liquefied caseum. Evidently,
they are attracted to all dying tissues (10). Are
PMN able to destroy or inhibit the tubercle
bacilli that they ingest? Do they carry the bacilli
to other sites and spread the disease? Do PMN
produce factors (e.g., hydrolytic enzymes and
cytokines) that aid the absorption of necrotic tis-
sue and promote healing (see reference 11)?
In dermal lesions produced in rabbits by
the topical application of dilute sulfur mustard,
PMN migrate right through the lesion and
accumulate on the surface where the epithe-
lium and previous PMN are dying (12).There,
they form a crust, or scab (12). In this sulfur
mustard model, the PMN attracted by the
dying tissues seem to prevent bacterial invasion
and aid the healing process. PMN may have
similar roles in caseous and liquefied tubercu-
lous lesions.
The role of eosinophils, basophils, and mast
cells has yet to be evaluated in tuberculosis (see
chapter 6). Do they play any role in DTH reac-
tions? Do any of their products activate
macrophages? Are they able to kill or inhibit
tubercle bacilli?
FORMATION OF LIQUEFIED
CASEUM AND CAVITIES AND
THEIR PREVENTION
Liquefaction of solid caseum often provides an
ideal “culture medium” for the extracellular
growth of tubercle bacilli. (Before liquefaction
occurs, tubercle bacilli only grow intracellu-
larly in nonactivated macrophages.) The large
numbers of actively growing bacilli in liquefied
caseum release high levels of tuberculin-like
products, which cause a tissue-damaging DTH
reaction. If the walls of bronchi are destroyed by
this reaction, a cavity is formed, and tubercle
bacilli are able to spread into the patient’s airways
and into the outside environment, where they
may infect other people (see chapter 4).
Studies on the formation of cavities and
their progression should be a major pri-
ority in tuberculosis research.Yet, at present,
few, if any, laboratory groups seem to be engaged
in such work. Understanding the host factors
involved would require:
1. Identifying the inhibitors that keep solid
caseum from liquefying. Numerous
enzyme inhibitors should exist in solid
caseum.Yet, none has been identified and
characterized.These inhibitors may be of
host or bacillary origin.
2. Determining the mechanism by which
inhibitors are removed during the lique-
faction of solid caseum.These inhibitors
may slowly diffuse from the caseum, or
they may be hydrolyzed or otherwise
inactivated.
3. Identifying and characterizing the spe-
cific enzymes that make solid caseum liq-
uefy. For the absorption of fluid into the
caseum, certain enzymes must be acti-
vated, components of the caseum must
be hydrolyzed, and an osmotic gradient
must be produced. Proteases, nucleases,
lipases, and glycolytic enzymes seem to be
involved (see chapter 4). Some of these
hydrolytic enzymes may exist as proen-
zymes that require activation. After the
enzymes causing liquefaction are identi-
fied, specific pharmacological inhibitors
 25.
could then be developed to stop this detri-
mental process.
4. Determining why some types of liquefied
caseum are more supportive of bacillary
growth than other types. The liquefied
caseum may require a maturation stage to
develop the right composition to support
bacillary growth.Therefore, the composi-
tion of caseum at various stages of the
liquefying process should be analyzed.
Nutrients different from those present in
laboratory culture medium may be found.
In fact, liquefied caseum may contain spe-
cific growth factors derived from host tis-
sues or even from the bacilli themselves.
5. Determining the role of DTH (tuber-
culin sensitivity) in liquefaction and cav-
ity formation (see chapter 4). DTH appar-
ently plays a role in cavity formation both
before and after the bacilli multiply extra-
cellularly. Before bacillary multiplication,
DTH increases the peripheral accumula-
tion of macrophages and their hydrolytic
enzymes (and possibly alters the ratio of
acid-acting to neutral-acting proteinases),
so that solid caseum becomes liquid.After
bacillary multiplication, enough antigen is
produced that the tissue-damaging DTH
reaction erodes a bronchial wall and forms
the cavity.
6. Developing a simple and convenient assay
system to measure the efficacy of new
methods that attempt to reduce liquefaction
and cavity formation. One such assay sys-
tem is described at the end of chapter 4.
That method uses the liquefaction and
ulceration of dermal lesions produced by
BCG (and/or virulent tubercle bacilli) as
surrogates for liquefaction and cavity for-
mation in the lungs.
PROGNOSTIC TESTS THAT
REFLECT DISEASE PROGRESSION
OR REGRESSION
Tests for identifying increases and decreases in
the number of tubercle bacilli in the host would
be quite useful in the care of tuberculosis
patients.
SUGGESTED FUTURE RESEARCH 䡵 377
1. When many bacilli grow extracellularly in
liquefied caseum and disseminate in the
lungs, the tuberculin skin test reaction may
increase because of the boosting effect of
the bacilli and their antigens. Tuberculin
sensitivity may also decrease, or even dis-
appear, when the host’s immune defenses
become exhausted by extensive disease (see
chapter 5). Changes in the tuberculin reac-
tion might be useful in monitoring the
course of the disease. However, careful clin-
ical correlations, including roentgeno-
graphic studies, would have to be made
before any prognostic meaning can be
attached to tuberculin skin reactivity.
2. Similarly, analyses of peripheral blood lym-
phocyte functions (including cell types
and their cytokines) will have to be cor-
related with clinical status to be meaning-
ful. Monitoring such peripheral changes to
reflect disease progression or regression is
further complicated by the fact that some
tuberculous lesions progress while others
regress in the same patient, and that each
lesion may have periods of exacerbation
and regression.Therefore, cells assayed in
the peripheral blood may not always reflect
the state of the entire disease.
3. During liquefaction, the blood may show
an increase in proteases or a decrease in
protease inhibitors. If so, assays for such
inhibitors might have prognostic value.
4. Antigen-antibody complexes in the blood
(13, 13a) may increase as tubercle bacilli
and their antigens increase in the host.
Assays of antigen-antibody complexes in
blood may be useful in determining the
extent of the disease and its prognosis, as
well as the effectiveness of antimicrobial
therapy (13, 13a). However, additional
studies of antigen-antibody complexes
must still be made before such assays are
adopted for general clinical use.
5. Chapter 5 discusses the progress made to
date in distinguishing tuberculin-positive
persons with early progressive tubercu-
lous lesions from persons with non-
progressive lesions. Only those with
378 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
progressive lesions need to be treated with
antimicrobial agents.The most promising
of these studies (14) measured the amount
of gamma interferon produced by periph-
eral blood mononuclear cells after 5 days
in culture and restimulation with the
Mycobacterium tuberculosis-specific antigen
ESAT-6.
6. Computed tomography scans might be
developed for rabbits so that the progress
of the disease could be followed without
killing the animal. Perhaps even tubercle
counts could be made by this method, but
our few attempts to do so have been
unsatisfactory (Y. C. Manabe, personal
communication).
COMPARISONS OF TUBERCULOUS
LESIONS PRODUCED BY LIVE AND
DEAD TUBERCLE BACILLI
Dead tubercle bacilli can produce lesions quite
similar to those produced by live bacilli (5).
Therefore, the number of dead bacilli, as well as
the number of live bacilli, determines the course
of the disease. Dead tubercle bacilli contain
some of the same antigens as live bacilli. How-
ever, secreted antigens from live tubercle bacilli
play a definite role in the development of DTH
and CMI (see chapter 22). More insight into the
immunology and pathology of the host response
to dead and live tubercle bacilli should be a
fertile field of investigation (see reference 15).
The dormancy of tubercle bacilli in the host is
discussed in chapters 6 and 10.
ROLE OF ANTIBODIES IN HOST
RESISTANCE TO REINFECTION
As discussed in chapter 5, an antigen-antibody
reaction can produce the chemotactic C5a
component of complement (and probably other
chemotactic factors) that hastens the accumu-
lation of an expanded antigen-specific Th1
lymphocyte population at the site of bacillary
lodgement. In other words, antibodies enhance
the local CMI response. Cytophilic antibodies
may attach to macrophages. If so, do they
enhance macrophage activation whenever anti-
gen is present? Do cytophilic antibodies cause
macrophage apoptosis when the macrophage
contains numerous bacilli? Answers to these
questions would provide new insights into host-
parasite interaction.
NEW RESISTANT AND SUSCEPTIBLE
RABBIT STRAINS
Since Lurie’s inbred rabbits are now extinct, it
behooves us to develop replacements. Existing
possibilities are described in chapter 14, but
they are not commercially available.
No one to date has evaluated the resistance
of any of the over 50 strains listed by the Amer-
ican Rabbit Breeders Association (16). Some of
these strains may have more uniform resistance
to tuberculosis than New Zealand White rab-
bits have. Some may be more susceptible than
New Zealand White rabbits. A few of these
strains are smaller than New Zealand White
rabbits, i.e., the size of large guinea pigs. The
smaller size would reduce the cost of caging,
food, and care.
We recommend that Lurie’s tubercle-count
method (described in chapter 11) be used to
evaluate the resistance of many of these rabbit
strains. If any were to prove more valuable than
New Zealand White rabbits, they could be
developed commercially.
PART II. SUGGESTED RESEARCH
ON THE BACILLUS
BACILLARY SURVIVAL
AND VIRULENCE
Virulent mycobacteria can survive (i) drying
in the outside environment, (ii) the effects of
ROIs (reactive oxygen intermediates), RNIs
(reactive nitrogen intermediates), and hydrolytic
enzymes within activated macrophages, and (iii)
the toxic fatty acids, low oxygen tension, and
reduced pH within solid caseum (see chapters
2 and 7) (9). They can multiply in liquefied
caseum.What genes are expressed by the tuber-
cle bacillus to enable it to survive (or multiply)
in each of these different local environments?
(i) Bacillary Survival in Air. A thick wax-
like glycolipid coat enables the bacilli to resist
desiccation in dry air. Is this coat altered when
the bacilli grow in macrophages? Are the thick-
ness and composition of this wax coat changed
 25.
when the bacilli grow extracellularly in the liq-
uefied caseum within cavities? Do bacilli grow-
ing in liquefied caseum have properties different
from bacilli growing in laboratory culture media?
Sigma factors (17) aid bacillary survival (see
chapter 1). Inhaled bacilli also resist the detergent
effects of surfactants in the airways of the lungs.
(ii) Bacillary Survival within Macro-
phages. Tubercle bacilli produce virulence
factors that interfere with the microbicidal func-
tions of macrophages (reviewed in chapter 6).
They impair the maturation of the macrophage
phagosome into its normal acidic, microbicidal,
and hydrolytic compartments, but allow nutri-
ents to enter it (15). The bacilli also slow the
development of the Th1 lymphocyte acquired
immune response.
What are the various microbial factors enabling
the bacillus to act in this way? Do tubercle bacilli
growing within macrophages produce antioxi-
dants that reduce the microbicidal effects of the
host’s ROIs and RNIs, or do such bacilli merely
inhibit the macrophage production of ROIs and
RNIs, or both? How do they resist and/or pre-
vent the lower pH normally found in lysosomes?
Are some of the genes activated for bacillary
growth within macrophages not active during
bacillary growth in laboratory culture media?
How are these various virulence mechanisms
integrated?
The multiplication of virulent tubercle bacilli
within macrophages is currently an active field
of investigation (see chapter 6). However, much
more insight is needed in order to develop new
drugs to inhibit such multiplication.
(iii) Bacillary Survival in Solid Caseum.
Dormancy genes and their products, e.g., sigma
factors (17–19), may be required for the bacil-
lus to survive for many years in solid caseum.
What activates these genes to enable the bacil-
lus to do so, and what turns dormancy genes off
when liquefaction occurs? Are dormancy genes
enabling survival in solid caseum different from
those enabling survival within macrophages
and survival in the outside environment? Are
dormancy genes that are active in solid caseous
tissue the same as those that are active in old
SUGGESTED FUTURE RESEARCH 䡵 379
laboratory cultures? See chapters 1, 6, and 10 for
additional information on bacillary persistence
and dormancy.
A recent experiment identified 10 such genes
associated with mycobacterial persistence in rab-
bit tuberculous lesions (Y. C. Manabe et al.,
unpublished data) (see chapter 6), but more work
needs to be done to identify the part of the
lesion in which such bacilli reside.The rabbit is
the only common laboratory animal in which
pulmonary lesions produced by M. tuberculosis
actually heal (similar to most early lesions in
humans).This latency (dormancy) model should
be further investigated in order to develop drugs
to prevent and/or treat the reactivation of tuber-
culous lesions.
(iv) Bacillary Growth in Liquefied
Caseum. Some of the mycobacterial genes
enabling bacillary growth in liquefied caseum are
probably different from those enabling growth
within macrophages.Are any of these genes dif-
ferent from those enabling bacillary growth in
laboratory culture medium? Are the genes that
are active in liquefied caseum before a cavity
forms different from the genes that are active
after a cavity forms when the oxygen tension is
higher and stimulates bacillary growth? (See
Fig. 20 in chapter 4.)
(v) Other Virulence Considerations.
How do the genes (and their products) of highly
virulent tubercle bacilli differ from those of
bacilli of reduced virulence (see references 15,
20, and 20a)? How do such differences enable
the bacillus to live in each of the environments
found in the host and in the outside environ-
ment? Fully virulent and semivirulent tubercle
bacilli may differ in cell wall composition (see
reference 21) and/or in the type and amount of
tuberculin-like substances that they produce.
Many virulence factors exist (discussed in
chapters 1 and 6). If a single gene is lacking, oth-
ers may compensate for it (21, 22). Also, the
virulence of the various mycobacterial strains is
not always the same in different animal species
(23, 24); e.g., rabbits are much more susceptible
to Mycobacterium bovis than to M. tuberculosis (5,
25) (see chapter 15). Despite the great progress
380 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
that has been made in explaining the virulence
of pathogenic tubercle bacilli, many additional
attributes remain to be discovered because of the
complexity of host-parasite interactions.
PART III. SUGGESTED RESEARCH ON
PROPHYLACTIC VACCINES AND
IMMUNOTHERAPY
MORE EFFECTIVE VACCINES
The prevention of clinical tuberculosis by a
more effective vaccine than currently available
BCG vaccines would be the most efficient way
to reduce the incidence of this disease in the
world. The protection by improved vaccines
may never be 100%. In fact, even the 80% effi-
cacy of BCG and Mycobacterium microti vaccines,
used in the 1950s, does not seem to be repro-
duced by the vaccines available today (26).We
should try to regain this efficacy and attempt to
increase it to 90 or 95%.
Perhaps adding genes for certain antigens to
BCG by molecular biology techniques would
increase its effectiveness. Such antigens might be
best given as a booster immunization, because
if they are incorporated into BCG, they might
substantially increase its virulence. (See chapter
22 for other possibilities.)
Fortunately, once an effective vaccine has
caused a major decline in the number of new
cases of active tuberculosis per year, the decline
would continue (because of decreased sources of
contagion) until the disease is no longer a major
concern.
To produce the most effective vaccine, certain
questions remain to be answered.Are live vaccines
required because they multiply, secrete, and release
antigens and persist longer in the host? Does
protection disappear if the vaccinating bacilli and
their antigens are eliminated from the host? Is
revaccination with the same or a different vaccine
required to boost immunity? Do effective vac-
cines need to produce a positive tuberculin skin
test reaction? What role does the adjuvanticity of
BCG play in its effectiveness (see chapter 19)? Are
recombinant BCG vaccines that produce
cytokines or costimulatory molecules more effec-
tive than the parent BCG? How can the number
of memory cells be increased? Different vaccines
may produce different Th1/Th2 lymphocyte
ratios. Are such ratios a major factor in vaccine
efficacy? If so, should Th1/Th2 ratios be used in
selecting vaccines for clinical trials?
IMMUNOTHERAPY TO SUPPLEMENT
ANTIMICROBIAL THERAPY AND TO
TREAT MULTIDRUG-RESISTANT
TUBERCULOSIS
Immunotherapy should be pursued more exten-
sively, because it would be of great benefit in
treating multidrug-resistant tuberculosis, and
because it could shorten the treatment of drug-
susceptible tuberculosis.
The main recent immunotherapy trials were
carried out with Mycobacterium vaccae (reviewed
in reference 27). These trials showed much
promise.Very few laboratory studies have been
made on the immunotherapy of cavitary tuber-
culosis with M. vaccae or any other immunizing
agent (see chapters 4 and 22)—probably because
the rabbit is the only common laboratory ani-
mal that readily produces cavities, and very few
investigators work with rabbits.
OTHER VACCINE POSSIBILITIES
Other vaccines, including DNA vaccines, are
discussed in chapter 22.
PART IV. DEVELOPMENT OF NEW
DRUGS FOR THE TREATMENT AND
PREVENTION OF TUBERCULOSIS
DRUGS ACTING ON THE HOST
Drugs to inhibit the hydrolytic enzymes that
cause liquefaction and cavity formation should be
evaluated in the rabbit model of tuberculosis.
Liquefied caseum and cavities are not readily
produced in other common laboratory animals.
Anticoagulants might also be a useful addition
to antimicrobial therapy. As discussed above,
they might decrease the amount of caseous
necrosis by reducing the thrombosis in the sur-
rounding vasculature.
DRUGS ACTING ON THE BACILLUS
New antimicrobial drugs are needed (i) to
shorten the present 6-month course of therapy
for drug-susceptible tuberculosis and (ii) to treat
multidrug-resistant disease. Chemotherapeutic
strategies that reduce the time needed for the
treatment of drug-susceptible tuberculosis would

have important effects on cost, compliance, drug
toxicity, and emergence of drug-resistant strains
of tubercle bacilli (18, 19, 28, 29). Most antimi-
crobial agents for tuberculosis are effective
against actively multiplying bacilli (18, 19, 30).
Tubercle bacilli in a dormant state are often
not killed by such antimicrobials, especially
bacilli in solid caseous tissues, which is the rea-
son why a long period of administration is
required. Such persistence is reviewed in refer-
ences 18, 19, and 31.Table 1 relates the effec-
tiveness of antimicrobials to the in vivo meta-
bolic states of the tubercle bacillus.
Pyrazinamide (PZA) is a prodrug that is con-
verted by the tubercle bacillus into pyrazinoic
acid, which is microbicidal (18). It is most active
at acid pH, at low oxygen concentrations, and
on nonreplicating (rather than actively growing)
tubercle bacilli (18). Therefore, unlike other
antimycobacterial drugs, it can kill a population
of persistent tubercle bacilli residing in solid
caseous tissues, thereby shortening the duration
of antimicrobial therapy. Since such caseum is
rich in lipid components, alteration of the PZA
structure (to make it more lipid soluble) might
increase its effectiveness against such dormant
bacilli. If so, one of the major difficulties in
controlling tuberculosis would be surmounted.
Perhaps incorporation into liposomes (see ref-
erence 32) would increase the penetration of the
drug into caseous areas.
New types of drugs are needed to prevent the
ability of the bacillus to enter each of the four
stages of its life cycle: (i) intracellular growth in
macrophages, (ii) dormancy in solid caseum,
TABLE 1 In vivo effectiveness of principal antituberculous agents on tubercle bacilli in various metabolic
statesa
Bacilli are actively
Drug metabolizing
Streptomycin ⫹⫹⫹
Isoniazid ⫹⫹⫹
Rifampin ⫹⫹
Ethambutol ⫾ ⫾
Pyrazinamide 0 ⫹⫹
a
⫹, ⫹⫹, and ⫹⫹⫹ represent increasing degrees of bactericidal activity; ⫾ represents bacteriostatic activity; 0 represents no
activity.Adapted from reference 30.
25. SUGGESTED FUTURE RESEARCH 䡵 381
(iii) extracellular growth in liquefied caseum
(often within cavities), and (iv) survival in the
outside environment, where the composition of
the protecting coat plays an important role.The
contagiousness of multidrug-resistant patients
might be reduced if a drug is developed that
reduces the thickness of the lipid coat of the
bacillus, even though the drug may have little
effect on intracellular bacillary multiplication
in vivo.
Drugs that target dormancy are especially
needed, e.g., drugs that inhibit DosR, RelA,
sigma factors, triacylglycerol synthase (33), and
isocitrate lyase (8) (reviewed in references 18, 19,
and 31; also see chapter 1). New drugs that
affect the viability and virulence of dormant
tubercle bacilli seem particularly pertinent. To
identify new drugs that are active on nonmul-
tiplying, persistent tubercle bacilli, drug screens
need to be developed that mimic dormancy in
vivo, including low oxygen, reduced pH, and
toxic fatty acids (18).
Three new drugs that have apparently differ-
ent targets from previous antimycobacterial drugs
are now in clinical trials.The first is a diarylquino-
line (R207910, developed by Janssen Pharma-
ceutica in Beerse, Belgium, a subsidiary of John-
son and Johnson). It inhibits the ATP synthase in
the mycobacterial membrane (34–36).The sec-
ond is a nitroimidazopyran (PA-824, developed
by PathoGenesis, Inc., a division of Chiron, Inc.,
Emeryville, Calif.) (36–38).The third is a pyrrole
derivative (LL-3858, developed by Lupin Labo-
ratories, Ltd., Pune, India) (S.Arora, Int. J.Tuberc.
Lung Dis. 8[Suppl. 1]:S29 [abstract], 2004). All
Bacilli are slowly Bacilli are slowly
metabolizing at acid pH metabolizing at neutral pH
0 0
⫹ 0
⫹ ⫹
0
0
382 䡵 PATHOGENESIS OF HUMAN PULMONARY TUBERCULOSIS
three have mycobacterial sterilizing activity in
mice.
However, it remains to be determined
whether (i) any of these new drugs will replace
one or more existing drugs in the standard treat-
ment of tuberculosis, (ii) the new drugs will
shorten the duration of therapy, e.g., from 6 to
3 months, or (iii) the new drugs will prevent
relapses caused by formerly dormant tubercle
bacilli located in solid caseous tissues.
In mice, when moxifloxacin (8-methoxy-
fluoroquinolone) (MXF) replaced isoniazid
(INH) in combinations with PZA and rifampin
(RIF), a greater number of tubercle bacilli were
killed than when all four drugs were present
(39). This suggests that INH had inhibitory
effects on one or more of the other drugs. In fact,
in 4 months, a regimen of PZA, RIF, and MXF
produced a stable cure of mice that were infected
by aerosol with virulent human-type tubercle
bacilli (H37Rv) (40). In contrast, the standard
regimen of PZA, RIF, and INH required 6
months of therapy. MXF is in clinical use in the
treatment of multidrug-resistant tuberculosis and
seems to be well tolerated (41).These and other
new drug candidates for the treatment of tuber-
culosis are reviewed in reference 36.
PART V. SOURCES OF ADDITIONAL
INFORMATION
Structure of mycobacterial cell walls (42).
Genomics of the M. tuberculosis complex (42
and 43).
Details on various antimicrobial agents and
strategies for developing new ones (28).
Mechanisms of drug resistance in M. tubercu-
losis (44).
Currently approved regimen for treating
tuberculosis (45).
Preventing the transmission of tuberculosis
(46) (see Appendix E).
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 APPENDIX A
AWARD OF THE TRUDEAU MEDAL FOR 1955†
The Trudeau Medal for 1955 was awarded to Dr. Max B.
Lurie at the annual meeting of the National Tuberculosis
Association, held in NewYork, N.Y., May 21–24, 1956.The
presentation was made by Dr.William H. Feldman.
DR. FELDMAN
In 1925 the Board of Directors of the National
Tuberculosis Association approved a suggestion of
the Committee on Medical Research that there be
awarded not oftener than once yearly a medal to
one who had made meritorious contributions to
the knowledge of the cause, prevention, or treat-
ment of tuberculosis.This symbol of recognition
was to be known as the Trudeau Medal.A year later
this suggestion was accepted by resolution and
adopted by the Executive Committee.
At the 23rd annual meeting of the National
Tuberculosis Association held in Washington,
October, 1926, the award was first presented to Dr.
Theobald Smith. Dr. Smith had been president of
the National Tuberculosis Association during the
preceding year and was one of the true giants of
American science.Thus was initiated a yearly cus-
tom, which has become an important tradition.
Today, we pause in our preoccupation with sci-
entific sessions, committee meetings, and the many
items that make a visit to New York exciting, to
honor one of our peers by making him the 30th
recipient of this enviable symbol of our respect and
esteem. The one who has been chosen unani-
mously to receive the Trudeau Award this year, and
whose name I shall disclose in a few moments, is
known to all of you, and his contributions to the
knowledge of tuberculosis are highly regarded
throughout the world.
Before proceeding further with the scientific
accomplishments that merited this award, I wish
to digress briefly and give a short “clinical history”
of our recipient. He was born in Lithuania,
endowed with little else than a priceless scholarly
heritage. He came to America at the age of fifteen.
After high school at Townsend Harris Hall, he
entered the College of the City of New York. His
†
Reprinted from Am. Rev.Tuberc. Pulm. Dis. 74:647–649,
1956, with slight modifications.
385
ambition was to study philosophy, but he soon real-
ized that this highly respected discipline offered lit-
tle that would provide a means of livelihood in the
immediate or near future.“In despair [and I use his
words] I determined to study medicine.” This
decision was influenced by “the idea that the most
one can do is to alleviate the pain of human life;
medicine offers that opportunity.”
He entered Cornell University Medical College
and served in the Student Army Training Corps.
During his medical training, he became distressed
at the thought of practicing medicine because of
what he considered would be his incompetence.
Always conscientious to a fault, he feared that his
lack of knowledge would endanger the lives of
those whom he was expected to treat. Finally, his
dilemma was resolved. Fate intervened and a highly
sensitive, reluctant young physician was spared the
agony and vicissitudes of practicing medicine,
eventually to become a highly productive and
brilliant investigator. In the words of our medal-
ist,“It was my good fortune to diagnose my own
tuberculosis in the fourth year of medical school.”
(He probably would have made a good physi-
cian.) He attempted to “take the cure” and at the
same time carry on an internship, with the pre-
dictable result, i.e., he finally had to spend full
time as a patient in a tuberculosis sanatorium. It
was there that he became interested in what was
to be his future career: research in tuberculosis. So
much for the “clinical” aspects of this gifted man.
What are some of the attributes that have char-
acterized him as a person and as a scientist? Above
all, he has a great compassion, a real concern for the
unfortunate, and an intense aversion to all that
denies freedom and justice to man.While he can be
militantly indignant concerning social evils, he
believes in and practices good will. He is a man of
much humility and utterly without pretense. He
places a high value on integrity, believing as he has
expressed it:“Of what avail is the acclaim of others
if you cannot countenance yourself.” He has a sense
of humor, enjoys an argument and, parenthetically
I would say, is a skillful debater. He considers dif-
ferences of opinion healthy and desirable. His extra-
scientific interests are social justice, philosophy and
386 䡵 APPENDIX A
music. The motivation of his labors has been to
seek a meaning and a justification for life. In his
quest he has succeeded admirably—to the envy of
us all.
He brought to his professional tasks a scholarly
insight, an ingenious talent for experimentation, a
masterful technique, and a fiery enthusiasm. Impor-
tantly, he has a fine concept of scientific evidence.
He has stated the underlying philosophy of his
research in the following words: “I have always
thought that it is better to attempt the solution of
a fundamental problem and fail than to undertake
the elaboration of minor details with great success.”
These words are worth remembering.
The scientific contributions of the recipient of
the Trudeau Medal for 1955 are sufficiently well
known to require no enumeration. Briefly, he has
been the sole or senior author of nearly 70 pub-
lications, including several of monographic dimen-
sion; and his name appears in joint authorship of
many more. His scientific writing, especially his
reviews, reveal a capacity to establish continuity of
new facts with those previously reported. This
bringing together of the new with the old enables
the reader to obtain a composite understanding of
the interrelationships of the various facets of the
subject discussed.
Although the main theme of his many investi-
gations has been resistance to tuberculosis, of out-
standing importance is his brilliant conception
and successful demonstration of genetic factors
in such resistance. One who knows him well has
said, “His early genetic work opened up a new
world.” He has continued to explore this world
successfully and with results that have brought
him international renown.
The recipient of the Trudeau Medal for 1955
has, in a word, accomplished work that has been
magnificent in its purpose.This work has, I am cer-
tain, been deeply satisfying and has brought this
devoted and dedicated servant of science a touch
of immortality.
On behalf of the Trudeau Award Medal Com-
mittee, I consider it an honor and a happy privi-
lege to present the Trudeau Medal Award for 1955
to Max B. Lurie.
DR. LURIE
Mr. President, the Committee on the Trudeau
Award, Dr. Feldman, ladies and gentlemen.
To merely say that I am profoundly moved by
the recognition that you have given to our labors
over the years would be an understatement. Indeed,
I see in it a vindication of my hope that my life has
not been altogether in vain. Even more, I take it as
evidence of the great generosity of spirit that char-
acterizes our democracy. For a poor immigrant
boy to have received the necessary training, to be
given the opportunity to pursue his craving to try
to understand even an iota of the mystery of life,
is a triumph of the human spirit.
Most of all, for the Trudeau Society to award this
distinguished honor to one who has achieved no
immediately practical solution to the problems on
hand—but to him who has endeavoured by tenac-
ity and devotion to penetrate ever so little into the
intricacies of the host-parasite relationships which
constitute native and acquired resistance to tuber-
culosis—is both a humbling thought and a warm-
ing of the heart. It is humbling because we all
know the ephemeral nature of even the loftiest and
most penetrating of scientific ideas.The centuries-
old sway of Newton’s laws over the universe has
yielded to the relativistic concepts of Einstein. It is
a sacrilege to mention these giants of the human
mind in the same breath with our feeble efforts.
But, the condition of human existence is the same
in the great and in the small; absolute knowledge
is beyond our capacity. It is in increasing under-
standing that the salvation of man lies. Not only in
the mastery of his environment and in the subju-
gation of the elements to his will (as we are wit-
nessing daily in ever increasing crescendo), but
even more in the control over himself and the
moral growth of mankind.The more man under-
stands himself, the more truly human will he
become. It is heartening that the Trudeau Society
deems it worthy to encourage these labors, assailed
as they often are by failure and doubt.
I wish to take this opportunity to express my
gratitude to the Henry Phipps Institute of the
University of Pennsylvania, to my teachers and
inspirers within and outside its walls, to my wife
who has stood unflinchingly by me in times of
trial, to the organizations that have supported
these endeavours over the years, especially to the
Commonwealth Fund, the Trudeau Society and
the Public Health Service, and above all to my col-
laborators and assistants, without whose devotion
even the little we did could not have been accom-
plished. On behalf of all those whose greatest joy
in life is the glimmer of understanding that comes
after searching and critical analysis, allow me to
thank you for your generosity.
 APPENDIX B
OBITUARY OF MAX B. LURIE, M.D. (1893–1966)†
Esmond R. Long, M.D., Ph.D., Sc.D.
Director of the Henry Phipps Institute for the Cure and Prevention
of Tuberculosis, University of Pennsy.lvania, Philadelphia
Max Bernard Lurie, experimental pathologist
and cultured scholar, well known for his investiga-
tions on heredity and constitutional factors in
tuberculosis, died in Philadelphia September 23,
1966. He had been in failing health for many
months, with increasing disability of the heart, but
maintained his studies and productivity to the end.
Lurie was born on September 12, 1893, in
Telshee, Lithuania, during the period of Russian
domination of the country. He emigrated with his
family to the United States in 1907. His parents
were of distinguished Jewish ancestry, with origins
traceable to Talmudic scholars in the eleventh cen-
tury. His early years in Lowell, Massachusetts, and
New York City were steeped in Jewish culture. In
spite of family tradition, however, he was not
inclined to enter his father’s profession, the rab-
binate. He was deeply interested in philosophy,
and would have liked to specialize in it, but for
practical reasons undertook the study of medi-
cine, which promised a more certain livelihood and,
more importantly, an opportunity for effective ser-
vice to mankind. He entered the medical school
of Cornell University, worked his way through,
often at poorly paying tasks, and graduated in
1921.
In his medical school days, it was recognized
that he had contracted tuberculosis, the disease
from which his mother died. Soon after graduation,
he entered the National Jewish Hospital for Con-
sumptives in Denver, Colorado, for a residency and
what was intended to be a period of rest, but soon
proved to be one of work and preparation for a life
of research on the disease from which he suffered.
After significant studies at the National Jewish
Hospital, first with H. J. Corper and soon inde-
pendently, he accepted an invitation in 1926 to
†
Reprinted from Am. Rev. Respir. Dis. 95:694–696,
1967. Dr. Lurie was at the Henry Phipps Institute from
1926 until his death. Dr. Long was director there dur-
ing much of that time.
387
become a Fellow in Pathology under Eugene L.
Opie at the Henry Phipps Institute of the Uni-
versity of Pennsylvania in Philadelphia. Here, he
stayed for forty years attaining the rank of full
professor in 1955, maintaining an unbroken line of
research from early studies on the fate of tubercle
bacilli in different organs of the animal body, to a
masterly comprehension of tuberculosis as a whole
and a position of eminence among investigators of
that disease.
The late 1920’s represented a period of general
and deep interest in pathologic and immunologic
differences between first infection and reinfec-
tion tuberculosis. Lurie’s chief, Dr. Opie, who
inspired Lurie in many ways, was much concerned
with the mechanism of transmission of the infec-
tion in man. Lurie became a pioneer in the devel-
opment of laboratory methods for study of the
problem. Beginning shortly before 1930, he set up
experimental models for air-borne infection in
guinea pigs, with ingenious arrangements of cages,
which not only demonstrated the mechanisms
and frequency of air-borne contagion, but even led
to determination of the particulate size of infect-
ing units.
In 1932, Lurie acquired a colony of highly
inbred rabbits from a stock raised for experiments
at the University of Chicago, from which he soon
separated out, by litter matings, two strains of ani-
mals with sharply differing degrees of resistance to
tuberculosis. These, and subsequent families of
intermediate character, proved invaluable for later
investigations on the heredity of resistance and
susceptibility to tuberculosis and the cellular mech-
anisms concerned.
In the ensuing years, Lurie steadily developed
the possibilities in this line of investigation, demon-
strating the varying role of the body’s macrophages
in the destruction of tubercle bacilli, a field in
which he was preeminent, and throwing light on
numerous aspects of the host-parasite relation,
extracellular factors in resistance, the role of age and
388 䡵 APPENDIX B
sex, and many other elements concerned in one
way or another in intrinsic resistance.
A practical application of his studies came about
through a cooperative investigation, in association
with W. F.Wells, of the germicidal effect of ultra-
violet light in the prevention of air-borne infection.
This has had extensive development since their
original reports.
In his later years, Lurie was much concerned
with the role of endocrine factors in resistance to
tuberculosis. His research was significant in demon-
strating the effect of cortisone in reducing pro-
tective inflammation and of thyroid hormones in
enhancing it.
The later years of Lurie’s life were devoted to
reflection and orderly arrangement of facts learned
during his years of research.The Commonwealth
Fund, which had been one of the major organi-
zations among the several sources of support for his
work, made it possible for him to devote a pro-
longed period to preparation of a comprehensive
book on Resistance to Tuberculosis, which covered his
experimental studies on native and acquired
defense mechanisms in relation to those of others.
This book is now a highly influential monograph
in the field.
Many honors were conferred on Dr. Lurie,
including the Dearholt lectureship in 1952,
the Claude Bernard Medal of the University of
Montreal in 1953, and the Trudeau Medal of the
National Tuberculosis Association in 1956. Over
many years, the Association furnished financial
support for his work. In 1960, the American Tho-
racic Society of the National Tuberculosis Associ-
ation honored him with senior membership.
Throughout those years, Lurie took part faithfully,
and with a vigor all his own, in both the presen-
tation of reports and assessment of research in the
meetings of the National Tuberculosis Associa-
tion. He was an excellent teacher, and a debater of
stature, in spite of his small physical frame, on all
problems in his chosen field. His published work
is widely quoted, and his influence will be lasting.
It was of great importance in transferring some
emphasis from other fields to the large one of
intrinsic constitution.
Lurie is survived by his wife, Rose Gamoran
Lurie, herself an educator and author of attain-
ment in Jewish history, and by his son Abraham
A. Lurie, an Associate Professor of Anesthesiology
at the School of Medicine of the University of
Rochester.
 APPENDIX C
PUBLICATIONS OF MAX B. LURIE
These publications are grouped somewhat chrono-
logically by subject. Comments follow many of the
publications to explain their contributions, which
are often not evident from their titles.These pub-
lications are mainly the ones listed and summarized
in Lurie’s book (see publication 68). Chapter 1 in
Lurie’s book reviews the basic pathogenesis of
tuberculosis, specifically, the cell and tissue response
to this infection, and his chapter 15 reviews the
entire book.
CULTIVATION OF TUBERCLE BACILLI
1. Lurie, M. B. 1923. A comparison of the
sodium hydroxide and anti-formin methods
for cultivating tubercle bacilli. Am. Rev.Tuberc.
7:332–343.
Sodium hydroxide treatment preserved the
viability of tubercle bacilli in sputum samples bet-
ter than did anti-formin treatment.
ORGAN RESISTANCE IN PRIMARY AND
REINFECTION TUBERCULOSIS
Various organs in the body differ in their ability to
stop the progression of tuberculous lesions. In rab-
bits, lesions progress in the lungs and kidneys but
heal in the liver, whereas in guinea pigs, lesions
progress in the lungs and liver but heal in the kid-
neys. Residual primary infection provides strong
acquired resistance against reinfection. Reviewed
in Lurie’s chapter 1 and, briefly, in chapter 7 in the
present volume.
2. Corper, H. J., and M. B. Lurie. 1926.The
variability of localization of tuberculosis in
the organs of different animals. I. Quantitative
relations in the rabbit, guinea pig, dog and
monkey. Am. Rev.Tuberc. 14:662–679.
3. Corper, H. J., and M. B. Lurie. 1926.The
variability of localization of tuberculosis in
the organs of different animals. II.The impor-
tance of the distribution of tubercle bacilli as
concerns differences of susceptibility of the
organs. Am. Rev.Tuberc. 14:680–705.
4. Corper, H. J., M. B. Lurie, and N. Uyei.
1927.The variability of localization of tuber-
culosis in the organs of different animals. III.
389
The importance of the growth of tubercle
bacilli as determined by gaseous tension. Am.
Rev.Tuberc. 15:65–87.
5. Corper, H. J., and M. B. Lurie. 1927.The
variability of localization of tuberculosis in
the organs of different animals. IV.The cellu-
lar factor in the susceptibility of the various
organs. Am. Rev.Tuberc. 15:237–269.
6. Corper, H. J., M. B. Lurie, and N. Uyei.
1927.The variability of localization of tuber-
culosis in the organs of different animals.V.
The significance of localization and devel-
opment of the bacilli and of the cellular reac-
tion in man and animals. Am. Rev. Tuberc.
15:389–398.
7. Lurie, M. B. 1928. The fate of human and
bovine tubercle bacilli in various organs of the
rabbit. J. Exp. Med. 48:155–182.
In rabbits, after an intravenous injection, both bacil-
lary strains are essentially cleared in 2 to 4 months
from the liver, spleen, and bone marrow, but may
grow extracellularly in the lungs (and kidneys)
where cavitation (ulceration) occurs.
8. Lurie, M. B. 1929.The fate of tubercle bacilli
in the organs of reinfected rabbits. J. Exp. Med.
50:747–765.
After an intravenous injection of virulent human-
type tubercle bacilli, the persisting infection (mostly
in cavitary lesions in the lungs and in ulcerated
lesions in the kidneys) markedly reduced the mul-
tiplication of virulent bovine-type tubercle bacilli
given intravenously 6 months later. Multiplication
of virulent human-type tubercle bacilli given intra-
venously 6 months later was reduced even fur-
ther. (The bovine type is much more virulent for
rabbits than the human type.) See chapter 2 in
Lurie’s book for more details.
9. Lurie, M. B. 1932.The correlation between
histological changes and fate of living tuber-
cle bacilli in the organs of tuberculous rabbits.
J. Exp. Med. 55:31–54.
Lesions in the lungs, liver, spleen, bone marrow, and
kidneys were produced in rabbits by an intravenous
injection of (i) BCG (an early strain that was more
virulent than current BCG),(ii) virulent human-type
bacilli, or (iii) virulent bovine-type bacilli.The rab-
bits responded to each type of tubercle bacillus in a
similar manner, but the bovine type was harder to
390 䡵 APPENDIX C
destroy and caused extensive progressive disease.
Lesions produced by all three types of tubercle bacil-
lus healed in the liver.
Mature epithelioid cells (described in detail)
were found to be associated with the destruction
of tubercle bacilli—a correlation that Lurie con-
sidered to be one of his major contributions.
Langhans’ giant cells occurred in the lesions
after the local multiplication of tubercle bacilli had
ceased. Giant cells frequently formed around bits
of caseous material.
Caseous necrosis was present when the host
became tuberculin positive and, therefore, delayed-
type hypersensitivity was considered to be the
cause of caseous necrosis.
Lymphocytes did not (directly) destroy tuber-
cle bacilli, whereas macrophages (epithelioid cells)
did so.
In expanding tuberculous lesions containing
many tubercle bacilli, some mononuclear cells were
multiplying (shown by the presence of mitotic
figures).
Chapter 1 in Lurie’s book reviews these subjects.
Chapter 10 in the present volume provides insights
concerning mononuclear cell division.
10. Lurie, M. B. 1933.A correlation between the
histological changes and the fate of living
tubercle bacilli in the organs of reinfected
rabbits. J. Exp. Med. 57:181–201.
Primary infection was produced by intravenous vir-
ulent human-type tubercle bacilli in 30 rabbits. Six
months later, the bacilli had almost disappeared
from the liver, spleen, and bone marrow, and their
number was usually reduced in the lungs and kid-
neys. At that time, 15 of these rabbits were rein-
fected intravenously with virulent human-type
bacilli, and 15 were reinfected intravenously with
virulent bovine-type bacilli. At that time, as con-
trols, 13 uninfected rabbits were injected intra-
venously with virulent human-type bacilli, and
13 uninfected rabbits were similarly injected with
virulent bovine-type tubercle bacilli.
In the reinfected rabbits, the formation of new
tubercles was accelerated, and a faster maturation of
epithelioid cells occurred. Also, both the human-
and bovine-type bacilli of reinfection were often
destroyed, while the human-type bacilli of the pri-
mary infection were still multiplying (extracellularly)
within cavitary lesions in the lungs and kidneys.
11. Lurie, M. B. 1934. The fate of BCG and
associated changes in organs of rabbits. J. Exp.
Med. 60:163–178.
In these experiments, BCG of normal virulence
was given intravenously.This BCG increased in the
organs of the rabbits for about 2 weeks and then
declined, similar to the more virulent BCG
(described in publication 9). The normal BCG
was almost eliminated by 4 weeks, whereas some
of the more virulent BCG persisted in the lungs
and kidneys for at least 4 months. Lurie quoted
from the literature that BCG persisted in the lymph
nodes of monkeys for at least 7 months and in the
lymph nodes of guinea pigs for at least 19 months.
Monkeys and guinea pigs are more susceptible to
virulent tubercle bacilli than are rabbits. See chap-
ter 1 in Lurie’s book and chapters 15 and 23 in the
present volume.
SPREAD OF TUBERCULOSIS FROM
INFECTED TO UNINFECTED ANIMALS
This series of reports documents the respiratory
and alimentary routes of infection among guinea
pigs and rabbits housed in an animal room.
12. Lurie, M. B. 1930. Experimental epidemi-
ology of tuberculosis: the effect of crowding
upon tuberculosis in guinea pigs, acquired by
contact and by inoculation. J. Exp. Med.
51:729–741.
13. Lurie, M. B. 1930. Experimental epidemi-
ology of tuberculosis: air-borne contagion in
tuberculosis in an animal room. J. Exp. Med.
51:743–751.
14. Lurie, M. B. 1930. Experimental epidemi-
ology of tuberculosis: the effect of eliminat-
ing exposure to enteric infection on the inci-
dence and course of tuberculosis acquired by
normal guinea pigs confined with tuberculous
cage mates. J. Exp. Med. 51:753–768.
15. Lurie, M. B. 1930. Experimental epidemi-
ology of tuberculosis: the route of infection in
naturally acquired tuberculosis of the guinea
pig. J. Exp. Med. 51:769–776.
NATURAL AIRBORNE INFECTION AND
REINFECTION WITH TUBERCLE BACILLI
In this series of reports (and those in the next two
sections), uninfected rabbits inhaled air in an ani-
mal room where other rabbits (caged separately)
were shedding virulent bovine-type tubercle bacilli
in their urine from infected kidneys.These bacilli
became airborne as the rabbits moved about on the
bedding.The following principles were established:
(i) Residual primary infection prevented or
reduced the severity of reinfection.These exper-
iments tell us how much immunity might be
expected in rabbits from a truly effective vaccine
(see chapters 22 and 25 in the present volume).

