Critical Reviews in Oral Biology and Medicine, 3(1/2):31-60 (1992)

Host Mediators in Gingival Crevicular Fluid:

Implications for the Pathogenesis of
Periodontal Disease
Ira B. Lamster
Division of Periodontics, School of Dental and Oral Surgery, Columbia University, 630 West 168th
Street, New York, NY 10032

M. John Novak
Department of Periodontics, Dental School, University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284

ABSTRACT: During the past few years, a considerable number of studies have examined different aspects of
the host response in gingival crevicular fluid (GCF), including the relationship of specific markers to the active
phases of periodontal disease. Various indicators of the acute inflammatory response (the lysosomal enzymes
P-glucuronidase and collagenase, the cytoplasmic enzyme aspartate aminotransferase, and the arachidonic acid
metabolite PGE2) have been shown to be associated with clinical attachment loss in chronic adult periodontitis
in man and experimental periodontitis in animal models. In contrast, the relationship of indicators of the humoral
immune response in GCF to active periodontal disease is equivocal. Furthermore, a number of indicators of the
cellular immune response have been identified recently in GCF (i.e., Interleukin-lat, IL-1P, tumor necrosis
factor-a), but their relationship to active phases of periodontal disease have not been studied.
The polymorphonuclear leukocyte (PMN) is the cellular hallmark of acute inflammation. Evidence from
the GCF studies suggests that hyperreactivity of these cells plays a critical role in the active phases of some
forms of periodontal disease. Metabolic activation of PMN can be associated with a number of potentially
destructive reactions. The major effector mechanism for tissue destruction that can be specifically identified
with the PMN is the synergistic effect of the release of PMN proteases and the generation of reactive oxygen
metabolites by these cells. Priming of the PMN, where the PMN response is enhanced by agents that do not
initiate the response, may be an important mechanism for PMN activation in the crevicular environment; for
example, cytokines such as IL- 1 p and TNF-a, and lipopolysaccharides released from subgingival Gram-negative
bacteria, can serve this function. The hypothesis proposed here argues that in addition to the severe forms of
periodontal disease that have been associated with qualitative or quantitative PMN defects, tissue destruction
in the periodontum can be observed with hyperreactivity of these cells. These differing conclusions do not create
a dilemma, but may represent opposite ends of a balance that is no longer in equilibrium.

KEY WORDS: gingival crevicular fluid (GCF), polymorphonuclear leukocyte (PMN), tissue destruction,
periodontal disease.

I. INDICATORS OF THE HOST pendent upon laboratory diagnostic procedures

RESPONSE IN GINGIVAL CREVICULAR for analysis of biological fluids and tissues. These
FLUID tests serve to identify specific biochemical me-
diators, microbial agents, or cellular perturba-
A. Introduction tions that are directly associated with different
pathological processes. As examples, exposure
The practice of medicine and the identifi- to the human immunodeficiency virus is deter-
cation of many systemic disorders is largely de- mined by the presence of an antibody titer in
1045-4411/92/$.50
© 1992 by CRC Press, Inc.
31
serum, the identification of pharyngeal strepto-
coccal infection can be made with an enzyme
immunoassay that identifies specific microbial
antigens, and damage to cardiac muscle follow-
ing myocardial infarction is initially detected by
elevations in cytoplasmic enzymes in serum.
The use of laboratory diagnostic procedures
by dental profession is currently not routine.
the
While immunofluorescent staining of biopsy
specimens is used to diagnose autoimmune dis-
eases affecting the gingiva and mucosa, labora-
tory procedures are not commonly employed for
the management of patients with dental caries or
periodontal disease. In reference to the perio-
dontal diseases, clinical studies published within
the past years have provided a better understand-
ing of the natural history of these common dis-
orders. Once thought to be continuous and slowly
progressive, some periodontal lesions are now
recognized as chronic conditions with relatively
brief periods of exacerbation and longer periods
of quiescence (Socransky et al., 1984). Further-
more, with the determination that classically em-
ployed clinical measures of periodontal disease
are poor identifiers or predictors of the active
phases (Haffajee et al., 1983), there has been
considerable interest in the development of di-
agnostic tests to aid in clinical management of
patients (Fine and Mandel, 1986). These tests
have focused on identification of the subgingival
plaque microbiota associated with the periodontal
diseases and the host response to that infection.
The host response can be examined in a number
of ways. The least invasive approach involves
analysis of gingival crevicular fluid (GCF), an
inflammatory exudate present in the gingival
crevice. This review examines what has been
learned about the relationship of different host
mediators in GCF to the progression of disease,
how the findings from these studies can help to
explain the pathologic basis of chronic adult per-
iodontitis, and discusses specific mechanisms that
can account for tissue destruction.
1. The Inflammatory and Immune
Response in Periodontal Disease
The host response in periodontal disease is
comprised of a complex series of responses to
32
the plaque microorganisms in the crevicular en-
vironment. Various components of the acute in-
flammatory, cellular immune, and humoral im-
mune responses have been demonstrated to occur
in periodontal disease. This response can be de-
tected both systemically and locally. For exam-
ple, lymphocyte transformation by peripheral T
cells in response to plaque antigens has been
demonstrated in patients with periodontal disease
(Ivanyi and Lehner, 1970, Horton et al., 1974),
and increased serum antibody titers to plaque mi-
croorganisms have also been detected in affected
individuals (for review, see Ebersole, 1990). In
addition, chemotactic defects in peripheral blood
polymorphonuclear leukocytes (PMN) from oth-
erwise healthy individuals are detected in patients
with juvenile periodontitis (Cianciola et al., 1977,
Van Dyke et al., 1980). Microscopic examina-
tion of tissue specimens collected from patients
with periodontal disease indicates that normal tis-
sue architecture is altered in association with the
accumulation of T and B lymphocytes, plasma
cells, and macrophages in the gingival connective
tissue, and the presence of large numbers of PMN
in the junctional and sulcular epithelium and gin-
gival crevice (Page and Schroeder, 1976). PMN
comprise better than 90% of the leukocytes pres-
ent in the crevice (Attstr6m, 1970; Attstrom and
Egelberg, 1970).
Of clinical significance, the study of the host
response in periodontal disease may provide a
mechanism to monitor the progression of disease
in humans. At this time, collection and analysis
of peripheral blood and gingival tissue does not
offer a practical diagnostic approach. In contrast,
analysis of an exudate originating from the gin-
gival crevice may provide a noninvasive means
of studying the host response by evaluation of
the constituents of the fluid. GCF is derived from
the periodontal tissues, and its analysis offers to
provide an early indication of biochemical changes
in the tissues that will ultimately manifest as a
clinical lesion. Another distinct advantage of us-
ing GCF analysis as the basis for a diagnostic
test is that the most popular collection system
(precut methylcellulose filter strips) allows mul-
tiple sites in a mouth to be sampled and analyzed.
This is a distinct advantage, since the severity of
human periodontal disease is variable within an
affected dentition.
2. Historical Background
The study of GCF can be traced back more
than 50 years (Bodecker, 1933), but comprehen-
sive examination of fluid production and con-
stituents began in the late 1950s with the studies
of Brill and co-workers (Brill and Krasse, 1958;
Brill and Bjorn, 1959). Those early studies, and
subsequent research during the following 20 years
focused on the mechanisms of GCF production
and cataloged a large number of constituents that
could be detected in the fluid. These studies did
not provide evidence that GCF analysis could be
clinically useful as a measure of periodontal dis-
ease progression (Cimasoni, 1983). This was due
to the absence of a meaningful clinical protocol
to study the relationship between specific GCF
mediators and disease progression. In the early
1980s clinical studies provided a mechanism for
studying periodontal disease progression in hu-
mans by assessing the loss of clinical attachment
on the root surface (Goodson et al., 1981; Haf-
fajee et al., 1983). In addition, the earlier de-
velopment of experimental periodontitis animal
models offered a test system for evaluating the
progression of periodontal disease (Kennedy and
Polson, 1973; Schroeder and Lindhe, 1975). Both
longitudinal trials as well as cross-sectional stud-
ies in human and animal models have been used
to evaluate the relationship of various mediators
in GCF to periodontal disease.
B. Indicators of Cellular and Humoral
Immunity in Gingival Crevicular Fluid
Both the cellular and humoral immune re-
sponses play important roles in the host response
to subgingival plaque microorganisms. Histo-
logic and immunofluorescent examination of bi-
opsy material has identified T cells and B
cells/plasma cells in the gingival connective tis-
sue, with immunoglobulin-bearing cells predom-
inating in established gingivitis or periodontitis
in adults (Longhurst et al., 1977; Mackler et al.,
1977, 1978; Seymour and Greenspan, 1979; Sey-
mour and Greaves, 1980). Antigen-induced blas-
togenesis by T cells from patients with perio-
dontitis was first identified by evaluating
leukocytes collected from peripheral blood (Ivanyi
and Lehner, 1970). T cell-blastogenesis was ob-
served to increase with the severity of the existing
periodontal disease (Horton et al., 1972; Patters
et al., 1980). In serum, antibody that is specific
for subgingival microorganisms has been iden-
tified in many laboratories. In general, the titer
of antibody is higher in patients with periodontitis
when compared with patients with gingivitis or
normal individuals (for review, see Ebersole,
1990). Mediators of the cellular immune and hu-
moral immune responses have been identified in
GCF. These mediators can provide an indication
of the nature of the immune response at the site
of the periodontal lesion.