(ii) Continuous exposure to low numbers of
tubercle bacilli had no effect on the course of the
primary infection.These experiments suggest that
hosts with active tuberculosis are well immunized
against low-dose exogenous reinfection. See pub-
lications 22, 23, 24, and 26 for additional insights
on immunity to reinfection.
16. Lurie, M. B. 1933. Experimental epidemi-
ology of tuberculosis: the effect of a primary
infection on contact tuberculosis in rabbits. J.
Exp. Med. 58:305–327.
During the first 6 months, subcutaneous vaccina-
tion with virulent human-type bacilli reduced the
incidence of natural infection with virulent bovine-
type bacilli in an animal room: 64% of the unvac-
cinated rabbits, whereas 37% of vaccinated rabbits
developed tuberculosis, and the disease was less
severe in the vaccinated group. During the next 6
months, no appreciable difference between the
two groups was found.
17. Lurie, M. B., and J. Becker. 1946. Effect of
exogenous superinfection on naturally
acquired bovine tuberculosis in inbred rabbits.
Proc. Soc. Exp. Biol. Med. 63:465–469.
The disease produced in inbred rabbits removed
from the source of natural contagion in the ani-
mal room as soon as they became tuberculin pos-
itive was compared to the disease in rabbits
remaining with the source. No appreciable dif-
ference was found. In other words, continuous
exogenous inhalation of low doses of bovine-type
bacilli did not affect the course of an active pri-
mary infection with virulent bovine-type bacilli.
Infection with the bovine type is uniformly fatal
in rabbits.
USE OF ULTRAVIOLET LIGHTS
TO PREVENT TUBERCULOSIS
These studies prove that UV lights are highly
effective in preventing tuberculosis. These lights
must be shielded to prevent damage to the eyes of
personnel from UV radiation.The shielding should
permit these lights to irradiate only the ceiling of
the room, and to irradiate both the ceiling and the
floor directly beneath doorways. See chapter 6 in
Lurie’s book (p. 159–164) and chapter 1 in the pre-
sent volume.
18. Lurie, M. B. 1944. Experimental epidemi-
ology of tuberculosis: the prevention of nat-
ural air-borne contagion of tuberculosis in
rabbits by ultraviolet irradiation. J. Exp. Med.
79:559–572.
APPENDIX C 䡵 391
This is Lurie’s main report on the efficacy of UV
light. See publication 21 for more experimental
details.
19. Lurie, M. B. 1946. Control of air-borne
contagion of tuberculosis. Am. J. Nurs. 46:808–
810.
This report discusses additional measures to prevent
the spread of the tubercle bacillus to human beings,
such as wearing masks and oiling the bed covers.
20. Lurie, M. B. 1947. Experimental air-borne
tuberculosis and its control. Am. Rev. Tuberc.
55:124–127.
This is a review of the subject.
RESISTANCE TO THE ESTABLISHMENT
OF TUBERCULOSIS VERSUS RESISTANCE
TO ITS PROGRESSION
21. Lurie, M. B. 1944. Experimental epidemi-
ology of tuberculosis: hereditary resistance to
attack by tuberculosis and to the ensuing dis-
ease, and the effect of the concentration of
tubercle bacilli upon these two phases of resis-
tance. J. Exp. Med. 79:573–589.
In these experiments, virulent bovine-type tuber-
cle bacilli were shed in the urine of infected rab-
bits, and uninfected inbred rabbits in nearby cages
contracted tuberculosis over a period of months.
As a group, the inbred resistant rabbits established
a primary pulmonary lesion (indicated by a con-
version of their tuberculin skin test) a few months
sooner than did the inbred susceptible rabbits.
However, the resistant rabbits controlled the
progress of their disease better and lived twice as
long. The alveolar macrophages of the resistant
strains apparently trapped inhaled bacilli more
effectively than those of susceptible strains (see
publication 61). Also, the resistant rabbits were
larger and therefore probably breathed more air.
See chapter 6 in Lurie’s book and chapter 12 in the
present volume.
SPREAD OF TUBERCULOSIS
FROM PRIMARY SITE OF
INFECTION IN THE SKIN
This series of reports covers many aspects of
acquired resistance to tuberculosis. Publication 22
is the primary study. It describes the fate of virulent
bovine-type tubercle bacilli injected subcutaneously
in agar into rabbits that (i) were immunized with
BCG intravenously, (ii) were immunized by a slowly
progressive tuberculosis produced by intravenous
bovine-type tubercle bacilli (Ravenel), or (iii) were
nonimmunized controls. Publication 24 describes
similar studies in guinea pigs.
392 䡵 APPENDIX C
In addition, it describes studies in rabbits in
which the reinfecting bacilli in agar were contained
in colloidin-coated silk bags placed intraperitoneally.
The walls of these bags allowed body fluids to enter
but prevented host cells, such as macrophages, from
doing so.
The subcutaneous agar (not in bags) had allowed
some cells to reach the bacilli in the agar. Both
experiments, however, produced the same results:
acellular body fluids from immunized hosts inhib-
ited bacillary growth. See chapter 3 in Lurie’s book.
22. Lurie, M. B. 1936. On the mechanism of
immunity in tuberculosis: the host-parasite
relationship under the conditions of a localized
agar focus of infection and the generalization
of the disease in normal and immunized rab-
bits. J. Exp. Med. 63:923–946.
The acellular body fluids of the immunized rab-
bits were markedly bacteriostatic in vivo when
compared with those of the nonimmunized con-
trols.The reason is not known, but antibodies were
probably not responsible.
The localization of tubercle bacilli at the portal
of entry is a minor component of acquired cellu-
lar resistance in tuberculosis. It occurred only with
low-dose reinfection, whereas with high-dose rein-
fection a more rapid dissemination of bacilli to
the draining lymph nodes occurred.
The activation of macrophages to destroy tuber-
cle bacilli (shown histologically by the presence of
mature epithelioid cells) is the major method by
which the host controls this disease.
Polymorphonuclear leukocytes were ineffective
at inhibiting the growth of tubercle bacilli and
soon died.
The immunity to reinfection produced by a
slowly progressive primary infection with viru-
lent bovine-type tubercle bacilli (Ravenel) was
greater than that produced by BCG.
23. Lurie, M. B. 1936. Further studies on the
mechanism of immunity to tuberculosis. J.
Bacteriol. 32:671–672.
24. Lurie, M. B. 1939. Studies on the mechanism
of immunity in tuberculosis.The role of extra-
cellular factors and local immunity in fixation
and inhibition of growth of tubercle bacilli.
J. Exp. Med. 69:555–578.
In guinea pigs, fibrin formed readily in tuberculous
lesions of reinfection.This fibrin was a fine mesh-
work that partly obstructed the lymphatics and
impeded the spread of tubercle bacilli to the drain-
ing lymph nodes.
In rabbits, relatively little fibrin formed in the
lesions of reinfection. The fibrin meshwork was
coarse and allowed the bacilli to pass into the
lymphatics. Therefore, many reinfecting bacilli
reached the draining lymph nodes in rabbits,
whereas only a few bacilli reached these lymph
nodes in guinea pigs.
Tuberculous humans are very tuberculin sensi-
tive (even more so than guinea pigs), so clotting of
the lymph should readily occur. However, the fib-
rin meshwork in humans is coarse (similar to that
in rabbits) and may not stop the spread of bacilli as
completely as does the fibrin of guinea pigs.
What matters, however, is the fate of these
bacilli after they reach the draining lymph nodes.
The bacilli in the draining lymph nodes of immu-
nized guinea pigs and rabbits were inhibited much
more than those in the draining lymph nodes of
the nonimmunized controls.
Immunity in tuberculosis is a local phenomenon,
with each lesion developing and healing (or pro-
gressing) somewhat independently of the other
lesions in the host (see chapter 5 in the present vol-
ume). Tubercle bacilli can be inhibited at sites of
reinfection and yet multiply in a lesion produced
by the primary infection.
25. Lurie, M. B. 1939. Relative spread of par-
ticulate and diffusible substances in the skin of
male and female rabbits. Proc. Soc. Exp. Biol.
Med. 42:741–744.
26. Lurie, M. B. 1950. Mechanisms affecting
spread in tuberculosis. Ann. N.Y.Acad. Sci. 52:
1074–1090.
This is a review of the publications in this section.
ROLE OF MONONUCLEAR
PHAGOCYTES IN TUBERCULOSIS
27. Lurie, M. B. 1939. Studies on the mechanism
of immunity in tuberculosis: the mobilization
of mononuclear phagocytes in normal and
immunized animals and their relative capaci-
ties for division and phagocytosis. J. Exp. Med.
69:579–609.
Exudate macrophages from tuberculous rabbits and
guinea pigs were more “activated” than were those
from normal controls: the rate of mobilization of
macrophages and their phagocytic abilities were
measured. Macrophages from tuberculous hosts also
responded to nonspecific irritants more vigorously
than did macrophages from uninfected hosts. Dis-
cussed in chapter 3 in Lurie’s book.
28. Lurie, M. B. 1942. Studies on the mechanism
of immunity in tuberculosis.The fate of tuber-
cle bacilli ingested by mononuclear phago-
cytes derived from normal and immunized
animals. J. Exp. Med. 75:247–268.

Mononuclear exudate cells that had ingested tuber-
cle bacilli in vitro were transferred into the ante-
rior eye chambers of a normal rabbit. The cells
(macrophages, lymphocytes, and probably den-
dritic cells) were from immunized (tuberculous)
and control rabbits in the presence of immune
and control serum. The macrophages from the
immune host inhibited the growth of phagocytized
tubercle bacilli, whereas those from the normal
host did not. Immune serum had no appreciable
effect. Described in chapter 4 in Lurie’s book and
interpreted in chapter 5 in the present volume.
TUBERCULOSIS IN INBRED RESISTANT
AND SUSCEPTIBLE RABBITS
29. Lurie, M. B. 1938. Role of inherited natural
resistance to tuberculosis. Proc. Soc. Exp. Biol.
Med. 39:176–181.
30. Lurie, M. B. 1938. Nature of inherited nat-
ural resistance to tuberculosis. Proc. Soc. Exp.
Biol. Med. 39:181–187.
31. Lurie, M. B. 1941. Heredity, constitution
and tuberculosis.An experimental study. Am.
Rev.Tuberc. 44(Suppl. 3):1–125.
This monograph describes the development of
Lurie’s inbred rabbit strains and the characteristics
of the tuberculosis produced in them. It is sum-
marized in chapter 6 in Lurie’s book, and its four
parts are:
I. Inherited Native Resistance and Natural Res-
piratory Contagion (p. 1–67). The origins and
breeding of Lurie’s susceptible and resistant strains
of rabbits are described, as well as the characteris-
tics of tuberculosis produced in each strain by vir-
ulent bovine-type bacilli inhaled in an animal room
over a period of months. The disease in the sus-
ceptible rabbits was the “childhood type,” with
caseous hilar lymph nodes and hematogenous spread
of the disease.The disease in the resistant rabbits was
the “adult type,” with cavity formation and
bronchial spread of the disease and minimal hilar
lymph node involvement.
II. Inherited Native Resistance and Artificial
Respiratory Contagion (p. 68–79).Tuberculosis was
produced in the inbred resistant and susceptible
strains of rabbits by a known dose of virulent
bovine-type bacilli given subcutaneously, intra-
venously, or by aerosol.After the aerosol infection,
multiple primary pulmonary lesions were produced.
In contrast, after natural contagion, only a single pri-
mary pulmonary lesion was produced. Otherwise,
the disease in each rabbit strain resembled that pro-
duced by natural contagion in the animal room.
III. Experimental Study of Certain Factors
Related to Hereditary Constitutional Resistance to
Tuberculosis (with P. Zappasodi) (p. 80–111).The
APPENDIX C 䡵 393
resistant strains of rabbits developed tuberculin
sensitivity and antibodies more rapidly than did the
susceptible strains of rabbits. Local immunity (fol-
lowing the intradermal injection of virulent
bovine-type tubercle bacilli) was most effective in
the resistant rabbits. Heat-killed virulent bovine-
type tubercle bacilli could immunize resistant
strains of rabbits, but could not immunize suscep-
tible strains to any appreciable degree.
IV. Heredity Constitution and Tuberculosis.
Summary and Discussion (p. 112–125).All of these
studies support the principle that, in tuberculosis,
acquired immunity is superimposed on and deter-
mined by the native resistance of the host. Host
resistance can be overwhelmed by large doses of
virulent tubercle bacilli.
EFFECTS OF ESTROGEN AND
CHORIONIC GONADOTROPIN
ON TUBERCULOSIS
These hormones affected the spread of the disease
from the site of bacillary injection in the skin: estro-
gens retarded the spread,and chorionic gonadotropin
enhanced it.However,these hormones had no effect
on the overall resistance of the host to tuberculosis.
Reviewed in chapter 7 in Lurie’s book and in chap-
ter 17 in the present volume.
32. Lurie, M. B., and P. Zappasodi. 1942.
Effect of chorionic gonadotropin on the
spread of particulate substances in the skin of
rabbits. Arch. Pathol. 34:151–166.
33. Lurie, M. B., S. Abramson, and M. J.
Allison. 1949. Constitutional factors in resis-
tance to infection. I.The effect of estrogen and
chorionic gonadotropin on the course of
tuberculosis in highly inbred rabbits. Am. Rev.
Tuberc. 59:168–185.
34. Lurie, M. B.,T. N. Harris, S. Abramson,
and M. J. Allison. 1949. Constitutional fac-
tors in resistance to infection. II.The effect of
estrogen on tuberculin skin sensitivity and on
the allergy of the internal tissues. Am. Rev.
Tuberc. 59:186–197.
35. Lurie, M. B., S. Abramson, A. G. Hep-
pleston, and M. J. Allison. 1949. Constitu-
tional factors in resistance to infection. III. On
the mode of action of estrogen and
gonadotropin on the progress of tuberculosis.
Am. Rev.Tuberc. 59:198–218.
This report analyzes other effects of these hor-
mones on the host. Unexpectedly, estrogen markedly
reduced the amount of amyloid degeneration in the
spleens of rabbits dying of chronic tuberculosis.
394 䡵 APPENDIX C
CHEMOTHERAPY OF TUBERCULOSIS
36. Lurie, M. B., and J. Stokes, Jr. 1943.The
effect of promin on experimental tuberculo-
sis in the rabbit. J. Bacteriol. 45:194–195.
37. Lurie, M. B. 1949.The use of the rabbit in
experimental chemotherapy of tuberculosis.
Ann. N.Y.Acad. Sci. 52:627–636.
In humans, antimicrobial-resistant strains of tuber-
cle bacilli develop during their inordinate extra-
cellular growth in the liquefied caseum in the walls
of pulmonary tuberculous cavities. Rabbits exposed
to aerosols of virulent bovine-type tubercle bacilli
readily form such cavities.Therefore, the rabbit is
the best commonly available laboratory animal in
which to study the development of antimicrobial
resistance within such cavities.
BOOK REVIEW
38. Lurie, M. B. 1944. Review of The Pathogen-
esis of Tuberculosis by Arnold R. Rich (Charles
C Thomas, Springfield, Ill.). Science 100:407–
408.
Lurie praises Rich’s book but admits that some of
Rich’s conclusions are not shared by others. How-
ever, there is wide acceptance of Rich’s claim that
tuberculin sensitivity causes caseous necrosis and that
tubercle bacilli are nontoxic before tuberculin sen-
sitivity develops.
QUANTITATIVE AIRBORNE INFECTION
Lurie and William F.Wells, a brilliant aerodynamic
engineer,constructed an apparatus that could expose
six rabbits (simultaneously and quantitatively) to
aerosols of tubercle bacilli.After 1941,Lurie used this
apparatus in almost all of his studies.
39. Wells,W. F., and M. B. Lurie. 1941. Exper-
imental airborne diseases: quantitative natural
respiratory contagion of tuberculosis. Am. J.
Hyg. 34(Sect. B):21–40.
The original quantitative aerosol apparatus for
infecting rabbits with tubercle bacilli is described.
The number of bacilli estimated to be inhaled by
the rabbits into the alveolar spaces was the same as
the number obtained by culturing their lungs 1 day
after infection.
40. Lurie, M. B.,A. G. Heppleston, S.Abram-
son, and I. B. Swartz. 1950. An evaluation
of the method of quantitative airborne infec-
tion and its use in the study of the pathogen-
esis of tuberculosis.Am. Rev.Tuberc. 61:765–797.
This report provides details on how these aerosol
experiments were performed, including how to
prepare suspensions containing only particles of 1
to 3 bacilli.A diagram of the modified Wells aerosol
apparatus that Lurie used is shown here and in pub-
lication 41.
41. Lurie, M. B., and S. Abramson. 1948.
Reproduction of human ulcerative pulmonary
tuberculosis in rabbits by quantitative natural
airborne contagion. Proc. Soc. Exp. Biol. Med.
69:531–537.
Cavities were produced in inbred strain III rab-
bits, which had recently been obtained from the
Jackson Laboratory in Bar Harbor, Me. These
rabbits were sensitized with heat-killed virulent
bovine-type tubercle bacilli (Ravenel) and chal-
lenged by aerosol with living tubercle bacilli of the
same type. Such sensitization hastened the for-
mation of cavities (see chapter 4 in the present
volume).
FATE OF INHALED HUMAN-TYPE
TUBERCLE BACILLI IN INBRED
RABBITS; THE TUBERCLE-COUNT
METHOD TO ASSESS HOST
RESISTANCE
42. Lurie, M. B., S. Abramson, and A. G.
Heppleston. 1952. On the response of
genetically resistant and susceptible rabbits to
the quantitative inhalation of human-type
tubercle bacilli and the nature of resistance to
tuberculosis. J. Exp. Med. 95:119–134.
Human-type tubercle bacilli are never fully vir-
ulent for rabbits. Rabbits usually recover from
infection with human-type bacilli, but not from
infection with bovine-type bacilli. To produce
one grossly visible primary tubercle 5 weeks after
infection, inbred susceptible rabbits must inhale 50
to 100 units of 1 to 3 human-type bacilli,
whereas inbred resistant rabbits must inhale 600
to over 1,000 units. At 24 weeks after infection
with human-type bacilli, inbred resistant rabbits
usually showed no evidence of disease, but the
inbred susceptible rabbits showed many residual
lesions.
In contrast, fully virulent bovine-type tubercle
bacilli produced one such grossly visible tubercle
for every 3 bacillary units inhaled, in both resistant
and susceptible inbred rabbits. Two-thirds of the
inhaled bacilli impinge on the bronchial mucosa
(which is quite resistant to tuberculosis).
The inhalation of human-type bacilli is the basis
of Lurie’s tubercle-count method to measure
mycobacterial virulence, genetic host resistance,
and vaccine efficacy.The number of grossly visible
pulmonary tubercles is dependent on the power of
pulmonary alveolar macrophages to initially destroy

the inhaled bacillus, and is also dependent on the
acquired immunity that prevents early microscopic
lesions from reaching grossly visible size. See chap-
ter 8 in Lurie’s book and chapter 11 in the present
volume.
THE TIME OF HEALING OF BCG LESIONS
MEASURES THE NATIVE RESISTANCE OF
THE HOST; THE IMMUNITY OF HOSTS
WITH POOR NATIVE RESISTANCE IS
NOT APPRECIABLY INCREASED BY BCG
VACCINATION
43. Lurie, M. B., P. Zappasodi, E. Cardona-
Lynch, and A. M. Dannenberg, Jr. 1952.
The response to the intracutaneous inocula-
tion of BCG as an index of native resistance
to tuberculosis. J. Immunology 68:369–387.
The rate of healing of dermal BCG lesions reflected
how Lurie’s inbred rabbit strains controlled the
progress of infection with virulent tubercle bacilli;
i.e., it measured their innate and acquired resistance
to this disease.These studies apply to groups of rab-
bits but were not precise for individuals. Rabbit
strains with poor innate resistance did not develop
as much immunity from BCG vaccination as did
rabbit strains with good innate resistance. See chap-
ter 9 in Lurie’s book and chapter 23 in the present
volume.
EFFECTS OF GLUCOCORTICOSTEROIDS
ON TUBERCULOSIS
Following the inhalation of virulent human-type
tubercle bacilli (H37Rv), rabbits treated with cor-
tisone developed 3 to 4 times as many grossly vis-
ible primary pulmonary tubercles as did the
untreated controls. However, the tubercles in the
cortisone-treated host were smaller and had more
compact caseous centers.These centers contained
dead macrophages just teeming with tubercle
bacilli.The dead macrophages were mostly within
alveoli, and very little perifocal inflammation
occurred in the surrounding alveolar walls.There-
fore, the spread of bacilli to the hilar nodes was
reduced. The tuberculin sensitivity of the corti-
sone-treated group was also reduced.
When the cortisone treatment was stopped after
6 to 9 weeks, massive pulmonary tuberculosis and
death occurred in Lurie’s inbred susceptible rab-
bits, and progressive disease with cavity forma-
tion occurred in Lurie’s inbred resistant rabbits. In
untreated susceptible and resistant rabbits, human-
type tubercle bacilli (H37Rv) never cause death.
APPENDIX C 䡵 395
See chapter 8 in Lurie’s book and chapter 11 in the
present volume.
The following publications contain some redun-
dancies, because Lurie participated in several sym-
posia on glucocorticosteroids that were subse-
quently published.
44. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and I. B. Swartz. 1951. Con-
stitutional factors in resistance to infection: the
effect of cortisone on the pathogenesis of
tuberculosis. Science 113:234–237.
45. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and E. Cardona-Lynch.
1952. Constitutional factors in resistance to
infection: the effect of cortisone on the patho-
genesis of tuberculosis and its implications
for nonspecific and allergic inflammations
and infectious diseases in general, p. 247–253.
In Advances in Medicine and Surgery (Graduate
School of Medicine of the University of
Pennsylvania). The W. B. Saunders Co.,
Philadelphia, Pa.
46. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and E. Cardona-Lynch. 1953.
The effect of cortisone and ACTH on the
pathogenesis of tuberculosis. Ann. N.Y.Acad. Sci.
56:779–792.
47. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and E. Cardona-Lynch. 1953.
Constitutional factors in resistance to infection:
the effect of cortisone on the pathogenesis of
tuberculosis, p. 84–99. In G. Shwartzman (ed.),
The Effect of ACTH and Cortisone upon Infection
and Resistance. Columbia University Press, New
York, N.Y.
48. Lurie, M. B., and P. Zappasodi. 1955. On
the mode of action of cortisone on the
pathogenesis of tuberculosis and its implica-
tions for the nature of genetic resistance to
the disease, p. 246–258. In Ciba Foundation
Symposium on Experimental Tuberculosis.
Churchill, London, U.K.
49. Lurie, M. B. 1955. On the role of hormones
in experimental tuberculosis. Adv.Tuberc. Res.
6:18–48.
50. Kass, E. H., O. Hechter, T. W. Mou, and
M. B. Lurie. 1955. Effects of adrenal steroids
on resistance to infection: differences in
the relative amounts of corticosterone and
396 䡵 APPENDIX C
hydrocortisone secreted and in their biologic
effects. Arch. Intern. Med. 96:397–402.
Corticosterone is the predominant adrenal steroid
secreted by normal rabbits. After adrenocorti-
cotropin treatment, they secrete predominantly
hydrocortisone. Hydrocortisone decreases resis-
tance to infection, whereas corticosterone does
not have this effect.
CROSSBREEDING INBRED RESISTANT
WITH INBRED SUSCEPTIBLE RABBITS
51. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and G. H.Weiss. 1952. On the
mechanism of genetic resistance to tubercu-
losis and its mode of inheritance. Am. J. Hum.
Genet. 4:302–314.
Crossbreeding of resistant and susceptible rabbits
produced an F1 hybrid of intermediate resistance.
Backcrossing this F1 hybrid with susceptible rab-
bits did not restore susceptibility in the offspring.
However, backcrossing this F1 hybrid with resistant
rabbits did restore high resistance in the offspring.
Therefore, resistant genes were dominant. See chap-
ter 10 in Lurie’s book and chapter 14 in the pre-
sent volume.
GROWTH CURVES OF VIRULENT
HUMAN-TYPE TUBERCLE BACILLI
IN RABBIT LUNGS FOLLOWING
AEROSOL INHALATION
52. Lurie, M. B., P. Zappasodi, and C.Tick-
ner. 1955. On the nature of genetic resistance
to tuberculosis in the light of the host-parasite
relationships in natively resistant and suscepti-
ble rabbits. Am. Rev.Tuberc. 72:297–329.
Rabbit lung homogenates were cultured at various
times after the inhalation of virulent human-type
tubercle bacilli (H37Rv).There was an early log-
arithmic increase in the number of viable tubercle
bacilli in the lungs. The logarithmic phase was
then followed by a stationary (plateau) phase.These
studies formed the basis of my concept that tissue-
damaging delayed-type hypersensitivity has a ben-
eficial role in tuberculosis. See chapters 2, 5, and 15
in the present volume.
COMPARISON OF TUBERCULOSIS
PRODUCED IN RABBITS BY AN
AEROSOL OF VIRULENT HUMAN-
TYPE TUBERCLE BACILLI WITH
THAT PRODUCED BY VIRULENT
BOVINE-TYPE TUBERCLE BACILLI
53. Allison, M. J., P. Zappasodi, and M. B.
Lurie. 1962. Host-parasite relationships in
natively resistant and susceptible rabbits on
quantitative inhalation of tubercle bacilli: their
significance for the nature of genetic resis-
tance. Am. Rev. Respir. Dis. 85:553–569.
The growth curves of viable virulent Mycobac-
terium tuberculosis (H37Rv) and Mycobacterium bovis
(Ravenel) in rabbit lungs showed the same pattern,
i.e., a logarithmic phase followed by a stationary
phase. M. bovis, however, grew for a longer period
of time in the logarithmic stage and reached higher
titers before the stationary stage occurred. See
chapter 15 in the present volume.
TISSUE RESPONSES TO MYCO-
BACTERIA: M.TUBERCULOSIS,
M. BOVIS, AND MYCOBACTERIUM
LEPRAE
54. Lurie, M. B. 1955.A pathogenetic relation-
ship between tuberculosis and leprosy: the
common denominators in the tissue response
to mycobacteria, p. 340–343. In Ciba Founda-
tion Symposium on Experimental Tuberculosis.
Churchill, London, U.K.
EFFECTS OF THYROID HORMONES ON
TUBERCULOSIS
Hyperthyroidism increased host resistance in only
certain inbred rabbit strains, but hypothyroidism
decreased host resistance in every rabbit strain
evaluated. See chapter 17 in the present volume.
55. Lurie, M. B., and G. S. Ninos. 1956.The
effect of triiodothyronine and propylthiouracil
on native resistance to tuberculosis. Am. Rev.
Tuberc. 73:434–437.
56. Lurie, M. B., P. Zappasodi, R. S. Levy,
and R. G. Blaker. 1959. On the role of the
thyroid in native resistance to tuberculosis. I.
The effect of hyperthyroidism. Am. Rev.Tuberc.
79:152–179.
57. Lurie, M. B., P. Zappasodi, R. S. Levy,
and R. G. Blaker. 1959. On the role of the
thyroid in native resistance to tuberculosis.
II.The effect of hypothyroidism: the mode of
action of thyroid hormones. Am. Rev.Tuberc.
79:180–203.
BIPHASIC IMMUNE RESPONSE
TO SUBCUTANEOUS BCG
58. Allison, M. J., P. Zappasodi, and M. B.
Lurie. 1962. The correlation of a biphasic
metabolic response with a biphasic response
in resistance to tuberculosis in rabbits. J. Exp.
Med. 115:881–890.