1. Cellular Immune Response in GCF
Evaluation of the cellular immune response
in GCF was hampered by our ignorance of spe-
cific mediators that have pathologic significance
and the lack of sensitive techniques to quantitate
these mediators in small volumes of fluid. With
the identification and description of the cyto-
kines, and development of monoclonal antibod-
ies to be used for their identification, cellular
immune activity in GCF can now be examined.
The first identification of cytokine activity in GCF
was provided by Charon et al. (1982) and Mer-
genhagen (1984), who studied interleukin-1 (IL-
1) activity in GCF. Using bioassays, the con-
centration of IL-1 in GCF increased when gin-
gival inflammation was present. IL-1-like activ-
ity in GCF enhanced mitogen-induced
blastogenesis by human T lymphocytes, in-
creased IL-2 production by T lymphocytes, and
enhanced fibroblast proliferation. These inves-
tigators could not identify IL-2 activity in GCF
using a lymphoproliferative bioassay. Recently,
enzyme-linked immunosorbent assays (ELISA)
have been used to identify both IL-1 and IL-2 in
GCF. Masada et al. (1990) measured IL-1a and
IL-1 3 in GCF. The biological specificity of these
assays was achieved with the use of monoclonal
antibodies to both molecules, with no cross-reac-
tivity to IL-2, IL-4, IL-6, or tumor necrosis fac-
tor-a (TNF-a). Patients with early onset perio-
dontitis or chronic adult periodontitis were
studied. The authors did not observe any corre-
lation of IL-1 levels with probing depth or vol-
33
ume of GCF collected on precut filter
strips.
However, differences in the level of IL-1 were
detected in samples from the same patient. The
absolute amount of IL-la and IL-1,3 in GCF de-
creased with periodontal therapy (root planing
and chlorhexidine). Also observed was a statis-
tically significant correlation between higher
amounts of IL- 13 in GCF and more IL- 13 mes-
senger RNA in gingival tissue. This correlation
was not observed for IL-la.
The correlation of levels of IL-113 in GCF
and tissues argues that this mediator is potentially
important in periodontal disease. IL-la and IL-
113 have similar proinflammatory effects and may
be important mediators in the periodontium. These
effects include increased binding of PMN and
monocytes to endothelial cells, increased pro-
duction of PGE2 by fibroblasts, induction of lytic
enzymes by lysosome-containing cells, and stim-
ulation of bone resorption in tissue culture (Di-
narello, 1990).
One important source of IL-1 in GCF may
be the macrophage (Masada et al., 1990). Never-
theless, as noted by these authors, other cell types
may be contributing to the IL-1 pool in GCF.
They suggest gingival epithelial cells, endothelial
cells, B lymphocytes, fibroblasts, and PMN. The
contribution of the PMN to the IL-1 pool in GCF
is noteworthy, especially since these cells are the
most numerous leukocytes in the gingival crevice
(Miller et al., 1984). Recently, production of IL-
lp3 by PMN has been demonstrated (Goh et al.,
1989). While these cells may not produce large
amounts of IL-113 on a per cell basis, the total
number of PMN in the crevicular environment
may make the PMN an important source of this
mediator in periodontal disease.
Recently, a preliminary report described the
presence of the cytokine IL-6 in GCF (Geivelis
et al., 1990). GCF samples from sites with deeper
probing depths contained more IL-6 than samples
from shallower sites. This cytokine may also play
a role in the pathogenesis of periodontal disease,
since it has similar, but less pronounced, proin-
flammatory effects as IL-1 (Dinarello 1990).
Early reports examining cytokine activity in
GCF could not identify IL-2 in the fluid (Mer-
genhagen, 1984). This may be due to the lack of
sensitivity of bioassays. Recently, an ELISA for
IL-2 in GCF has been described (Gur-Yosef and
34
Cox, 1986; Cox and Mendoza, 1987). In a pre-
liminary report, these investigators determined
that the IL-2 concentration in GCF from patients
with periodontitis is lower than the IL-2 concen-
tration in GCF from normal sites. This finding
was attributed to increased IL-2 receptor density
at periodontitis sites. While this proposal is in-
triguing, the increased fluid volume collected from
periodontitis sites compared to normal sites makes
interpretation difficult. We have previously ad-
dressed the manner in which GCF constituent
data are reported (Lamster et al., 1985a) and
observed that reporting total activity of the me-
diator, as well as concentration, may be useful
(Lamster et al., 1986).
The presence of the cytokine TNF-a in GCF
has also been investigated recently (Rossomando
et al., 1990). TNF-a is a monocyte-derived cy-
tokine that potentially could be involved in the
pathogenesis of the periodontal lesion due to its
ability to activate PMN, to cause alterations in
endothelial cell function, and to induce the pro-
duction of collagenase and PGE2 by fibroblasts
(Beutler and Cerami, 1990). Using an ELISA,
Rossomando and co-workers detected TNF-a in
only 21% of samples. While TNF-a was found
in sites with a variety of clinical findings, the
presence of TNF-a in GCF was not related to
gingival inflammation, plaque, or existing prob-
ing depth. In fact, total TNF-a in GCF appeared
greater when the tissue was less inflamed. The
concentration of TNF-a in serum is low, and
when detected in GCF, the higher amounts of
TNF-a in GCF suggest that this mediator is re-
leased locally. A longitudinal evaluation of TNF-
a in GCF was not part of this study. Furthermore,
we have recently described a sensitive ELISA for
the detection of gamma interferon (IFN--y) in GCF
(Grbic et al., 1991). Among its other biological
activities, IFN-y has been demonstrated to inhibit
the bone-resorbing action of IL-1 13 in vitro (Go-
wan and Mundy, 1986). IFN-y, therefore, may
play an important regulatory role in inflammatory
and immune reactions. The relationship of IFN-
y in GCF to active periodontal disease is cur-
rently being investigated, but preliminary find-
ings suggest that active attachment loss is asso-
ciated with a reduction of IFN-y in GCF.
In summary, the ability to detect and quan-
titate cytokines in GCF is now possible using
immunoassays. These assays have provided a new
approach to the study of T-cell and macrophage
function in periodontal disease and ultimately may
aid in the clinical management of patients. None
of the cytokines that have been identified in GCF
have been comprehensively evaluated for their
relationship to the active phase of periodontal
disease. Nevertheless, both the presence in GCF
and the varied biological activities of a number
of the cytokines (i.e., IL-1 3, IL-2, TNF-a) sug-
gest that these mediators may play an important
role in human periodontal disease.
2. Humoral Immune Response in GCF
In general, the presence of an increased serum
antibody response to periodontal microorganisms
is a well-characterized part of the host response
in periodontal disease (for review see Ebersole,
1990). Nevertheless, the specific relationship of
this increased antibody titer to the development
of disease is still a subject of controversy. A
review of early studies examining the presence
of serum antibody titers in patients with different
periodontal diseases indicated that an increased
titer to the microorganisms associated with dis-
ease could not always be detected (Genco et al.,
1974). Furthermore, with identification of im-
munoglobulins in GCF (Brandtzaeg, 1965;
Holmberg and Killander, 1971), the importance
of the local humoral immune response was
recognized.
When considering the small volume of GCF
that is present in an undisturbed crevice, intro-
duction of sensitive immunoassays (ELISA) for
the study of antibody in GCF allowed the spec-
ificity of the response to be analyzed (Ebersole
et al., 1984). Using ELISA, the relationship of
specific GCF antibody concentration to the con-
centration of the antibody in serum was examined
(Ebersole et al., 1985a). The antibody analyzed
in GCF was chosen based on an elevated serum
response. It was determined that 9% of GCF sam-
ples had an antibody titer greater than that seen
in serum, while 91% of samples had antibody
levels equal to or less than that in serum. In a
related paper, a correlation between the concen-
tration of specific antibody in GCF and the amount
of that antibody in a homogenate of tissue col-
lected from the same site was demonstrated (Smith
et al., 1985). In addition to the antibody titer in
serum, these data suggested the importance of
locally produced antibody to the pool of antibody
in GCF. Local production of antibody by cells
in the periodontium had been suggested in a num-
ber of earlier studies (Lally et al., 1980; Steubing
et al., 1982).
The issue of the relative levels of local (GCF)
to systemic (serum) antibody in periodontal dis-
ease, and how these titers relate to active disease,
are important in understanding the pathogenesis
of periodontal disease. Naito et al. (1985) ex-
amined antibody to Gram-negative subgingival
bacteria. For periodontitis patients, IgG antibody
levels to one strain of Bacteroides gingivalis (381)
were higher in GCF than serum. In contrast, the
antibody titers to a strain of B. intermedius (24)
and Fusobacterium nucleatum were markedly
lower in GCF. Serum antibody titers from healthy
controls and three groups of periodontitis patients
stratified by disease severity were compared. It
is interesting to note that while serum antibody
was higher in periodontitis patients than healthy
controls, the serum antibody titers to five of the
seven microorganisms were lower in the ad-
vanced periodontitis group compared to the mod-
erate periodontitis group. In a similar study, Tew
et al. (1985) examined serum and GCF antibody
levels in localized juvenile periodontitis and se-
vere periodontitis. They found great variability
in GCF antibody to specific microorganisms, both
between patients and from different sites within
the same patient. This variability was seen pri-
marily as different degrees of reduced antibody
levels in GCF relative to the serum level. How-
ever, these reductions in GCF antibody levels
relative to serum tended to disappear when the
concentration of serum albumin was factored.