Rabbits were given a primary intradermal injection
of BCG (30 ⫻ 106) 6 weeks before the same dose
was given subcutaneously as a booster.When chal-
lenged by aerosol with virulent human-type tuber-
cle bacilli (H37Rv) 4 days after the BCG booster,
the rabbits had twice as many grossly visible pri-
mary tubercles (5 weeks after challenge) than did
the unvaccinated controls.When challenged with
H37Rv 28 days after the BCG booster, the rabbits
had half as many visible tubercles (5 weeks later)
than did the controls. In other words, the BCG
booster made the host more susceptible to exoge-
nous infection during the first week and more
resistant thereafter.
These experiments should be repeated with
only a single intradermal primary BCG injection
and no booster—similar to what is commonly
used in humans. The negative phase of resistance
to challenge by virulent bacilli (just described)
was at a time when the host was tuberculin pos-
itive and the immune forces were actively elim-
inating the rather large dose of BCG in the vac-
cine booster. After a single primary intradermal
BCG injection, the host immune forces are
mobilized more gradually. Therefore, the negative
phase of host resistance would probably not
occur.
METABOLIC STUDIES ON INTACT
OIL-INDUCED PERITONEAL MONO-
NUCLEAR CELLS (MACROPHAGES)
FROM INBRED RESISTANT AND
SUSCEPTIBLE RABBITS
Various metabolic activities of oil-induced peri-
toneal macrophages were sometimes higher in
Lurie’s inbred resistant rabbit strain III than in his
susceptible strain CaC. However, peritoneal
macrophages induced by mineral oil are only
weakly activated when compared with pulmonary
alveolar macrophages and mature epithelioid cells
within tuberculous lesions (see publications 17, 26,
32, and 96 in appendix D).Alveolar macrophages
become highly activated by the continuous inges-
tion of dust and microorganisms, and macrophages
(epithelioid cells) within tuberculous lesions
become highly activated by cytokines from anti-
gen-specific lymphocytes. Evidently, mineral oil is
a rather poor macrophage activator.
59. Allison, M. J., P. Zappasodi, and M. B.
Lurie. 1961.Metabolic studies of mononuclear
cells from rabbits of varying genetic resistance
to tuberculosis. I. Studies on cells of normal
non-infected animals. Am. Rev. Respir. Dis. 84:
364–370.
APPENDIX C 䡵 397
60. Allison, M. J., P. Zappasodi, and M. B.
Lurie. 1962. Metabolic studies of mono-
nuclear cells from rabbits of varying genetic
resistance to tuberculosis. II. Studies on cells
from BCG-vaccinated animals. Am. Rev. Respir.
Dis. 85:364–372.
Note:Allison continued these studies as parts III
and IV of his series: III. Studies on variation of thy-
roid function. Am. Rev. Respir. Dis. 86:513–517,
1962. IV. Studies on cortisone-treated animals.
Am. Rev. Respir. Dis. 87:384–388, 1963.
In general, compared to controls, hyperthy-
roid rabbits had more activated cells in the
oil-induced peritoneal exudates, whereas
cortisone-treated rabbits had fewer activated
cells in these exudates.
PHAGOCYTIC ABILITIES OF
ALVEOLAR MACROPHAGES
FROM INBRED RESISTANT
AND SUSCEPTIBLE RABBITS
61. Henderson, H. J., A. M. Dannenberg,
Jr., and M. B. Lurie. 1963. Phagocytosis of
tubercle bacilli by rabbit pulmonary alveolar
macrophages and its relation to native resis-
tance to tuberculosis. J. Immunol. 91:553–556.
Alveolar macrophages from inbred resistant rabbits
ingested twice as many tubercle bacilli in vitro than
did those from inbred susceptible rabbits.Perhaps,the
greater phagocytic ability of alveolar macrophages
from the resistant rabbits enabled them to trap more
bacilli from inhaled air in the animal room:they con-
verted their tuberculin skin test several months
sooner than did the susceptible rabbits. See chapter
6 in Lurie’s book, publication 21 in this appendix,
and chapter 12 in the present volume.
VALY MENKIN: A BIOGRAPHICAL
SKETCH
62. Lurie, M. B. 1961. On the work of Valy
Menkin: a biographical sketch. Anat. Rec. 140:
234–236.
Valy Menkin pioneered the cytokine field, but the
purification methods available at that time were not
sufficient to identify individual cytokines. Menkin
called the chemotactic fraction of his inflammatory
exudates “leukotaxin.” See V. Menkin, Biochemical
Mechanisms in Inflammation, 2nd ed. Charles C
Thomas, Springfield, Ill., 1956.
398 䡵 APPENDIX C
REVIEW ARTICLES ON THE EFFECTS
OF VARIOUS HORMONES ON THE
PATHOGENESIS OF TUBERCULOSIS
63. Lurie, M. B. 1960. The reticuloendothelial
system, cortisone, and thyroid function: their
relation to native resistance to infection. Ann.
N.Y.Acad. Sci. 88:83–98.
64. Lurie, M. B. 1965. Immunological aspects of
steroid therapy. Arch. Environ. Health 11:235–
241.
This review covers some of the literature that does
not appear in Lurie’s other reports on this subject.
REVIEW ARTICLES ON NATIVE
AND ACQUIRED RESISTANCE
TO TUBERCULOSIS
65. Lurie, M. B. 1950. The Cyclopedia of Medicine,
Surgery and Specialties, vol. 7. Immunology of
Tuberculosis, p. 1–30. F. A. Davis Company,
Philadelphia, Pa.
The literature on immunology of tuberculosis up
to 1950 is reviewed. Unfortunately, the reader must
refer to Lurie’s article in the 1945 edition of the
Cyclopedia for all references before 1940.
66. Lurie, M. B. 1950. Native and acquired resis-
tance to tuberculosis. Am. J. Med. 9:591–610.
This is an excellent review of the subject up to
1950.
67. Lurie, M. B., and A. M. Dannenberg, Jr.
1965. Macrophage function in infectious dis-
ease with inbred rabbits. Bacteriol. Rev. 29:466–
476.
This review briefly describes the characteristics of
innate and acquired resistance to tuberculosis,as well
as other characteristics, found in Lurie’s inbred rab-
bits. See chapters 13 and 14 of the present volume.
LURIE’S BOOK
68. Lurie, M. B. 1964. Resistance to Tuberculosis.
Experimental Studies in Native and Acquired
Defensive Mechanisms. Harvard University
Press, Cambridge, Mass.
This book summarizes and interprets most of
Lurie’s publications.
BIOGRAPHY OF MAX B. LURIE
69. Fahy,A. 1966. Scientists at work: Max Lurie,
M.D. Bull. Natl.Tuberc.Assoc. 52:10.
 APPENDIX D
PUBLICATIONS OF ARTHUR M. DANNENBERG, JR.
These publications are grouped somewhat chrono-
logically by subject. Comments follow many of the
publications to explain their contributions, which
are often not evident from their titles. In this appen-
dix,“Lurie’s book” refers to his 1964 text (publica-
tion 68 in appendix C). Chapters not attributed to
Lurie’s book refer to the present volume. Publica-
tion numbers refer to this appendix (D) unless
stated otherwise.
PART I. RESEARCH ARTICLES
RHEUMATOID ARTHRITIS
1. Harris,T. N., S. Harris, A. M. Dannen-
berg, Jr., and J. L. Hollander. 1950. Strep-
tococcal antihyaluronidase titers in the sera
of patients with rheumatoid arthritis and
glomerular nephritis. Ann. Intern. Med. 32:
917–922.
I helped the Harrises with this study during my
1-year research residency at the Children’s Hos-
pital of Philadelphia (University of Pennsylvania).
STUDIES ON THE PATHOGENESIS
OF TUBERCULOSIS WITH LURIE’S
INBRED RABBIT STRAINS
Comments on the following glucocorticosteroid
studies (publications 2 through 6) are in Lurie’s
publication list (appendix C, publications 44
through 48).These studies are reviewed in chap-
ter 11 in Lurie’s book and in chapter 16 in the pre-
sent volume.
2. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and I. B. Swartz. 1951. Con-
stitutional factors in resistance to infection: the
effect of cortisone on the pathogenesis of
tuberculosis. Science 113:234–237.
3. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and E. Cardona-Lynch.
1952. Constitutional factors in resistance to
infection: the effect of cortisone on the
pathogenesis of tuberculosis and its implica-
tions for nonspecific and allergic inflamma-
tions and infectious diseases in general,
p. 247–253. In Advances in Medicine and
Surgery (Graduate School of Medicine of
399
the University of Pennsylvania). The W. B.
Saunders Co., Philadelphia, Pa.
4. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and E. Cardona-Lynch.
1953. The effect of cortisone and ACTH
on the pathogenesis of tuberculosis. Ann.
N.Y.Acad. Sci. 56:779–792.
5. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and E. Cardona-Lynch.
1953. Constitutional factors in resistance to
infection: the effect of cortisone on the
pathogenesis of tuberculosis, p. 84–99. In G.
Shwartzman (ed.), The Effect of ACTH and
Cortisone upon Infection and Resistance. Colum-
bia University Press, New York, N.Y.
6. Lurie, M. B., and P. Zappasodi. 1955. On
the mode of action of cortisone on the
pathogenesis of tuberculosis and its implica-
tions for the nature of genetic resistance to
the disease, p. 246–258. In Ciba Foundation
Symposium on Experimental Tuberculosis.
Churchill, London, U.K.
I participated in many of the studies described
here.
7. Lurie, M. B., P. Zappasodi, E. Cardona-
Lynch, and A. M. Dannenberg, Jr. 1952.
The response to the intracutaneous inocu-
lation of BCG as an index of native resistance
to tuberculosis. J. Immunol. 68:369–387.
The rate of healing of dermal BCG lesions
reflected how inbred rabbits controlled the
progress of infection with virulent tubercle bacilli;
i.e., it indicated the amount of their innate resis-
tance to this disease.These studies apply to groups
of rabbits, but were not precise for individual
rabbits.
Hosts with poor innate resistance did not
develop the amount of immunity from the vac-
cine that hosts with good innate resistance did.Yet,
hosts with poor resistance need such immuniza-
tion the most. See chapter 23.
8. Lurie, M. B., P. Zappasodi, A. M. Dan-
nenberg, Jr., and G. H. Weiss. 1953. On
the mechanism of genetic resistance to
tuberculosis and its mode of inheritance.
Am. J. Hum. Genet. 4:302–314.
400 䡵 APPENDIX D
Crossbreeding of resistant and susceptible rabbits
produced an F1 hybrid of intermediate resistance.
Backcrossing this F1 hybrid with susceptible rab-
bits did not regain susceptibility in the offspring.
However, backcrossing this F1 hybrid with resis-
tant rabbits did regain high resistance in the off-
spring.Therefore, resistance was dominant, or sus-
ceptibility was lacking some of the resistant genes.
See chapter 10 in Lurie’s book and chapter 14 in
the present volume.
9. Lurie, M. B., and A. M. Dannenberg, Jr.
1965. Macrophage function in infectious
disease with inbred rabbits. Bacteriol. Rev.
29:466–476.
This report briefly reviews the native and acquired
characteristics of Lurie’s inbred rabbits. I wrote it
at Johns Hopkins while Dr. Lurie was still alive,
and he checked its accuracy. See chapters 13 and
14 in the present volume.
10. Colberg, J. E., and A. M. Dannenberg,
Jr. 1965.An evaluation of inbred rabbit pop-
ulations by skin homotransplantation. Nature
207:777–778.
Lurie’s C and AD rabbit strains were only partly
inbred: the C strain to a greater extent than the
AD strain.The C strain rejected a skin graft from
an outbred Flemish strain of rabbits in a normal
fashion, despite its poor immune response to the
tubercle bacillus.
IDENTIFICATION AND
CHARACTERIZATION
OF LUNG PROTEINASES
I performed these studies as a postdoctoral fellow
in the laboratory of Professor Emil L. Smith at the
University of Utah, Salt Lake City.
11. Dannenberg,A. M., Jr., and E. L. Smith.
1955. Proteolytic enzymes of lung. J. Biol.
Chem. 215:545–554.
Macrophage cathepsin D (proteinase I) was partly
purified from beef lung. (A typographical error
appeared in this report. It should read:“Two vol-
umes of 50% acetone in water [not 100% acetone]
was used in the Waring blender.”) Publication
42 describes the complete purification of this
enzyme from rabbit and beef lungs.
12. Dannenberg,A. M., Jr., and E. L. Smith.
1955.Action of proteinase I of bovine lung.
Hydrolysis of the oxidized B chain of insulin;
polymer formation from amino acid esters.
J. Biol. Chem. 215:55–66.
Macrophage proteinase I hydrolyzed the oxi-
dized B chain of insulin at sites similar to those
hydrolyzed by pepsin. However, another protease
was present in the original proteinase I prepara-
tion (see publication 43). This second protease
polymerized amino acid esters that were sub-
strates for chymotrypsin.
RESPIRATORY MELIOIDOSIS
These studies were performed at the Naval Bio-
logical Laboratories in Oakland, Calif. (under Dr.
Clara Nigg) while I was a naval officer attached to
Naval Medical Research Unit #1 in Berkeley, Calif.
13. Dannenberg,A. M., Jr., and E. M. Scott.
1956. Determination of respiratory LD50
from a number of primary lesions as illus-
trated by melioidosis. Proc. Soc. Exp. Biol.
Med. 92:571–575.
With virulent strains of Malleomyces pseudomallei,
every established pulmonary lesion was always
fatal in mice.Therefore, if mice inhaled sufficient
bacilli to produce 10 to 50 primary pulmonary
lesions, one could calculate the inhaled dose
required to produce one lesion (the LD50), using
Poisson’s distribution.This method could actually
determine an LD50 by exposing only one animal.
However, more than one animal was usually used,
to allow for variations between individual mice.
Pulmonary lesion counts can greatly reduce the
number of animals needed to obtain an LD50,
because such counts provide a mean value of
many lesions in each animal, whereas a single
(lethal) lesion might not represent the mean.
14. Dannenberg,A. M., Jr., and E. M. Scott.
1958. Melioidosis: pathogenesis and immu-
nity in mice and hamsters. I. Studies with
virulent strains of Malleomyces pseudomallei.
J. Exp. Med. 107:153–166.
The characteristics of peracute, acute, and chronic
melioidosis in mice are described. In general, the
type of disease produced depended on the dose
of the bacilli inhaled.The acute form of melioi-
dosis was a septicemic disease similar to the acute
forms of plague and anthrax.
15. Dannenberg,A. M., Jr., and E. M. Scott.
1958. Melioidosis: pathogenesis and immu-
nity in mice and hamsters. II. Studies with
avirulent strains of Malleomyces pseudomallei.
Am. J. Pathol. 34:1099–1121.
These avirulent strains cannot multiply well in the
host. However, if very large numbers of avirulent
strains were administered by any route, the disease
produced was similar to that produced by fully
virulent strains.With these avirulent strains, the
disease was exacerbated in mice by cortico-
steroids or by fasting.

16. Dannenberg,A. M., Jr., and E. M. Scott.
1960. Melioidosis: pathogenesis and immunity
in mice and hamsters. III.The effect of vac-
cination with avirulent strains of Pseudomonas
pseudomallei on the resistance to the estab-
lishment and the resistance to the progress of
respiratory melioidosis caused by virulent
strains: all-or-none aspects of this disease.
J. Immunol. 84:233–246.
Vaccination could prevent the establishment of
melioidosis after the inhalation of virulent strains
of Pseudomonas pseudomallei but had little or no
effect on its progress. See chapter 12.
ENZYME HISTOCHEMISTRY
OF MACROPHAGES, POLY-
MORPHONUCLEAR LEUKOCYTES
(PMN), AND PULMONARY
ALVEOLAR MACROPHAGES
17. Dannenberg,A. M., Jr., M. S. Burstone,
P. C.Walter, and J.W. Kinsley. 1963.A his-
tochemical study of phagocytic and enzy-
matic functions of rabbit mononuclear and
polymorphonuclear exudate cells and alve-
olar macrophages. I. Survey and quantitation
of enzymes, and states of cellular activation.
J. Cell Biol. 17:465–486.
This report illustrates that pulmonary alveolar
macrophages contain higher levels of hydrolytic
and oxidative enzymes than do macrophages and
PMN. Cytochrome oxidase, aminopeptidase, suc-
cinic dehydrogenase, acid phosphatase, esterase,
and alkaline phosphatase were evaluated histo-
chemically with respect to stability, activators,
inhibitors, and pH optimum. Only PMN con-
tained alkaline phosphatase.
18. Yarborough, D. J., O. T. Meyer, A. M.
Dannenberg, Jr., and B. Pearson. 1967.
Histochemistry of macrophage hydrolases.
III. Studies on -galactosidase, -glu-
curonidase and aminopeptidase with indolyl
and naphthyl substrates. J. Reticuloendothel.
Soc. 4:390–408.
These enzymes, in addition to those in publica-
tion 17, confirmed that pulmonary alveolar
macrophages are highly activated cells.
The possible role of enzymes that remove
only one sugar or one amino acid at a time is dis-
cussed. Such enzymes may downregulate cell-
receptor functions and may also alter the surfaces
of invading microorganisms so that they are less
virulent.
APPENDIX D 䡵 401
HYDROLASES OF CULTURED
MACROPHAGES
19. Dannenberg, A. M., Jr., P. C. Walter,
and F. A. Kapral. 1963. A histochemical
study of phagocytic and enzymatic func-
tions of rabbit mononuclear and polymor-
phonuclear exudate cells and alveolar
macrophages. II.The effect of particle inges-
tion on enzyme activity; two phases of in
vitro activation. J. Immunol. 90:448–465.
“Protoplasmic excitation” is the first phase of
phagocyte activation. It causes an increase in the
phagocyte’s oxygen and glucose consumption,
lipid turnover, pseudopod formation, and, prob-
ably, motility.With excitation, cell enzymes are not
increased but are utilized to nearer capacity.After
excitation, “protoplasmic adaptation” occurs:
many of the initial changes remain, and many of
the enzymes utilized in excitation are increased
in amount, so that the phagocyte can remain in
the activated state.
20. Mizunoe, K., and A. M. Dannenberg, Jr.
1965. Hydrolases of rabbit macrophages. III.
Effect of BCG vaccination, tissue culture, and
ingested tubercle bacilli. Proc. Soc. Exp. Biol.
Med. 120:284–290.
Rabbit mineral oil-induced peritoneal macro-
phages were assayed for proteinase, chymotrypsin-
like esterase, lipase, lysozyme, and acid phosphatase.
These macrophages came from BCG-vaccinated
rabbits and controls, with or without added tuber-
cle bacilli. The macrophages were cultured for
2 days.
Differences in macrophage enzymes occurred
in culture among these various groups. How-
ever, cell function in culture cannot be considered
equivalent to cell function in vivo. Tuberculous
lesions in vivo have a continuous blood supply,
enabling the local accumulation of dendritic cells,
macrophages and lymphocytes, and serological
components, none of which can be adequately
reproduced in vitro.
PHAGOCYTOSIS BY PULMONARY
ALVEOLAR MACROPHAGES OF
LURIE’S INBRED RABBITS
21. Henderson, H. J., A. M. Dannenberg,
Jr., and M. B. Lurie. 1963. Phagocytosis of
tubercle bacilli by rabbit pulmonary alveo-
lar macrophages and its relation to native
resistance to tuberculosis. J. Immunol. 91:553–
556.
Alveolar macrophages from inbred resistant rab-
bits ingested twice as many tubercle bacilli in
402 䡵 APPENDIX D
vitro than did those from inbred susceptible rab-
bits. Perhaps, the greater phagocytic ability of
alveolar macrophages from the resistant rabbits
enabled them to trap more bacilli from inhaled air
in the animal room, because they converted their
tuberculin skin test several months sooner than
did the susceptible rabbits. See chapter 6 (p. 164–
168) in Lurie’s book and chapter 12 in the pre-
sent volume.
INFLAMMATION CAUSED BY A
LIPOPOLYSACCHARIDE FROM
TUBERCLE BACILLI
22. Dannenberg, A. M., Jr., K. Mizunoe,
M. Peace, and P. Zappasodi. 1965. Der-
mal response to the lipopolysaccharide
PmKo from tubercle bacilli as an index of
resistance to tuberculosis. Bull. Johns Hopkins
Hosp. 117:174–194.
PmKo is a lipopolysaccharide extracted and puri-
fied from heat-killed virulent tubercle bacilli.
Used as a skin test in tuberculous humans, it was
meant to predict whether the disease was pro-
gressing or regressing. However, in several strains
of Lurie’s inbred resistant and susceptible rabbits
with active tuberculosis, we could not show any
prognostic differences between the PmKo and
tuberculin skin tests.
HYDROLYTIC ENZYMES OF RABBIT
MACROPHAGES (AND PMN)
These reports describe the properties of various
hydrolytic enzymes in rabbit peritoneal exudate
cells (not containing tubercle bacilli).
The major activation of macrophages occurs
locally within tuberculous lesions where the bacilli
and their products exist. Therefore, the macro-
phages in peritoneal exudates from tuberculous
and control rabbits (where no tubercle bacilli are
present) showed little or no difference in their
enzyme content (publications 23 and 26), except
for lysozyme (publications 20 and 35).
23. Dannenberg, A. M., Jr., and W. E. Ben-
nett. 1963. Hydrolases of mononuclear
exudate cells and tuberculosis. I. Exudate
characteristics, esterases, proteinases and
lipase. Arch. Pathol. 76:581–591.
24. Dannenberg, A. M., Jr., and W. E. Ben-
nett. 1964. Hydrolytic enzymes of rabbit
mononuclear exudate cells. I. Quantitative
assay and properties of certain proteases,
nonspecific esterases and lipases of mononu-
clear and polymorphonuclear cells and ery-
throcytes. J. Cell Biol. 21:1–13.
25. Carson, M. E., and A. M. Dannenberg,
Jr. 1965. Hydrolytic enzymes of rabbit
mononuclear exudate cells. II. Lysozyme:
properties and quantitative assay in tubercu-
lous and control inbred rabbits. J. Immunol.
94:99–104.
26. Meyer, O. T., A. M. Dannenberg, Jr.,
and K. Mizunoe. 1970. Hydrolytic
enzymes of rabbit mononuclear and poly-
morphonuclear exudate cells and pulmonary
alveolar macrophages. III. Deoxyribonucle-
ase and ribonuclease: properties and quanti-
tative assay in macrophages from tuberculous
and control inbred rabbits. J. Reticuloendothel.
Soc. 7:15–31.
Some of these assays were made on the peri-
toneal exudates, described in publication 23, that
had been stored at –20°C for 5 to 7 years with lit-
tle or no apparent loss of activity.
RADIATION AND MACROPHAGE
FUNCTION
These reports are summarized in chapter 18.
27. Kambara, T., S. Chandrasekhar, A. M.
Dannenberg, Jr., and O.T. Meyer. 1970.
Radiation, infection and macrophage func-
tion. I. Effects of whole body radiation on
dermal tuberculous lesions in rabbits: devel-
opment, histology and histochemistry. J. Retic-
uloendothel. Soc. 7:53–78.
28. Meyer, O.T., and A. M. Dannenberg, Jr.
1970. Radiation, infection and macrophage
function. II. Effect of whole body radiation
on the number of pulmonary alveolar
macrophages and their levels of hydrolytic
enzymes. J. Reticuloendothel. Soc. 7:79–90.
29. Dannenberg, A. M., Jr.,W. G. Roessler,
O. T. Meyer, S. Chandrasekhar, and T.
Kambara. 1970. Radiation, infection and
macrophage function. III. Recovery from
the effects of radiation illustrated by dermal
BCG lesions: resistance of pulmonary alve-
olar macrophages to radiation illustrated by
tuberculosis produced by the airborne route.
J. Reticuloendothel. Soc. 7:91–108.
30. Chandrasekhar, S., K. Shima,A. M. Dan-
nenberg, Jr.,T. Kambara, J. I. Fabrikant,
and W. G. Roessler. 1971. Radiation, infec-
tion and macrophage function. IV.The effect
of radiation on the proliferative abilities of
mononuclear phagocytes in tuberculous
lesions of rabbits. Infect. Immun. 3:254–259.

MACROPHAGE PROTEINASE
ACTIVATES COMPLEMENT
COMPONENTS
31. Snyderman, R., H. S. Shin, and A. M.
Dannenberg, Jr. 1972. Macrophage pro-
teinase and inflammation: the production of
chemotactic activity from the fifth compo-
nent of complement by macrophage pro-
teinase. J. Immunol. 109:896–898.
Both partly purified beef lung cathepsin D and
rabbit macrophage homogenates hydrolyzed the
C5 component of complement to produce the
active chemotaxin C5a.Therefore, complement,
in addition to chemokines, apparently plays a
role in the accumulation of mononuclear cells in
tuberculous lesions. See chapter 5.
THE LOCAL NATURE OF
CELL-MEDIATED IMMUNITY
32. Dannenberg, A. M., Jr., O. T. Meyer,
J. R. Esterly, and T. Kambara. 1968.The
local nature of immunity in tuberculosis,
illustrated histochemically in dermal BCG
lesions. J. Immunol. 100:931–941.
This seemed to be the first histochemical demon-
stration of the local nature of acquired cellular
resistance (macrophage activation). Macrophages
were most activated (for -galactosidase, -
glucuronidase, succinic dehydrogenase, and
cytochrome oxidase) within the tuberculous
lesion at sites where the bacillus and its antigens
are located. Elsewhere in the lesion, macrophages
were less activated.
This report also describes other important
contributions. (i) Macrophages staining darkest
for -galactosidase contained fewer tubercle
bacilli than those staining lighter, and their shape
resembled that of Lurie’s mature epithelioid cells.
(ii) DNase and RNase (demonstrated by the
Daoust substrate film technique) were active in
intact and necrotic cells within the liquefying
areas of the lesions.This technique also showed
that rabbit eosinophils had very high RNase
activity.
Our review of local and systemic immunity in
tuberculosis (publication 96) was published shortly
after this report. Chapter 5 presents the concepts
therein. Macrophages are locally activated for
many different functions (see chapter 6 and pub-
lication 54).
33. Ando, M., A. M. Dannenberg, Jr., M.
Sugimoto, and B. S. Tepper. 1977. His-
tochemical studies relating the activation of
macrophages to the intracellular destruction
of tubercle bacilli. Am. J. Pathol. 86:623–634.
APPENDIX D 䡵 403
Dermal lesions were produced by 14C-labeled
BCG.The most mature epithelioid cells (which
Lurie associated with bacillary destruction) stained
histochemically the darkest for -galactosidase
(see publication 32).This publication (number 33)
showed that these mature epithelioid cells con-
tained 14C-labeled bacillary components but few
intact bacilli, confirming that these cells had
destroyed the bacilli that they once contained.
MACROPHAGE KINETICS: THEIR
TURNOVER AND ACTIVATION IN
TUBERCULOUS LESIONS
A continuous turnover of macrophages occurs in
the viable tuberculous granulation tissue that sur-
rounds the caseous centers of the lesions. This
turnover occurs even in arrested lesions, although
at a reduced rate.
The continuous entry of nonactivated mono-
cytes/macrophages into viable areas around the
caseous center provides an opportunity for
renewed intracellular bacillary growth.The caseous
center contains dormant tubercle bacilli. In arrested
lesions, such bacilli could escape from the edges of
the caseum and grow in nonactivated macrophages
before they are destroyed by activated macro-
phages. Therefore, even in arrested lesions, some
bacilli are probably not dormant. True bacillary
dormancy may only occur within solid caseous tis-
sue itself.
This series of reports is summarized in publi-
cation 119 and in chapter 10.
34. Shima, K., A. M. Dannenberg, Jr.,
M.Ando, S. Chandrasekhar, J.A. Seluz-
icki, and J. I. Fabrikant. 1972. Macro-
phage accumulation, division, maturation,
and digestive and microbicidal capacities in
tuberculous lesions. I. Studies involving their
incorporation of tritiated thymidine and
their content of lysosomal enzymes and
bacilli. Am. J. Pathol. 67:159–180.
35. Ando, M., A. M. Dannenberg, Jr., and
K. Shima. 1972. Macrophage accumula-
tion, division, maturation and digestive and
microbicidal capacities in tuberculous lesions.
II. Rate at which mononuclear cells enter
and divide in primary BCG lesions and those
of reinfection. J. Immunol. 109:8–19.
36. Dannenberg, A. M., Jr., M. Ando, and
K. Shima. 1972. Macrophage accumula-
tion, division and immunity in primary BCG
404 䡵 APPENDIX D
lesions and in those of reinfection. J. Immunol.
109:1109–1121.
37. Ando, M., and A. M. Dannenberg, Jr.
1972. Macrophage accumulation, division,
maturation and digestive and microbicidal
capacities in tuberculous lesions. IV. Macro-
phage turnover, lysosomal enzymes and
division in healing lesions. Lab. Investig. 27:
466–472.
38. Ando, M. 1973. Macrophage activation in
tuberculin reactions of rabbits with primary
BCG infection and reinfection. J. Reticu-
loendothel. Soc. 14:132–145.
From our laboratory.
39. Ando, M., A. M. Dannenberg, Jr., E.
Courtade, and K. Shima. 1976.Turnover
of tritiated-thymidine-labeled mononuclear
cells in tuberculous lesions of rabbits.A com-
parison of primary dermal BCG lesions and
those of reinfection. Proc. Soc. Exp. Biol. Med.
151:491–494.
40. Dannenberg, A. M., Jr., M. Sugimoto,
L. P. Fay, and A. L. Massaquoi. 1976. In
vivo labeling effectiveness of tritiated thymi-
dine of high and low specific activities in
rabbits. Radiat. Res. 67:98–103.
41. Tsuda, T., A. M. Dannenberg, Jr., M.
Ando, H.Abbey, and A. R. Corrin. 1976.
Mononuclear cell turnover in chronic
inflammation. Studies on tritiated-thymi-
dine-labeled cells in blood, tuberculin traps
and dermal BCG lesions of rabbits. Am. J.
Pathol. 83:255–268.
PURIFICATION OF MACROPHAGE
PROTEASES FROM RABBIT LUNGS
42. Rojas-Espinosa, O.,A. M. Dannenberg,
Jr., P. A. Murphy, P. A. Straat, P. C.
Huang, and S. P. James. 1973. Purification
and properties of the cathepsin D-type pro-
teinase from beef and rabbit lung and its
identification in macrophages. Infect. Immun.
8:1000–1008.
Cathepsin D was purified from rabbit and beef
lungs. Four isoenzymes were obtained from each
source.An antibody to one rabbit isoenzyme was
produced in goats for the immunohistochemical
studies described in publication 44. The anti-
body inhibited the proteolytic activity of rabbit
peritoneal and pulmonary macrophages.
43. Rojas-Espinosa, O., P. Arce-Paredez,
A. M. Dannenberg, Jr., and R. L.
Kamenetz. 1975. Macrophage esterase:
identification, purification and properties of
a chymotrypsin-like esterase from lung that
hydrolyzes and transfers nonpolar amino acid
esters. Biochim. Biophys.Acta 403:161–179.
VISUALIZATION OF MACROPHAGE
PROTEINASE, RIBONUCLEASE,
AND PHOSPHOLIPASE A2 IN TISSUE
SECTIONS OF TUBERCULOUS LESIONS
BY PEROXIDASE–ANTIPEROXIDASE
TECHNIQUES
44. Rojas-Espinosa, O.,A. M. Dannenberg,
Jr., L. A. Sternberger, and T. Tsuda.
1974. Role of cathepsin D in the patho-
genesis of tuberculosis.A histochemical study
employing unlabeled antibodies and the per-
oxidase-antiperoxidase complex. Am. J.
Pathol. 74:1–17.
Cathepsin D was visualized in rabbit peritoneal
and pulmonary macrophages and in dermal BCG
lesions. It was present at the border of the lique-
fied caseous centers of these lesions. See publica-
tion 54 for additional histochemical studies.
45. Namba, M., M. Suga, F.Tanaka, A. M.
Dannenberg, Jr., A. T. Hastie, and R.
C. Franson. 1983. Immunocytochemical
demonstration of rabbit ribonuclease and
phospholipase A 2 by the peroxidase-
antiperoxidase technique in professional
phagocytes (pulmonary alveolar macro-
phages and granulocytic and mononuclear
peritoneal exudate cells) and in glycol
methacrylate sections of dermal tubercu-
lous (BCG) lesions. J. Reticuloendothel. Soc.
34:425–435.
Ribonuclease and phospholipase A2 (and cathep-
sin D, publication 44) are increased together as
macrophages become activated.
Anti-RNase serum stained alveolar macro-
phages more intensely than PMN and macro-
phages in oil-induced exudates, whereas
anti-RNase serum stained PMN and macro-
phages more intensely than alveolar macrophages
(see publication 56). RNase evidently increases
with macrophage activation.
PEPSTATIN: A HIGHLY SPECIFIC
INHIBITOR OF MACROPHAGE
CATHEPSIN D
46. McAdoo, M. H., A. M. Dannenberg,
Jr., C. J. Hayes, S. P. James, and J. H.
Sanner. 1973. Inhibition of the cathepsin

D-type proteinase of macrophages by pep-
statin, a specific pepsin inhibitor, and other
substances. Infect. Immun. 7:655–665.
Pepstatin, discovered in H. Umezawa’s laboratory
( J.Antibiot. [Japan] 23:259–262, 1970), is a highly
specific inhibitor of macrophage cathepsin D.
Other inhibitors were identified, but they were not
nearly as specific as pepstatin. The structure of
pepstatin, how it works, and the other proteinases
that it inhibits (including pepsin) are reviewed. Its
in vivo applications to date have been disap-
pointing, even for the treatment of peptic ulcers.
Perhaps an analog of pepstatin would be more
effective. See chapter 4 for the possible use of
such inhibitors in preventing liquefaction and the
formation of cavities in tuberculosis.
HISTOCHEMICAL VISUALIZATION OF
HYDROLYTIC ENZYMES IN
TUBERCULOUS LESIONS BY
SUBSTRATE FILM TECHNIQUES
47. Tsuda, T., A. M. Dannenberg, Jr., M.
Ando, O. Rojas-Espinosa, and K.
Shima. 1974. Enzymes in tuberculous
lesions hydrolyzing protein, hyaluronic acid
and chondroitin sulfate: a study of isolated
macrophages in developing and healing rab-
bit BCG lesions with substrate film tech-
niques; the shift of enzyme pH optima
towards neutrality in “intact” cells and tissues.
J. Reticuloendothel. Soc. 16:220–231.
In cryostat-prepared tissue sections of rabbit
tuberculous lesions (produced by BCG), these
enzymes were most active after delayed-type
hypersensitivity (DTH) and cell-mediated immu-
nity (CMI) had developed.They remained active
long after the cells containing them had died
(similar to the RNase and DNase described in
publication 32).
The pH optimum of these “acid-acting” hydro-
lases in these tissue sections was closer to neutral-
ity than their pH optimum in tissue homogenates.
48. Smokovitis, A., M. Sugimoto, A. M.
Dannenberg, Jr., and T. Astrup. 1976.
A histochemical study of the fibrinolytic
activity in dermal tuberculous lesions pro-
duced by BCG in rabbits. Exp. Mol. Pathol.
25:236–241.
The microvasculature in tuberculous granulation
tissue of BCG lesions showed high fibrinolytic
activity, probably due to plasminogen activator.
The fibrinolytic activity was highest between 11
and 18 days when the microvasculature was pro-
liferating. See publication 49.
APPENDIX D 䡵 405
CAPILLARY DENSITY IN DEVELOPING
AND HEALING TUBERCULOUS
LESIONS
49. Courtade, E.T.,T.Tsuda, C. R.Thomas,
and A. M. Dannenberg, Jr. 1975. Capillary
density in developing and healing tuberculous
lesions produced by BCG in rabbits.A quan-
titative study. Am. J. Pathol. 78:243–260.
This is the only study of this type on tuberculous
lesions.The entire microvasculature in the rabbit
was made visible by injecting (under deep ter-
minal anesthesia) the aorta with a warm gela-
tinized colloidal carbon suspension. After the
gelatin had solidified in 10% formalin, thick, 250-
m tissue sections of the BCG lesions were made.
The microvasculature density seemed to peak in
15-day BCG lesions and remained about 60%
above normal throughout the healing process.
Chapter 8 provides more details on this experi-
ment, including photographs.
THE COLLECTION AND
MEASUREMENT OF EXTRACELLULAR
HYDROLYTIC ENZYMES IN
DEVELOPING AND HEALING BCG
LESIONS BY USING SKIN CHAMBERS
50. Sugimoto, M., A. M. Dannenberg, Jr.,
L. M. Wahl, W. H. Ettinger, Jr., A. T.
Hastie, D. C. Daniels, C. R.Thomas, and
L. Demoulin-Brahy. 1978. Extracellular
hydrolytic enzymes of rabbit dermal tuber-
culous lesions and tuberculin reactions col-
lected in skin chambers. Am. J. Pathol.
90:583–608.
Macrophages are known to secrete collagenase,
secrete and store lysozyme, and release DNase,
RNase, and lactic dehydrogenase after they die.
We measured these enzymes in skin chambers
placed over developing and healing BCG lesions
(with the epidermis removed).The concentration
of all enzymes in the chamber fluids peaked
between 11 and 18 days—a time when CMI and
DTH produced the highest percentage of acti-
vated macrophages in the chamber beds.
EFFECT OF GLUCOCORTICOSTEROIDS
IN DEVELOPING AND HEALING BCG
LESIONS
51. McCue, R. E.,A. M. Dannenberg, Jr., S.
Higuchi, and M. Sugimoto. 1978. The
effect of cortisone on the accumulation,
activation, and necrosis of macrophages in
tuberculous lesions. Inflammation 3:159–176.
In rabbit dermal BCG lesions, cortisone reduced
the number of infiltrating mononuclear cells, the
406 䡵 APPENDIX D
amount of caseous necrosis and ulceration, and the
percentage of the macrophage population that
became activated (i.e., became positive for -
galactosidase).These effects readily explain why
glucocorticosteroids reduce host resistance to
tuberculosis.The effects of glucocorticosteroids on
the inflammatory response are reviewed in pub-
lication 85. (See chapter 16.)
EFFECTS OF BCG VACCINATION ON
THE PATHOGENESIS OF SYPHILIS
52. Hardy, P. H., Jr., D. J. Graham, E. E.
Nell, and A. M. Dannenberg, Jr. 1979.
Macrophages in immunity to syphilis: sup-
pressive effect of concurrent infection with
Mycobacterium bovis BCG on the develop-
ment of syphilitic lesions and growth of
Treponema pallidum in tuberculin-positive
rabbits. Infect. Immun. 26:751–763.
This report clearly demonstrates the nonspecific
nature of local acquired resistance (see chapter 5).
BCG was injected into intradermal sites where
Treponema pallidum had been injected a few min-
utes earlier. In rabbits sensitized by BCG 3 weeks
earlier, these mixed lesions showed more activated
macrophages and fewer treponemes.
EFFECTS OF THE IRRITANT SULFUR
DIOXIDE ON THE AIRWAYS OF
CHICKENS
53. Okuyama, H., Y. Majima, A. M. Dan-
nenberg, Jr., M. Suga, B. G. Bang, and
F. B. Bang. 1979. Quantitative histological
changes produced in the tracheal mucosa of
young chickens by the inhalation of sulfur
dioxide in low concentrations. J. Environ.
Sci. Health C13(4):267–300.
Following the inhalation of SO2, the mucosa of the
trachea was thickened and contained more infil-
trating leukocytes, more acid phosphatase-con-
taining mononuclear cells, and more cells secret-
ing sialidase-sensitive sialomucins (when compared
with controls).The change in the mucus compo-
sition was apparently a protective mechanism.
HETEROGENEITY OF MACROPHAGES
IN TUBERCULOUS LESIONS
54. Suga, M.,A. M. Dannenberg, Jr., and S.
Higuchi. 1980. Macrophage functional het-
erogeneity in vivo: macrolocal and microlocal
macrophage activation, identified by double-
staining tissue sections of BCG granulomas
for pairs of enzymes. Am. J. Pathol. 99:305–
324.
Many macrophages within tuberculous lesions
were activated for different functions, even though
they were next to each other.Why and how this
occurs are matters of conjecture.This report and
chapter 6 review the possibilities.
PERSISTENCE OF MYCOBACTERIAL
ANTIGENS IN TISSUES
55. Higuchi, S., M. Suga, A. M. Dannen-
berg, Jr., L. F. Affronti, I. Azuma,T. M.
Daniel, and J. P. Petrali. 1981. Persistence
of protein, carbohydrate and wax compo-
nents of tubercle bacilli in dermal BCG
lesions. Am. Rev. Respir. Dis. 123:397–401.
Antibodies were used to visualize these bacillary
components in tissue sections with the peroxi-
dase–antiperoxidase technique. These compo-
nents disappeared from the BCG lesions at dif-
ferent rates: protein B was undetectable at 21
days, polysaccharide I was undetectable at 35
days, but wax D could still be found at 56 days if
the lesions were not completely healed.Antisera
against intact tubercle bacilli produced results
similar to antibodies to wax D.
PURIFICATION AND PROPERTIES OF
RIBONUCLEASE FROM LUNG
56. Hastie, A.T. 1981. Monospecific antibod-
ies to rabbit lung ribonucleases. J. Biol. Chem.
256:12553–12560.
(From our laboratory.) RNases were purified
from rabbit lungs by chromatographic techniques
and antibodies to them made in goats. These
antibodies were then used to visualize the RNases
in macrophage suspensions and in tuberculous
lesions (see publication 45). The anti-RNase
and anti-RNase sera were obtained by immu-
noelectrophoresis and immunoabsorbent chro-
matography.These sera inhibited their respective
RNase activities.
HISTOLOGY AND HISTOCHEMISTRY
IN PLASTIC-EMBEDDED TISSUE
SECTIONS
57. Higuchi, S., M. Suga, A. M. Dannen-
berg, Jr., and B. H. Schofield. 1979. His-
tochemical demonstration of enzyme activ-
ities in plastic- and paraffin-embedded tissue
sections. Stain Technol. 54:5–12.
58. Namba, M.,A. M. Dannenberg, Jr., and
F. Tanaka. 1983. Improvement of the his-
tochemical demonstration of acid phos-
phatase, -galactosidase and nonspecific
esterase in glycol methacrylate tissue sec-
tions by cold temperature embedding. Stain
Technol. 58:207–213.
Polymerization of glycol methacrylate generates
heat equivalent to boiling.When polymerization

was performed in cracked ice, the structure of cells
in the 1- to 2-m tissue sections was markedly
improved. See publication 60.
59. Vogt, R. F., Jr., N.A. Hynes,A. M. Dan-
nenberg, Jr., S. Castracane, and L.Weiss.
1983. Improved techniques using Giemsa-
stained glycol methacrylate tissue sections to
quantitate basophils and other leukocytes in
inflammatory skin lesions. Stain Technol.
58:193–205.
The granules of basophils and mast cells are beau-
tifully preserved in these thin plastic-embedded
tissue sections (see publication 60)—so much so
that the amount of degranulation can be quan-
titated (see publication 67).
PATHOGENESIS AND SERUM
TURNOVER OF ACUTE DERMAL
INFLAMMATORY LESIONS
PRODUCED IN RABBITS BY
DILUTE SULFUR MUSTARD
60. Vogt, R. F., Jr., A. M. Dannenberg, Jr.,
B. H. Schofield, N. A. Hynes, and B.
Papirmeister. 1984. Pathogenesis of skin
lesions caused by sulfur mustard. Fund.Appl.
Toxicol. 4:S71–S83.
In rabbits, basophils constituted 32% of the gran-
ulocyte population that infiltrated (during the
first 2 h) skin lesions produced by dilute sulfur
mustard. In guinea pigs, basophils were 21% of this
population. In guinea pigs, eosinophils were as
common as basophils at that time, but eosinophils
cannot always be distinguished from heterophils
(PMN) in rabbits, because both are eosinophilic.
These results suggest that basophils and
eosinophils play an important role in the early
inflammation that follows mild or slowly devel-
oping epidermal injury. Basophils would release
histamine, and eosinophils would destroy the
released histamine. Light abrasion, UV light, oxa-
zolone, and sometimes mild heat produced a sim-
ilar early basophil infiltration.
The role of basophils in the initiation of inflam-
mation needs further study. The granules of
basophils are not well preserved in paraffin-
embedded tissue sections but are very well pre-
served and easily recognized in cold-embedded
glycol methacrylate sections stained with Giemsa.
61. Dannenberg, A.M., Jr., P. J. Pula, L.
Liu, S. Harada, F.Tanaka, R. F.Vogt, Jr.,
A. Kajiki, and K. Higuchi. 1985. Inflam-
matory mediators and modulators released in
organ culture from rabbit skin lesions pro-
duced in vivo by sulfur mustard. I. Quanti-
tative histopathology; PMN, basophil and
mononuclear cell survival; and unbound
APPENDIX D 䡵 407
(serum) protein content. Am. J. Pathol.
121:15–27.
These dermal lesions contained mostly mononu-
clear cells, because as soon as the epithelium died,
the PMN migrated in large numbers through
the tissues to form the crust, i.e., the scab.Within
the deep parts of the lesion, basophils and PMN
were about equal in number. Perhaps PMN are
attracted into the crust by the bacteria that
invaded the area after surface epithelium died.
The full-thickness rabbit skin explants survived
rather well in culture for at least 3 days. In fact,
the epithelium migrating under the crust in 3-,
6- and 10-day lesions continued to do so in cul-
ture. Publication 121 reviews this organ culture
series.
62. Harada, S., A. M. Dannenberg, Jr., A.
Kajiki, K. Higuchi, F. Tanaka, and P. J.
Pula. 1985. Inflammatory mediators and
modulators released in organ culture from
rabbit skin lesions produced in vivo by sul-
fur mustard. II. Evans blue dye experiments
that determined the rates of entry and
turnover of serum protein in developing
and healing lesions. Am. J. Pathol. 121:28–38.
One-day sulfur mustard lesions are quite ede-
matous due to extravasated serum. The serum
that was unbound, i.e., extractable into the cul-
ture fluids, had a turnover rate of once every 8 h.
In other words, the extravasated serum in ede-
matous lesions was not static. In 3- and 6-day
sulfur mustard lesions that were not grossly ede-
matous, the unbound serum had a turnover rate
of once every 35 h. In normal skin, the unbound
serum had a turnover rate of once every 80 h.
MEDIATORS AND MODULATORS OF
ACUTE DERMAL INFLAMMATORY
LESIONS PRODUCED IN RABBITS BY
DILUTE SULFUR MUSTARD
63. Harada, S.,A. M. Dannenberg, Jr., R. F.
Vogt, Jr., J. E. Myrick, F. Tanaka, L. C.
Redding, R. M. Merkhofer, P. J. Pula,
and A. L. Scott. 1987. Inflammatory medi-
ators and modulators released in organ
culture from rabbit skin lesions produced
in vivo by sulfur mustard. III. Electro-
phoretic protein fractions, trypsin-inhibitory
capacity, 1-proteinase inhibitor, and 1- and
2-macroglobulin proteinase inhibitors of
culture fluids and serum. Am. J. Pathol.
126:148–163.
In acute inflammatory reactions, the proteinase
inhibitors in serum are probably major factors in
reducing tissue damage from the proteinases of
408 䡵 APPENDIX D
extravasated serum, infiltrating leukocytes, and
activated fibroblasts.
64. Kajiki, A., K. Higuchi, M. Nakamura,
L. H. Liu, P. J. Pula, and A. M. Dan-
nenberg, Jr. 1988. Sources of extracellular
lysosomal enzymes released in organ cul-
ture by developing and healing inflammatory
lesions. J. Leukoc. Biol. 43:104–116.
We evaluated acid phosphatase, -glucuronidase,
-galactosidase, lysozyme, and lactic dehydroge-
nase in organ culture fluids from rabbit dermal
sulfur mustard lesions.The enzyme levels increased
as the lesions healed. Many fibroblasts in the heal-
ing lesions were shown histochemically to be
activated for acid phosphatase and -galactosidase.
These cells and the large number of dead PMN
in the crust seemed to be the source of the
increased enzyme levels in the culture fluids of
healing lesions.
65. Higuchi, K.,A. Kajiki, M. Nakamura, S.
Harada, P. J. Pula,A. L. Scott, and A. M.
Dannenberg, Jr. 1988. Proteases released in
organ culture by acute dermal inflamma-
tory lesions produced in vivo in rabbit skin
by sulfur mustard: hydrolysis of synthetic
peptide substrates for trypsin-like and chy-
motrypsin-like enzymes. Inflammation 12:
311–334.
Again, higher levels of these enzymes were found
in culture fluids from healing lesions.
66. Woessner, J. F., Jr., A. M. Dannenberg,
Jr., P. J. Pula, M. G. Selzer, C. L. Rup-
pert, K. Higuchi, A. Kajiki, M. Naka-
mura, N. M. Dahms, J. S. Kerr, and
G.W. Hart. 1990. Extracellular collagenase,
proteoglycanase and products of their activ-
ity, released in organ culture by intact dermal
inflammatory lesions produced by sulfur
mustard. J. Investig. Dermatol. 95:717–726.
Collagenase and proteoglycanase and products
of their hydrolytic activities (hydroxyproline-
containing peptides and glycosaminoglycans)
were measured in culture fluids as these lesions
developed and healed. The activity of these
enzymes was highest during healing when
remodeling of the connective tissue occurred.
At that time, there was a decrease in the extrava-
sation of serum with a concomitant decrease in
serum proteinase inhibitors (such as 1-proteinase
inhibitor and the -macroglobulins; see publica-
tion 63). Fibroblasts seemed to be a major source
of both collagenase and proteoglycanase.
67. Rikimaru, T., M. Nakamura, T. Yano,
G. Beck, G. S. Habicht, L. L. Rennie,
M. Widra, C. A. Hirshman, M. G.
Boulay, E.W. Spannhake, G. S. Lazarus,
P. J. Pula, and A. M. Dannenberg, Jr.
1991. Mediators, initiating the inflamma-
tory response, released in organ culture by
full-thickness human skin explants exposed
to the irritant, sulfur mustard. J. Investig. Der-
matol. 96:888–897.
Angiotensin-converting enzyme, trypsin-like and
chymotrypsin-like proteases, acid phosphatase,
-glucuronidase, -galactosidase, lysozyme,
deoxyribonuclease, ribonuclease, interleukin 1,
and lactic dehydrogenase were released in simi-
lar amounts (into the culture fluids) from sulfur
mustard-exposed full-thickness skin explants and
from controls. However, histamine, prostaglandin
E2, and plasminogen activators (which are medi-
ators of the early inflammatory response) were
increased in the culture fluids from the toxicant-
exposed explants.
The amount of mast-cell degranulation was
correlated with the amount of histamine released.
68. Tanaka, F., A. M. Dannenberg, Jr., K.
Higuchi, M. Nakamura, P. J. Pula,T. E.
Hugli, R. G. DiScipio, and D. L.
Kreutzer. 1997. Chemotactic factors are
continuously released by cultured intact
developing and healing skin lesions pro-
duced in rabbits by sulfur mustard. Inflam-
mation 21:251–267.
The culture fluids were chemotactic for both
PMN and mononuclear cells and contained
leukotriene B4, interleukin 8, and proteases pro-
ducing the complement fragment C5a. Fluids
from cultured (as well as frozen-and-thawed)
macrophages, granulocytes (PMN), and fibroblasts
were also chemotactic for both macrophages and
PMN.The chemokines MCP-1 and GRO were
probably also present; see publication 75.
Note: In vivo, dying cells release chemotaxins,
whereas living cells and dead cells do not (see
Majno, G., and I. Joris. 1996. Cells,Tissues, and
Disease: Principles of General Pathology, p. 390.
Blackwell Science, Cambridge, Mass.). Perhaps
frozen and thawed cells are similar to dying cells.
IN VITRO TOXICITY TO SKIN,
ASSESSED IN ORGAN CULTURE BY
PARANUCLEAR VACUOLIZATION IN
1- TO 2-␮m TISSUE SECTIONS AND BY
[14C]LEUCINE INCORPORATION
BY LIVE CELLS IN THE EXPLANT
69. Moore, K. G., B. H. Schofield,
K. Higuchi, A. Kajiki, K.-W. Au, P. J.
Pula, D. P. Bassett, and A. M. Dannen-
berg, Jr. 1986.Two sensitive in vitro mon-

itors of chemical toxicity to human and
animal skin (in short-term organ culture):
I. Paranuclear vacuolization in glycol meth-
acrylate tissue sections. II. Interference with
14
C-leucine incorporation. J. Toxicol. Cuta-
neous Ocular Toxicol. 5:285–302.
Two sensitive in vitro methods were developed to
determine the toxicity of chemicals applied to
full-thickness human, rabbit, and guinea pig skin
explants. In both methods, the toxicants were
topically applied to the outer surface of each
explant. Then, the explant was organ cultured
for 24 h in shallow petri dishes.
For the first method, cold-embedded plastic tis-
sue sections were prepared (see publication 58),
and the paranuclear vacuoles in the basal epider-
mal cells were counted microscopically.The num-
ber of these vacuoles increased with the toxicant
concentration. Electron microscopy suggested
that the vacuoles were a bleb in the cell’s nuclear
membrane.
For the second method, the explants were cul-
tured in the presence of [14C]leucine.The incor-
poration of the [14C]leucine into the proteins of
the explants decreased as the toxicant concentra-
tion increased.
Both methods indicate injury to the cells in the
explant.
Organ culture itself enables studies on the ini-
tiation of the inflammatory response by the res-
ident cells in the explant, which include epider-
mal cells, mast cells, vascular endothelial cells,
histiocytes, and fibroblasts.
70. Dannenberg,A. M., Jr., K. G. Moore, B.
H. Schofield, K. Higuchi, A. Kajiki, K.-
W.Au, P. J. Pula, and D. P. Bassett. 1987.
Two new in vitro methods for evaluating
toxicity to skin (employing short-term organ
culture): I. Paranuclear vacuolization, seen
in glycol methacrylate tissue sections; II.
Inhibition of 14C-leucine incorporation, p.
115–127. In A. M. Goldberg (ed.), Alternative
Methods in Toxicology, vol. 4 (Proceedings of
the 1986 Symposium of the Center for
Alternatives to Animal Testing). Mary Ann
Liebert, Inc., New York, N.Y.
This report is similar to publication 69.
71. Nakamura, M., T. Rikimaru, T. Yano,
K. G. Moore, P. J. Pula, B. H. Schofield,
and A. M. Dannenberg, Jr. 1990. Full-
thickness human skin explants for testing
the toxicity of topically applied chemicals.
J. Investig. Dermatol. 95:325–332.
Details of the paranuclear vacuolization test on
full-thickness human skin explants are described.
APPENDIX D 䡵 409
After 7 days of storage at 4°C, these explants
were still viable and could be used in this test.
Electron microscope studies suggest that the
paranuclear vacuoles were invaginations of
the cell’s plasma membrane.
STUDIES ON CONTACT
SENSITIVITY OF THE SKIN
72. Moore, K. G., and A. M. Dannenberg,
Jr. 1992. Antigen-specific IgG1-mediated
epidermal cell injury: a component of con-
tact hypersensitivity reactions in guinea pigs,
measurable in vitro in full-thickness skin
explants. J. Investig. Dermatol. 98:929–935.
Full-thickness skin explants were used with our
paranuclear vacuolization test (see publication
69) to measure contact sensitivity reactions to
dinitrochlorobenzene and oxazolone. Studies on
mast cell degranulation, microblistering, and
[14C]leucine incorporation were also made.The
intradermal injection of IgG1 from sensitized
guinea pigs could passively sensitize normal skin
explants to these contact sensitizers.
These studies support the “threshold hypoth-
esis” that chemical sensitizers must be applied in
slightly toxic or irritating concentrations to elicit
an antigen-specific reaction in the skin of a sen-
sitized host. Nonspecific irritants can lower the
threshold of antigen-specific irritants. Publica-
tion 121 reviews these studies.
Intradermal DTH reactions in cattle infected
with virulent Mycobacterium bovis showed a sim-
ilar threshold response to antigens, such as ESAT-
6. In this case, the lipopeptide Pam3CSK4 was used
as the nonspecific irritant (Whelan et al. 2003.
Infect. Immun. 71:6420–6425).
73. Moore, K. G., and A. M. Dannenberg,
Jr. 1993. Immediate and delayed (late-phase)
dermal contact sensitivity reactions in guinea
pigs: passive transfer by IgG1 antibodies, ini-
tiation by mast cell degranulation, and sup-
pression by soybean proteinase inhibitor. Int.
Arch.Allergy Immunol. 101:72–81.
This report describes the pathogenesis of imme-
diate and delayed (late-phase) dermal contact
sensitivity reactions. The immediate phase seems
to be due to mast cells releasing histamine and
other quick-acting mediators. The late phase
seems to be due to mast cells releasing cyto-
kines, including chemokines. The late phase has
some components of a cutaneous basophil hyper-
sensitivity reaction. Soybean proteinase inhibitor
(which inhibits mast cell degranulation) sup-
pressed both the immediate and delayed skin
reactivities mediated by antigen-specific IgG1.
410 䡵 APPENDIX D
HISTOCHEMISTRY OF HYDROGEN
PEROXIDE
74. Dannenberg,A. M., Jr., B. H. Schofield,
J. B. Rao,T.T. Dinh, K. Lee, M. Boulay,
Y. Abe, J.Tsuruta, and M. J. Steinbeck.
1994. Histochemical demonstration of
hydrogen peroxide production by leuko-
cytes in fixed-frozen tissue sections of inflam-
matory lesions. J. Leukoc. Biol. 56:436–443.
In lightly fixed frozen tissue sections of dermal
inflammatory reactions, PMN and eosinophils
still produced H2O2, which did not injure adja-
cent tissues.The H2O2 was demonstrated histo-
chemically by the diaminobenzidine reaction.
CYTOKINES AND VASCULAR
ADHESION MOLECULES IN
ACUTE AND CHRONIC
INFLAMMATORY LESIONS
Cytokines (including chemokines) are major
mediators of the inflammatory response.Vascular
adhesion molecules enable leukocytes to leave the
bloodstream and enter inflammatory lesions.
75. Tsuruta, J., K. Sugisaki, A. M. Dannen-
berg, Jr., T. Yoshimura, Y. Abe, and
P. Mounts. 1996. The cytokines NAP-1
(IL-8), MCP-1, IL-1 beta, and GRO in rab-
bit inflammatory skin lesions produced by
the chemical irritant sulfur mustard. Inflam-
mation 20:293–318.
In tissue sections of these lesions, the mRNAs of
these cytokines were hybridized with 35S-radio-
labeled antisense riboprobes and autoradiographed.
This technique demonstrated histochemically the
“factories” for cytokine production. In these tissue
sections, the distribution of cells producing
chemokines was correlated with the distribution
of cells that these chemokines are known to attract.
76. Abe, Y., K. Sugisaki, and A. M. Dan-
nenberg, Jr. 1996. Rabbit vascular endothe-
lial adhesion molecules: ELAM-1 is most
elevated in acute inflammation, whereas
VCAM-1 and ICAM-1 predominate in
chronic inflammation. J. Leukoc. Biol. 60:692–
703.
Summarized in chapter 21.
77. Sugisaki, K., A. M. Dannenberg, Jr.,
Y. Abe, J. Tsuruta, W.-J. Su, W. Said,
L. Feng, T. Yoshimura, P. J. Converse,
and P. Mounts. 1998. Nonspecific and
immune-specific up-regulation of cytokines
in rabbit dermal tuberculous (BCG) lesions.
J. Leukoc. Biol. 63:440–450.
Cytokine production in dermal BCG lesions is
biphasic: an early adjuvant-type response within
2 days followed by an immune-specific response
at 9 days. See chapter 19.
ROLE OF ANTIBODIES,
LYMPHOCYTES, CYTOKINES,
AND VASCULAR ADHESION
MOLECULES IN TUBERCULOUS
LESIONS OF REINFECTION
78. Shigenaga, T., A. M. Dannenberg, Jr.,
D. B. Lowrie, W. Said, M. J. Urist,
H. Abbey, B. H. Schofield, P. Mounts,
and K. Sugisaki. 2001. Immune responses
in tuberculosis: antibodies and CD4-CD8
lymphocytes with vascular adhesion mole-
cules and cytokines (chemokines) cause a
rapid antigen-specific cell infiltration at sites
of Bacillus Calmette-Guérin reinfection.
Immunology 102:466–479.
This report clearly documents that antibodies play
an important role in hosts already possessing
acquired resistance to the tubercle bacillus: antigen-
antibody reactions produce chemotactic factors
that rapidly bring the expanded antigen-specific
lymphocyte population to sites of bacillary lodge-
ment.Therefore, antibodies increase the effective-
ness of CMI and DTH by hastening the local
accumulation of cells that produce the antigen-spe-
cific Th1 response. The rapidity of the cellular
response to the bacilli of reinfection is a major fac-
tor in the ability of an immunized host to prevent
clinically apparent disease. See chapter 20.
After sufficient cells had accumulated at sites of
bacillary deposition, the chemokines that attracted
them were downregulated. In other words, the
number of cells in an inflammatory site is evi-
dently under tight control.
PATHOGENESIS OF PULMONARY
TUBERCULOSIS, PRODUCED BY
INHALED BACILLI
These two reports seem to be the first in over 30
years to produce chronic progressive cavitary
tuberculosis in rabbits by the aerosol method.
Chapter 4 presents the details.
79. Converse, P. J., A. M. Dannenberg, Jr.,
J. E. Estep, K. Sugisaki, Y. Abe, B. H.
Schofield, and M. L. M. Pitt. 1996. Cav-
itary tuberculosis produced in rabbits by
aerosolized virulent tubercle bacilli. Infect.
Immun. 64:4776–4787.
80. Converse, P. J., A. M. Dannenberg, Jr.,
T. Shigenaga, D. N. McMurray, S. W.
Phalen, J. L. Stanford, G. A. W. Rook,

T. Koru-Sengul, H. Abbey, J. E. Estep,
and M. L. M. Pitt. 1998. Pulmonary
bovine-type tuberculosis in rabbits: bacillary
virulence, inhaled dose effects, tuberculin
sensitivity, and Mycobacterium vaccae immuno-
therapy. Clin. Diagn. Lab. Immunol. 5:871–881.
This report analyzes more deeply the data pre-
sented in publication 79.We could not detect any
effect of Mycobacterium vaccae immunotherapy,
because the disease varied too much between
individual rabbits.
BACILLARY VIRULENCE AND VACCINE
EFFICACY EVALUATED IN RABBITS BY
LURIE’S TUBERCLE-COUNT METHOD
81. Dannenberg, A. M., Jr. 1998. Lurie’s
tubercle-count method to test TB vaccine
efficiency in rabbits. Front. Biosci. 3:c27–33.
http://www.bioscience.org/1998/v3/c/
dannenbe/list.htm.
This article presents the details on the tubercle-
count method and its use in assessing bacillary
virulence, host genetic resistance, and vaccine
efficacy. Since its publication, we have gained
further insight into this method’s applicability to
other laboratory species. See chapter 11.
Major insights into primary tuberculous lesions
in human beings came from Lindgren’s necropsy
studies (see chapter 3). In Finland at a time when
infection with Mycobacterium tuberculosis was com-
mon, he found with a special roentgenographic
technique that every human who became tuber-
culin positive from a natural infection with M.
tuberculosis had at least a minute grossly visible cal-
cified primary pulmonary tuberculous lesion at
death, even though death usually occurred years
later from other causes. BCG immunization did
not reduce the number of such lesions found,
but did reduce lesion size and lymph node
involvement.
Humans infected with virulent tubercle bacilli
develop high tuberculin sensitivity. Therefore,
each primary tubercle develops a caseous center
that absorbs calcium and becomes grossly visible
before its progress is arrested.
In mice and guinea pigs, each microscopic pri-
mary pulmonary lesion usually progresses until the
animal dies. In these animals, because of their high
susceptibility, immunization with BCG would
probably not reduce the number of grossly visible
primary lesions, but would reduce the progression
of the lesions and increase the survival time of the
host. Because of these considerations, the tubercle-
count method is best used in rabbits, a species that
is more resistant than mice and guinea pigs to vir-
ulent human-type tubercle bacilli. See chapters 11
and 15.
APPENDIX D 䡵 411
82. Bishai, W. R., A. M. Dannenberg, Jr.,
N. Parrish, R. Ruiz, P. Chen, B. C.
Zook,W. Johnson, J.W. Boles, and M. L.
M. Pitt. 1999.Virulence of Mycobacterium
tuberculosis CDC1551 and H37Rv in rabbits
evaluated by Lurie’s pulmonary tubercle
count method. Infect. Immun. 67:4931–4934.
See chapter 11 and publication 128.
83. Dannenberg, A. M., Jr.,W. R. Bishai, N.
Parrish, R. Ruiz,W. Johnson, B. C. Zook,
J.W. Boles, and M. L. M. Pitt. 2000. Effi-
cacies of BCG and vole bacillus (Mycobac-
terium microti) vaccines in preventing clini-
cally apparent pulmonary tuberculosis in
rabbits: a preliminary report. Vaccine 19:796–
800.
Reviewed in chapter 23.
PART II. REVIEW ARTICLES
ENVIRONMENTAL EFFECTS
ON THE LUNG
84. Dannenberg,A. M., Jr. 1977. Influence of
environmental factors on the respiratory
tract. Summary and perspectives. J. Reticu-
loendothel. Soc. 22:273–290.
This review briefly describes the effects of inhaled
environmental and industrial substances on the
airways and peripheral lung, including clearance
mechanisms, carcinogenesis, asbestosis, silicosis,
and allergic pulmonary reactions.
EFFECTS OF
GLUCOCORTICOSTEROIDS ON
INFLAMMATION
85. Dannenberg, A. M., Jr. 1979. The anti-
inflammatory effects of glucocorticosteroids:
a brief review of the literature. Inflammation
3:329–343.
This article describes the effects of glucocorti-
costeroids on (i) infections, (ii) lymphocytes,
macrophages, and other leukocytes, (iii) cell recep-
tors and transduction pathways, (iv) host metab-
olism, (v) cell membranes, (vi) connective tissue,
and (vii) inflammation.To obtain a more complete
orientation in this field, consult the two classic
articles by Henry N. Claman in Hospital Practice
(18:123–134 and 143–151, 1983).
MACROPHAGES AND THEIR
FUNCTIONS
86. Dannenberg, A. M., Jr., M. Ando,
O. Rojas-Espinosa, K. Shima, and
T. Tsuda. 1974. Macrophage activation in
412 䡵 APPENDIX D
tuberculous lesions, p. 223–235. In W. H.
Wagner and H. Hahn (ed.), Activation of
Macrophages. Excerpta Medica International
Congress Series 325. Excerpta Medica,
Amsterdam,The Netherlands.
87. Dannenberg, A. M., Jr., M. Ando,
K. Shima, and T. Tsuda. 1975.
Macrophage turnover and activation in
tuberculous granulomata, p. 959–980. In
R. van Furth (ed.), Mononuclear Phagocytes in
Immunity, Infection and Pathology. Blackwell
Scientific Publications, Oxford, U.K.
88. Dannenberg, A. M., Jr. 1975. Macro-
phages in inflammation and infection.
N. Engl. J. Med. 293:489–493.
89. Dannenberg,A. M., Jr., and S. Higuchi.
1979. Chronic inflammation involving cel-
lular hypersensitivity. Chest 75(Suppl. 2):265–
266.
90. Dannenberg, A. M., Jr. 1980. Macro-
phages and monocytes, p. 1–17. In J.A. Spit-
tell, Jr. (ed.), Clinical Medicine, vol. 5. Harper
& Row, Publishers, Inc., New York, N.Y.
See publication 94 for the update in the next
edition.
91. Dannenberg, A. M., Jr. 1980. Macro-
phages and monocytes, p. 137–153. In J. L.
Spivak (ed.), Fundamentals of Clinical Hema-
tology. Harper & Row, Publishers, Inc., New
York, N.Y.
Republication of publication 90. See publication
95 for the update in the next edition.
92. Dannenberg, A. M., Jr., and M. Suga.
1981. Histochemical stains for macro-
phages in cell smears and tissue sections:
-galactosidase, acid phosphatase, nonspe-
cific esterase, succinic dehydrogenase, and
cytochrome oxidase, p. 375–396. In D. O.
Adams, P. J. Edelson, and H. S. Koren (ed.),
Methods for Studying Mononuclear Phagocytes.
Academic Press, Inc., New York, N.Y.
This article describes the methodology we used.
93. Dannenberg, A. M., Jr., M. Suga, and
J. E. Garcia-Gonzalez. 1981. Macrophages
in granulomas: histochemical evidence sug-
gesting local control of heterogeneous func-
tions. Haematol. Blood Transfusion 27:109–119.
This is a concise review of various macrophage
functions.
94. Dannenberg, A. M., Jr. 1983. Macro-
phages and monocytes, p. 1–21. In J.A. Spit-
tell, Jr. (ed.), Clinical Medicine, vol. 5 (revised
ed.). Harper & Row, Publishers, Philadelphia.
This article reviews many aspects of macrophage
function, including (i) chemotaxis, (ii) phagocy-
tosis, (iii) their origin, life span, differentiation, and
turnover, (iv) their heterogeneity, (v) their secre-
tory and microbicidal functions, and (vi) their
roles in bacterial and viral infections, neoplasia,
and endotoxin inactivation. This review is an
update of publication 90.
95. Dannenberg, A. M., Jr. 1984. Macro-
phages and monocytes, p. 153–173. In J. L.
Spivak (ed.), Fundamentals of Clinical Hema-
tology, 2nd ed. Harper & Row, Publishers,
Inc., New York, N.Y.
This review is a republication of publication 94.
PATHOGENESIS OF TUBERCULOSIS
96. Dannenberg, A. M., Jr. 1968. Cellular
hypersensitivity and cellular immunity in
the pathogenesis of tuberculosis: specificity,
systemic and local nature, and associated
macrophage enzymes. Bacteriol. Rev. 32:85–
102.
This review puts local and systemic immunity to
tuberculosis in their proper perspective. See
chapter 5.
97. Dannenberg, A. M., Jr., and M. Sugi-
moto. 1976. Liquefaction of caseous foci in
tuberculosis. Editorial. Am. Rev. Respir. Dis.
113:257–259.
This review briefly describes what little is known
about the causes of liquefaction and cavity for-
mation, including some of our own unpublished
experiments. It is summarized in chapter 4.
98. Dannenberg, A. M., Jr. 1978. Pathogen-
esis of pulmonary tuberculosis in man and
animals; protection of personnel against
tuberculosis, p. 65–75. In J. L. Montali (ed.),
Mycobacterial Infections of Zoo Animals. Smith-
sonian Institution Press,Washington, D.C.
This review compares the disease caused by var-
ious mycobacteria in different animal species,
including birds.Also see publication 103.
99. Dannenberg, A. M., Jr. 1980. Pathogene-
sis of tuberculosis, p. 1264–1281. In A. P. Fish-
man (ed.), Pulmonary Diseases and Disorders.
McGraw-Hill Book Co., New York, N.Y.
This is the first edition of this textbook. See pub-
lications 105 and 115 for updated versions.
100. Dannenberg, A. M., Jr., M. Suga, and
J. E. Garcia-Gonzalez. 1980. Cellular