With this calculation, the concentration of anti-
body to B. gingivalis and Actinobacillus acti-
nomycetemcomitans Y4 in GCF tended to be
higher than serum. Nevertheless, the sites with
elevated levels of antibody to these microorgan-
isms displayed no differences in clinical param-
eters compared to sites in which the GCF anti-
body titers was not elevated. Genco et al. (1985)
also examined antibody to A. actinomycetem-
comitans in GCF and serum from patients with
various forms of periodontitis. Patients with lo-
35
calized juvenile periodontitis had high serum an-
tibody titers to A. actinomycetemcomitans, and
the titers in GCF generally reflected the serum
level. Occasionally, markedly elevated levels of
antibody to these organisms were seen in GCF,
suggesting local production.
Ebersole et al. (1985b) have evaluated the
correlation of antibody activity in GCF and serum,
and the presence of the microorganisms in the
crevice. To determine the nature of this relation-
ship, they defined a positive correlation as ele-
vated levels of GCF antibody to the microorgan-
ism and the presence of the microorganisms at
the site, or levels of antibody equivalent to or
less than the serum titer and the absence of the
microorganisms at the local site. For patients with
advanced destructive periodontitis, agreement was
seen at 78% of sites. For localized juvenile per-
iodontitis patients, agreement was 71% and for
adult periodontitis agreement at 54% of sites was
observed. A lack of agreement was most fre-
quently seen when the microorganisms were
present, but elevated local antibody was not. The
development of an antibody response to an in-
fection is a normal and appropriate part of the
immune response to that infection (Ebersole,
1990). While a number of potentially destructive
reactions can occur in association with that an-
tibody (Genco et al., 1974), the literature also
supports the concept that the lack of an appro-
priate antibody response in an individual with
periodontal disease can be detrimental to the in-
dividual. Ranney et al. (1982) evaluated the serum
antibody response in young individuals with ju-
venile periodontitis or severe periodontitis. Pa-
tients with the more limited juvenile periodontitis
displayed a greater amount of precipitating an-
tibody to A. actinomycetemcomitans than pa-
tients with the more generalized severe perio-
dontitis. This inverse relationship of antibody titer
and attachment loss was confirmed in other stud-
ies by this group (Gunsolley et al., 1987, 1990).
In addition, Rowland et al. (1989) have dem-
onstrated that patients in the early stage of acute
necrotzing ulcerative gingivitis had a lower serum
antibody to four strains of B. intermedius when
compared with healthy controls. Recently, Taub-
man (personal communication) examined the re-
lationship of antibody concentrations in GCF and
serum. Their large database indicated that the
36
absolute GCF to serum ratio was 0.15 to 0.27,
and only 3% of GCF samples had levels of an-
tibody higher than serum. This ratio was still well
below 1.0 if albumin ratios are considered. He
has concluded that the concentration of antibody
in GCF is lower than the concentration in serum,
and this represents local immunoregulatory
activity.
Considering the heterogeneous nature of the
subgingival microflora, and the resultant heter-
ogeneous nature of the antibody response to those
microorganisms, the data available on the role of
antibody in the pathogenesis of periodontal dis-
ease are difficult to interpret. The emerging con-
clusion is that in a patient with existing perio-
dontal disease a reduction in antibody activity in
serum and GCF is detrimental. Data from our
laboratory have provided supporting evidence for
the protective role of antibody in periodontal dis-
ease (Lamster et al., 1990). We examined the
relationship of antibody activity in serum to a
panel of putative periodontal pathogens to dif-
ferent mediators in GCF, and found that a neg-
ative correlation existed between specific IgG an-
tibody in serum and lysosomal enzyme activity
in the crevice. This study will be reviewed in
greater detail in the next section.
C. Indicators of the Acute Inflammatory
Response in Gingival Crevicular Fluid
The very earliest investigations of the con-
stituents of GCF focused on markers of the acute
inflammatory response. As noted by Cimasoni
(1983), 30 years ago Gustafsson and Nilsson
(1961) detected products of the fibrinolytic sys-
tem in GCF. Subsequently, numerous studies have
reported the presence of serum, leukocyte, and
bacteria-derived enzyme activities in the fluid
(Cimasoni, 1983; Lamster, 1989). During the past
few years, lytic enzyme activity in GCF has been
studied for its role in tissue destruction in the
periodontal environment and as a potential marker
of active periodontal disease. In addition, anal-
ysis of other markers of the acute inflammatory
response in GCF have provided an emerging pic-
ture of the changes in GCF chemistry that char-
acterize the destructive phases of adult
periodontitis.
 The presence of complement components in
GCF was first reported by Attstr6m et al. (1975).
The activation of the complement cascade has
the potential for inducing many changes associ-
ated with the periodontal lesion, including re-
cruitment of PMN, cellular disruption and lysis,
and increased vascular permeability. Niekrash and
Patters (1985a) have described a sensitive crossed-
immunoelectrophoresis technique to allow eval-
uation of complement components in a volume
of GCF as small as 0.2 p1l. Patients with chronic
adult periodontitis were examined before and after
root planing and scaling (Niekrash and Patters,
1985b). Therapy significantly decreased conver-
sion of C3, but no C4 cleavage products were
observed in samples before or after therapy. In-
terestingly, the levels of factor B, indicative of
the alternate pathway, did not change with ther-
apy. They concluded that while complement ac-
tivation in GCF could occur by the classical path-
way, alternate pathway, or proteolytic enzymatic
activation, the data only supported the latter two
mechanisms. For cascade the alternate pathway,
endotoxin was suggested to be an important ac-
tivator. Subsequently, these investigators ex-
amined complement cleavage in GCF collected
from patients with different periodontal condi-
tions (Niekrash and Patters, 1986). Conversion
of C3 was minimal in GCF samples from healthy
individuals (12.6%), followed by gingivitis
(39.8%), chronic adult periodontitis (69.0%),
rapidly progressive periodontitis (76.6%), and
juvenile periodontitis (90.2%). C4 cleavage was
only observed in juvenile periodontitis samples,
suggesting the activation of the classical pathway
only in juvenile periodontitis.
Among its other effects, the activation of the
complement cascade in the periodontal lesion may
be one very important mechanism for recruitment
of PMN into the crevicular environment. The
presence of PMN into the crevice is a normal
part of the host's response to the accumulation
of subgingival bacteria, and these cells are the
host's first line of defense against the expanding
plaque mass (Garant, 1976). It has long been
recognized that qualitative and quantitative
changes in PMN function are associated with early
loss of alveolar bone (Miller et al., 1984; Van
Dyke and Hoops, 1990). In contrast, studies of
enzyme activity in GCF from experimental per-
iodontitis models and longitudinal evaluations of
humans with existing chronic adult periodontitis
suggest the association of exuberant PMN activ-
ity with the destructive phase of periodontal
disease.
The development of a model system to study
the conversion of gingivitis to periodontitis offers
an opportunity to study the clinical, microbiol-
ogical, and biochemical events that characterize
the progression of periodontal disease. Kennedy
and Polson (1973) described a silk ligature-in-
duced periodontitis model in squirrel monkeys,
while Schroeder and Lindhe (1975) described a
similar model in dogs. Analysis of the histologic
changes accompanying the conversion of gingiv-
itis to periodontitis in the squirrel monkey was
described by Heijl et al. (1976). Animals were
sacrificed 1 to 14 d following ligation. They ob-
served that periods of tissue destruction were
characterized by the accumulation of spirochetes
and Gram-negative microorganisms in the crev-
ice, ulceration of the crevicular epithelium, and
a shift in the population of cells in the underlying
connective tissue from plasma cell-rich infiltrate
to a predominance of PMN. Schroeder and Lindhe
(1980) studied the histologic findings in the dog
model and described similar findings, including
ulceration of the sulcular epithelium and devel-
opment of a PMN-rich infiltrate.
The dog model was subsequently used to
evaluate the relationship of collagenolytic en-
zymes and enzyme inhibitors in GCF to the de-
velopment of active disease (Kryshtalskyj et al.,
1986). Using a radioactive collagen fibril assay,
an increase in active collagenolytic activity in
GCF was observed with the development of at-
tachment loss. Samples with greater amounts of
active collagenase also tended to have lower
amounts of enzyme inhibitor activity. Conse-
quently, these investigators (Kryshtalskyj and
Sodek, 1987) studied the nature of the collagen-
olytic activity in GCF collected from these ani-
mals. Analysis of the degradation products using
sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis detected the cleavage pattern char-
acteristic of mammalian, and not bacterial, col-
lagenase. These and other investigators (Larivee
et al., 1986; Villela et al., 1987a, 1987b; Hak-
karainen et al., 1988) studied collagenolytic ac-
tivity in GCF from humans with various forms
37
of periodontal disease. Using similar assays that
allow for detection of collagen cleavage prod-
ucts, GCF from healthy, gingivitis, chronic adult
periodontitis, and localized juvenile periodontitis
patients demonstrated the 3/4, /4 collagen frag-
ments characteristic of the cleavage pattern of
mammalian collagenase. Degradation by bacte-
rial collagenase was either not seen or was ob-
served in only small amounts. These investiga-
tors proposed that the origin of the mammalian
collagenase was either PMN, macrophages, fi-
broblasts, or epithelial cells. The origin of the
collagenase in human GCF has been studied sub-
sequently by Sorsa et al. (1988). The PMN was
the primary source of collagenase in GCF, while
collagenase activity from gingival explants was
derived primarily from fibroblasts.