composition of the tuberculous (BCG) gran-
uloma: local differentiation and turnover of
macrophages, p. 21–32. In D. L. Boros and
T.Yoshida (ed.), Basic and Clinical Aspects of
Granulomatous Diseases. Elsevier/North-
Holland, Inc.,Amsterdam,The Netherlands.
See chapters 5 and 10.
101. Dannenberg, A. M., Jr. 1982. Pathogen-
esis of pulmonary tuberculosis. Am. Rev.
Respir. Dis. 125(no. 3 Suppl.):25–29.
Koch Centennial Issue (by invitation).
102. Dannenberg, A. M., Jr. 1982. Pathogen-
esis of pulmonary tuberculosis: basic princi-
ples. Indian J. Chest Dis. 24:68–77.
Koch Centennial Issue.
103. Dannenberg, A. M., Jr. 1984. Pathogen-
esis of tuberculosis: native and acquired resis-
tance in animals and humans, p. 344–354. In
Microbiology—1984. American Society for
Microbiology,Washington, D.C.
This review is similar to publication 98. It repro-
duces (with slight modifications) Francis’s table
that compares the characteristics of tuberculosis
in 23 different animal species. (Francis, J. 1958.
Tuberculosis in Animals and Man.A Study in Com-
parative Pathology, p. 294. Cassell and Co., Ltd.,
London, U.K.).
104. Dannenberg, A.M., Jr. 1984. Chemical
and enzymatic host factors in resistance to
tuberculosis, p. 721–760. In G. P. Kubica and
L. G.Wayne (ed.), The Mycobacteria: a Source-
book. Marcel Dekker, Inc., New York, N.Y.
Our chapter (with 290 references) reviews our
understanding of the pathogenesis of tuberculo-
sis, with special emphasis on macrophage func-
tions and their enzymes.
The Kubica and Wayne book is an excellent
source of information on mycobacteria up to
the time of its publication.
105. Dannenberg, A. M., Jr., and J. F.
Tomashefski, Jr. 1988. Pathogenesis of pul-
monary tuberculosis, p. 1821–1842. In A. P.
Fishman (ed.), Pulmonary Diseases and Disor-
ders, 2nd ed., vol. 3. McGraw-Hill Book Co.,
New York, N.Y.
See publication 115 for the updated version of this
chapter in the next edition.
106. Dannenberg, A. M., Jr. 1989. Immune
mechanisms in the pathogenesis of pul-
monary tuberculosis. Rev. Infect. Dis.
11(Suppl. 2):S369–S378.
APPENDIX D 䡵 413
107. Dannenberg,A. M., Jr. 1990. Controlling
tuberculosis: the pathologist’s point of view.
Fifth Forum in Microbiology on “Killing
Intracellular Mycobacteria: Dogmas and
Realities.” Res. Microbiol. 141:192–196,
262–263.
Here, we describe some of the areas in patho-
genesis of tuberculosis that need further research.
See chapter 25 for an update.
108. Dannenberg, A. M., Jr. 1991. Delayed-
type hypersensitivity and cell-mediated
immunity in the pathogenesis of tuberculo-
sis. Immunol.Today 12:228–233.
This report describes for the first time the ben-
eficial role of caseation in tuberculosis. It explains
why DTH developed throughout mammalian
evolution, despite the tissue damage it produces.
Included are photomicrographs of various
stages in the pathogenesis of this disease. Publi-
cation 109 contains our revised numbering sys-
tem for these stages.
109. Dannenberg, A. M., Jr. 1993. Immuno-
pathogenesis of pulmonary tuberculosis.
Hosp. Pract. 28:51–58.
This review was written for clinicians. It contains
innovative drawings of the various stages in the
pathogenesis of tuberculosis in a revised num-
bering sequence. We should have used “tissue-
damaging DTH” or “cytotoxic DTH” in many
places of the report, but it was too late when this
was called to our attention. See publication 112
and chapter 2.
DTH is not damaging when the local con-
centration of tuberculin is low. DTH damages tis-
sues only when the tuberculin concentration is
high, e.g., when many tubercle bacilli are rapidly
multiplying intracellularly within macrophages.
By killing such macrophages, bacillary multipli-
cation is stopped, because tubercle bacilli do not
multiply in the solid caseous tissue that results.
110. Dannenberg, A. M., Jr. 1994. Pathogen-
esis and immunology: basic aspects, p. 17–39.
In D. Schlossberg (ed.), Tuberculosis, 3rd ed.
Springer-Verlag, New York, N.Y.
This is our first chapter in Schlossberg’s Tubercu-
losis. It was updated in the next two editions,
publications 116 and 120.
111. Dannenberg, A. M., Jr. 1994. Rabbit
model of tuberculosis, p. 149–156. In B. R.
Bloom (ed.), Tuberculosis: Pathogenesis, Protec-
tion, and Control. ASM Press, Washington,
D.C.
Here, we reviewed the pathology of the disease
produced in rabbits by virulent bovine-type
414 䡵 APPENDIX D
tubercle bacilli, by virulent human-type tubercle
bacilli, and by BCG. It is similar to publication 9.
See chapter 13.
112. Dannenberg, A. M., Jr., and G. A. W.
Rook. 1994. Pathogenesis of pulmonary
tuberculosis: an interplay of tissue-damaging
and macrophage-activating immune
responses—dual mechanisms that control
bacillary multiplication, p. 459–483. In
B. R. Bloom (ed.), Tuberculosis: Pathogenesis,
Protection, and Control. ASM Press,Washing-
ton, D.C.
Our review presents the five stages of pulmonary
tuberculosis with cartoons (see chapter 2) as well
as with photographs.
Bloom’s Tuberculosis was written by numerous
authorities in the field. It is one of the most com-
plete books on this subject in existence. It cov-
ers tuberculosis history, epidemiology, diagnosis,
pathogenesis, immunology, prevention, and treat-
ment, as well as animal models, the cultivation of
the bacillus, mycobacterial physiology and genet-
ics, and laboratory safety. Cole et al. (Cole, S.T.,
K. D. Eisenach, D. N. McMurray, W. R.
Jacobs, Jr. [ed.]. 2005. Tuberculosis and the Tuber-
cle Bacillus.ASM Press,Washington, D.C.) update
a lot of it.
113. Dannenberg, A. M., Jr. 1994. Roles of
cytotoxic delayed-type hypersensitivity and
macrophage-activating cell-mediated immu-
nity in the pathogenesis of tuberculosis.
Immunobiology 191:461–473.
114. McMurray, D. N., F. M. Collins, A. M.
Dannenberg, Jr., and D.W. Smith. 1996.
Pathogenesis of experimental tuberculosis
in animal models. Curr. Top. Microbiol.
Immunol. 215:157–179.
I wrote the section on rabbit tuberculosis.
115. Dannenberg, A. M., Jr., and J. F.
Tomashefski, Jr. 1998. Pathogenesis of pul-
monary tuberculosis, p. 2447–2471. In A. P.
Fishman (ed.), Fishman’s Pulmonary Diseases
and Disorders, 3rd ed., vol. 2. McGraw-Hill
Co., Inc., New York, N.Y.
Our chapter in this edition was updated and con-
tains additional references.
Our chapters in previous editions are publica-
tions 99 and 105.
116. Dannenberg, A. M., Jr. 1999. Pathophys-
iology: basic aspects. I. Pathogenesis of tuber-
culosis. II. Immunology of tuberculosis, p.
17–47. In D. Schlossberg (ed.), Tuberculosis and
Nontuberculous Mycobacterial Infections, 4th ed.
The W. B. Saunders Co., Philadelphia, Pa.
This is a rather complete review of the patho-
genesis and immunology of tuberculosis. It was
updated for the next edition. See publication 120.
117. Dannenberg, A. M., Jr. 2001. Pathogen-
esis of pulmonary Mycobacterium bovis infec-
tion: basic principles established by the rab-
bit model. Presented at Third International
Conference on Mycobacterium bovis, Cam-
bridge, U.K., July 13–16, 2000. Tuberculosis
81:87–96.
This review is a succinct summary of the patho-
genesis of tuberculosis.
118. Dannenberg, A. M., Jr., and F. M.
Collins. 2001. Progressive pulmonary tuber-
culosis is not due to increasing numbers of
viable bacilli in rabbits, mice and guinea
pigs, but is due to a continuous host response
to mycobacterial products. Tuberculosis
(Edinb.) 81:229–242.
This review compares the disease in rabbits, mice,
guinea pigs, and monkeys. See chapter 15.
119. Dannenberg,A. M., Jr. 2003. Macrophage
turnover, division and activation within
developing, peak and “healed” tuberculous
lesions produced in rabbits by BCG. Tuber-
culosis (Edinb.). 83:251–260.
This review gathers in one place our many reports
on macrophage kinetics in tuberculous lesions
(produced by BCG) and offers additional inter-
pretations of the data. See publications 34 to 41.
Chapter 10 is essentially the same.
In brief, nonactivated monocytes/macrophages
still enter arrested “healed” lesions and provide an
opportunity for renewed intracellular bacillary
growth. Such growth is then stopped by the
immune forces of the host.Tubercle bacilli may
only be truly dormant in solid caseous tissue, but
they are probably not fully dormant in the tuber-
culous granulation tissue that surrounds this
caseum.These studies explain why immunosup-
pression, such as in HIV/AIDS, often activates a
formerly well-controlled and inapparent tuber-
culous lesion.
120. Manabe,Y. C., and A. M. Dannenberg,
Jr. 2006. Pathophysiology: basic aspects. Part
I. Pathogenesis of tuberculosis. Part II.
Immunology of tuberculosis, p. 18–51. In
D. Schlossberg (ed.), Tuberculosis and Nontu-
berculous Mycobacterial Infections, 5th ed.,
McGraw-Hill, New York, N.Y.
Our chapter in publications 110 and 116 was
updated again. Much of the recent understand-
ing about the immunology of tuberculosis appears
in chapters 5 and 6 in the present volume.

OUR STUDIES ON IRRITANT
INFLAMMATION AND CONTACT
SENSITIVITY
121. Dannenberg,A. M., Jr., and K. G. Moore.
1994.Toxic and allergic skin reactions, evalu-
ated in organ-cultured full-thickness human
and animal skin explants, p. 351–366. In A.
Rougier,A. M. Goldberg, and H. I. Maibach
(ed.),In Vitro Skin Toxicology (Alternative Meth-
ods in Toxicology Series, vol. 10). Mary Ann
Liebert, Inc, New York, N.Y.
This review briefly summarizes most of these
studies and includes the pertinent literature on
(i) dermal inflammatory reactions produced by
the chemical toxicant dilute sulfur mustard, and
(ii) dermal contact sensitivity reactions produced
by dinitrochlorobenzene and oxazolone. See pub-
lications 60 through 73.
In many of these studies, full-thickness skin
explants of inflammatory lesions in various stages
of development and healing were organ cultured,
and the locally produced inflammatory mediators
(extracted by the culture fluids) were correlated
with the histological changes found in the lesion.
In this review, unfortunately, some section head-
ings were not printed correctly. Specifically, the
summaries of parts 4 and 5 were not so designated.
PART III. COLLABORATIVE
PROJECTS MAINLY FROM
OTHER LABORATORIES
122. Gertner, A., B. Bromberger-Barnea,
A. M. Dannenberg, Jr., R. Traystman,
and H. Menkes. 1983. Responses of the
lung periphery to 1.0 ppm ozone. J. Appl.
Physiol. 55:770–776.
123. Emmett, E. A., P. G. Lewis, F. Tanaka,
M. Bleecker, R. Fox, A. C. Darlington,
D. R. Synkowski,A. M. Dannenberg, Jr.,
W. J. Taylor, and M. S. Levine. 1985.
Industrial exposure to organophosphorus
compounds: studies of a group of workers
with a decrease in esterase-staining mono-
cytes. Occup. Med. 27:905–914.
124. Kleeberger, S. R., E. M. Wagner, G. K.
Adams III, A. M. Dannenberg, Jr., and
E.W. Spannhake. 1985. Effect of repeated
antigen exposure on antigen- and mediator-
induced bronchospasm in sheep. J.Appl. Phys-
iol. 59:1866–1873.
125. Berzon, D. M., H. Menkes, A. M. Dan-
nenberg, Jr., A. Gertner, P. Terry,
D. Plump, and B. Bromberger-Barnea.
APPENDIX D 䡵 415
1986. Interstitial fibrosis and collateral ven-
tilation. J.Appl. Physiol. 61:300–303.
126. Feldman, G. M., A. M. Dannenberg,
Jr., and J. L. Seed. 1990. Physiologic oxy-
gen tensions limit oxidant-mediated killing
of schistosome eggs by inflammatory cells
and isolated granulomas. J. Leukoc. Biol.
47:344–354.
127. Manabe, Y. C., A. M. Dannenberg, Jr.,
and W. R. Bishai. 2000.What we can learn
from the Mycobacterium tuberculosis genome
sequencing projects. Int. J.Tuberc. Lung Dis.
4:S18–S23.
The complete genome of M. tuberculosis (H37Rv)
is now known. It is being used to identify viru-
lence factors for the development of new drugs
and new vaccines.
128. Manabe,Y. C.,A. M. Dannenberg, Jr., S.
K. Tyagi, C. L. Hatem, M.Yoder, S. C.
Woolwine, B. C. Zook, M. L. M. Pitt,
and W. R. Bishai. 2003. Different strains of
Mycobacterium tuberculosis cause various spec-
trums of disease in the rabbit model of tuber-
culosis. Infect. Immun. 71:6004–6011.
With the tubercle-count method, Erdman,
H37Rv, and CDC1551 (Oshkosh strain) had
decreasing virulence for rabbits, in that order.
129. Dorman, S., C. L. Hatem, S. Tyagi, K.
Aird, J. Lopez-Molina, M. L. M. Pitt, B.
C. Zook, A. M. Dannenberg, Jr., W. R.
Bishai, and Y. C. Manabe. 2004. Suscep-
tibility to tuberculosis: clues from studies
with inbred and outbred New Zealand
White rabbits. Infect. Immun. 72:1700–1705.
The Thorbecke inbred New Zealand White rab-
bits were more susceptible to tuberculosis than
commercial outbred rabbits. Following the inhala-
tion of virulent human-type tubercle bacilli
(H37Rv), the inbred rabbits produced 3 times as
many grossly visible tubercles, and these tubercles
were larger, showed more caseous necrosis, and
contained more bacilli.The tubercle counts in the
inbred rabbits also showed less variation. The
inbred rabbits had reduced tuberculin sensitivity,
and their macrophages produced decreased
amounts of tumor necrosis factor alpha (a primary
cytokine).
These were the only inbred rabbits that were
commercially available in 2003, but unfortunately
(because of a fire), they were no longer available
in 2006 (see chapter 14).
The photographs of the granulomas in Fig. 3
in this publication were inadvertently published
vertically instead of horizontally.They should be
416 䡵 APPENDIX D
rotated clockwise and counterclockwise (respec-
tively) to make the figure descriptions match
what is portrayed.
130. Kesavan,A. K., S. Mendez, C. L. Hatem,
J. Lopez-Molina, K.Aird, M. L. M. Pitt,
A. M. Dannenberg, Jr., and Y. C. Man-
abe. 2005. Effects of dexamethasone and
transient malnutrition on rabbits infected
with aerosolized M. tuberculosis CDC1551.
Infect. Immun. 73:7056–7060.
Transient malnutrition decreased the resistance of
these rabbits in a manner similar to that of dex-
amethasone.
131. Manabe,Y. C., A. K. Kesavan, J. Lopez-
Molina, C. L. Hatem, M. Brooks,
R. Fujiwara, K. Hochstein, M. L. M.
Pitt, D. N. McMurray, J.-A. Tufariello,
J. Chan, A. M. Dannenberg, Jr., W. R.
Bishai, and S. Mendez. Mycobacterial
gene expression in the resistant rabbit model
of latency, reactivation and immune recon-
stitution tuberculosis. In preparation.
Rabbits were aerosol infected with M. tuberculosis
(H37Rv). From 10 to 15 weeks after infection,
they were given dexamethasone.Then, the dex-
amethasone was discontinued, and the rabbits
were euthanized 5 weeks later. This regimen
reproduced (i) the paucibacillary stage of this dis-
ease, (ii) the latent stage, and (iii) the early stage of
reactivation, which are commonly found in
humans. Some of the mycobacterial genes that are
upregulated in each of these stages were identified.
 APPENDIX E
GUIDELINES FOR PREVENTING THE
TRANSMISSION OF MYCOBACTERIUM
TUBERCULOSIS IN HEALTH-CARE SETTINGS†
Department of Health and Human Services, Centers for
Disease Control and Prevention,Atlanta, Georgia

CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
HCWs Who Should Be Included in a TB Surveillance Program . . . . . . . . . 3
Risk for Health-Care-Associated Transmission of M. tuberculosis . . . . . . . . . . 6
Fundamentals of TB Infection Control . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Relevance to Biologic Terrorism Preparedness . . . . . . . . . . . . . . . . . . . . . . 8
Recommendations for Preventing Transmission of M. tuberculosis in
Health-Care Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
TB Infection-Control Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
TB Risk Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Risk Classification Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Managing Patients Who Have Suspected or Confirmed TB Disease:
General Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Managing Patients Who Have Suspected or Confirmed Disease:
Considerations for Special Circumstances and Settings . . . . . . . . . . . 19
Training and Educating HCWs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
TB Infection-Control Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Problem Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Collaboration with the Local or State Health Department . . . . . . . . . . . . 36
Environmental Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Respiratory Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Cough-Inducing and Aerosol-Generating Procedures . . . . . . . . . . . . . . . . 40

†
From Recommendations and Reports, Morb. Mortal.Wkly. Rep. 54(RR-17):1-141, December
30, 2005. Available on the Internet at http://www.cdc.gov/mmwr/PDF/rr/rr5417.pdf or
http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5417a1.htm.

417
418 䡵 APPENDIX E

Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Estimating the Infectiousness of a TB Patient . . . . . . . . . . . . . . . . . . . . . . 42
Diagnostic Procedures for LTBI and TB Disease . . . . . . . . . . . . . . . . . . . . 44
Treatment Procedures for LTBI and TB Disease . . . . . . . . . . . . . . . . . . . . 53
Surveillance and Detection of M. tuberculosis Infections in
Health-Care Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Environmental Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Respiratory Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Cleaning, Disinfecting, and Sterilizing Patient-Care Equipment
and Rooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Frequently Asked Questions (FAQs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Terms and Abbreviations Used in This Report . . . . . . . . . . . . . . . . . . . . . . 103
Glossary of Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Continuing Education Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CE-1
 APPENDIX F
COLLECTED ABSTRACTS IN THIS VOLUME
SECTION 1. PATHOGENESIS
OF TUBERCULOSIS
CHAPTER 1. OVERVIEW
Tuberculosis is still one of the major diseases of the
world, especially in developing countries. It kills
over 2 million people each year, more than any
other infectious disease.
Childhood-type pulmonary tuberculosis is a
disease of susceptible hosts, such as infants and
immunosuppressed individuals. From the primary
parenchymal lesion, the bacilli frequently spread via
lymphatics and cause caseous lesions of the hilar
lymph nodes.The bacilli may also spread via the
bloodstream and cause lesions elsewhere in the
host.The primary lesion, as well as the metastatic
lesions, often progresses until the host succumbs.
Adult-type pulmonary tuberculosis is a disease
of innately resistant hosts, a category that includes
most immunocompetent persons. The active
parenchymal lesion (often subapical) frequently
forms a cavity, in which the bacilli may multiply
extracellularly. If so, the bacilli may spread via the
bronchial tree to other parts of the lung. In such
a resistant host, many metastatic microscopic lesions
do not progress, but those that do may form new
cavities. Also, caseous bronchopneumonia may
occur when an appreciable amount of liquefied
caseum enters the bronchial tree. Cavity formation
perpetuates tuberculosis in humankind because
coughing spreads bacilli from the lungs into the
environment, where they may infect other people.
Contracting clinical tuberculosis depends on
(i) the size and physiological state of the bacillary
particle, (ii) its virulence, and (iii) the native and
acquired resistance of the host.
How to protect personnel against tuberculosis is
also discussed in this chapter. UV lights (shielded
to protect people’s eyes) or HEPA-filtered air puri-
fiers should be used more frequently in hospital
areas where tubercle bacilli are likely to be present.
CHAPTER 2. STAGES IN THE
PATHOGENESIS OF HUMAN
AND RABBIT TUBERCULOSIS
After the inhalation of tubercle bacilli by rabbits
and humans, the disease may progress through the
419
following stages. In humans, the disease begins
with the establishment of only a single primary
pulmonary tubercle.
Stage 1: Ingestion and often destruction of
bacilli by pulmonary alveolar macrophages.
Stage 2: Logarithmic growth of bacilli within
nonactivated macrophages that entered the devel-
oping tubercle from the bloodstream.
Stage 3: Arrest of the logarithmic bacillary
growth by delayed-type hypersensitivity, which
kills the bacilli-laden macrophages and often forms
a solid caseous center in the tubercle.
Stage 4a: In hosts with weakly developed cell-
mediated immunity, enlargement of the tubercle
and its caseous center with hematogenous dis-
semination of the bacilli.
Stage 4b: In hosts with strongly developed cell-
mediated immunity, stabilization or regression of
the tubercle.
Stage 5: Liquefaction of the caseous center,
extracellular bacillary growth, cavity formation, and
bronchial dissemination of the bacilli.
These stages are not distinct but blend into
each other.Also, stages 3, 4, and 5 may occur in the
same lung and even in different parts of the same
lesion, depending on the local concentration of
bacilli and their tuberculin-like products. Native
and acquired resistance is never absolute, because
a large number of tubercle bacilli (which have
grown extracellularly in a cavity) can overwhelm
even the best-developed host resistance and cause
secondary pulmonary lesions.
CHAPTER 3. TYPES OF HUMAN
PULMONARY TUBERCULOSIS
Human tuberculosis most frequently occurs as a
tiny inapparent lesion that stays dormant through-
out the life of the host. If clinical disease is pro-
duced, it varies from the rapidly progressing,
hematogenously spread disease that occurs in
infants and immunosuppressed individuals to a
chronic, slowly progressing cavitary disease that is
commonly found in immunocompetent adults.
A brief overview of both the childhood and adult
types of pulmonary tuberculosis is presented in
chapter 1.
420 䡵 APPENDIX F
In this chapter, the gross and histopathological
characteristics of each type are described in more
detail. Included are the possible causes of subapi-
cal localization in adult-type tuberculosis, the loca-
tion of multidrug-resistant tubercle bacilli in the
lungs, the characteristics of tuberculosis in
immunocompromised individuals, and a compar-
ison of inapparent arrested primary lesions in
humans and rabbits. For further discussion of many
of the topics in this chapter, see reference 1.
CHAPTER 4. LIQUEFACTION
OF CASEOUS FOCI AND
CAVITY FORMATION
Part I is a review of the literature on liquefaction
and cavity formation. Liquefaction seems to be a
delayed-type hypersensitivity reaction to the tuber-
culin-like products of the bacillus. It seems to be
carried out by hydrolytic enzymes from the sur-
rounding host cells and possibly by enzymes within
the caseum after inhibitors have dissipated. Liq-
uefied caseum and cavities occur frequently in
rabbits inhaling virulent bovine-type tubercle
bacilli. They also occur occasionally in rabbits
inhaling virulent human-type tubercle bacilli. Part
II describes experiments of long duration in which
pulmonary cavities were produced in commercial
New Zealand White rabbits by aerosolized viru-
lent bovine-type bacilli (Mycobacterium bovis,
Ravenel strain).After the inhalation of a low num-
ber of these bacilli, liquefied caseum and cavities
occurred in 6 to 10 weeks.After the inhalation of
a high number of these bacilli, liquefied caseum
and cavities occurred sooner. Details on the gross
pathology and histopathology of these pulmonary
lesions are presented. Part III describes two attempts
to reduce liquefaction and cavity formation in
tuberculous rabbits. One was immunotherapy
with Mycobacterium vaccae. The other was therapy
with Ritonavir, a proteinase inhibitor. Neither
treatment (as administered) had any observable
effect.
SECTION 2. IMMUNOLOGY
OF TUBERCULOSIS
CHAPTER 5. DELAYED-TYPE
HYPERSENSITIVITY, CELL-
MEDIATED IMMUNITY, AND
ANTIBODIES IN TUBERCULOSIS
Both delayed-type hypersensitivity (DTH) and
cell-mediated immunity (CMI) are T-lympho-
cyte responses to bacillary antigens presented
mainly by dendritic cells. In tuberculous lesions,
DTH kills (nonactivated) macrophages that con-
tain numerous tubercle bacilli when these bacilli
release tissue-damaging local concentrations of
tuberculin-like products. In the resulting (solid)
caseous necrosis, bacillary growth is inhibited and
many bacilli die because of low oxygen tension
and other factors. Therefore, tissue-damaging
DTH has apparently evolved in mammals to stop
continuing bacillary growth within the nonacti-
vated macrophages that have permitted such
growth.
In tuberculous lesions, CMI activates macro-
phages so that they can inhibit and destroy ingested
tubercle bacilli. DTH can also activate macro-
phages if only low local concentrations of tuber-
culin-like products are present. In this respect,
DTH (the host reaction to tuberculin-like prod-
ucts) is part of the CMI response.
Nonactivated macrophages continuously enter
every tuberculous lesion and may ingest tubercle
bacilli (see chapter 10).To stop the progression of
the disease, macrophages containing a few bacilli
must be activated by CMI to prevent further intra-
cellular bacillary multiplication, and macrophages
containing many bacilli must be killed by the
DTH process. The interplay of CMI and tissue-
damaging DTH seems to explain the entire spec-
trum of the disease found in tuberculous hosts.
Both DTH and CMI exert their control locally.
Their main systemic manifestation is to provide an
expanded antigen-specific lymphocyte popula-
tion to infiltrate local sites of bacillary lodgement.
Antibodies that aid phagocytosis apparently play
little or no role in the destruction of the tubercle
bacillus. The bacillus readily enters macrophages
without being opsonized by antibodies and evi-
dently can multiply intracellularly within nonacti-
vated macrophages in the presence of antibodies.
However, in immunized hosts, antibodies seem to
be an important host defense against the develop-
ment of clinically apparent tuberculosis. Antigen-
antibody reactions at sites of bacillary lodgement
result in the production of chemotactic factors,
including the C5a component of complement. In
immunized hosts, such chemotaxins cause a rapid
local accumulation of dendritic cells, macrophages,
and antigen-specific T cells—all of which would
accumulate more slowly without the local antigen-
antibody reaction. In other words, in immunized
hosts the antigen-antibody reaction enables the
local cell-mediated immune response to occur so

rapidly that the bacillus is often inhibited before it
multiplies extensively.
CHAPTER 6. MACROPHAGES AND
OTHER CELLS IN TUBERCULOUS
LESIONS
The main types of cells participating in rabbit
tuberculous lesions are dendritic cells, macrophages,
natural killer cells, lymphocytes, and granulocytes.
The role of most of these cells is discussed only
briefly in this chapter because details are available
in textbooks of immunology.The role of macro-
phages, however, is presented more fully because
their rates of turnover, their states of activation, their
extracellular and intracellular enzymes, and their
heterogeneity have been extensively studied in my
laboratory in the rabbit model of tuberculosis.
SECTION 3. TUBERCULOUS LESIONS
CHAPTER 7. STRUCTURAL
COMPONENTS OF
TUBERCULOUS LESIONS
This chapter describes the structural components
of tuberculous lesions: the surrounding granulation
tissue, solid caseous necrosis, liquefied caseum,
cavities, and the fibrosis and calcification of heal-
ing lesions.
CHAPTER 8. MICROVASCULAR
DENSITY IN TUBERCULOUS LESIONS
The vasculature plays an important role in the
pathogenesis of tuberculous lesions. Blood vessels
bring in the host defense cells, and vascular throm-
bosis is a major cause of caseous necrosis. This
chapter describes the study of microvascular den-
sity in tissue sections of developing and healing
dermal BCG lesions and in 48-h dermal tuberculin
reactions.
Rabbits were placed under deep terminal anes-
thesia,and their entire vasculature was perfused (via
the aorta) with a gelatin-colloidal carbon suspen-
sion.Then, serial 250-µm-thick tissue sections of
the dermal BCG lesions were prepared, and the
total length of the microvasculature in the whole
BCG lesion was calculated from measurements
made with a microscope containing an ocular grid.
By 3 days, the vascular density in BCG lesions
had increased to roughly 1.6 times that found in
normal skin. It remained at this level for at least 6
to 7 weeks. The vascular density in tuberculin
reactions showed a similar increase.
APPENDIX F 䡵 421
It was concluded that the local microvasculature
increases relatively little during the course of this
slowly healing infection. A greater blood flow
through existing capillaries evidently provides most
of the nourishment needed by the infiltrating cells.
These studies also demonstrated that microvas-
cular thrombosis is a major cause of the caseous
necrosis that occurs during the course of this
disease.
CHAPTER 9. EARLY PULMONARY
LESIONS IN RABBITS
In rabbits, 1- to 7-day pulmonary tuberculous
lesions produced by aerosols are difficult to find
because the inhaled dose of tubercle bacilli cannot
be made large enough. A large intravenous dose,
however, readily produces many such tubercles.
This chapter describes their characteristics and
provides information on the activation and mul-
tiplication of macrophages within such lesions.
To produce these early lesions, we injected rab-
bits intravenously with 108 to 109 tubercle bacilli
(BCG).The blood-borne macrophages that entered
the developing tubercles became partly activated
during the first day. These entering macrophages
retained their ability to divide, i.e., incorporate
[3H]thymidine ([3H]TdR), even though they had
ingested tubercle bacilli. In contrast, fully activated
macrophages within tuberculous lesions lose their
ability to divide (see chapter 10).
Pulmonary alveolar macrophages did not seem
to participate in early pulmonary lesions produced
by the intravenous route, but accumulated in the
surrounding alveolar spaces. However, even though
these alveolar macrophages were highly activated,
they retained their ability to divide.
CHAPTER 10. MACROPHAGE
TURNOVER, DIVISION, AND
ACTIVATION IN TUBERCULOUS
LESIONS
In rabbit BCG lesions, the turnover of mono-
nuclear cells was most rapid in BCG lesions at
2 to 3 weeks, when the lesion size peaked and
tuberculin sensitivity and acquired cellular resis-
tance were well developed. (The mononuclear
cells were mostly macrophages, with some medium
and large lymphocytes and probably some den-
dritic cells.) At this 2- to 3-week peak, more
macrophages entered, more died or left, more
remained at the site, and more became activated
than before or afterward. Before this time, the
422 䡵 APPENDIX F
host had neither delayed-type hypersensitivity nor
cell-mediated immunity, so no antigen-specific
enhancement of the inflammatory response
occurred.After this time, the bacilli and their anti-
genic products had decreased, so antigen-specific
stimuli for cell infiltration and activation were
reduced. In “healed” lesions, the mononuclear cell
turnover still occurred but was decreased.
The continuous entry of live nonactivated
macrophages into the viable parts of tuberculous
lesions provides fresh intracellular sites where
tubercle bacilli can multiply before they are again
inhibited by the delayed-type hypersensitivity and
cell-mediated immunity of the host. In tubercu-
losis, bacillary dormancy of long duration can
only be present in caseous necrotic tissue where no
live host cells exist.
CHAPTER 11. LURIE’S PULMONARY
TUBERCLE-COUNT METHOD
Lurie’s tubercle-count method consists of counting
the number of grossly visible primary pulmonary
tubercles, present 5 weeks after an aerosol infection
of rabbits with virulent human-type tubercle bacilli.
It is a quantitative measure of clinically apparent dis-
ease.At 5 weeks, the grossly visible primary tuber-
cles are easily recognized, and many microscopic
tubercles have regressed. Since human-type tuber-
cle bacilli are not fully virulent for rabbits, the pul-
monary-count method has a sensitivity that is not
possible with fully virulent strains.
The number of grossly visible pulmonary tuber-
cles produced by human-type bacilli decreases (i)
when rabbits are infected with bacilli of reduced
virulence, (ii) when rabbits of high genetic (innate)
resistance are used, and (iii) when rabbits are effec-
tively immunized, so that they can rapidly activate
macrophages and stop the development of early
tubercles while they are still microscopic in size.
Therefore, the pulmonary tubercle-count method
can be used to assess (i) bacillary virulence, (ii) the
genetic resistance of the host, and (iii) the efficacy
of vaccines for tuberculosis.
SECTION 4. TUBERCULOSIS IN
RABBITS AND OTHER COMMON
LABORATORY ANIMALS
CHAPTER 12. NATURAL AIRBORNE
INFECTION
Using natural airborne infection of virulent
bovine-type tubercle bacilli over many months,
Lurie showed that resistance to the establishment
of tuberculosis and resistance to its progress are sep-
arate phenomena: his inbred resistant rabbits con-
verted their tuberculin skin tests an average of 2.7
months sooner than did his inbred susceptible
rabbits.Yet, once established, the disease progressed
slowly in the resistant rabbits and rapidly in the sus-
ceptible rabbits.The separation of the establishment
and progress of tuberculosis is only applicable to
experiments in which occasional fully virulent
tubercle bacilli are inhaled over many months. It
does not seem applicable to rabbits or humans
inhaling human-type tubercle bacilli, which are
never fully virulent in these hosts.
Airborne infection of laboratory animals over
many months has, however, established other
concepts directly applicable to tuberculosis in
humans. (i) Only a single grossly visible primary
pulmonary lesion will be produced, despite the
continuous presence of virulent tubercle bacilli
in the air.The immunity developed in response
to the primary lesion is evidently sufficient to
prevent other occasionally inhaled tubercle bacilli
from causing grossly visible lesions. (ii) Some
animals (and perhaps a few humans) may convert
their dermal tuberculin reactions, and yet show
no grossly visible primary lesions in their lungs
at necropsy.This occurrence may be due to the
early spread of inhaled bacilli out of the lungs to
the hilar lymph nodes, where the growth of
tubercle bacilli can be more easily controlled.
These concepts are consistent with what Riley
found when he exposed guinea pigs for months
to air from a ward containing sputum-positive
tuberculous patients.
CHAPTER 13. RESPONSE OF RABBITS
TO INHALED TUBERCLE BACILLI
Virulent bovine-type and human-type tubercle
bacilli and BCG are of decreasing virulence for
rabbits, in that order.The host uses the same type
of immune response to control each of these infec-
tions, but the response is more effective with bacil-
lary strains of reduced virulence.
With fully virulent bovine-type tubercle bacilli,
only 3 bacillary units of 1 to 3 bacilli must be
inhaled to cause one grossly visible tubercle at 5
weeks. In Lurie’s inbred susceptible rabbits, bovine-
type bacilli produced the childhood form of tuber-
culosis with hematogenous dissemination. In
Lurie’s resistant rabbits, bovine-type bacilli pro-

duced the adult form of tuberculosis with pul-
monary cavities and bronchial dissemination.
Human-type tubercle bacilli are not fully viru-
lent in rabbits. In Lurie’s inbred resistant rabbits and
in commercially available New Zealand White
rabbits, 300 to 1,900 bacillary units must be inhaled
to produce one grossly visible primary pulmonary
tubercle at 5 weeks, depending on the virulence of
the infecting strain and on variations among the
individual rabbits. In Lurie’s susceptible rabbits,
such tubercles often gave rise to secondary tuber-
cles. In Lurie’s resistant rabbits and commercial
New Zealand White rabbits, human-type tubercle
bacilli usually healed in a few months, except when
they formed cavities, which persisted much longer.
However, in all rabbits, lesions produced by human-
type bacilli eventually heal and are never fatal.
BCG is avirulent in all common laboratory
species. In commercial rabbits, a large inhaled dose
of aerosolized BCG produces few, if any, tiny non-
progressive tubercles. Most of the inhaled BCG
bacilli are apparently destroyed by the alveolar
macrophages before they can multiply appreciably.
Without such multiplication, the degree of immu-
nization would be negligible. Similar to rabbits,
humans should be less immunized by the inhala-
tion of BCG than by parenteral administration, in
which higher doses of BCG can be injected and
greater bacillary multiplication can occur (because
the alveolar macrophages are bypassed). In mice
and guinea pigs (which are more susceptible to
Mycobacterium tuberculosis), inhaled BCG would
immunize more effectively, because their alveolar
macrophages do not destroy inhaled BCG as read-
ily (see chapter 22).
CHAPTER 14. CHARACTERISTICS OF
RESISTANCE AND SUSCEPTIBILITY
TO TUBERCULOSIS IN LURIE’S
INBRED RABBITS
Lurie’s rabbits were inbred for either susceptibil-
ity or resistance to the progress of tuberculosis.
When infected with virulent bovine-type tuber-
cle bacilli, the susceptible rabbits developed a
rapidly progressing, hematogenously spreading
“childhood type” of tuberculosis, and the resistant
rabbits developed a slowly progressing, cavitary,
bronchial-spreading “adult type” of tuberculosis.
In lesions produced by virulent bovine-type
bacilli, by human-type bacilli, and by BCG, the
same manifestations of genetic resistance to tuber-
APPENDIX F 䡵 423
culosis were evident histologically: mature epithe-
lioid cells (now known as highly activated
macrophages) were always more numerous in the
lesions of resistant rabbits than in the lesions of sus-
ceptible rabbits, irrespective of the differences in vir-
ulence of the infecting bacillary strains.
The genetic resistance of these rabbits resides in
their ability to activate macrophages to control the
growth of tubercle bacilli, both nonspecifically
and immune-specifically. Crossbreeding showed
that the genetic resistance to tuberculosis is mul-
tifactorial, with genes associated with resistance
being dominant over susceptibility genes.
The commercially available outbred New
Zealand White rabbits seem almost as resistant as
Lurie’s inbred resistant strain III rabbits. Thor-
becke inbred rabbits were distinctly more suscep-
tible than commercial outbred rabbits, but appar-
ently not as susceptible as Lurie’s inbred C and FC
rabbits.Van Zutphen’s inbred rabbits (which are
hypo- and hyperresponsive to dietary cholesterol,
respectively) have not been adequately studied for
resistance to tuberculosis.
CHAPTER 15. COMPARISONS OF
TUBERCULOSIS IN RABBITS, MICE,
AND GUINEA PIGS
In recent times, mice have been by far the most fre-
quently used animal for the study of tuberculosis.
Guinea pigs and rabbits are used less often, and
monkeys are used only occasionally.
Rabbits are highly susceptible to bovine-type
tubercle bacilli and are the only common labora-
tory species in which chronic cavitary tuberculo-
sis with bronchial spread is readily produced. Rab-
bits develop grossly visible pulmonary tubercles
following the inhalation of virulent human-type
tubercle bacilli, but these tubercles usually regress,
as they do in most humans.Rabbit pulmonary
lesions caused by virulent human-type tubercle
bacilli sometimes form nonprogressive cavities.
Rabbits die after an infection with virulent bovine-
type tubercle bacilli, but eventually heal an infec-
tion with virulent human-type tubercle bacilli.
Mice develop slowly progressing pulmonary
tubercles with both bovine and human strains of
tubercle bacilli, but the disease progresses more
rapidly with the bovine strain. With virulent
human-type bacilli, the tubercles of mice contain
a larger number of viable bacilli than do the tuber-
cles of rabbits and guinea pigs.Apparently, the low
424 䡵 APPENDIX F
levels of delayed-type hypersensitivity (DTH) in
mice and the rarity of caseous necrosis allow the
bacillus to grow to higher titers in the logarithmic
stage.After these titers are reached, the good cell-
mediated immunity (CMI) developed by mice
reduces the intracellular multiplication of the
majority of the bacilli to almost a dormant state.
However, the low percentage of nondormant
bacilli causes the disease to slowly progress until the
animal dies.
Guinea pigs are highly susceptible to both
human and bovine strains of tubercle bacilli.They
usually develop a rapidly progressing, hematoge-
nously spread form of tuberculosis, similar to that
developed by infants and immunosuppressed indi-
viduals. Because guinea pigs develop relatively
high sensitivity to tuberculin, which causes con-
siderable caseous necrosis, their lesions contain a
rather low number of viable bacilli. In other
words, despite the rather poor CMI developed by
guinea pigs, their good DTH effectively reduces
the number of viable bacilli in their lesions. How-
ever, because of the extensive lung destruction
caused by tissue-damaging DTH, guinea pigs
often die in less time than do rabbits and mice.
Rhesus monkeys are very susceptible to tuber-
culosis, but cynomolgus monkeys are more resis-
tant. Some cynomolgus monkeys can even stop the
progression of the disease.
SECTION 5. EFFECTS OF HORMONES
AND X-IRRADIATION ON
TUBERCULOSIS
CHAPTER 16. EFFECTS OF CORTISONE
AND ADRENOCORTICOTROPIC
HORMONE ON TUBERCULOSIS
Pharmacological amounts of glucocorticoids are
frequently given as therapy for a variety of aller-
gic, autoimmune, and inflammatory conditions,
such as asthma and rheumatoid arthritis. When
such drugs are continued for long periods of time,
latent tuberculosis may reactivate.
In tuberculous rabbits infected with human-type
tubercle bacilli, pharmacological amounts of glu-
cocorticoids decreased cell-mediated immunity and
delayed-type hypersensitivity. Macrophages were
poorly activated, and tubercle bacilli grew to large
numbers within these phagocytes.After glucocor-
ticoid administration was stopped, the tuberculin
sensitivity returned, and (because of the large num-
bers of bacilli) liquefaction, cavity formation, tuber-
culous bronchopneumonia, and hematogenous dis-
semination occurred in some of the rabbits.
One of Lurie’s inbred rabbit strains was evi-
dently deficient in glucocorticoid production. In
these rabbits, the administration of physiological
doses of adrenocorticotropic hormone increased
their resistance to tuberculosis.
CHAPTER 17. EFFECTS OF ESTROGEN,
CHORIONIC GONADOTROPIN, AND
THYROID HORMONES ON
TUBERCULOSIS
Since estrogen increased the hyaluronic acid and
water content of the skin, it decreased the spread
of intradermally injected virulent bovine-type
tubercle bacilli in rabbits. Chorionic gonadotropin
had the reverse effect.These two sex hormones had
no appreciable effect on the innate or acquired
ability of the host to control the progression of
tuberculosis, because neither hormone apprecia-
bly changed the number of primary pulmonary
tubercles generated by aerosols of virulent human-
type tubercle bacilli. However, estrogen markedly
suppressed the development of amyloid in the
spleens of rabbits dying of the more chronic form
of tuberculosis caused by bovine-type bacilli.
In rabbits, triiodothyronine or thyroxine
increased host resistance in that they decreased
the number of grossly visible primary pulmonary
tubercles produced by the inhalation of virulent
human-type tubercle bacilli, whereas thyroidec-
tomy or propylthiouracil treatment increased the
number of such primary tubercles.Thyroid hor-
mones were most beneficial in inbred rabbits of
intermediate resistance.The resistance of the most
susceptible inbred C rabbits and that of the
most resistant III(r) rabbits were not appreciably
increased by thyroid hormones.
CHAPTER 18. EFFECTS OF WHOLE-
BODY X-IRRADIATION ON
TUBERCULOSIS
Commercial rabbits were irradiated with 400 rads
of whole-body X-irradiation—a sublethal dose.At
2 or 10 days thereafter, they were injected intra-
dermally with BCG. Between 2 and 4 weeks after
irradiation, the BCG lesions and 48-h tuberculin
reactions in the irradiated group were smaller than
those of the nonirradiated controls. The BCG

lesions in the irradiated group also contained more
bacilli.
This dose of whole-body X-irradiation evi-
dently decreased the supply of macrophages and
lymphocytes from “cell factories” in the bone
marrow and lymphoid tissues, so that fewer cells
were available to infiltrate the BCG lesions.These
cell factories had apparently recovered 4 to 5
weeks after irradiation, because BCG lesions start-
ing at this time were the same size as those in the
nonirradiated controls and contained the same
number of bacilli.
Pulmonary alveolar macrophages (AM) recov-
ered by bronchoalveolar lavage (BAL) from irra-
diated rabbits contained higher levels of hydrolytic
enzymes than did AM from nonirradiated controls.
The AM from the irradiated group were apparently
an older (more activated) cell population, because
they had ingested inhaled particles for a longer
period of time. The irradiation evidently had
reduced the young macrophage population that
replenishes the AM population.
When heat-killed tubercle bacilli in oil were
injected intravenously 1 day after irradiation, the
BAL specimens obtained 9 and 10 days later (from
the resulting granulomatous lungs) contained about
half as many macrophages as did those from non-
irradiated controls.This finding indicated that the
irradiation reduced the supply of macrophages
from the bone marrow.When the heat-killed tuber-
cle bacilli were injected 4 weeks after irradiation,
BAL specimens from the granulomatous lungs
of the irradiated and the nonirradiated animals
contained similar numbers of macrophages, which
indicated that the supply of these macrophages
had recovered.
Rabbits were infected by aerosol with virulent
human-type tubercle bacilli (H37Rv) at 12 or 30
days after irradiation. In each case, 5 weeks after
infection, the number of primary pulmonary
tubercles in their lungs was the same in both the
irradiated and the nonirradiated groups. Also,
the number of viable bacilli in these tubercles
was the same. Therefore, this sublethal dose of
irradiation had no appreciable effect on the devel-
opment and progress of primary pulmonary
tubercles in rabbits.
In brief, X-irradiation reduces the bone mar-
row’s capacity to provide defense cells to protect
the host against infection.When the host is chal-
lenged by inhaled virulent human-type tubercle
APPENDIX F 䡵 425
bacilli, an adequate supply of defense cells is avail-
able. However, with acute infections (requiring
many more defense cells), the irradiated host would
have an inadequate supply.
SECTION 6. CYTOKINES AND
VASCULAR ADHESION MOLECULES
IN TUBERCULOUS LESIONS
CHAPTER 19. CYTOKINE PRODUCTION
IN PRIMARY BCG LESIONS
A sequential histochemical study of cytokines in
developing and healing rabbit tuberculous (BCG)
lesions is described. In tissue sections, interleu-
kin-1 (IL-1), tumor necrosis factor alpha
(TNF-), macrophage chemoattractant (activating)
protein 1 (MCP-1), and IL-8 were evaluated for
cytokine mRNA by in situ hybridization tech-
niques and for cytokine protein by immunohis-
tochemical techniques. In tissue homogenates,
gamma interferon (IFN-) mRNA was evaluated
by reverse transcription-PCR.
In the BCG lesions, the percentage of mononu-
clear cells that contained the mRNAs of these
cytokines showed a biphasic pattern. At 1 to 3
days, a peak occurred, which was apparently a non-
specific inflammatory response caused by the tuber-
cle bacilli in the BCG vaccine.At 5 days, the per-
centage of mononuclear cells containing the
cytokine mRNAs was significantly reduced, but by
9 days, the percentage had again increased, and the
rabbits had become tuberculin positive.This second
peak was apparently antigen specific.With IFN-,
the two mRNA peaks were delayed by 2 days.
Mononuclear cells containing IL-1 and
IL-8 mRNAs were more numerous surrounding
the caseous center. These cytokines evidently
recruited the polymorphonuclear leukocytes that
were common in this location. Mononuclear cells
containing MCP-1 mRNA were more numerous
in the outer third of the lesion where new
macrophages and lymphocytes were being re-
cruited.
Both the nonspecific and antigen-specific
cytokine responses of BCG vaccines are evidently
synergistic. The early nonspecific cytokine
(chemokine) response causes a local accumulation
of antigen-presenting cells and lymphocytes,which
explains, at least in part, why tubercle bacilli are
good immunological adjuvants. This adjuvant
effect should be considered in developing
426 䡵 APPENDIX F
improved vaccines for the prevention of tuber-
culosis, because vaccines producing a strong early
nonspecific cytokine (chemokine) response should
be more immunogenic than vaccines with simi-
lar antigens producing a weak response.
CHAPTER 20. CYTOKINE PRODUCTION
IN REINFECTION BCG LESIONS AND
IN TUBERCULIN REACTIONS
Reinfection BCG lesions provide a simple model
of how tuberculosis vaccines would affect an
exogenous infection with virulent tubercle bacilli.
Therefore, rabbit dermal primary and reinfection
BCG lesions were produced and evaluated during
the first 5 days of their existence.Tissue sections
of the lesions were prepared, and the types of cells
and their cytokine mRNAs and proteins were
analyzed by histochemical methods.The cytokines
studied were interleukin-1, macrophage
chemoattractant (activating) protein (MCP-1),
interleukin-8, and tumor necrosis factor alpha (see
chapter 19).
Our most informative findings were with MCP-
1, one of the main chemokines attracting mononu-
clear cells (MN). (In tuberculous lesions, MN are
mostly macrophages but also contain dendritic
cells and lymphocytes.) At 3 h, both the reinfection
lesions and the primary lesions contained the same
percentage of MN labeled for MCP-1 mRNA.
However, the reinfection lesions were 400 to 500
times larger and therefore contained many more of
these MN. This high cell number alone would
cause the total chemokine production to exceed by
far that occurring in the primary lesions.
By 1 day, the percentage of MN containing
MCP-1 mRNA (and protein) had markedly
decreased in the reinfection lesions, but remained
high for at least 2 days in the primary lesions,
which were beginning to increase in size. This
finding suggests that chemokine production is
turned off when sufficient MN have accumu-
lated. In other words, the local accumulation of
MN is carefully regulated, so that excessive cell
infiltration into the lesions is prevented.
The rapid local accumulation of MN (macro-
phages, dendritic cells, and antigen-specific lym-
phocytes) in the early reinfection BCG lesions
seemed to be due to the presence of antibodies that
developed during the first BCG infection (see
chapter 5). The antigen-antibody complexes
formed at the site of reinfection evidently produced
chemotactic factors that markedly hastened the
cell infiltration.
In general, cytokine production in tuberculin
reactions showed the same pattern as that found in
the early reinfection BCG lesions.
CHAPTER 21.VASCULAR ADHESION
MOLECULES IN TUBERCULOUS
LESIONS
Vascular adhesion molecules enable host defense
cells to leave the bloodstream and enter tubercu-
lous lesions.After the inhalation of tubercle bacilli,
Lurie’s resistant rabbits had a larger number of
mononuclear cells within developing lesions than
did his susceptible rabbits. Therefore, the rapid
local accumulation of mononuclear cells seems
to be one of the factors associated with resis-
tance to the progress of this disease.Vascular adhe-
sion molecules enable such an accumulation to
occur.
With immunohistochemical techniques, we
evaluated the rise and fall of three major vascular
adhesion molecules as rabbit dermal BCG lesions
developed and healed. ICAM-1 (intercellular adhe-
sion molecule 1) is important for the adherence of
polymorphonuclear leukocytes (PMN), mono-
cytes, and lymphocytes to activated vascular
endothelium before they emigrate from the blood-
stream into sites of inflammation and infection.
VCAM-1 (vascular cell adhesion molecule 1) is a
major factor in monocyte, lymphocyte, and
eosinophil emigration. ELAM-1 (endothelial-
leukocyte adhesion molecule 1, now called E-
selectin) aids the emigration of granulocytes (and
some monocytes and T lymphocytes).
In primary BCG lesions, ICAM and VCAM
peaked at 1 to 2 weeks and decreased as the lesions
healed. In reinfection BCG lesions, ICAM and
VCAM were upregulated much sooner, beginning
at 3 to 12 h and peaking at 1 to 2 days.The upreg-
ulation of these two adhesion molecules apparently
caused the rapid infiltration of mononuclear cells
into sites of BCG reinfection. ELAM-1 seemed to
be less involved.
In tuberculosis, epithelioid cells are macrophages
that adhere to one another in an epithelial-like
pattern.This adherence seems to be due in part to

the ICAM-1 of one macrophage’s binding to its
ligand LFA-1 (lymphocyte function-associated
antigen 1) (CD11a/CD18) on a neighboring
macrophage. Whether or not this epithelial-like
pattern benefits the host remains to be determined.
From these studies of vascular adhesion mole-
cules, we developed a theory of why so much tis-
sue destruction occurs in tuberculosis: ICAM,
VCAM, and ELAM are markers for activated vas-
cular endothelial cells. In tuberculous lesions, such
activated endothelial cells can capture and present
local mycobacterial antigens and therefore may be
killed by antigen-specific cytotoxic T lympho-
cytes.When the vascular endothelium is no longer
intact, thrombosis occurs, and the local tissues (now
lacking a blood supply) undergo caseous necrosis.
SECTION 7. TUBERCULOSIS
VACCINES
CHAPTER 22. PRINCIPLES AND
GUIDELINES FOR DEVELOPING
BETTER TUBERCULOSIS VACCINES
Tuberculosis vaccines have little or no effect on the
establishment of a microscopic pulmonary lesion
produced by the inhalation of a virulent tubercle
bacillus. Such a lesion is established only when the
pulmonary alveolar macrophages fail to destroy the
inhaled bacillus. Alveolar macrophages do not
expand their population in response to specific
antigens.Therefore, the establishment of a micro-
scopic pulmonary tubercle is not affected by vac-
cination. Effective tuberculosis vaccines may, how-
ever, stop the progression of a tiny established
lesion, because the vaccination has expanded anti-
gen-specific lymphocyte populations.These lym-
phocytes enter the early lesion, where they cause
a rapid local delayed hypersensitivity (DTH) and
cell-mediated immunity (CMI) response that often
prevents progression of the disease.
When comparing their relative efficacies, two or
more live vaccines should be standardized for
equal numbers of live and dead bacilli, equal num-
bers of log-phase and dormant bacilli, and equal
numbers of clumps and isolated bacilli.
Vaccines will probably never be 100% effective
in preventing active tuberculosis in humans,
because humans with arrested tuberculous lesions
are able to be reinfected with exogenous tubercle
bacilli. However, the efficacy of BCG could be
APPENDIX F 䡵 427
improved by recombinant constructs that contain
major protective antigens.Also, the immunity pro-
vided by BCG could be increased by booster
injections of such antigens.
If vaccines more effective than BCG are ever
developed, they would probably produce in the
host a higher CMI/DTH ratio, i.e., an expanded
antigen-specific lymphocyte population capable of
producing increased numbers of activated macro-
phages and decreased amounts of tissue necrosis.To
do this, the improved vaccine would probably con-
tain increased bacillary glycolipid-protein compo-
nents and decreased tissue-damaging tuberculin-like
protein components.The vaccine should also con-
tain components that increase the Th1/Th2 ratio.
CHAPTER 23. CHARACTERISTICS OF
RABBIT BCG LESIONS AND EFFICACIES
OF BCG AND MYCOBACTERIUM
MICROTI VACCINES
In rabbits (and humans), dermal BCG lesions have
the same components as lesions caused by virulent
tubercle bacilli, i.e., a caseous liquefied center sur-
rounded by tuberculous granulation tissue.There-
fore, BCG lesions can be used as a model in which
to study the host response to this disease.
In immunocompetent hosts, BCG lesions do
not progress and always heal.The rate of healing
of dermal BCG lesions reflected the native and
acquired resistance of Lurie’s susceptible and resis-
tant inbred rabbit strains. Similarly, the rate of
healing of BCG lesions has been shown to reflect
the resistance to tuberculosis of human popula-
tions. However, the healing of BCG lesions in
individual rabbits and humans may vary consid-
erably from the mean.
In Lurie’s natively resistant rabbits, immuniza-
tion with BCG was quite effective in preventing
many grossly visible primary pulmonary tubercles
(i.e., clinically apparent tuberculosis). In his natively
susceptible rabbits, BCG was hardly effective at all.
In other words, the rabbits that needed it the least
were helped by BCG vaccination the most, and the
rabbits that needed it the most were helped the
least.This principle also applies to human popu-
lations: persons with poor resistance to clinical
tuberculosis would develop less immunity from
BCG administration than persons with strong
resistance.
428 䡵 APPENDIX F
The efficacies of various BCG and Mycobacterium
microti (the vole bacillus) vaccines were evaluated in
commercial outbred rabbits by the tubercle-count
method. Both of these vaccine types were effective.
However,the variations in resistance among outbred
rabbits prevented our rating any one vaccine above
another. Recombinant BCG vaccines that contain
additional antigenic components may be more
promising than currently used BCG vaccines (see
chapter 22).
 APPENDIX G
ACKNOWLEDGMENTS
I wish to acknowledge the many associates who
have carried out our laboratory’s experiments dur-
ing their two-year sojourns in my laboratory.
Their names appear as coauthors in my publica-
tion list in Appendix D. Rena Ashworth and Ilse
Moeller Harrop have assisted faithfully in the
preparation of this entire book. Many friends have
read and improved various chapters.Their help is
acknowledged below.
Most of the photographs in this book were
carefully reproduced by Jon R. Christofersen in the
Photography Laboratory of the Dept. of Pathol-
ogy, Johns Hopkins Medical Institutions, and some
photographs were similarly reproduced by Nor-
man J. Barker, director of this laboratory.
Finally, I wish to express my deep appreciation
to the editorial staff of ASM Press for the dedicated
care that they gave to the production of this book,
particularly to Eleanor Tupper, Senior Production
Editor; Mary McKenney, the copyeditor; and Jen-
nifer Adelman, Marketing Director. This book
could never have been published in its present
form without the faith in its value of Jeff Holt-
meier (Director,ASM Press) and Ellie Tupper and
their many concessions to variations from the
usual ASM format.
Partial financial support for the preparation of this
book came from the Aeras (formerly Sequella)
Global TB Vaccine Foundation; from Ellison Med-
ical Foundation; from grant HL-71554 from the
National Institutes of Health (NIH) to William R.
Bishai and Yukari C. Manabe; and from Johns Hop-
kins Environmental Health Science Center, Dept.
of Environmental Health Sciences, Bloomberg
School of Public Health, Johns Hopkins University
(grant ES-03819 from the National Institute of
Environmental Health Sciences, NIH).
CHAPTER 1. OVERVIEW
The Virulence section of this chapter was reviewed
by William R. Bishai, M.D., Ph.D., Professor of
Medicine at the Johns Hopkins Center for Tuber-
culosis Research. The Prevention section of this
chapter was reviewed by Byron S.Tepper, Ph.D.,
former Director of Environmental Health and
429
Safety; Edward J. Bernacki, M.D., M.P.H., Execu-
tive Director of Health Safety and Environment;
and John A. Schaefer, M.S., Associate Director of
Health Safety and Environment.The entire chap-
ter was reviewed by Carlton Evans, M.D., Ph.D.,
Associate in the Dept. of International Health.All
of these reviewers are at the Johns Hopkins Med-
ical Institutions, Baltimore, Md. Edward A. Nardell,
M.D. (Associate Professor, Harvard School of Pub-
lic Health, Boston, Mass.), also reviewed this entire
chapter and provided the information on his cur-
rent studies of the transmission and infectiousness
of tubercle bacilli aerosolized by patients with
tuberculosis.
CHAPTER 3. TYPES OF HUMAN
PULMONARY TUBERCULOSIS
I am indebted to the late Walter H. Sheldon, M.D.
(Professor of Pathology, Johns Hopkins School of
Medicine), and to Joseph F.Tomashefski, Jr., M.D.
(Professor of Pathology, Case-Western Reserve
School of Medicine), for their help in preparing
the original publication of reference 1 from which
most of this chapter was derived. Dr.Tomashefski
supplied the text and figures for the part of this
chapter describing tuberculosis in immunocom-
promised persons. Richard E. Chaisson, M.D., and
Jacques H. Grosset, M.D., Professors of Medicine;
Ralph H. Hruban, M.D., Professor of Pathology;
Robert Frank, M.D., Professor of Physiology; and
Noreen A. Hynes, M.D., Assistant Professor of
Medicine—all at Johns Hopkins Medical Institu-
tions—reviewed parts or all of this chapter.
CHAPTER 4. LIQUEFACTION OF
CASEOUS FOCI AND CAVITY
FORMATION
Raymond E. Lund, Norman J. Barker, Jon R.
Christofersen, and Rick M.Tracey (Johns Hopkins
Medical Institutions, Baltimore, Md.) made many
of the photographs in this chapter.Walter Johnson
(George Washington University School of Medi-
cine,Washington, D.C.) administered the Riton-
avir orally to the rabbits.Yukari C. Manabe, M.D.,
Paul J. Converse, Ph.D., and Norman E. Morrison,
430 䡵 APPENDIX G
Ph.D. (Johns Hopkins Center for Tuberculosis
Research), reviewed this chapter and offered sev-
eral important suggestions for its improvement.
CHAPTER 5. DELAYED-TYPE
HYPERSENSITIVITY, CELL-MEDIATED
IMMUNITY, AND ANTIBODIES IN
TUBERCULOSIS
I am indebted to Alan L. Scott, Ph.D. (Professor of
Immunology, School of Public Health), and Mark
J. Soloski, Ph.D. (Professor of Immunology, School
of Medicine), both at Johns Hopkins University,
for reviewing the immunology in this chapter.
CHAPTER 6. MACROPHAGES
AND OTHER CELLS IN
TUBERCULOUS LESIONS
I am indebted to Alan L. Scott, Ph.D. (Professor of
Immunology, School of Public Health), and Mark
J. Soloski, Ph.D. (Professor of Immunology, School
of Medicine), both at Johns Hopkins University,
for reviewing the immunology in this chapter,
and also to Yukari C. Manabe, M.D. (Assistant
Professor, Division of Infectious Diseases, Johns
Hopkins University School of Medicine), for her
contributions to some sections of this chapter.
CHAPTER 11. LURIE’S PULMONARY
TUBERCLE-COUNT METHOD
Edward A. Nardell, M.D. (Associate Professor, Har-
vard School of Public Health, Boston, Mass.), pro-
vided the information on current studies of the
transmission and infectiousness of tubercle bacilli
from patients with active tuberculosis.
CHAPTER 15. COMPARISONS OF
TUBERCULOSIS IN RABBITS, MICE,
AND GUINEA PIGS
For reviewing the parts of this chapter concern-
ing tuberculosis in mice, I am indebted to Pere-
Joan Cardona, M.D., Ph.D. (Unitat de Tuberculosi
Experimental, Dept. of Microbiology, Hospital
Universitari “Germans Trias i Pujol” and Univer-
sitat Autònoma de Barcelona, Catalonia, Spain), to
Robert J. North, Ph.D. (Trudeau Institute, Saranac
Lake, N.Y.), to JoAnne L. Flynn, Ph.D. (Dept. of
Molecular Genetics and Biochemistry, University
of Pittsburgh School of Medicine, Pittsburgh, Pa.),
and to Jacques H. Grosset, M.D. (Johns Hopkins
School of Medicine, Baltimore, Md.). David N.
McMurray, Ph.D. (Dept. of Microbiology,Texas A
and M University, College Station), reviewed the
parts concerning tuberculosis in guinea pigs. Dr.
Flynn also reviewed the section on tuberculosis in
monkeys. Dr. Cardona kindly provided Fig. 5, 6,
7, 8, and 9.
CHAPTER 19. CYTOKINE PRODUCTION
IN PRIMARY BCG LESIONS
I am indebted to Pere-Joan Cardona, M.D., Ph.D.
(Unitat de Tuberculosi Experimental, Dept. of
Microbiology, Hospital Universitari “Germans
Trias i Pujol” and Universitat Autònoma de
Barcelona, Catalonia, Spain), and to JoAnne L.
Flynn, Ph.D. (Dept. of Molecular Genetics and
Biochemistry, University of Pittsburgh School of
Medicine, Pittsburgh, Pa.), for reviewing this chap-
ter, especially the parts concerning tuberculosis
in mice.
CHAPTER 22. PRINCIPLES AND
GUIDELINES FOR DEVELOPING
BETTER TUBERCULOSIS VACCINES
Jacques H. Grosset, M.D.,William R. Bishai, M.D.,
Richard E. Chaisson, M.D., and Carlton Evans,
M.D., reviewed parts of this chapter and provided
helpful suggestions.
CHAPTER 25. SUGGESTED FUTURE
RESEARCH AND UNANSWERED
QUESTIONS
Mark A. Chambers, Ph.D. (Veterinary Laboratories
Agency,Weybridge, United Kingdom), suggested
that this chapter be written. He also suggested
that anticoagulants might decrease the amount of
caseous necrosis in tuberculous lesions.William R.
Bishai, M.D., Ph.D., Jacques H. Grosset, M.D.,
Eric L. Nuermberger, M.D., and Ying Zhang,
M.D., Ph.D. (all at Johns Hopkins Center for
Tuberculosis Research), reviewed this chapter.
GLOSSARY
I am indebted to Alan L. Scott, Ph.D., Professor of
Immunology in the Johns Hopkins Bloomberg
School of Public Health, and Mark J. Soloski,
Ph.D., Professor of Immunology in the Johns
Hopkins School of Medicine, for reviewing the
immunology in this glossary.
 GLOSSARY
This glossary includes definitions and abbreviations
and is arranged in two parts: first, a listing of the
immunology terms most frequently used in this
book, and then an alphabetical listing of the
remaining terms. For details on cytokines,
chemokines, and adhesion molecules, see refer-
ences 1, 2, and 3, respectively. Some of the defin-
itions are from references 4 and 5.
PART I. FREQUENTLY USED
IMMUNOLOGY TERMS
Acquired cellular resistance (ACR). ACR
occurs locally in tuberculous lesions. It is charac-
terized by a population of macrophages that have
been activated by cytokines from nearby antigen-
specific Th1 lymphocytes. Such activated macro-
phages can inhibit and/or destroy ingested tuber-
cle bacilli.Cell-mediated immunity (CMI) produces
local acquired cellular resistance (ACR), and both
terms are frequently used interchangeably. However,
we prefer to use ACR for macrophages that have
been already activated in tuberculous lesions, and
to use CMI for an expanded antigen-specific
T-lymphocyte population that is able to activate
macrophages if the antigen is present, whether or
not it actually does so.
Antigen-presenting cells (APCs). Dendritic
cells (DCs) (see chapter 6) are the most effective
antigen-presenting cells, but macrophages and B
lymphocytes can also present antigens. DCs are
present both in the tissues and in the circulation.
Tissue DCs migrate from sites of infection to the
draining lymph nodes, where they present antigens
to lymphocytes that continuously circulate from
the blood to lymph nodes and back to the blood
(via efferent lymphatics). In the lymph nodes
draining tuberculous lesions, antigen-specific lym-
phocytes clonally expand their population, enter
the bloodstream, and then enter tuberculous
lesions. Once there, these antigen-specific lym-
phocytes produce macrophage-activating cytokines
and continue to proliferate.
Cell-mediated immunity (CMI). CMI in
tuberculosis is characterized by an expanded pop-
ulation of recirculating antigen-specific Th1 lym-
phocytes. In sites where tubercle bacilli and their
431
antigens are located, these lymphocytes produce
cytokines that activate macrophages so that they
can inhibit or destroy ingested tubercle bacilli.
Delayed-type hypersensitivity (DTH). In
the field of tuberculosis, the terms DTH and tuber-
culin sensitivity are used interchangeably. DTH
gets its name because the skin reaction (to tuber-
culin) usually peaks in 1 to 3 days rather than in the
minutes or hours that characterize the more imme-
diate hypersensitivities. Like CMI, DTH is char-
acterized by an expanded population of recircu-
lating antigen-specific Th1 lymphocytes. The
difference from CMI is the amount of antigen that pro-
duces each reaction. In a highly tuberculin-positive
person, a positive skin test can be elicited with an
intradermal injection of 1 tuberculin unit of puri-
fied protein derivative, i.e., 0.1 ml containing 0.02
g (see “PPD”). If greater concentrations are
injected intradermally into such a person, necrosis
may develop at the site of injection. In immunized
hosts, antigens eliciting CMI usually require a
higher concentration than antigens eliciting DTH.
Innate and adaptive (acquired) immunity.
Innate immunity is present before the host is
infected by the tubercle bacillus. The host will
recognize the foreignness of an invader, but will not
recognize its exact nature. For example, the
lipopolysaccharides (endotoxins) of many differ-
ent bacteria are recognized as foreign by receptors
on the host’s phagocytes.Adaptive immunity to the
tubercle bacillus is specific for its antigens. It
involves the clonal expansion of lymphocytes that
have specific receptors for these antigens. Lurie and
I use the term “acquired immunity” for what is
now called adaptive immunity, and the term
“native resistance” for what is now called innate
immunity.
Lymphocytes (T cells and B cells). T cells
and B cells provide immunologic specificity to the
host defense against tubercle bacilli.
T cells originate in the bone marrow and enter
the thymus, where they develop antigen-specific
receptors (by DNA rearrangements). They then
enter the bloodstream and recirculate in and out of
peripheral lymphoid tissues, including the spleen.
T cells are the immunocytes responsible for the
432 䡵 GLOSSARY
cell-mediated immunity and delayed-type hyper-
sensitivity found in tuberculosis.Antigen-activated
T cells produce cytokines that activate nearby
macrophages. T cells have been subdivided in a
variety of ways based on (i) their surface markers
(CD4 and CD8 T cells), (ii) their antigenic recep-
tors (/, /, and CD1), (iii) the cytokines they
produce (Th1 and Th2 cells), and (iv) their func-
tions (helper, regulatory, and cytotoxic T cells).
Th1 cells produce gamma interferon and other
cytokines that activate macrophages, so that these
macrophages can inhibit the growth of the tuber-
cle bacilli that they ingest.Th2 cells produce inter-
leukin 4 (IL-4), IL-5, and other cytokines that
activate B lymphocytes (B cells) to produce anti-
body.
B cells also originate in the bone marrow of
mammals and recirculate in a manner similar to
that of T cells. Antigen-activated B cells produce
antibodies, especially when they terminally dif-
ferentiate into plasma cells.Antibodies enhance the
cell-mediated immune response in a variety of
ways (see chapter 5).
Macrophages. Macrophages are the effector
cells of the mononuclear phagocyte system.They
are produced in the bone marrow, circulate as
monocytes in the bloodstream, and are called
macrophages after they emigrate from the blood
into the tissues. Virulent tubercle bacilli readily
multiply in nonactivated macrophages, whereas
they are inhibited (and often destroyed) in highly
activated macrophages.
Pulmonary alveolar macrophages (AM).
Pulmonary alveolar macrophages possess high lev-
els of innate immunity, because most are strongly
activated nonspecifically by a variety of inhaled par-
ticles that the AM ingest and digest.AM are usu-
ally capable of destroying numerous inhaled viru-
lent tubercle bacilli. In humans and rabbits, only a
rare alveolar macrophage—probably one that has
recently migrated there from the bloodstream—is
so poorly activated it will allow an ingested human-
type tubercle bacillus to multiply and establish an
infection (thereby converting the tuberculin skin
test). Human-type tubercle bacilli do not cause
progressive disease in most humans and rabbits.
Tissue-damaging DTH. Tissue-damaging
DTH kills macrophages in which more than a few
tubercle bacilli are multiplying, thereby causing
solid caseous necrosis in which tubercle bacilli can-
not grow. It kills macrophages and the surrounding
tissues, because multiplying intracellular tubercle
bacilli produce high local concentrations of
tuberculin-like products. In brief, tissue-damaging
DTH is a fail-safe immune response to stop intra-
cellular bacillary growth when CMI cannot do so.
DTH carries out this function at the expense of host
tissues, and is the cause of almost all of the tissue
damage found in tuberculosis.Tubercle bacilli do not
usually damage host tissues before DTH develops.
Tissue-damaging DTH also damages vascular
endothelial cells, and the resulting thrombosis causes
much of the tissue necrosis found in humans, rab-
bits, and guinea pigs with tuberculosis.
PART II. ALPHABETICAL LISTINGS
Acquired cellular resistance (ACR). See
definition in part I.
Acute-phase reaction. An early system host
response to infection in which the liver increases
or decreases several proteins in the blood. These
changes aid the host in combating the infection.
The acute-phase reaction is mediated by IL-1,
IL-6, tumor necrosis factor alpha (TNF-), and
probably other cytokines. (See references 4 and 6).
Antibody-dependent cell-mediated cyto-
toxicity (ADCC). The killing of cells (that
bind antibody) by cells recognizing that antibody’s
Fc region, especially natural killer (NK) cells.
Antigen-presenting cells (APC). See defin-
ition in part I.
Apoptosis (programmed cell death). An
internal cell death program whereby cells eliminate
themselves without causing an inflammatory
response.Apoptosis removes cells that are no longer
needed during embryological development and
repair processes. It also keeps the immune responses
from becoming excessive. In apoptosis, a series of
caspases (cysteine-aspartic acid proteases) are acti-
vated, DNA is degraded and condensed, and the
cell residue is phagocytized by macrophages. (See
reference 6).
In necrosis, cells are killed by external (not inter-
nal) forces (e.g., microorganisms and their products,
trauma, or thrombosis of their blood supply), and
an inflammatory response occurs. During necrosis,
some cells may die from apoptosis.Therefore, these
two types of cell death can exist together. (See
reference 6).