In summary, animal models to study the de-
velopment of periodontitis have suggested that
the active phase of tissue destruction is related
to the development of a PMN infiltrate in the
crevicular environment. When evaluated con-
currently, tissue destruction and increased col-
lagenolytic activity in GCF were observed in the
dog experimental periodontitis model. The col-
lagenolytic activity in GCF was subsequently
demonstrated, both in animal and human GCF
samples, to display the 3/4, /4 cleavage pattern
of mammalian collagenase. The collagenase in
human GCF was shown to be derived from PMN.
Data from the animal studies provide one line
of evidence that exuberant PMN activity in the
crevicular environment is related to the destruc-
tive phase of periodontal disease. Additional evi-
dence is provided by studying the longitudinal
relationship of enzyme activity in GCF to the
development of clinical attachment loss in
humans.
To evaluate the relationship of different as-
pects of the host response in GCF to the natural
history of human periodontal disease, a biochem-
ical profile for GCF was introduced (Lamster et
al., 1985a). This collection and processing tech-
nique is based on collection of fluid for a stan-
dardized period of time (30 s). Precut filter paper
strips are introduced into the crevice until mild
resistance is felt. The entire mouth can be sur-
veyed in a reproducible manner as samples are
collected from the mesial surfaces of all teeth.
After collection of the samples, the small vol-
38
umes of crevicular fluid collected (generally 1.0
1p1) are then eluted into a larger volume of diluent.
Aliquots of the GCF/diluent solution can then be
assayed for different mediators of the host
response.
Utilizing the human experimental gingivitis
model, different lysosomal enzymes in GCF were
evaluated for their relationship to the develop-
ment of gingival inflammation. In addition to a
measure of gingival inflammation and the volume
of GCF collected, the activity of the ground sub-
stance-degrading enzymes P-glucuronidase (BG)
and arylsulfatase (AS), and collagenase-like ac-
tivity were assayed using modified spectropho-
tometric procedures (Lamster et al., 1985a,
1985b). The collagenase-like activity was as-
sayed using a dinitrophenyl peptide that mimics
the cleavage site of mammalian collagenase on
the collagen molecule. This peptide is also sus-
ceptible to the actions of other collagen-degrad-
ing enzymes, including various gelatinases (Hak-
karainen et al., 1988). In this assay
phenylmethylsulfonyl fluoride was used to in-
hibit substrate hydrolysis by serine proteases, and
aminophenylmercuric acetate was added to a par-
allel assay to activate any latent enzyme in order
to determine the percent of collagenase-like ac-
tivity in the active form. The clinical measure of
tissue inflammation and the volume of GCF in-
creased steadily during the 4-week trial. The in-
crease in active collagenase-like activity also rose
steadily during the entire course of the trial. In
contrast, the activity of the two ground-sub-
stance-degrading enzymes increased during the
first 2 or 3 weeks of the trial, and then remained
at that level. This plateau in BG and AS activity
in GCF may indicate the biochemical stabiliza-
tion of the gingivitis lesion. This preliminary,
short-term longitudinal trial suggested that dif-
ferent host mediators in GCF behaved differently
when followed longitudinally, and perhaps of
greater significance, that analysis of GCF con-
stituents may provide information about the per-
iodontal lesion that is otherwise not available.
Subsequently, patients with chronic adult
periodontitis were monitored longitudinally to
determine the relationship of clinical attachment
loss to different host mediators in GCF (Lamster
et al., 1988). The first analysis of the data ex-
amined the findings for the relationship of the
lysosomal enzymes BG and AS, and the cyto-
plasmic enzyme lactate dehydrogenase (LDH) to
clinical attachment loss in 36 patients monitored
for 6 months. The data from that study suggested
that the enzyme BG in GCF could both identify
clinical attachment loss and predict the occur-
rence of future attachment loss. The mammalian
form of BG is an acid hydrolase that participates
in the degradation of the glycosaminoglycan
component of proteoglycans. Of greater signifi-
cance in GCF, BG is considered a marker for
primary granule release from PMN (Taichman et
al., 1977; Miller and Russel, 1986). The rela-
tionship of BG activity in GCF to clinical at-
tachment loss was confirmed in an analysis of
59 patients monitored for 1 year (Lamster et al.,
1991). In this study, a persistently elevated level
of BG in GCF could identify patients at risk for
clinical attachment loss 3 to 6 months in the fu-
ture. This study also reported on the relationship
of the protease inhibitor a-2-macroglobulin (a-
2-M), IgG and IgM in GCF to clinical attachment
loss. An increase in a-2-M in GCF was observed
in patients experiencing disease activity, and this
paralleled the increase in BG in GCF. In contrast,
compared to patients who did not experience dis-
ease activity, the amount of IgG in GCF was
somewhat lower in patients experiencing clinical
attachment loss. The relationship of IgM activity
in GCF to clinical attachment loss was different
from that seen for IgG. For example, patients
who were experiencing clinical attachment loss
displayed higher levels of IgM at the initiation
of monitoring and at 3 months following root
planing and scaling. No differences between
groups were detected at 2 weeks and 6 months
following therapy.
The identification of BG as a potential marker
for clinical attachment loss in humans, and the
implication that PMN may be involved in the
pathology of periodontal disease, led to other
studies to examine the relationship of BG in GCF
to other aspects of the periodontal lesion. The
relationship of the subgingival microflora to BG
and other enzymes of GCF was studied (Harper
et al., 1989). Both darkfield and cultural analysis
of the subgingival microflora, and the activity of
the enzymes BG, AS, and LDH were evaluated
in patients with chronic adult periodontitis. To
minimize the effect of probing depth, sampled
sites were selected on the presence of variable
enzyme activity and similar probing depth (i.e.,
all sites were 4 to 6 mm). A total of 54 sites in
13 patients were evaluated. The results indicate
that BG activity in GCF displayed a significant
positive correlation with spirochetes, Bacteroides
intermedius, lactose-negative, black-pigmented
Bacteroides, and B. gingivalis, and a significant
negative correlation with coccoid cells. LDH dis-
played a significant positive correlation with lac-
tose-negative, black-pigmented Bacteroides and
B. gingivalis, and AS displayed a significant po-
sitive correlation with B. gingivalis. These data
indicated that enzyme activity was elevated, and,
in the case of BG, PMN activity was elevated,
in association with a more prominent microbial
challenge. It is interesting to note, however, that
the maximum correlation was only in the range
of r = 0.5. This implies that individual variation
is important to this response.
In a subsequent study, the relationship of
different host mediators in GCF to the serum IgG
antibody titers to putative periodontal pathogens
was evaluated (Lamster et al., 1990). The serum
IgG antibody titer to a panel of 17 microorgan-
isms was studied. Patients with elevated levels
of IgG in GCF tended to have elevated levels of
serum IgG antibody to the putative periodontal
pathogens. This relationship was particularly
strong for IgG in GCF and serum IgG antibody
titers to a strain of B. intermedius and Capno-
cytophaga ochraceous. A similar but less pro-
nounced trend was seen for the relationship of
IgM in GCF to the serum antibody titer. In con-
trast, the correlation of BG in GCF to the serum
antibody titer was negative. This negative cor-
relation was pronounced for three different strains
of A. actinomycetemcomitans, one strain of Ei-
konella corrodens, and Wolinella recta. No re-
lationship was observed between the amount of
a-2-M in GCF and the serum IgG antibody titer
to the periodontal pathogens.
The findings from this study are intriguing
and suggest that the appearance of an increased
serum IgG antibody titer is essentially protective.
The absence of this serum antibody response,
which will be reflected in GCF, may predispose
to the accumulation of pathogens in the subgin-
gival plaque. In turn, the accumulation of path-
ogenic microorganisms in the crevice would be
39
associated with an increased influx of PMN into
the crevicular region (Harper et al., 1989).
Many other PMN lysosomal constituents have
been identified in GCF, including myeloperoxi-
dase (Smith et al., 1986) and lactoferrin and ly-
sozyme (Friedman et al., 1983). Nevertheless,
the relationship of these indicators in GCF to
active periodontal disease has not been studied.
A preliminary report examining the relationship
of PMN elastase to clinical attachment loss has
been published recently (Palcanis et al., 1990).
Using a sensitive automated probe, sites expe-
riencing clinical attachment loss were observed
to also display significantly higher levels of PMN
elastase when compared with sites that did not
experience disease activity. These data confirm
the finding that large accumulations of PMN in
the gingival crevice are associated with a loss of
clinical attachment along the root surface.
The studies examining changes in GCF
chemistry during experimental periodontitis in
dogs (Kryshtalskyj et al., 1986; Kryshtalskyj and
Sodek, 1987), and changes in host mediators in
GCF associated with attachment loss in humans
(Lamster et al., 1991), suggest the importance
of protease-inhibitor activity in crevicular fluid.
oa-1-antitrypsin (a-l-A) and a-2-M are the two
major protease inhibitors in serum (Sandholm,
1986), and both have been examined in GCF.
The relationship of oa-1-A in GCF to serum was
evaluated by Asman et al. (1981). Using the a-
1-A/transferrin ratio, the concentration of a- -A
was higher in GCF than in serum. While no dif-
ferences were detected between inflamed and
healthy sites from patients with different degrees
of periodontal disease, it was suggested that pa-
tients with periodontal disease had less xa--A in
GCF than healthy patients. The presence of a-
2-M in GCF has been studied more intensely.
This protease inhibitor was first identified in GCF
in 1960 (Brill and Bronnestam, 1960). Condacci
et al. (1982) detected an elevation in the con-
centration of total a-2-M in GCF during the de-
velopment of experimental gingivitis in human
volunteers. Furthermore, the concentration of to-
tal a-2-M in GCF from periodontitis patients was
higher before treatment than after treatment. Us-
ing crossed immuno-electrophoresis, fluid col-
lected from periodontitis patients before treat-
ment displayed peaks of both free and bound a-
40
2-M. After therapy, only bound a-2-M was de-
tected in GCF. Findings from our laboratory agree
with the data reported by Condacci et al. (1982).
We observed that a greater amount of immuno-
logically identified a-2-M was present in GCF
samples from patients with periodontitis prior to
treatment compared with after therapy (Sengupta
et al., 1989). In addition, as noted earlier, pa-
tients with active periodontitis displayed more
total a-2-M in GCF than patients who did not
experience disease activity (Lamster et al., 1991).
In contrast to these findings, Skaleric et al. (1986)
performed a cross-sectional analysis of the re-
lationship of a-2-M in GCF to existing perio-
dontal pathology. They determined that the con-
centration of total a-2-M in GCF was lower in
sites with more inflammation, more bone loss,
and greater probing depth. These findings agree
with the data from the dog experimental perio-
dontitis model (Kryshtalskyj et al., 1986; Krysh-
talskyj and Sodek, 1987), where reduced func-
tional inhibitor activity was associated with
elevated collagenolytic activity in GCF from dogs
experiencing active disease.
While differences exist between studies ex-
amining a-2-M activity in GCF, these discrep-
ancies are likely due to the clinical protocols (i.e.,
longitudinal vs. cross-sectional studies) and the
methods of assaying protease inhibitor activity
(immunologic identification vs. functional anal-
ysis). Nevertheless, the importance of protease-
inhibitor activity in GCF will be considered later
in this review when pathologic mechanisms are
discussed.
Other approaches have been utilized to study
the biochemical events associated with the pro-
gression of periodontitis. Chambers et al. (1984)
used the dog experimental periodontitis model to
study the relationship of the cytoplasmic enzyme
aspartate aminotransferase (AST) to clinical at-
tachment loss. This cytoplasmic enzyme is used
as a marker of cell death, as seen, for example,
following myocardial infarction. In their report,
Chambers et al. (1984) demonstrated that the
AST concentration in GCF was 10 to 100 times
higher in GCF when compared with serum. At-
tachment loss was observed to begin after liga-
tion, and approximately one half of the total at-
tachment loss was realized during the first 2 weeks
following ligation. A pronounced peak in AST
concentration in GCF was observed at the 2-week
GCF collection, indicating that this measure of
cell death was observed soon after the initial burst
of tissue destruction. Subsequently, the relation-
ship of AST in GCF to both tissue inflammation
and clinical attachment loss in humans was as-
sessed (Persson et al., 1990a, 1990b). Using the
human experimental gingivitis protocol, a statis-
tically significant relationship existed between
AST in GCF and clinical measures of tissue in-
flammation and sulcular bleeding (Persson et al.,
1990a). When studying the relationship of AST
in GCF to clinical attachment loss at individual
sites, they determined that elevated AST levels
either preceded or occurred at the time of detec-
tion of disease activity. Unfortunately, these in-
vestigators did not suggest the source of the en-
zyme in GCF. Their data indicate, however, that
this enzyme is derived locally, and that eryth-
rocytes do not make a major contribution to the
AST pool in GCF. Likely sources include the
PMN and epithelial cells.
A series of reports examining prostaglandin
E2 (PGE2) in GCF also offer information about
the nature of the destructive lesion in human per-
iodontal disease. The first identification of PGE2
in gingival tissue exudates was by Goodson et
al. (1974). They observed levels of PGE2 of 10-6
to 10-7 M. At these concentrations PGE2 can
inhibit collagen synthesis and induce bone re-
sorption in in vitro models. Offenbacher et al.
(1981) described a method for detection of PGE2
in GCF. Their radioimmunoassay was sensitive
to 4 pg of PGE2. This preliminary report dem-
onstrated that the concentration of PGE2 in GCF
from periodontitis patients was approximately five
times greater than that seen in samples from pa-
tients with gingivitis. The sources of PGE2 in
GCF were suggested to be epithelial cells, en-
dothelial cells, and fibroblasts, and perhaps of
greater significance, PMN and macrophages. It
was later demonstrated that the concentration of
PGE2 in GCF correlates with the concentration
of PGE2 in the adjacent tissues was related to
existing attachment loss and was approximately
three times higher in GCF samples from patients
with juvenile periodontitis compared to adult per-
iodontitis (Offenbacher et al., 1984). Subse-
quently, these investigators studied patients with
adult periodontitis to determine the longitudinal
relationship between the concentration of PGE2
in GCF and clinical attachment loss (Offenbacher
et al., 1986). Collecting and analyzing GCF from
all mesiobuccal sites, a whole-mouth PGE2 con-
centration was determined. Patients were moni-
tored every 3 months until one site in the mouth
displayed a significant amount of clinical attach-
ment loss. Patients who experienced disease ac-
tivity displayed significantly higher mean con-
centrations of PGE2 compared to patients who
did not demonstrate active disease. Elevated lev-
els of PGE2 in GCF occurred 6 months prior to
the detection of disease activity. In addition, ex-
amination of the site displaying attachment loss
revealed that the PGE2 concentration at the active
site was elevated approximately fivefold at the
visit when disease activity was detected. The con-
centration of PGE2 in GCF collected from the
active sites decreased dramatically 1 month after
root planing and scaling.
The relationship of elevated PGE2 concen-
tration in GCF to clinical attachment loss pro-
vides yet another indication of an association be-
tween an exuberant acute inflammatory response
in the crevicular environment and the destructive
phases of periodontal disease. The cellular hall-
mark of the inflammatory response is the PMN.
Mechanisms associated with the PMN that may
account for tissue destruction include (1) release
of lysosomal constituents to degrade both the fi-
brillar components and ground substance of the
connective tissue; (2) the proinflammatory ef-
fects of certain products of the arachidonic acid
cascade, most notably PGE2; and (3) other mech-
anisms not as extensively examined in the crev-
icular environment, including release of cyto-
kines such as IL-liP, and generation of reactive
oxygen metabolites, such as hypochlorous acid
(HOCI), superoxide anion (02-), and hydrogen
peroxide (H202).
The first part of this review has presented
evidence to implicate the PMN in the destructive
phases of periodontal disease. While a number
of mechanisms associated with tissue destruction
were described, some of the mediators that could
account for this destruction would not be specific
to an exuberant response by PMN to the subgin-
gival microbial plaque. Current biomedical re-
search into the pathogenesis of tissue destruction
associated with a PMN-dominated inflammatory
41
response suggests that this destruction may be
due to the synergistic effect of PMN protease
activity and the generation of reactive oxygen
metabolites. This is probably the major effector
mechanism for tissue destruction that can be spe-
cifically identified with the PMN. The next sec-
tion of this article will focus on the physiological
and biochemical mechanisms of PMN-mediated
tissue destruction. Attention will be directed to
mechanisms of tissue destruction associated with
the release of proteases and oxygen metabolites
that may be implicated in the pathogenesis of
periodontal disease. The hypothesis that will be
developed suggests that periodontal tissue de-
struction may, in some instances, be associated
with an enhanced PMN response.
II. THE PHYSIOLOGY AND
BIOCHEMISTRY OF THE HUMAN
NEUTROPHIL AND ITS POTENTIAL
ROLE IN TISSUE DESTRUCTION IN
PERIODONTAL DISEASE
A. Introduction
The importance of the PMN in the inflam-
matory response and the subsequent protection
of the host against microbial invasion has been
appreciated for over 100 years. In the early 20th
century, Metchnikoff, in his studies of phago-
cytes and their role in the inflammation, con-
cluded, "The diapedesis of the white corpus-
cles . . . is one of the principal means of defense
possessed by an animal. As soon as the infective
agents have penetrated into the body, a whole
army of white corpuscles proceeds towards the
menaced spots, there entering into a struggle with
the micro-organisms. The leukocytes, having ar-
rived at the spot where the intruders are found,
seize them . . . and within their bodies subject
them to intracellular digestion" (Metchnikoff
1905, cited in Weissmann, 1988).