Arthus reaction. An inflammatory reaction
commonly produced by injecting an antigen intra-
dermally into a host that has circulating antigen-
specific (IgG) antibodies. The resulting antigen-
antibody reaction activates complement, which
causes a slowly developing cell infiltration that
usually peaks in 1 day. In contrast, DTH reactions
are caused by antigen-specific T lymphocytes.
However, because antibodies usually circulate in
tuberculin-positive individuals, the typical skin
tests produced by tuberculin almost always have an
Arthus component.
B cells, B lymphocytes. See “Lymphocytes (T
cells and B cells)” in part I.
B7.1 and B7.2. Cell surface molecules on
antigen-presenting cells that costimulate antigen-
specific T cells.
BCG. Bacille Calmette-Guérin, the live atten-
uated strain of Mycobacterium bovis used world-
wide as a vaccine for tuberculosis.
C5a. A chemotactic peptide released when the
fifth component of complement is activated.
C-C and C-X-C chemokines. See “Inter-
leukin 8.”
CD (cluster of differentiation). CD numbers
are the names given to cell surface markers that
have been identified by specific monoclonal or
polyclonal antibodies. Over 200 CD antigens have
so far been identified, and many are listed in ref-
erences 1 and 7. Most helper T lymphocytes are
CD4 positive, and most cytotoxic T lymphocytes
are CD8 positive (see chapter 6).
Cell-mediated immunity (CMI). See defin-
ition in part I.
Chemokines. Chemotactic cytokines that
attract various types of leukocytes into sites of
inflammation. See “Interleukin 8” and “Mono-
cyte chemoattractant (activating) protein 1.” (See
references 2 and 4).
Colony-forming units (CFU). CFU arise
from bacteria deposited on solid culture medium.
Cytokines. Locally produced proteins that affect
the behavior of cells in their vicinity. Some
cytokines (e.g., IL-1, IL-6, and TNF-) enter the
bloodstream and cause an acute-phase reaction,
including fever. (See reference 6).
Cytotoxic T lymphocytes (CTLs). CTLs
can kill other cells, especially those containing
viruses and other intracellular microorganisms,
GLOSSARY 䡵 433
such as tubercle bacilli. Most CTLs are major his-
tocompatibility complex 1-restricted CD8 T cells,
but some are CD4 cells.
Delayed-type hypersensitivity (DTH). See
definition in part I.
Dendritic cells (DC). See “Antigen-present-
ing cells” in part I.
Endothelial-leukocyte adhesion molecule
(ELAM). ELAM,or E-selectin,enables polymor-
phonuclear leukocytes (and some monocytes) to
emigrate from the microvasculature. (See refer-
ence 3).
Epithelioid cells. Large macrophages within
tuberculous lesions in various states of activation (⫹
to ⫹⫹⫹⫹). They received their name because
they frequently adhere to one another to form
sheets that resemble epithelium. In rabbits, mature
epithelioid cells have rounded borders with rather
homogeneous cytoplasm caused by finely dispersed
lipid material. Immature epithelioid cells have irreg-
ular borders with a vacuolated cytoplasm.
Fas and Fas ligand. Fas is a receptor on cer-
tain cells that makes such cells undergo apoptosis
(programmed cell death) when Fas ligand on a
“killer” cell combines with this receptor. Fas is a
member of the same receptor family as the recep-
tor for TNF, and Fas ligand is a member of the
TNF family itself.
Gamma interferon (IFN-). IFN- is a major
macrophage-activating cytokine produced by T
lymphocytes and natural killer cells. IFN- inhibits
the multiplication of tubercle bacilli within
macrophages.All interferons enable cells to inhibit
viral replication.
Gamma interferon-inducible protein 10 (IP-
10). IP-10 is a C-X-C chemokine (see “Inter-
leukin 8”) that attracts monocytes and NK cells.
Glycol methacrylate (GMA). Tissues embed-
ded in polymerized GMA can be cut at 1 to 2 µm.
Heat shock protein 65 (Hsp 65). A consti-
tutive antigen released from dead tubercle bacilli
and other microorganisms. Heat shock proteins
enable microorganisms and cells of higher organ-
isms to survive adverse environmental conditions.
HEPA filters. High-efficiency particle air
(HEPA) filters will not let airborne bacteria pass
through.
HIV/AIDS. Human immunodeficiency virus/
acquired immunodeficiency syndrome.
434 䡵 GLOSSARY
[3H]TdR. Tritiated thymidine.
IFN-. See “Gamma interferon.”
Immunoglobulin G (IgG). IgG is the most
abundant class of circulating immunoglobulins
(antibodies).
Inducible nitric oxide synthase (iNOS).
iNOS is upregulated as macrophages become acti-
vated.This synthase produces nitric oxide (NO),
which in turn forms other reactive nitrogen inter-
mediates. Reactive nitrogen intermediates and
reactive oxygen intermediates (ROIs) enable
macrophages to kill intracellular tubercle bacilli.
Intercellular adhesion molecules (ICAMs).
ICAMS of vascular endothelium bind to inte-
grins on leukocytes (e.g., the leukocyte functional
antigen 1 [LFA-1]). This binding enables leuko-
cytes to leave the circulation and enter sites of
inflammation. ICAMs also play a role in all cell-
cell interactions, including the binding of lym-
phocytes to dendritic cells. (See reference 3).
Interleukin 1-beta (IL-1). IL-1 is a primary
cytokine that upregulates the production of other
cytokines.
Interleukin 2 (IL-2). IL-2 causes antigen-
specific T lymphocytes to clonally expand their
population in tuberculous lesions. It is produced
by the T lymphocytes in this population.
Interleukin 8 (IL-8). A C-X-C chemokine
(same as neutrophil attractant-activating protein 1).
C ⫽ cysteine; X ⫽ any single amino acid that sep-
arates the two cysteines. IL-8 attracts all granulo-
cytes and a subpopulation of lymphocytes into
sites of inflammation.
Interleukin 10 (IL-10). IL-10 is an inhibitor
of the Th1 cytokine response and an inducer of
the Th2 cytokine response. It is produced by
Th2 lymphocytes and other cells (including
macrophages). IL-10 suppresses macrophage
functions.
Interleukin 12 (IL-12). IL-12 is the major
cytokine that induces natural killer cells and lym-
phocytes to produce IFN-. Macrophages that
have ingested tubercle bacilli produce large
amounts of IL-12. Dendritic cells exposed to bacil-
lary antigens also do so.
Knockout mice. Mice in which portions of
their DNA have been removed or inactivated. For
example, a TNF- knockout mouse does not pro-
duce the cytokine TNF-, and a CD8 knockout
mouse does not produce CD8 lymphocytes.
Koch phenomenon. Classically, the Koch
phenomenon is the local necrotic skin reaction
produced in a tuberculous guinea pig by the intra-
dermal injection of tubercle bacilli. Details are pre-
sented in chapter 15.
Macrophage inflammatory protein 1 (MIP-
1). MIPs are chemokines attracting mono-
cytes and T lymphocytes.
Major histocompatibility complex (MHC).
A set of genes encoding glycoproteins in cell
membranes called MHC molecules (4). These
molecules are major histocompatibility antigens
that enable the host to distinguish self from non-
self tissues, and they are involved in antigen pro-
cessing and host defense functions. MHC class I
molecules present peptides generated in the cytosol
to CD8 T cells, and MHC class II molecules pre-
sent peptides degraded in intracellular vesicles to
CD4 T cells (4).
Miliary tubercles. Tubercles of hematogenous
origin, often 1 to 2 mm in diameter, or about the
size of a millet seed.
Monocyte chemoattractant (activating) pro-
tein 1 (MCP-1). MCP-1 is a C-C chemokine
(see “Interleukin 8”). It attracts into sites of inflam-
mation monocytes/macrophages,T lymphocytes,
dendritic cells, and NK cells, as well as eosinophils,
basophils, and mast cells.
Mononuclear cells (MN). Histological term
for a cell infiltrate containing macrophages, den-
dritic cells, and medium to large lymphocytes, in
which each cell type is not specifically identified
by immunohistochemical methods. In general, the
MN population of tuberculous lesions consists
mostly of macrophages, a substantial proportion of
lymphocytes, and some dendritic cells.We did not
include small lymphocytes as members of the
mononuclear cell population that we counted in
tuberculous lesions, because their size enabled us
to distinguish them from macrophages. In this
book, I often use the term macrophages rather than
mononuclear cells to describe the cell population
in tuberculous lesions, which is only precise when
we have identified the macrophages histochemi-
cally by their -galactosidase (or acid phosphatase)
content.The percentage of the macrophage pop-
ulation in the lesions that are really dendritic cells
is not known. Both cell types have the same ori-
gin and each may possibly convert to the other
type.

Natural killer (NK) cells. NK cells (both local
and circulating) are large, granular, non-T, non-B
lymphocytes.They are an important early defense
against intracellular microorganisms (viruses, bac-
teria, fungi, and protozoa). In tuberculosis, NK
cells can kill bacilli-laden macrophages and can
produce IFN-, which activates macrophages and
stimulates a Th1-cytokine immune response.
Neutrophil attractant-activating protein 1
(NAP-1). Same as IL-8.
Old Tuberculin. An unfractionated filtrate of
autoclaved Mycobacterium tuberculosis cultures. It
contains more antigens than PPD does.
Opsonins. Molecules, such as antibodies, the
C3b component of complement, mannose-binding
protein, and fibronectin, that attach to the surface
of microorganisms so that they can be phagocytized
(usually by granulocytes and macrophages).
PCR (polymerase chain reaction). PCR is
a procedure that amplifies a specific sequence in
DNA by repeated cycles of synthesis driven by
pairs of reciprocally oriented primers. (See
“RT-PCR,” below.)
Peripheral blood mononuclear cells (PBMC).
Mostly lymphocytes, but they include mono-
cytes/macrophages, dendritic cells, and NK cells.
Phosphate-specific transport protein 1 (PstS-
1). An antigen secreted from live tubercle bacilli.
Plasma cells. Antibody-producing cells of B-
lymphocyte origin. See “Lymphocytes (T cells
and B cells)” in part I.
Plethismograph. The apparatus that we used
to measure the volume of air breathed by a given
rabbit.This measurement enabled us to calculate
the volume of the aerosol of tubercle bacilli sub-
sequently breathed by this rabbit (8).
PPD (purified protein derivative). PPD is
the purified protein derivative of M. tuberculosis
used for intradermal tuberculin testing. It con-
tains several of the many mycobacterial antigens.
The original PPD standard (PPD-S) was pro-
duced by Florence B. Seibert of the Henry Phipps
Institute of the University of Pennsylvania (9).
First-strength PPD-S is 1 TU (tuberculin unit), an
injection of 0.02 g. It is used when the patient
is expected to have a positive reaction. Second-
strength PPD-S is 250 TU (5.0 g). It can be
used in persons testing negative with the first
strength. As a general test for human tuberculin
positivity, intermediate-strength PPD-S (5 TU
GLOSSARY 䡵 435
[0.1 g]) is commonly used in the United States.
The worldwide standard today is PPD-RT23,
produced by the Statens Serum Institut in Copen-
hagen, Denmark. PPD-RT23 is used as 2 TU
(0.04 g) for first strength and 10 TU (0.20 g)
for second strength.
The dose of PPD for laboratory animals has not
been established. P.-J. Cardona’s laboratory uses
0.04 g of PPD-RT23 (2 TU) in humans, 0.50 g
(25 TU) in guinea pigs, and 5.0 g (250 TU) in
mice. D. N. McMurray’s laboratory uses 100 TU
of PPD-RT23 in rabbits and 50 TU of PPD-
RT23 in guinea pigs. (The PPD from Mycos
Research LLC, Loveland, Calif., is used in similar
concentrations.) Lurie used Old Tuberculin, diluted
1:10 in rabbits, and diluted 1:20 in guinea pigs. Our
laboratory found that 250 TU of PPD-S gave a
skin test reaction in rabbits comparable to the
1:10 dilution of Old Tuberculin.
The above concentrations can only serve as
guidelines, because the size of the dermal tuber-
culin reaction does not decrease in direct propor-
tion to the tuberculin concentration. In BCG-
vaccinated rabbits, the size of the tuberculin
reactions produced by a 1:20 dilution of Old
Tuberculin was only slightly smaller than the size
of those produced by a 1:10 dilution, not half the
size.
Pulmonary alveolar macrophages (AM).
See definition in Part I.
RANTES (regulated upon activation, nor-
mal T cell expressed and secreted). A
chemokine attracting monocytes, T cells, den-
dritic cells, natural killer cells, eosinophils, and
basophils.
Reactive oxygen intermediates (ROIs) and
reactive nitrogen intermediates (RNIs).
ROIs and RNIs are produced by activated
macrophages. These reactive intermediates enable
macrophages to kill (or inhibit) the tubercle bacilli
within them.
Regulatory (suppressor) T lymphocytes (Treg).
Treg produce cytokines and probably other factors
that keep the T-cell immune response from becom-
ing excessive. They also prevent autoimmune
diseases.
Reverse transcription-polymerase chain
reaction (RT-PCR). Reverse transcription
enables mRNA to form the complementary
DNA. Then, DNA polymerase is used with
436 䡵 GLOSSARY
specific primers in a series of heating and cool-
ing steps to make multiple copies of comple-
mentary sense and antisense DNAs. RT-PCR is
used to identify genes from the mRNA that such
genes produce. In time, the genes of tubercle
bacilli that enable (i) their multiplication in non-
activated macrophages, (ii) their dormancy in
solid caseous tissue, and (iii) their multiplication
in liquefied caseum might be identified by this
method. Real-time PCR is a more quantitative
version of the polymerase chain reaction, because
it monitors the time course of the DNA accu-
mulation.
Shwartzman phenomenon. Produced by
endotoxins, i.e., bacterial lipopolysaccharides (LPS),
either locally in the skin or systemically in many
organs. It occurs only after a second injection of
endotoxin (usually at 24 h), but not after the first
injection.The systemic reaction is characterized by
intravascular thrombosis that may cause ischemic
necrosis (6, 10). The first LPS injection was evi-
dently detoxified well, but the second injection was
not detoxified, apparently because the detoxifica-
tion mechanisms were exhausted by the first LPS
injection (6, 11, 12). The second LPS injection
probably activates the clotting cascade, so that
thrombosis occurs. Other possibilities have also
been described (6, 10, 11). Mycobacterial tuber-
culin-like antigens have an endotoxin-like effect
in hosts with active tuberculosis (13).Therefore, the
Shwartzman phenomenon may cause some of the
thrombosis produced by tissue-damaging DTH in
this disease.
T cells (Th1 and Th2). T lymphocytes [see
“Lymphocytes (T cells and B cells)” in part I].Th1
cells are T-helper lymphocytes that produce IFN-
 and other cytokines that activate macrophages.
Th2 cells are T-helper lymphocytes that produce
IL-4, IL-5, and other cytokines that activate B
lymphocytes (B cells) to produce antibody.
T-cell immunoglobulin mucin gene (TIM)
family. TIM proteins are expressed by T cells
and regulate their function.
Transforming growth factor beta (TGF-).
A cytokine that is a major downregulator of
inflammatory and immune processes. It is also a
major cytokine stimulating fibroblasts to produce
connective tissue for repair and healing.
Tubercles. Accumulations of lymphocytes and
macrophages (along with some other cell types) in
organs of the tuberculous host.Tubercles range in
size from microscopic to almost a centimeter and
may or may not contain caseous centers.
Tumor necrosis factor alpha (TNF-). A
primary cytokine,like IL-1,that upregulates the pro-
duction of other cytokines.TNF- and IFN- are
major macrophage activators.TNF- is the defin-
ing member of the TNF family of cytokines (see
“Fas and Fas ligand”). It is both cell associated and
secreted.
von Willebrand factor (vW factor). vW fac-
tor is stored in vascular endothelial cells and
released when they are injured. Then, vW factor
binds to the exposed vascular basement mem-
brane, where it helps platelets stick and initiate the
clotting cascade.
Vascular cell adhesion molecule 1 (VCAM-1).
A member of the immunoglobulin supergene fam-
ily. It helps monocytes/macrophages, lymphocytes,
and eosinophils emigrate from the bloodstream
into sites of inflammation.
Vascular endothelial growth factor (VEGF).
A cytokine, produced by monocytes/macrophages,
keratinocytes, and other cells, that stimulates vas-
cularization.
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 INDEX

A hemoptysis, 80, 81
Abstracts, of text chapters,Appendix F, 419–428 lymphocytes and plasma cells, 82–83, 88
Acknowledgments,Appendix G, 429–430 metaplastic alveolar epithelium and chemotaxis
Acquired cellular resistance (ACR) of alveolar macrophages, 83–84, 88, 89
definition, 431 number of bacilli, 81, 83, 84, 85
duration and specificity, 102 AIDS/HIV, 59–60, 433
overview, 102 Airborne infections, natural, 215–227
recall upon reinfection, 111–112 Lurie’s experiments, 215–224
ACTH (adrenocorticotropic hormone), 280–282 resistance to establishment of lesions by bovine-
Acute infections, general characteristics of, 371 type bacilli, 216–218
Acute inflammatory lesions, vascular adhesion mole- resistance to establishment of lesions by human-
cules in, 331 type bacilli, 223–224
Acute-phase reaction, 432 resistance to progress of pulmonary tuberculosis
Adaptive immunity by bovine-type bacilli, 218–223
definition, 431 resistance to progress of pulmonary tuberculosis
innate immunity relation to, 112–114 by human-type bacilli, 224
nonspecific and antigen-specific immune responses Riley’s experiments, 224–226
in, 114 Alveolar macrophages
ADCC (antibody-dependent cell-mediated cytotoxi- activation, 54–55
city), 432 chemotaxis, 83–84
Adjuvant, vaccine, 345–346 definition, 432
Adrenocorticotropic hormone (ACTH), 280–282 division of activated in early lesions in rabbits, 175
Adult-type tuberculosis, 11, 12, 13, 14, 15, 51–52 ingestion of tubercle bacilli, 16, 22–23, 36
AED (androstenediol), 282 innate immunity and, 98, 114
Aerosolized infection, see also Inhalation of bacilli location in lesion, 37
bacillary titers in stationary stage in rabbits, mice, logarithmic growth of bacilli within nonactivated,
and guinea pigs, 261–262 24–25
fate of BCG in rabbits, mice, and guinea pigs, 262 microbicidal power of, 16
histopathology of liquefaction of caseous foci and role in establishment of grossly visible primary
cavity formation by virulent M. bovis in rab- tubercles, 200
bits, 79–84 susceptibility versus resistance, 37–38
cavity formation, 78, 79–80 vaccine effects, 342
epithelioid cells, 80–81 X-irradiation, effects of, 293–296
fibroblasts, 82, 87, 88 Androstenediol (AED), 282
granulated macrophages, 80, 82 Angiogenesis, regulators of, 167

439
440 䡵 INDEX
Antibody
enhancement of local DTH and CMI reactions,
106–107, 108, 110
host resistance to reinfection, role in, 378
role, as elucidated by Lurie’s eye chamber experi-
ments, 107, 109, 111
Antibody-dependent cell-mediated cytotoxicity
(ADCC), 432
Antigen-presenting cells (APCs), 99, 113–114, 121–
123; see also Dendritic cells
definition, 431
published reviews on presentation, 123
T cell activation, 121–122
Antimicrobials, resistance to, 31, 43, 57–59
Apoptosis, 142–143, 193, 263, 432
Arrested lesions, 28, 37, 38, 60–62
CMI and DTH required for, 101–102
Arthus reaction, 432–433
Atelectasis, 44
Azathioprine, cavity prevention by, 66
B rates of activation in primary and reinfection
B cells, 433
BAL (bronchoalveolar lavage), 296
BALT (bronchial-associated lymphoid tissue), 343
Bar Harbor rabbits, 241–242
Basophils, 144
B7.1/B7.2, 433
BCG, see also Mycobacterium bovis BCG
cytokines in primary lesions, 301–309
cell types containing MCP-1, IL-1, IL-8, and
TNF- mRNAs, 304
comparison among cytokines, 306–307
cytokine mRNA in cells within tissue sections,
301–304
IFN- mRNA identified by RT-PCR, 305
immunohistochemical studies, 305
overview, 301–304
cytokines in reinfection lesions, 312–323
lesion size, ulceration, and healing, 313
local cell infiltration, 313
mononuclear cells and granulocytes in lesions,
315–319
number of tubercle bacilli in, 319
definition, 433
dermal lesions in rabbits
BCG preparations for, 354–355
development and healing, 355–356
healing as measure of host resistance, 359
histopathology, 356–357
number of bacilli in lesions, 357–358
pulmonary lesions compared, 358–359
fate of aerosolized BCG in rabbits, mice, and
guinea pigs, 262
intradermal injection to produce caseous necrosis
and liquefaction, 68
intravenous in rabbits, Lurie’s experiments with,
359–360
macrophages
activation, 127–128
enzymes released extracellularly, 128–129
heterogeneity in lesions, 129, 131, 133
turnover, activation, and division in healing der-
mal lesions, 189, 191
in vitro division of activated in BCG lesions, 179
in vitro division of cells containing bacilli in
BCG lesions, 179
in vivo [3H]TdR labeling, 179, 180
microvasculature of lesions
histopathology, 163–166
production of developing and healing lesions,
161–162
mononuclear cells
activation in tuberculin reactions and in lesions
caused by nonspecific irritants, 192–193
in blood, labeled, 185–186
disappearance from lesions, 182–185
division rate within lesions, 184–185
entry into lesions, 180, 182
lesions, 187, 189
pulmonary lesions in rabbits, production by intra-
venous injection, 170–175
rabbit response to inhaled bacilli, 233–234
vaccination
combination vaccines, 349
inapparent lesions and, 61–62
in mice and guinea pigs, 343–344
in monkeys, 258, 260
nonspecific irritants, effect on, 193
recombinant technology improved, 349
strong CMI and weak DTH, 344–345
systemic nature in humans, 360–361
vascular adhesion molecules, 327–336
activation of microvascular endothelium and
caseous necrosis, 334–335
in acute inflammatory lesions, 331
in epithelioid cells, 333
functions, 333–334
identification of microvessels in tissue sections of
BCG lesions, 329
leukocyte ligands in BCG lesions, 333
overview in, 328–329
in primary BCG lesions, 329–330
quantitation in tissue sections, 329
questions to be answered, 335–336
in reinfection BCG lesions, 331, 332
virulence, 13, 14
X-irradiation effects on dermal BCG lesions,
293
Blood supply
activation of microvascular endothelium and
caseous necrosis, 334–335
angiogenesis, regulators of, 167
capillary density, determined by gelatin-colloidal
carbon perfusion, 162–163

microvascular patency, in mouse pulmonary lesions,
254
microvessels in tissue sections of BCG lesions, 329
pathophysiology of, 167–168
vascular thrombosis role in causing caseous necro-
sis, 375–376
Bronchial spread
adult-type tuberculosis, 11, 14
cavity formation and liquefaction, 29, 30–31,
42–46
contagiousness, 11–12
Bronchial-associated lymphoid tissue (BALT), 343
Bronchoalveolar lavage (BAL), 296
Burkholderia pseudomallei, 226–227
C tion, 68, 71
C5a, 24, 433
Calcification of nodules, 37, 38, 45
Capillary density, determined by gelatin-colloidal
carbon perfusion, 162–163
Caseous necrosis
activation of microvascular endothelium, 334–335
advanced fibrocaseous tuberculosis, 46–47
caseous tissue as structural component of lesions,
155–156
causes
table of, 156
vascular thrombosis, role of, 375–376
cavity formation, 65–91
healed lesions, 45, 158
liquefaction, 29, 30–31, 42–46, 65–91
progressive lesions, 46
radiography of encapsulated nodules, 38
research, suggested future, 375–376
bacillary growth in liquefied caseum, 379
bacillary survival in solid caseum, 379
stages of tuberculosis and
stage 3, early stage, 25–28
stage 4, progression or arrest of lesion, 28
stage 5, cavity formation, 29, 30–31
Caseous pneumonia, 31, 45, 53, 57
Cathepsin D, 68, 128
Cavity formation, 65–91; see also Liquefaction
aerosolized virulent M. bovis in rabbits, 71–84
background, 71
high-dose experiments, 73, 74–78
histopathology, 79–84
low-dose experiments, 71–72, 74
tuberculin sensitivity, 78–79
analysis of lesions, 59
bronchoscope production in rabbits, 68
delayed-type hypersensitivity (DTH), role of, 66
in guinea pigs, 256, 263
histopathology, rabbit, from aerosolized virulent M.
bovis
cavity formation, 78, 79–80
epithelioid cells, 80–81
fibroblasts, 82, 87, 88
INDEX 䡵 441
granulated macrophages, 80, 82
hemoptysis, 80, 81
lymphocytes and plasma cells, 82–83, 88
metaplastic alveolar epithelium and chemotaxis
of alveolar macrophages, 83–84, 88, 89
number of bacilli, 81, 83, 84, 85
hydrolytic enzymes, role of, 66
literature review, 66–71
bronchoscope production of cavities in rabbits,
68
dermal BCG lesions in pilot studies on caseation
and liquefaction, 68
distinction between caseation and liquefaction,
67–68
effects of large numbers of bacilli on liquefac-
role of delayed-type hypersensitivity, 66
role of hydrolytic enzymes, 66–67
measuring factors affecting, proposed method for,
90–91
in mice, 263
in monkeys, 263
pathogenesis, 29, 30–31, 42–46, 157–158
in rabbits, 263
recent experiments attempting to reduce, 84–87
Mycobacterium vaccae, studies with, 84, 86
ritonavir, studies with, 86–87
research, suggested future, 375–376
summary, 87, 90–91
tuberculin sensitivity in rabbits and, 78–79
C-C chemokines, 433
CD4, 138, 139, 140, 319, 356
CD8, 99–100, 138, 139, 140, 319, 356
CD (cluster of differentiation), 433
CD1 proteins, 122–123
Cell-mediated immunity (CMI)
acquired cellular resistance (ACR) compared, 102
antibody enhancement of local reactions, 106–107
arrested lesions, required for, 101–102
CMI/DTH ratio
favorable, 100–101
ideal, 374
research, suggested future, 374–375
species variations, 375
control of bacillary growth by, 26–28, 247, 249
definition, 99, 431
in guinea pigs, 263–264
kinetic studies in rabbits, insights on provided by,
193–194
local nature of, 102–104
macrophage turnover, 375
in mice, 259–260, 263–264
Mycobacterium vaccae, effect of, 84
overview, 99, 100
in rabbits, 263–265
similarities to DTH in tuberculosis, 99–100, 263
summary of role in pathogenesis of tuberculosis,
369–370
442 䡵 INDEX
Cell-mediated immunity (CMI) (Continued)
synergism with DTH, 27, 104–105
vaccines producing strong CMI, 344–345
Chemokines, 433; see also Cytokines
Chemotaxins, 24
Chemotaxis, of alveolar macrophages, 83–84
Childhood-type tuberculosis, 8, 9–11, 49–51
Chorionic gonadotropin, effects of, 286
Chronic infections, general characteristics of, 371
CMI, see Cell-mediated immunity (CMI)
Complement system, 112–113
Contagiousness, 11–12
Cord factor, 157
Cortisone
development of disease, effects on, 273–275
withdrawal, effects of, 275–279
CTLs (cytotoxic T lymphocytes), 141–142, 433
C-X-C chemokines, 433
Cynomolgus monkeys, tuberculosis in, 258–259, 263
Cytokines
from CMI-producing lymphocytes, 99
definition, 433
downregulation, causes of, 321, 323
in humans, 307–308
in mice, 308–309
mRNAs, 319, 321
networks, 323
non-antigen-specific nature of, 102
nonspecific and antigen-specific production, 309
in primary BCG lesions, 301–309
cell types containing MCP-1, IL-1, IL-8, and
TNF- mRNAs, 304
comparison among cytokines, 306–307
cytokine mRNA in cells within tissue sections,
301–304
IFN- mRNA identified by RT-PCR, 305
immunohistochemical studies, 305
overview, 301–304
in reinfection BCG lesions, 312–323
lesion size, ulceration, and healing, 313
local cell infiltration, 313
mononuclear cells and granulocytes in lesions,
315–319
number of tubercle bacilli in, 319
in tuberculin reactions, 312–323
Cytotoxic T lymphocytes (CTLs), 141–142, 433
D Diapedesis, 328
Dannenberg,Arthur M., Jr.
contributions of, 2–3
publications,Appendix D, 399–416
Dehydroepiandrosterone (DHEA), 282
Delayed-type hypersensitivity (DTH)
antibody enhancement of local reactions, 106–107
arrested lesions, required for, 101–102
caseous necrosis and, 155–157
cavity formation, role in, 30–31
CMI/DTH ratio
favorable, 100–101
ideal, 374
research, suggested future, 374–375
species variations, 375
control of bacillary growth by, 26–28, 247
definition, 98, 431
in guinea pigs, 263–264
kinetic studies in rabbits, insights on provided by,
193–194
liquefaction and cavity formation, role in, 66
local nature of, 102–104
macrophage turnover, 375
in mice, 259–260, 263–264
Mycobacterium vaccae, effect of, 84
overview, 98–99
in rabbits, 193–194, 263–265
similarities to CMI in tuberculosis, 99–100, 263
summary of role in pathogenesis of tuberculosis,
368–370
synergism with CMI, 27, 104–105
tissue-damaging, 26–27, 98–99, 155–157, 432
tuberculin sensitivity, 98
tuberculin skin reaction and, 105–106
vaccines producing weak DTH, 344–345
Dendritic cells
as adjuvants, 122
as antigen-presenting cells (APCs), 99, 121–123
CD1 proteins, 122–123
conventional, 122
major histocompatibility complex (MHC),
122–123
organ effects on, 122
pattern recognition receptors (PRR), 98
plasmacytoid, 122
published reviews on antigen presentation, 123
T-cell activation, 121–122
tolerogenic, 122
as vaccine carriers, 347
Dermal lesions, in rabbits
BCG preparations for, 354–355
development and healing, 355–356
healing as measure of host resistance, 359
histopathology, 356–357
number of bacilli in lesions, 357–358
pulmonary lesions compared, 358–359
Dexamethasone, reactivation of healing pulmonary
tubercles by, 279–280
DHEA (dehydroepiandrosterone), 282
DNA vaccines, 349
Dormancy, 104–105, 124, 126, 263–264
Drugs, suggested future research
acting on bacillus, 380–381
acting on host, 380
DTH, see Delayed-type hypersensitivity (DTH)
E
ELAM (endothelial-leukocyte adhesion molecule),
327–333, 433; see also Vascular adhesion
molecules
 INDEX 䡵 443

Emphysematous bleb or bulla, 45 Granulation tissue, 44, 59, 155

Empyema, 49 Granulocytes, 143–144, 376
Encapsulated nodules, 38, 40 in primary and reinfection BCG lesions, 315–319
Eosinophils, 144 Granuloma, macrophage turnover in mouse, 193
Epithelioid cells, 133; see also Langhans’ giant cells Growth curves, bacillary
adhesion molecules in, 333 guinea pig, 254–255
definition, 433 mouse, 247
histopathology in aerosolized virulent M. bovis in Guidelines for Preventing the Transmission of Mycobac-
rabbits, 80–81 terium tuberculosis in Health-Care Settings, CDC,
immature, 28, 103, 133 Appendix E, 417–418
mature, 26, 38, 49, 80–81, 103, 133 Guinea pigs
in miliary tubercles, 42 bacillary titers in stationary stage after aerosol
ESAT-6 antigen, 106 infection, 261–262
Establishment of infection, 35–38 establishment and progress of tuberculosis, 224
airborne infections, natural immunization of, 256, 343–344
resistance to establishment of lesions by bovine- inbred strains, 256
type bacilli, 216–218 organ resistance, 262
resistance to establishment of lesions by human- pulmonary tubercle counts in, 207, 210
type bacilli, 223–224 tuberculin sensitivity, 262
factors influencing, 15–16 tuberculosis in, 254–257
subapical localization, 54–55 bacillary growth curves, 254–255
vaccine, effects of cavity formation, 256, 263
macroscopic tubercle, 342–343 cell-mediated immunity (CMI), 263–264
microscopic tubercle, 342 characteristics of lesions, 260
Estrogen, effects of, 285–286 comparison to mice, humans, and rabbits,
Extracellular dormancy, 263–264 259–260
Exudative lesions, 39, 41, 42 delayed-type hypersensitivity (DTH), 263–264
fate of aerosolized BCG, 262
F fibrin meshworks, 256
Fas, 433 gross pathology, 255–256
Fas ligand, 433 histopathology, 256
Fatty acids, toxic, 31 immunization, 256
Feldman,William H., 385–386 Koch phenomenon, 257
Fibrin meshworks, 256 virulence of inhaled bacilli, 260–261
Fibroblasts, 144 Gut-associated lymphoid tissue (GALT), 343
Fibrocaseous tuberculosis, advanced, 46–47, 54
Fibrosis, 45 H
Filters, HEPA, 16, 17, 18, 19 Head cover, 17, 18, 19
N-Formyl-methionyl-leucyl-phenylalanine, 24 Healing
Freezing and thawing, effect on infectivity, 203–204 caseous necrosis, 45, 158
cytokines in reinfection BCG lesions, 313
G dermal BCG lesions in rabbits, 355–356
-Galactosidase, 67, 102–104, 127 macrophage turnover, activation, and division in
GALT (gut-associated lymphoid tissue), 343 BCG lesions, 189, 191
Gamma interferon (IFN-), 99, 106, 124, 141, 305– as measure of host resistance, 241, 359
308, 433 reactivation of healing pulmonary tubercles by
Gelatin-colloidal carbon perfusion, 162–163 glucocorticoids, 279–280
Genetic resistance, 37–38 Heat shock protein 65, 433
in mice, 251, 253 Hematogenous spread
tubercle-count method, determination by, 204, 207 in childhood-type tuberculosis, 8, 9–11
Ghon complex, 11, 37, 49–52, 58 miliary tuberculosis, 40–42
Glossary, 431–436 Hemoptysis
Glucocorticoids cavity formation and, 44
ACTH effects, 280–282 histopathology, 80, 81
development of disease, effects of cortisone on, HEPA filters, 16, 17, 18, 19
273–275 Histopathology
reactivation of healing pulmonary tubercles by, aerosolized virulent M. bovis lesions in rabbits,
279–280 79–84
withdrawal, effects of, 275–279 dermal BCG lesions in rabbits, 356–357
444 䡵 INDEX

Histopathology (Continued) Immunocompromised hosts, 59–60, 123

guinea pig, tuberculosis in, 256 Immunoglobulin G (IgG), 433
hemoptysis, 80, 81 Immunohistochemical studies for cytokine proteins,
microvascular-cell interactions, 163–167 305
BCG lesions, 163–166 Immunology of tuberculosis, 95–145
tuberculin reactions, 164, 167 acquired cellular resistance (ACR)
mouse pulmonary tuberculosis lesions, 253–254 definition, 431
HIV/AIDS, 59–60, 433 duration and specificity, 102
Hoods, 17, 18, 19 overview, 102
Hormones recall upon reinfection, 111–112
ACTH effects, 280–282 adaptive immunity
androstenediol (AED), 282 definition, 431
chorionic gonadotropin, effects of, 286 innate immunity relation to, 112–114
dehydroepiandrosterone (DHEA), 282 nonspecific and antigen-specific immune
estrogen, effects of, 285–286 responses in, 114
glucocorticoids antibody enhancement of local DTH and CMI
ACTH effects, 280–282 reactions, 106–107, 108, 110
development of disease, effects of cortisone on, antibody role, as elucidated by Lurie’s eye chamber
273–275 experiments, 107, 109, 111
reactivation of healing pulmonary tubercles by, antigen-presenting cells (APCs), 99, 113–114,
279–280 121–123
withdrawal, effects of, 275–279 arrested lesions, CMI and DTH required for,
thyroid hormones, effects of, 286–291 101–102
Host-parasite interactions cell-mediated immunity (CMI)
host response, suggested research on local control acquired cellular resistance (ACR) compared,
of, 373 102
principles of, 370–372 antibody enhancement of local reactions,
summary of, 367 106–107
Human disease, types of, 9–11 arrested lesions, required for, 101–102
adult-type tuberculosis, 11, 12, 13, 14, 15 control of bacillary growth by, 26–28, 247, 249
childhood-type tuberculosis, 8, 9–11 definition, 99, 431
Hydrolytic enzymes, 31 in guinea pigs, 263–264
in activated macrophages, 128 kinetic studies in rabbits, insights on provided by,
liquefaction and cavity formation, role in, 66 193–194
local nature of, 102–104
I macrophage turnover, 375
ICAM (intercellular adhesion molecule), 327–333, in mice, 259–260, 263–264
433–434; see also Vascular adhesion molecules Mycobacterium vaccae, effect of, 84
IFN-, 99, 106, 124, 141, 305–308, 433 overview, 99, 100
Immunity, see also Cell-mediated immunity (CMI) in rabbits, 263–265
adaptive similarities to DTH in tuberculosis, 99–100, 263
definition, 431 summary of role in pathogenesis of tuberculosis,
innate immunity relation to, 112–114 369–370
nonspecific and antigen-specific immune synergism with DTH, 27, 104–105
responses in, 114 vaccines producing strong CMI, 344–345
innate cells involved, 120–145
definition, 431 antigen-presenting cells, 99, 113–114, 121–123
overview, 98 epithelioid cells, 133
relation to acquired (adaptive) immunity, granulocytes, 143–144
112–114 Langhans’ giant cells, 133, 136
systemic, 104 lymphocytes, 137–140
Immunization, see also Vaccine macrophages, 104, 123–133
degree produced, tubercle-count method for deter- mononuclear cell turnover, 136–137
mining, 204 NK cells, 140–141, 142
guinea pig, 256 table of, 121
monkey, 258, 259 CMI/DTH ratios, 100–101
postinfection, 349 favorable, 100–101
resistance to melioidosis and tuberculosis com- ideal, 374
pared, 226–227 research, suggested future, 374–375
 INDEX 䡵 445

species variations, 375 L

delayed-type hypersensitivity, 98–99 Labeling of mononuclear cells
DTH and CMI similarities, 99–100 in vitro [3H]TdR labeling, 178–179
DTH and CMI synergism, 27, 104–105 in vivo [3H]TdR labeling, 179, 180
innate immunity Langhans’ giant cells, 38, 39, 40, 42, 133, 136
definition, 431 Laryngitis, tuberculous, 56, 71
overview, 98 Latency, 126; see also Dormancy
relation to acquired (adaptive) immunity, Leukocyte ligands in BCG lesions, 333
112–114 Leukocytes, diapedesis of, 328
macrophage activation, 104 Leukotriene B4, 24
systemic immunity, 104 LFA-1, 333, 335
tuberculin skin test Liquefaction, 65–91
booster phenomenon in repeated testing, aerosolized virulent M. bovis in rabbits, 71–84
105–106 background, 71
size of reaction, prognostic value of, 105 high-dose experiments, 73, 74–78
Immunosuppression, effects of, 59–60 histopathology, 79–84
Immunotherapy low-dose experiments, 71–72, 74
dendritic cells, 347 tuberculin sensitivity, 78–79
Mycobacterium vaccae, 17, 77, 347 bronchogenic spread and, 42–46
research, suggested future, 380 caseation compared, 67–68
Inapparent lesions causes and results, 67, 157–158
in humans, 60–62 histopathology, rabbit, with aerosolized virulent
in rabbits, 62 M. bovis
Inducible nitric oxide synthase (iNOS), 433 cavity formation, 78, 79–80
Infectivity, freezing and thawing effect on, 203–204 epithelioid cells, 80–81
Inhalation of bacilli fibroblasts, 82, 87, 88
bacillary titers in stationary stage after aerosol granulated macrophages, 80, 82
infection, 261–262 hemoptysis, 80, 81
fate of BCG in rabbits, mice, and guinea pigs, lymphocytes and plasma cells, 82–83, 88
262 metaplastic alveolar epithelium and chemotaxis
Lurie’s tubercle-count, 206, 208–209 of alveolar macrophages, 83–84, 88, 89
number inhaled, 15–16 number of bacilli, 81, 83, 84, 85
rabbit response to, 233–234 literature review, 66–71
virulence of bacilli in rabbits, mice, and guinea bronchoscope production of cavities in rabbits,
pigs, 260–261 68
X-irradiation effects on virulent human-type dermal BCG lesions in pilot studies on caseation
bacilli, 296–297 and liquefaction, 68
Innate immunity distinction between caseation and liquefaction,
definition, 431 67–68
overview, 98 effects of large numbers of bacilli on liquefac-
relation to acquired (adaptive) immunity, 112–114 tion, 68, 71
iNOS (inducible nitric oxide synthase), 433 role of delayed-type hypersensitivity, 66
Intercellular adhesion molecule (ICAM), 327–333, role of hydrolytic enzymes, 66–67
433–434; see also Vascular adhesion molecules measuring factors affecting, proposed method for,
Interleukins 90–91
definition, 434 in pathogenesis process, 29, 30–31
IL-2, 307, 434 recent experiments attempting to reduce,
IL-8, 304, 306, 307, 319, 321–323, 434 84–87
IL-10, 113, 307, 434 Mycobacterium vaccae, studies with, 84, 86
IL-12, 113–114, 141, 307, 434 ritonavir, studies with, 86–87
IL-1, 304, 306, 307, 319, 321–323, 434 research, suggested future, 376–377
Th cell production of, 138 summary, 87, 90–91
Intracellular dormancy, 263–264 Logarithmic growth
Isocitrate lyase, 124 end of by DTH and CMI, 25–28, 247, 249
within macrophages, 24–25
K Long, Esmond R., 387–388
Knockout mice, 434 Lurie, Max B.
Koch phenomenon, 257, 434 antibody role, as elucidated by eye chamber experi-
Koch, Robert, 26 ments, 107, 109, 111
446 䡵 INDEX
Lurie, Max B. (Continued)
award of Trudeau medal for 1955,Appendix A,
385–386
contributions of, 1–2
intravenous BCG experiments, 359–360
natural airborne infection experiments, 215–224
obituary,Appendix B, 387–388
publications,Appendix C, 389–398
pulmonary tubercle-count method, 196–210
Lurie’s inbred rabbits
adrenocorticotropic hormone (ACTH), 280–282
airborne infections, natural, 215–224
resistance to establishment of lesions by bovine-
type bacilli, 216–218
resistance to establishment of lesions by human-
type bacilli, 223–224
resistance to progress of pulmonary tuberculosis
by bovine-type bacilli, 218–223
resistance to progress of pulmonary tuberculosis
by human-type bacilli, 224
characteristics of lesions in, 260
fate of, 241
genetic experiments, 238–241
healing rate of dermal lesions as method of deter-
mining resistance, 241
history and description of, 235–236
intradermal BCG vaccine in Lurie’s inbred rabbit
strains
lesions, 361
protection, amount of, 361–363
vaccine, 361
relative resistance of strains, 236
resistance and susceptibility to tuberculosis,
237–238
Lymphatic spread, in childhood-type tuberculosis, 9,
49–51
Lymphatics in tuberculous lesions, 144–145
Lymphocytes
apoptosis and, 142
CD4, 138, 139, 140
CD8, 99–100, 138, 139, 140
cell-mediated immunity and, 99
cytotoxic T cells, 141–142, 433
definition, 431–432
delayed-type hypersensitivity and, 98
gamma-delta () T cells, 138–140
histopathology of aerosolized virulent M. bovis in
rabbits, 82–83, 88
memory T cells, 140
NK cells, 140–141, 142, 434
overview, 137–138
regulatory T cells, 138, 435
Th cells, 138, 435–436
turnover, 136–137
Lysosomes, 123–124
M in pulmonary lesion in rabbits, early
Mac-1, 333
Macaca fascicularis, 258–259
Macaca mulatta, 257–258
Macrophage inflammatory proteins (MIPs), 434
Macrophages, 123–133; see also Epithelioid cells
activation, 54–55, 99, 100, 104, 126–128
in healing dermal BCG lesions, 189, 191
in vitro division of activated in BCG lesions, 179
alveolar
activation, 54–55
chemotaxis, 83–84
definition, 432
division of activated in early lesions in rabbits,
175
ingestion of tubercle bacilli, 16, 22–23, 36
innate immunity and, 98, 114
location in lesion, 37
logarithmic growth of bacilli within nonacti-
vated, 24–25
microbicidal power of, 16
role in establishment of grossly visible primary
tubercles, 200
susceptibility versus resistance, 37–38
vaccine effects, 342
X-irradiation, effects of, 293–296
bacillary survival within, 379
blood-borne, 24–25, 36–37, 170–175
in caseous tissues, 155–156
chemotaxins, 24
definition, 432
delayed-type hypersensitivity (DTH) killing of,
26–28
division
of alveolar activated in early lesions in rabbits,
175
in early pulmonary lesions in rabbits, 155–156
in healing dermal BCG lesions, 189, 191
in vitro division of activated in BCG lesions,
179
in vitro division of cells containing bacilli in
BCG lesions, 180
enzymes released extracellularly in BCG lesions,
128–129
fate of mycobacteria within, 123–124
granulated
histopathology of aerosolized virulent M. bovis in
rabbits, 80, 82
heterogeneity
in BCG lesions, 129, 131, 133
causes, 131, 133
in vitro division
of activated in BCG lesions, 179
of cells containing bacilli in BCG lesions, 179
labeling of
in vitro [3H]TdR labeling, 178–179
in vivo [3H]TdR labeling, 180
location in lesion, 37, 59
nonspecific activation, 22–23, 54
activation and division of blood-borne, 170–175
division of activated pulmonary alveolar, 175

receptors, 123
turnover, 136–137
in healing dermal BCG lesions, 189, 191
in mouse tuberculous granulomas, 193
research, suggested future, 375
Major histocompatibility complex (MHC)
activation of microvascular endothelium contribu-
tion to caseous necrosis, 334–335
cytotoxic T cells and, 142
dendritic cells, 122–123
NK cells, 140
Masks, 16, 19
Mast cells, 144
MCP-1 (monocyte chemoattractant [activating] pro-
tein 1), 304–306, 308, 319, 321–323, 434
Medical radiation, public health consequences of,
297–298
Melioidosis, 226–227
Meningitis, tuberculous, 58
Metaplastic alveolar epithelium
histopathology of aerosolized virulent M. bovis in
rabbits, 83–84, 88, 89
Metaplastic bronchial epithelium, 45
MHC, see Major histocompatibility complex (MHC)
Mice
cytokines, 308–309
establishment and progress of tuberculosis, 224
immunization of, 343–344
melioidosis, immunization and resistance to,
226–227
pulmonary lesions
histopathology, 253–254
microvascular patency, 254
pulmonary tubercle counts in, 207, 210
tuberculin sensitivity, 262
tuberculosis in, 247–254
apoptosis, 263
bacillary growth curves and type of disease,
247
bacillary titers in stationary stage after aerosol
infection, 261–262
bovine-type bacilli, 251
cavity formation, 263
cell-mediated immunity (CMI), 263–264
characteristics of lesions, 260
comparison to rabbits, humans, and guinea pigs,
259–260
delayed-type hypersensitivity (DTH), 263–264
fate of aerosolized BCG, 262
genetic differences in resistance, 251, 253
human-type bacilli, 251
logarithmic stage of growth, ending, 247, 249
stationary stage of growth, maintaining, 249
tissue necrosis, 249–251, 263
virulence of inhaled bacilli, 260–261
Microbicidal power, of pulmonary alveolar
macrophages, 16
Microvascular density in tuberculous lesions, 161–168
angiogenesis, regulators of, 167
INDEX 䡵 447
blood supply, pathophysiology of, 167–168
capillary density, determination of, 162–163
histopathology of microvascular-cell interactions,
163–167
BCG lesions, 163–166
tuberculin reactions, 164, 167
overview, 161
production of BCG lesions and tuberculin reac-
tions, 161–162
Microvascular endothelium, activation and caseous
necrosis, 334–335
Microvascular patency, in mouse pulmonary lesions,
254
Microvessels, identification in tissue sections of BCG
lesions, 329
Miliary tuberculosis, 40–42, 43, 44, 61
MIPs (macrophage inflammatory proteins), 434
Monkeys, tuberculosis in, 257–259, 263
Monocyte chemoattractant protein 1, 24
Monocyte chemoattractant (activating) protein 1
(MCP-1), 304–306, 308, 319, 321–323, 434
Monocytes, symbiotic growth of bacilli within non-
activated, 24–25
Mononuclear cells, see also Macrophages
BCG lesions
activation in tuberculin reactions and in lesions
caused by nonspecific irritants, 192–193
in blood, labeled, 185–186
disappearance from lesions, 182–184
division rate within lesions, 185
entry into lesions, 180, 182
rates of activation in primary and reinfection
lesions, 187, 189
definition, 434
labeling
in vitro [3H]TdR labeling, 178–179
in vivo [3H]TdR labeling, 180
in primary and reinfection BCG lesions, 315–319
turnover, 136–137
Mycobacterium bovis, aerosolized virulent in rabbits,
79–84
histopathology
cavity formation, 78, 79–80
epithelioid cells, 80–81
fibroblasts, 82, 87, 88
hemoptysis, 80, 81
lymphocytes and plasma cells, 82–83, 88
metaplastic alveolar epithelium and chemotaxis
of alveolar macrophages, 83–84, 88, 89
number of bacilli, 81, 83, 84, 85
Mycobacterium bovis BCG
intradermal injection to produce caseous necrosis
and liquefaction, 68
macrophage activation, 127–128
macrophage enzymes released extracellularly,
128–129
macrophage heterogeneity in lesions, 129, 131,
133
virulence, 13, 14
448 䡵 INDEX

Mycobacterium microti role of delayed-type hypersensitivity, 66

vaccine efficacy in New Zealand White rabbits, role of hydrolytic enzymes, 66–67
363 summary, 87, 90–91
virulence, 14 overview, 7–19
Mycobacterium tuberculosis contagiousness, 11–12
Guidelines for Preventing the Transmission of Mycobac- establishment of pulmonary lesion, factors influ-
terium tuberculosis in Health-Care Settings, CDC, encing, 15–16
Appendix E, 417–418 prevention of clinical disease, 17–19
mutants as possible new vaccines, 349 resistance, factors influencing, 16–17
strain virulence, 13–15 size of infectious particles, 12–13
virulence by Lurie tubercle-count method types of human disease, 9–11
CDC1551 (Oshkosh) strain, 201, 203, 204 virulence of bacillary strains, 13–15
Erdman strain, 203 stages, 22–31
H37Rv strain, 201, 202, 203, 204 stage 1, ingestion of bacilli by macrophages,
Mycobacterium vaccae 22–23
effect on cavity formation, 84, 86 stage 2, logarithmic growth of bacilli within
immunotherapy, 17, 347 macrophages, 24–25
for active tuberculosis from inhalation of viru- stage 3, end of logarithmic growth by DTH and
lent bovine-type bacilli, 77 CMI, 25–28
stage 4, progression or arrest of caseous lesions,
N 28
Necropsies, safety precautions for, 18 stage 5, cavity formation and bronchial spread of
Necrosis, see Caseous necrosis disease, 29, 30–31
Neutrophil attractant-activating protein 1 (NAP-1), summary, 367–368
434 table of, 30
Nitric oxide synthase (NOS), 123, 124 types of human pulmonary tuberculosis, 9–11,
NK (natural killer) cells, 140–141, 142, 434 34–62
adult-type tuberculosis, 11, 12, 13, 14, 15, 51–52
O advanced fibrocaseous, 46–47
Old tuberculin, 434 cavitary lesions, analyses of, 59
Opsonins, 434 childhood-type tuberculosis, 8, 9–11, 49–51
Organ resistance, 102–104, 158–159 early primary lesion, 38
guinea pigs, 262 encapsulated caseous or calcified nodules, 38
rabbits, 262 establishment of infection, 35–38
research, suggested future, 373–374 exudative lesions, 39, 41, 42
Ossification, 38, 45 in immunocompromised host, 59–60
inapparent lesions, 60–62
P liquefied caseous lesions, 42–46
Parasite-host interactions miliary, 39–42
host response, suggested research on local control multidrug-resistant bacilli, 57–59
of, 373 pneumonia and pleurisy, 47–49
principles of, 370–372 progressive, locally destructive lesions, 46
summary of, 367 proliferative lesions, 38–39, 40, 42
Pathogenesis subapical localization, 51–57
liquefaction of caseous foci and cavity formation, table, 35
65–91 Pathogenicity, virulence of bacillary strain and,
aerosolized virulent M. bovis in rabbits, 71–84 13–15, 24
bronchoscope production of cavities in rabbits, Pattern recognition receptors (PRR), 98, 113–114
68 PBMC, see Peripheral blood mononuclear cells
dermal BCG lesions in pilot studies on caseation Pepstatin, 67
and liquefaction, 68 Perforin, 142
distinction between caseation and liquefaction, Pericarditis, 48
67–68 Peripheral blood mononuclear cells (PBMC), 307–
effects of large numbers of bacilli on liquefac- 308, 434
tion, 68, 71 Persistence, of tubercle bacilli, 158
literature review, 66–71 Phagosomes, 123–124
measuring factors affecting, proposed method Phosphate-specific transport protein 1 (PstS-1), 435
for, 90–91 Plasma cells
recent experiments attempting to reduce, 84–87 definition, 435

histopathology of aerosolized virulent M. bovis in
rabbits, 82–83, 88
Plasmacytoid dendritic cells, 122
Plethismograph, 435
Pleurisy, 48–49
Pneumonia
caseous pneumonia, 31, 45, 53, 57
multidrug-resistant, 59
overview, 47–49
rupture of liquefied lesion and, 44
Polymorphonuclear cells (PMNs), 143–144
interleukin mRNAs in, 321
in primary and reinfection BCG lesions, 315–319
PPD (purified protein derivative), 435; see also Tuber-
culin skin test
Prevention of clinical disease, 17–19
Primary lesion, early, 38
Prognostic tests, 377–378
Programmed cell death, see Apoptosis
Progression, prognostic tests for, 377–378
Progressive lesions, 46
Proliferative lesions, 38–39, 40, 42
Propylthiouracil, 289–291
PRR, see Pattern recognition receptors (PRR)
PstS-1 (phosphate-specific transport protein 1),
435
Public health consequences of medical radiation,
297–298
Pulmonary alveolar macrophage, see Alveolar
macrophages
Pulmonary lesion
caseous necrosis
cavity formation, 29, 30–31
early stage, 25–28
progression or arrest of lesion, 28
dermal BCG lesions compared in rabbits, 358–359
factors influencing establishment of, 15–16
establishment of infection, 16
microbicidal power of pulmonary alveolar
macrophages, 16
number of bacilli inhaled, 15–16
mouse
histopathology of, 253–254
microvascular patency of, 254
in rabbits, early, 170–175
macrophages, activation and division of blood-
borne, 170–175
macrophages, division of activated pulmonary
alveolar, 175
production by intravenous injection of bacilli,
advantages of, 170
reactivation of healing pulmonary tubercles by glu-
cocorticoids, 279–280
resistance to progress in Lurie’s rabbits
bovine-type bacilli, 218–223
human-type bacilli, 224
Pulmonary lesion-count method, Lurie’s, 196–210
Purified protein derivative (PPD), 435; see also Tuber-
culin skin test
INDEX 䡵 449
R
Rabbits
airborne infections, natural, 215–227
Lurie’s experiments, 215–224
Riley’s experiments, 224–226
Bar Harbor inbred, 241–242
BCG, intravenous, 359–360
BCG dermal lesions
BCG preparations for, 354–355
development and healing, 355–356
healing as measure of host resistance, 359
histopathology, 356–357
number of bacilli in lesions, 357–358
pulmonary lesions compared, 358–359
commercial inbred, 242
comparison of tuberculosis in humans, 226
histopathology of lesions from aerosolized virulent
M. bovis, 79–84
Lurie’s inbred
adrenocorticotropic hormone (ACTH),
280–282
airborne infections, natural, 215–224
characteristics of lesions in, 260
fate of, 241
genetic experiments, 238–241
healing rate of dermal lesions as method of
determining resistance, 241
history and description of, 235–236
intradermal BCG vaccine in, 361–363
relative resistance of strains, 236
resistance and susceptibility to tuberculosis,
237–238
Lurie’s tubercle-count method, 196–210
model of tuberculosis, advantages of, 199–200
organ resistance, 262
pulmonary lesion, early, 170–175
macrophages, activation and division of blood-
borne, 170–175
macrophages, division of activated pulmonary
alveolar, 175
production by intravenous injection of bacilli,
advantages of, 170
response to inhaled bacilli
BCG, 233–234
bovine-type bacilli, 231–233
human-type bacilli, 233
Lurie’s resistant rabbits, 231–233
Lurie’s susceptible rabbits, 231
New Zealand White rabbits, 233
strains, new resistant and susceptible, 378
Thorbecke, 242–244
tuberculin reactions, 358
tuberculin sensitivity, 262
tuberculosis in, 247, 248
bacillary titers in stationary stage after aerosol
infection, 261–262
cavity formation, 263
cell-mediated immunity (CMI), 263–265
characteristics of lesions, 260
450 䡵 INDEX
Rabbits (Continued)
comparison to mice, humans, and guinea pigs,
259–260
delayed-type hypersensitivity (DTH), 263–265
fate of aerosolized BCG, 262
virulence of inhaled bacilli, 260–261
X-irradiation effects, 297
vaccine
efficacy of BCG in New Zealand White rabbits,
363
efficacy of M. microti in New Zealand White
rabbits, 363
maximal effectiveness, 348–349
van Zutphen, 241
Radiation (X-irradiation), 292–298
alveolar macrophage from granulomatous lungs,
effects on, 294–296
dermal BCG lesions, effects on, 293
inhalation of virulent human-type bacilli, effects
on, 296–297
lethal dose, 293
public health consequences of medical radiation,
297–298
pulmonary alveolar macrophage populations,
effects on, 293–294
in rabbits, 297
recovery from effects of, 296
Radiographs
of encapsulated nodules, 38
lesion size, 38
Reactivation, 52, 59, 279–280
Reactive oxygen intermediates (ROIs), 435
Recombinant technology improved vaccines, 349
Regression, prognostic tests for, 377–378
Regulatory (suppressor) T lymphocytes, 138, 435
Reinfection, 51–52
acquired cellular resistance (ACR) recall, 111–112
antibody role in resistance to, 378
cytokines in BCG lesions, 312–323
lesion size, ulceration, and healing, 313
local cell infiltration, 313
mononuclear cells and granulocytes in lesions,
315–319
number of tubercle bacilli in, 319
rates of activation of mononuclear cells in BCG
lesions, 187, 189
vascular adhesion molecules in BCG lesions, 331,
332
Research, suggested future, 373–384
on the bacillus
growth in liquefied caseum, 379
survival in air, 378–379
survival in solid caseum, 379
survival within macrophages, 379
virulence, 379–380
on drugs
acting on bacillus, 380–381
on the host
antibody role in host resistance to reinfection,
378
CMI/DTH ratio, 374–375
DTH and CMI and macrophage turnover, 375
granulocytes, role of, 376
host response, local control of, 373
lesions produced by live and dead tubercle
bacilli, comparisons of, 378
liquefaction and cavity formation and preven-
tion, 375–376
organ resistance and its implications, 373–374
prognostic tests that reflect disease progression
and regression, 377–378
rabbit strains, new resistant and susceptible, 378
vascular thrombosis, role in caseous necrosis and
inhibition of bacillary growth, 375–376
on immunotherapy, 380
on vaccines, 380
Resistance
acquired cellular resistance (ACR), 102, 111–112
in adults versus children, 51
airborne infections, natural
to establishment of lesions by bovine-type
bacilli, 216–218
to establishment of lesions by human-type
bacilli, 223–224
to progress of pulmonary tuberculosis by
bovine-type bacilli, 218–223
to progress of pulmonary tuberculosis by
human-type bacilli, 224
antibody role in, 378
bovine-type and human-type bacilli, 159
factors influencing, 16–17
genetic, 37–38, 204, 207, 251, 253
healing as measure of host resistance, 241, 359
host
healing as measure of, 241, 359
tubercle-count to determine, 204
to melioidosis, 226–227
mouse strains, 251, 253
new rabbit strains, 378
organ, 102–104, 158–159, 263, 373–374
species, 158–159
versus susceptibility, 24–25, 37–38
symbiotic growth of bacilli within macrophages,
24–25, 37–38
of tubercle bacilli to antimicrobials, 31, 57–59
Respirators, 16, 17
Reverse transcription-polymerase chain reaction, see
RT-PCR
Rhesus monkeys, tuberculosis in, 257–258, 259, 263
Riley, R. L., 224–226
Ritonavir, studies with, 86–87
RNase, 68, 128
ROIs (reactive oxygen intermediates), 435
Room air sampling, 226
RT-PCR (reverse transcription-polymerase chain
reaction)
 INDEX 䡵 451

definition, 435 Titers, in stationary stage in rabbits, mice, and guinea

IFN- mRNA identified by, 305 pigs, 261–262
TNF- (tumor necrosis factor alpha), 99, 141, 304–
S 308, 320–321, 323, 436
Satellite lesions, 46 Tolerogenic dendritic cells, 122
Shwartzman phenomenon, 435 Toll-like receptors, 112, 113, 123
Simon foci, 53 Transforming growth factor beta (TGF-), 436
Size, of infectious particles, 12–13 Triiodothyronine, effects of, 286–291
Stages in pathogenesis, 22–31 Trudeau medal for 1955, 385–386
stage 1, ingestion of bacilli by macrophages, Tubercle bacilli
22–23 dormancy, 124, 126, 263–264
stage 2, logarithmic growth of bacilli within ending of logarithmic growth by DTH and CMI,
macrophages, 24–25 25–28
stage 3, end of logarithmic growth by DTH and ingestion by macrophages, 16, 22–23, 36, 123–124
CMI, 25–28 inhaled, number, 15–16
stage 4, progression or arrest of caseous lesions, 28 inhibition of growth, 375–376
stage 5, cavity formation and bronchial spread of intracellular versus extracellular growth, 43
disease, 29, 30–31 logarithmic growth within macrophages, 24–25
table of, 30 number in dermal rabbit BCG lesions, 357–358
STAT-1, 124 persistence, 158
Strain, virulence of, 13–15 research, suggested future
Subapical localization growth in liquefied caseum, 379
in adult-type tuberculosis, 51–52 survival in air, 378–379
causes of, 52–54 survival in solid caseum, 379
physiology of apical versus basal pulmonary survival within macrophages, 379
regions, 53–54 virulence, 379
source of bacilli, 52–53 resistance, 31, 43
establishment of lesions, effect on, 54–55 virulence
progress of lesions, effect on, 55–57 research, suggested future, 378–380
Summary strain, 13–15, 24
host-parasite interactions, 367 Tubercle-count method, Lurie’s, 196–210
pathogenesis, role of DTH and CMI in, 368–370 advantages of rabbit model of tuberculosis,
pathogenesis, stages, 367–368 199–200
Suppressor T-cells, 138 aerosol infections versus intravenous infections,
Surfactant proteins, 113 comparison of, 206–207, 210
Symbiotic growth of bacilli within nonactivated bacillary virulence, 207
macrophages, 24–25 host genetic resistance, 207
Systemic immunity, 104 vaccine efficacy, 207
alveolar macrophage role in establishment of
T grossly visible primary tubercles, 200
T cell comparison of counts, 204–206
activation by dendritic cells, 121–122 genetic resistance of host, determination of, 204
cytotoxic, 141–142, 433 in guinea pigs and mice, 207, 210
gamma-delta (), 138–140 immunization degree, determination of, 204
helper, 138, 435–436 inhaled dose, effect of, 206, 208–209
memory, 140 overview, 197–198
regulatory, 138, 435 variability of counts, 205, 206
TGF- (transforming growth factor beta), 436 virulence, determination of
Th cells, 138, 435–436 bovine-type bacilli, 201
Thorbecke rabbits, 242–244 clinical Mycobacterium tuberculosis isolate, 201,
Thyroid hormones, effects of, 286–291 203, 204
Thyroidectomy, 289–291 freezing and thawing, effect on infectivity, 203–
Thyroxine, effects of, 286–291 204
Tissue damage human-type bacilli (H37Rv), 201, 202, 203, 204
causes, 156–157 overview, 200–201
delayed-type hypersensitivity (DTH), 26–27, 98– Tubercles
99, 155–157 alveolar macrophage role in establishment of
Tissue necrosis in mice, 249–251, 263 grossly visible primary tubercles, 200
452 䡵 INDEX

Tubercles (Continued) production by intravenous injection of bacilli,

definition, 436 advantages of, 170
development in humans, 197 resistance to establishment in Lurie’s rabbits
in rabbits caused by inhaled BCG, 233–234 bovine-type bacilli, 216–218
reactivation of healing pulmonary tubercles by glu- human-type bacilli, 223–224
cocorticoids, 279–280 structural components, 155–159
Tuberculin reactions caseous tissues, 155–157
cytokines in, 312–323 granulation tissue, 155
microvasculature of lesions healing, 158
histopathology, 164, 167 liquefied caseum and cavities, 157–158
production of developing and healing lesions, organ and species resistance, 158–159
161–162 overview, 155
mononuclear cell activation in, 192–193 persisting viable bacilli, 158
in rabbits, 358 vascular adhesion molecules in, 327–336
Tuberculin sensitivity; see also Delayed-type hypersen- activation of microvascular endothelium and
sitivity (DTH) caseous necrosis, 334–335
cavity formation in rabbits and, 78–79 in acute inflammatory lesions, 331
disease control and, 346 in epithelioid cells, 333
in laboratory animals, 262 functions, 333–334
Tuberculin skin test identification of microvessels in tissue sections of
anergy, 105 BCG lesions, 329
booster phenomenon in repeated testing, 105–106 leukocyte ligands in BCG lesions, 333
early positives, 38 overview in, 328–329
size of reaction, prognostic value of, 105 in primary BCG lesions, 329–330
Tuberculin traps, 185 quantitation in tissue sections, 329
Tuberculin unit, 99 questions to be answered, 335–336
Tuberculous lesions, 155–210; see also Pathogenesis in reinfection BCG lesions, 331, 332
arrested, 28, 37, 38, 60–62 Tumor necrosis factor alpha (TNF-), 99, 141, 304–
CMI and DTH required for, 101–102 308, 320–321, 323, 436
early in humans, 263 Types of human disease, 9–11
inapparent, 60–62 adult-type tuberculosis, 11, 12, 13, 14, 15
Lurie’s pulmonary lesion-count method, 196–210 childhood-type tuberculosis, 8, 9–11
lymphatics in, 144–145 Types of human pulmonary tuberculosis, 9–11,
macrophages, 177–194 34–62
activation, 189, 191 adult-type tuberculosis, 11, 12, 13, 14, 15, 51–52
division, 189, 191 advanced fibrocaseous, 46–47
in vitro division of activated in BCG lesions, 179 cavitary lesions, analyses of, 59
in vitro [3H]TdR labeling, 178–179 childhood-type tuberculosis, 8, 9–11, 49–51
in vivo [3H]TdR labeling, 180 early primary lesion, 38
overview, 178 encapsulated caseous or calcified nodules, 38
turnover in healing dermal BCG lesions, 189, establishment of infection, 35–38
191 exudative lesions, 39
turnover in mouse tuberculous granulomas, 193 in immunocompromised host, 59–60
microvascular density, 161–168 inapparent lesions, 60–62
angiogenesis, regulators of, 167 liquefied caseous lesions, 42–46
blood supply, pathophysiology of, 167–168 miliary, 39–42
capillary density, determination of, 162–163 multidrug-resistant bacilli, 57–59
histopathology of microvascular-cell interactions, pneumonia and pleurisy, 47–49
163–167 progressive, locally destructive lesions, 46
overview, 161 proliferative lesions, 38–39
production of BCG lesions and tuberculin reac- subapical localization, 51–57
tions, 161–162 table, 35
produced by live and dead bacilli, comparisons of,
378 U
pulmonary lesions in rabbits, early, 170–175 Uric acid, 24
macrophages, activation and division of blood- UV (ultraviolet) lights, for disease prevention, 18–19
borne, 170–175
macrophages, division of activated pulmonary V
alveolar, 175 Vaccine, 341–350

BCG
combination vaccines, 349
inapparent lesions and, 61–62
in mice and guinea pigs, 343–344
in monkeys, 258, 260
nonspecific irritants, effect on, 193
recombinant technology improved, 349
strong CMI and weak DTH, 344–345
systemic nature in humans, 360–361
combination, 349
composition of improved
adjuvant composition, 345–346
antigen composition, 345
degree of immunization produced, tubercle-count
method for determining, 204
DNA vaccines, 349
efficacy
in aerosol infections versus intravenous infec-
tions, comparison of, 207
BCG in New Zealand White rabbits, 363
M. microti in New Zealand White rabbits, 363
standardization for comparing relative, 346–347
future possibilities, 349–350
intradermal BCG in Lurie’s inbred rabbit strains
lesions, 361
protection, amount of, 361–363
vaccine, 361
intradermal in Lurie’s inbred rabbit strains,
361–363
M. tuberculosis mutants, 349
maximal effectiveness
factors influencing, 348
in humans, 347–348
in rabbits, 347–348
mice and guinea pigs, immunization of, 343–344
postinfection immunization, 349
pulmonary tubercle establishment, effects on
macroscopic tubercle, 342–343
microscopic tubercle, 342
recombinant technology improved vaccines, 349
research, suggested future, 380
reviews of possible new, 349–350
routes of administration, 343
systemic nature of BCG vaccination in humans,
360–361
van Willebrand factor, 436
van Zutphen rabbits, 241
Vascular adhesion molecules, 327–336
activation of microvascular endothelium and
caseous necrosis, 334–335
INDEX 䡵 453
in acute inflammatory lesions, 331
in epithelioid cells, 333
functions, 333–334
identification of microvessels in tissue sections of
BCG lesions, 329
leukocyte ligands in BCG lesions, 333
overview in, 328–329
in primary BCG lesions, 329–330
quantitation in tissue sections, 329
questions to be answered, 335–336
in reinfection BCG lesions, 331, 332
Vascular endothelial growth factor (VEGF), 436
Vascular thrombosis, role in caseous necrosis and
inhibition of bacillary growth, 375–376
VCAM-1 (vascular cell adhesion molecule-1), 327–
333, 436
VEGF (vascular endothelial growth factor), 436
Virulence
of bacillary strain, 13–15, 24, 207
inhaled bacilli in rabbits, mice, and guinea pigs,
260–261
tubercle-count method, determination by
bovine-type bacilli, 201
clinical Mycobacterium tuberculosis isolate, 201,
203, 204
freezing and thawing, effect on infectivity,
203–204
human-type bacilli (H37Rv), 201, 202, 203,
204
overview, 200–201
VLA-4, 333
W
Whole-body irradiation, see X-irradiation
X
X-irradiation, 292–298
alveolar macrophage from granulomatous lungs,
effects on, 294–296
dermal BCG lesions, effects on, 293
inhalation of virulent human-type bacilli, effects
on, 296–297
lethal dose, 293
public health consequences of medical radiation,
297–298
pulmonary alveolar macrophage populations,
effects on, 293–294
in rabbits, 297
recovery from effects of, 296