The recognition by van Leeuwenhoek some
300 years ago that the oral cavity was colonized
extensively by a variety of microorganisms (or
"animalcules") led to the beginnings of modem-
day microbiology and an understanding of the
importance of anaerobic bacteria in human oral
infections. It has taken 300 years to clarify that
42
inflammatory periodontal disease, one of the most
common chronic disorders affecting adults, is in-
itiated by the presence of specific bacteria on the
teeth and gingival tissues (L6e et al., 1965).
Moreover, we have recently begun to understand
the important role of the PMN in defending the
oral tissues against microbial invasion (Van Dyke
et al., 1985). In health, an intricate balance exists
between the host, its protective inflammatory and
immune systems, and the colonizing microor-
ganisms of the oral cavity, many of which gain
their nutritional requirements from their host. The
integument lining the gingival crevice is pro-
tected by a constant efflux of phagocytic cells
and soluble antimicrobial factors flowing from
the subepithelial vascular plexus into the gingival
crevice. However, as was described by Metch-
nikoff exactly 100 years ago when studying the
effects of inserting a rose thorn into a starfish
larva, any penetration of the integument by bac-
teria or their products leads to an inflammatory
response characterized by the accumulation of a
large number of leukocytes at the point of in-
vasion. Since then, many of the physiological
and biochemical mechanisms by which PMN mi-
grate to sites of infection, the mechanisms by
which they ingest and kill invading bacteria, and
the amplification system for the inflammatory
response have been identified. However, as
Weissmann has expressed so succinctly, "one
might perhaps wonder why, since PMNs have
probably been evolving for several hundred mil-
lion years at least, they do not operate more 'ef-
ficiently'. Granted that they are necessary parts
of the body's defenses, but why, in defending
the body, must they at times damage the very
tissues they are defending" (Weissmann 1978).
In reference to periodontal disease, it has become
apparent that, in some individuals, the normal
protective response of PMNs to the accumulation
of microbial plaque adjacent to the gingival tis-
sues has the ability, under certain conditions, to
cause extensive destruction of the periodontium.
The following sections review the origin and life
history of the PMN, the physiology and bio-
chemistry of activation of these important host
defense cells, and how changes in the normal
physiologic and biochemical responses may re-
sult in tissue destruction. The biologic basis for
a new hypothesis will be presented that suggests
that the tissue destruction associated with inflam-
matory periodontal disease may, in some in-
stances, be due to the PMN hypereactivity, and
not necessarily to PMN hyporeactivity, as pre-
viously suggested (Van Dyke and Hoops, 1990).
B. The Polymorphonuclear Leukocyte
1. Origin and Life History
The life of the PMN is spent in three major
compartments of the body: the bone marrow,
where it begins life; the circulatory blood system,
in which it travels around the body; and its final
location and destiny, the tissues of the body.
PMNs are produced in the bone marrow and re-
leased at a rate of 80 to 160 x 107 cells/kg body
weight/d. This means that approximately 1011
PMNs are released each day from the marrow of
a 70-kg individual (Cartwright et al., 1964; Dan-
cey et al., 1976). The bone marrow comprises
approximately 4.5% of total adult body weight.
About 55% of the bone marrow is dedicated to
the production of PMN, which, during their life
in the marrow, pass through three major com-
partments of differentiation. They begin life as
a pluripotent stem cell, which forms the center
of a self-maintaining hemopoietic clone. This
clone has the potential for self-renewal, as well
as for the generation of multiple hemopoietic cel-
lular lineages. The pluripotent stem cell is ca-
pable of producing erythroid, granulocytic, mon-
ocytic, thrombocytic, or mixed colonies. The
pluripotent stem cell has been termed the colony
forming unit-spleen (CFU-S), since the first assay
system of Till and McCulloch (1961) demon-
strated that single stem cells from mouse bone
marrow, when injected into irradiated mice,
formed hemopoietic colonies in the spleen. One
of the progenitor cells, derived from the stem
cell, gives rise to colonies containing granulo-
cytes and monocytes. This progenitor cell is
termed the granulocyte-macrophage colony
forming unit (CFU-GM), and it subsequently dif-
ferentiates into the CFU-G and CFU-M to pro-
duce granulocytes and monocytes. The produc-
tion by the CFU-G of the myeloblast initiates the
granulocyte differentiation pathway, with sub-
sequent formation of the promyelocyte, myelo-
cyte, metamyelocyte, band cell, and, finally, the
mature PMN. All differentiation, proliferation,
and maturation phases of the PMN life cycle oc-
cur in the bone marrow, with the subsequent re-
lease of the mature PMN into the circulation.
Proliferation, consisting of approximately five
mitoses over 5 d, results in the stem cell maturing
through the myeloblast, promyelocyte, and mye-
locyte stages. At the myelocyte stage the cell is
no longer capable of mitosis and so becomes an
"end cell", entering a large storage pool of cells
that undergo further differentiation over a period
of the next 5 d (Cronkite and Vincent, 1969;
Athens, 1970). This differentiating pool is made
up of metamyelocytes, band cells, and the mature
segmented PMN. The differentiation from the
stem cell to the band cell takes approximately 2
weeks (Bainton et al., 1971), following which
the mature cells are released into the circulation.
Studies of neutrophil disappearance from the cir-
culation yield a half-life of approximately 6.7 h.
The circulating pool makes up approximately 50%
of the blood PMN, as determined by counting,
with the other 50% on the blood vessel walls to
form the so-called marginating pool (Athens et
al., 1961). The mature PMN lasts about 1 to 2
d in the tissues (Peters et al., 1985).
2. The Morphologic Stages of PMN
Maturation and Differentiation
The myeloblast constitutes about 1% of mar-
row cells, is 15 to 20 ,xm in diameter, and is
never seen in the peripheral blood of healthy sub-
jects. It is a relatively undifferentiated cell, with
a high nuclear-to-cytoplasmic ratio, prominent
pale blue nucleoli, and, occasionally, some im-
mature peroxidase positive azurophil, or pri-
mary, granules. The promyelocyte is character-
ized by the accumulation of peroxidase-positive
azurophil granules. The peroxidase is located
throughout the rough endoplasmic reticulum, the
Golgi complex, and small transport vesicles, in-
dicating the synthetic nature of peroxidase for-
mation and later storage in the granules. Cessa-
tion in the production of peroxidase positive
granules marks the end of the promyelocyte stage,
and the myelocyte stage is marked by production
of the peroxidase-negative specific, or second-
43
ary, granules. The specific granules are formed
by the Golgi, and during the myelocyte stage
approximately three divisions occur, with azur-
ophil and specific granules being divided equally
between daughter cells (Cronkite and Vincent,
1969). In contrast to the peroxidase-positive na-
ture of the azurophil granules, the specific gran-
ules are negative for peroxidase but positive for
alkaline phosphatase. Proliferation ceases at the
myelocyte stage, and differentiation into the ma-
ture PMN continues with development of the me-
tamyelocyte. At this stage, the cell assumes the
uniformly pink cytoplasm of the mature PMN,
and the nucleus begins its segmentation process
by becoming bean shaped or indented. The band
cell is the next stage. Band cells are similar to
mature PMN, but do not have fully segmented
nuclei. This cell is found in the peripheral blood
of healthy subjects at about 3 to 5% of the dif-
ferential count, but this number increases during
infection. The mature PMN is characterized by
a segmented nucleus of two to five lobes that are
joined by thin strands of chromatin. The mature
PMN has numerous granules, of which 80 to 90%
are secondary granules and 10 to 20% are primary
granules. This preponderance of secondary gran-
ules is due to the fact that primary granule for-
mation ends at the promyelocyte stage, and their
numbers are subsequently diluted by cell division
during the myelocyte stage when secondary gran-
ules are formed.
3. PMN Granules
Morphologic heterogeneity among PMN
granules has been recognized for some time and
is characteristic of these cells (Bainton et al.,
1971). As noted, primary and secondary granules
appear at different stages of PMN development
(Bainton et al., 1971; Bainton and Farquhar,
1968), are cytochemically distinct due to differ-
ences in their content (Bainton et al., 1971; Bain-
ton and Farquhar, 1968), have different sites of
origin at the Golgi (Bainton and Farquhar, 1966),
and differ in their degranulation kinetics during
phagocytosis (Bainton, 1973). In the late 19th
century, leukocyte biologists were divided in their
opinions on the function of PMN granules. When
describing phagocytosis, Metchnikoff(1905) dis-
44
cussed the intracellular role of "cytases" in the
phagocytosis and intracellular digestion of mi-
croorganisms. He concluded that these "cy-
tases" were not released from the cell, except
following cell death. In contrast, in 1900 Paul
Ehrlich concluded that PMN granules were se-
cretory products that the cell released to the ex-
ternal environment (Hirsch and Hirsch, 1980).
Support for the intracellular role of granules dur-
ing phagocytosis was provided by the studies of
granule fusion with phagocytic vacuoles during
ingestion of microorganisms (Hirsch and Cohn,
1960; Cohn and Hirsch, 1960; Hirsch, 1962). It
also became evident that granule products could
be released into the extracellular environment
during phagocytosis by the incomplete closure of
the phagocytic vesicle during ingestion. This was
termed regurgitation while feeding (Weissmann
et al., 1972). A second phenomenon, termed
frustrated phagocytosis, was reserved for the ex-
tracellular release of granule products due to the
inability of PMN to ingest opsonized particles
that were too big or too plentiful (Henson, 1971a;
Henson, 1971b). It was proposed that the extra-
cellular release of granule contents led to path-
ologic tissue changes during PMN-dominated in-
flammation (Weissmann et al., 1972; Goldstein,
1976; Weissmann et al., 1973; Henson, 1972).
Considerable evidence has been accumulated from
in vitro studies of bacterial-PMN interactions and
from in vivo studies of crevicular fluid contents
during active phases of periodontal destruction
to support a role for the extracellular release of
granule contents in the pathogenesis of perio-
dontal disease. This concept is discussed further
in Section D.
In 1900, Ehrlich had noted that migrating
PMNs lost their granules during emigration from
tissues and concluded that this reflected the "ex-
trusion" of granules by the cell (Hirsch and
Hirsch, 1980). Leffel and Spitznagel observed
that during phagocytosis, secondary granules were
directed to the periphery of the cell, while pri-
mary granules fused with the phagocytic vacuole
(Leffell and Spitznagel, 1974; Leffell and Spitz-
nagel, 1975). In vivo support for this observation
did not come until the study of Wright and Gallin
(1979), who examined PMNs and exudates from
sterile skin lesions and postsurgical wound ab-
scesses. Constituents of secondary granules ap-
peared in the exudates, and PMN from these le-
sions were depleted of their secondary granules
when compared with peripheral blood PMN.
There was little or no depletion in primary gran-
ules, as determined by ultrastructural analysis and
granule separation by ultracentrifugation on su-
crose density gradients. These in vivo studies sup-
ported earlier in vitro observations that selective
release of secondary granule constituents could
be induced with soluble stimuli, such as phorbol
myristate acetate (Goldstein et al., 1975; White
and Estensen, 1974; Wright et al., 1977), the
lectin concanavalin A (Con A; Hoffstein et al.,
1976), and with certain concentrations of the di-
valent cation ionophore A23187 (Wright et al.,
1977). This cumulative evidence led to the con-
clusion that the PMN acts as both a phagocyte
and as a secretory cell that can release granule
constituents to modify its surroundings. Conse-
quently, the PMN has been termed a secretory
organ of inflammation and host defense (Weiss-
mann et al., 1973, Wright 1982).
With increasing knowledge of the role of the
PMN as a secretory organ, investigators have
paid considerable attention to the heterogeneity
of the granule population and its influence on
PMN function in health and disease (Boxer and
Smolen, 1988). Table 1, taken from a recent
review by Boxer and Smolen (1988), provides
our current understanding of granule heteroge-
neity and content. In addition to the original gran-
ules described during differentiation (primary and
secondary granules), two other types of granules
have been identified on the basis of density cen-
trifugation and enzyme activity. The tertiary
granule has a density below that of the secondary
granule and contains the enzyme gelatinase (De-
wald et al., 1982). Its secretion may be associ-
ated with increased membrane expression of the
adhesion glycoprotein Mo 1, suggesting that re-
lease of tertiary granule constituents at the cell
membrane, and the renewal of the membrane that
occurs during granule fusion and release, is in-
volved in PMN attachment and migration (Pe-
trequin et al., 1987; Todd et al., 1984). A fourth
granule, termed the replenishsome, has been
characterized recently (Borregaard et al., 1987)
and also may serve to replenish the cell mem-
brane with essential receptors and modulators re-
quired for appropriate cellular function. How-
ever, the tertiary granules and the replenishsomes
share many characteristics with respect to the
transport of adhesion molecules Mo 1 and C3bi
to the cell surfaces, and, although they appear
distinct from the secondary granules, their
uniqueness can only be determined following ad-
ditional study.
It is evident that the early morphologic ob-
servations of Leffel and Spitznagel (Leffell and
Spitznagel, 1974; Leffell and Spitznagel, 1975)
appear to be supported by the later studies dem-
onstrating sequential degranulation of secondary,
tertiary, and replenishsome granules to the ex-
tracellular environment. These granules appear
to make up the "secretory" component of the
PMN. This secretory activity leads to the release
of antimicrobial agents, such as lysozyme and
lactoferrin (Wright, 1982), as well as replenish-
ing of membrane receptors, enzymes, and cy-
tochromes essential for mobilization of the PMN
to the site of infection and prolonged activation.
The expression of surface receptors in the human
PMN is crucial for normal physiologic function.
Abnormalities of receptor expression have been
defined in PMN from individuals with leukocyte
adhesion deficiency syndrome and localized ju-
venile periodontitis, where altered PMN function
has been associated with advanced, early onset,
periodontal destruction (reviewed in Todd and
Freyer, 1988; Van Dyke and Hoops, 1990).
Much of our current understanding of granule
function has been gained by studying PMN from
patients with genetic granule abnormalities. These
disorders of altered PMN function are often as-
sociated with recurrent or prolonged infection.
The deficiencies described to date appear to be
inherited as autosomal recessive traits. Five pa-
tients with a secondary granule deficiency have
been identified (Gallin, 1985), and, although no
individuals with an absence of primary granules
have been described, myeloperoxidase (MPO)
deficiency is observed in approximately 1 in 2000
individuals (Nauseef, 1988).
Secondary granule deficiency is associated
with recurrent infections of the skin, upper res-
piratory tract, middle ear, and lung. This disorder
is associated with abnormal PMN chemotaxis in
vivo and in vitro, and abnormal killing by PMN
(Gallin et al., 1982). These observations may be
due to the fact that there is a defect in the up-
45
TABLE 1
PMN Granule Constituents
Primary Secondary
(azurophilic) (specific) Tertiary
granules granules granules Replenishsomes
Myeloperoxidase Lysozyme Gelatinase C3bi receptors
Lysozyme Collagenase Cytochrome b
Elastase Lactoferrin Alkaline phosphatase
Cathepsin G Vitamin B,2 binding protein
Proteinases Cytochrome b /
N-Acetyl glucuronidase Histaminase
Cathepsin B Complement activator
Cathepsin D Monocyte chemoattractant
B glucuronidase Plasminogen activator
B glycerophosphate Protein kinase C Inhibitor
Mannosidase FMLP receptors
Defensins C3bi receptors
Antibacterial cationic
proteins
Kinin-generating
enzymes
C5a inactivating factor
Adapted from Boxer, L. A. and Smolen, J. E., Hematol./Oncol. North Am., 2(1), 101, 1988. With permission.
regulation of chemotactic receptors (such as N- plification of the respiratory burst may also be
formyl methionyl leucyl phenylalanine: FMLP), affected by lactoferrin depletion, since lactoferrin
and for the adhesion glycoproteins (such as C3bi), has been demonstrated to enhance the generation
in activated cells (Gallin et al., 1982). In addi- of hydroxyl radicals (Ambruso and Johnson,
tion, approximately two thirds of the lysozyme 1981).
recovered from PMN is contained in the second- MPO-deficient PMN show delayed killing of
ary granules, and levels of this enzyme in plasma internalized bacteria, an absence of killing of
and urine have been related to the rate of PMN Candida species, and a prolonged respiratory burst
turnover in the body. Similarly, the iron-binding (Nauseef, 1988). The inability of the PMN to
protein lactoferrin, which in its unsaturated form form the MPO-H202-halide complex that is bac-
may restrict microbial growth by competing for tericidal to many bacteria will delay bacterial kill-
available iron, is located in the secondary gran- ing. However, other bactericidal components of
ules. The deficiency in circulating lysozyme and PMN, such as defensins contained in the primary
lactoferrin may decrease the host's bactericidal granules and the hydroxyl radical, can exert bac-
and bacteriostatic activity at mucosal surfaces tericidal activity.
(Wright, 1982). It is also possible that, in ad-
dition to defective PMN function observed in
these individuals, there is an inability to amplify 4. The Biochemistry and Physiology of
the inflammatory response. This is due to the fact PMN Activation
that secondary granules have been shown to con-
tain a constituent that can generate the major PMN released from the bone marrow into the
chemotactic component of complement, C5a, circulation display minimal metabolic activity and
from C5 (Wright and Gallin, 1977). In addition, are considered "resting" cells. With initiation of
lactoferrin can affect amplification of the PMN an inflammatory response following the local re-
response by inhibiting the release of granulocyte- lease of vasoactive substances, vascular changes
macrophage colony stimulating factor (GM-CSF) result in reduction in velocity of blood passing
from monocytes (Broxmeyer et al., 1980). Am- through the vessels, subsequent margination of

46
PMN, and attachment of these cells to the en-
dothelial cells lining the blood vessels. The con-
tact between the cell surface of the PMN and the
endothelial cell is mediated by a series of adhe-
sion glycoproteins expressed on the PMN cell
surface. This group of closely related glycopro-
teins, known as the CD11/CD18 glycoprotein
family, are responsible for mediating PMN adhe-
sion to cellular and particulate surfaces, as well
as between PMN during aggregation of these cells
(Todd and Freyer, 1988). The three glycoproteins
are heterodimers with a common beta subunit,
CD18, and variable alpha subunits. They are
CD1 la/CD18 (previously LFA-1), CD1 lb/CD18
(previously Mo 1), and CD1 c/CD 18 (previously
gp 150, 95), all of which have been characterized
in patients demonstrating leukocyte adhesion de-
ficiency syndrome (Todd and Freyer, 1988). Mol
functions as CR3, the PMN receptor for the com-
plement component C3bi. As such, this glyco-
protein mediates PMN binding to C3bi opsonized
particles (Todd and Freyer, 1988) and to a CR3
binding site (ICAM-1) on human endothelial cells
(Smith et al., 1988). It has been demonstrated
that this initial adhesion results in degranulation
of secondary and tertiary granule products (Wright
and Gallin, 1979), which are also necessary for
upregulating the adhesion molecules (Petrequin
et al., 1987; Todd et al., 1984). The movement
of PMN from the point of adhesion on the en-
dothelial cell through the blood vessel wall and
to the site of infection is a chemotactic response.
Chemotaxis refers to the directed locomotion
of cells along a concentration gradient of a chem-
otactic factor or chemotaxin. Numerous chem-
otactic factors have been identified (Wilkinson,
1982), but the most potent appear to be the fifth
component of complement (C5a), the synthetic
bacterial peptide FMLP, and leukotriene B4
(LtB4), a lipoxygenase product of arachidonic
acid (Weissmann et al., 1973). The response of
PMN to chemotactic stimuli has been extensively
studied using FMLP. The FMLP receptor is an
integral membrane protein with a molecular
weight of 50 to 70,000 Da (Niedel et al., 1980)
and with approximately 6 x 104 receptors per
cell (Niedel et al., 1979). Binding of the ligand
FMLP to the receptor initiates a process termed
stimulus-response coupling, in which the re-
sponses of the cell are directly and temporally
linked to binding of the stimulus through a de-
fined activation pathway. Multiple secondary
messengers are involved in the transduction of
the stimulatory signal from the cell surface to the
relevant cellular effector mechanisms, and many
of these are common to several activation path-
ways, either working independently or in con-
cert. The chemotactic response is initiated by
binding of FMLP to the receptor, and it appears
that subsequent responses are affected through
the action of guanine triphosphate (GTP) binding
proteins whose activity is inhibitable by pertussis
toxin. Treatment of PMN with pertussis toxin
results in the adenine diphosphate ribosylation of
the alpha subunit of the 41,000 Da GTP-depen-
dent regulatory component (G1) responsible for
inhibition of adenylate cyclase (Matsumoto et al.,
1986; Okajima and Ui, 1984). Inhibition by per-
tussis toxin has implicated this particular G pro-
tein as an early second messenger in the chem-
otactic peptide-stimulated activation of PMN.
Pertussis toxin treatment has been shown to in-
hibit FMLP-induced elevation in intracellular
calcium (Goldman et al., 1985), superoxide an-
ion production (Lad et al., 1985a; Okamura et
al., 1985), lysosomal enzyme release (Goldman
et al., 1985; Lad et al., 1985a), aggregation
(Krause et al., 1985; Lad et al., 1985b), chem-
otaxis (Goldman et al., 1985; Krause et al., 1985),
cytoskeletal changes (Becker et al., 1985), and
protein phosphorylation (Volpe et al., 1985).
Although stimulus-response coupling has
been described most extensively for the chemo-
tactic peptide FMLP, multiple pathways of ac-
tivation appear to exist in PMN, and the re-
sponses initiated in the PMN are characteristic
of the stimulus used (McPhail et al., 1984). The
chemotactic stimuli FMLP, C5a, and LtB4 are
able to initiate chemotaxis, but at physiologic
concentrations are poor activators of the microb-
icidal respiratory burst. However, nonchemotac-
tic stimuli, such as Con A, the opsonic third
component of complement C3b, and the Fc frag-
ment of antibody, are able to activate the mi-
crobicidal respiratory burst when bound to spe-
cific PMN receptors. Increases in the intracellular
pools of "free" calcium by the mobilization of
calcium from intracellular pools or from extra-
cellular sources is important in the organization
of the cytoskeleton (Sklar et al., 1985). This
47
event is required for the activation of the calcium-
dependent protein kinase C, which catalyzes the
phosphorylation of a number of proteins at serine
and threonine residues. Approximately 90% of
the protein kinase C activity is cytosolic in resting
PMN (Wolfson et al., 1985). Stimulation by ac-
tivators of the respiratory burst, such as the phor-
bol esters (Wolfson et al., 1985; Gennaro et al.,
1986), or chemoattractants such as FMLP at ap-
propriate concentrations (Pike et al., 1986), re-
sult in the redistribution of protein kinase C to
the plasma membrane.
The mobilization of the cytosolic protein ki-
nase C to the membrane or particulate fraction
of the cell has been associated with the activation
of a membrane-bound NADPH oxidase (Wolfson
et al., 1985; Gennaro et al., 1986). This acti-
vation occurs following stimulation of PMN by
agents known to activate the "respiratory burst",
so named because of the sharp increase in oxygen
uptake that takes place upon stimulation of the
cells (Baldridge and Gerard, 1933). Although
stimulation results in an increase in oxygen up-
take (Sbarra and Karnovsky, 1959), the oxygen
is not essential for phagocytosis, nor is there an
increase in mitochondrial respiration. It was ob-
served that almost all of the oxygen was con-
verted to H202 by activated PMN (Iyer et al.,
1961). PMN stimulation also results in an in-
crease in glucose oxidation, through the activity
of the hexose monophosphate shunt, using NADP
as an electron acceptor (Sbarra and Karnovsky,
1959). The availability of NADP is the rate-lim-
iting step in glucose oxidation. This step is reg-
ulated by both the speed at which NADPH can
be oxidized to NADP by the NADPH oxidase
(Beck, 1958) and by the regeneration of NADP
from NADPH as part of the glutathione-peroxi-
dase-glutathione reductase system that protects
PMN from internal oxidative damage by inter-
nally generated H202 (Reed 1969). Activation of
the hexose monophosphate shunt is therefore
closely linked to the oxidation of NADPH by the
NADPH oxidase. Studies have demonstrated that
PMN produce large quantities of superoxide an-
ion (02-) by the one-electron reduction of oxy-
gen (Babior et al., 1973; Drath and Karnovsky,
1975; Weening et al., 1975). Furthermore, all of
the oxygen taken up during the respiratory burst
is converted to O2-, with 80% of this being dis-
48
mutated to H202 either spontaneously or by su-
peroxide dismutase (Root and Metcalf, 1977).
The reactions are
202 + NADPH--202 + NADP+ + H+
202 + 2H+ -
02 + H202
C. Oxygen Metabolites and Tissue
Destruction
The electron transport chain that comprises
the NADPH oxidase in human PMN utilizes ox-
ygen as the terminal electron acceptor. The ac-
ceptance of electrons and subsequent reduction
of oxygen initiates the production of a series of
highly reactive oxidants. In PMN, the NADPH
oxidase is located within the plasma membrane
and is activated following stimulation of the cell.
The flow of electrons is from the cytosolic side
of the membrane, where NADPH is oxidized to
NADP, to the external surface of the PMN, where
the one electron reduction of oxygen results in
the production of 02- on the outer surface of the
PMN cell membrane (McCord and Fridovich,
1978). Oxygen, by the acceptance of four elec-
trons, can be reduced to water. However, highly
reactive, partially reduced intermediates can be
formed from 02-. Superoxide is rapidly con-
verted to H202, either by spontaneous dismuta-
tion of two molecules of 02- (202- + 2H + -
H202 + 02) or through a reaction that is cata-
lyzed by superoxide dismutase. It has been pro-
posed that the highly reactive oxidant hydroxyl
radical (OH) can be formed from the iron-cat-
alyzed interaction of H202 and 02- by the Haber-
Weiss reaction.
The effects of oxygen metabolites on the cel-
lular and extracellular components of human tis-
sues has received considerable attention due to
the extensive tissue destruction that accompanies
a PMN-dominated inflammatory infiltrate (Hal-
liwell, 1988). Superoxide and H202 are the pri-
mary oxygen metabolites formed during the PMN
respiratory burst, but neither of these molecules
is as reactive an oxidant as OH-, which may be
formed from these species in the presence of metal
ions. In vitro studies have demonstrated that the
release of these reactive oxygen species may alter
proteins in such a way as to make them more
susceptible to proteolysis (Davies, 1987a). This
may occur through a modification of the primary,
secondary, and tertiary structure of proteins
(Davies et al., 1987a; Davies and Delsignore,
1987; Davies et al., 1987b). Reactive oxygen
species also modify cell viability by affecting
breaks in DNA, decreasing mitochondrial activ-
ity, decreasing the activity of intracellular en-
zymes, and peroxidation of membrane lipids
(Cochrane et al., 1988). The release of 02- into
extracellular fluids has also been shown to acti-
vate a latent chemotactic factor that acts to rap-
idly recruit more PMN to the site of infection
(Petrone et al., 1980).
The rapid dismutation of 02- at neutral pH
results in the production of large quantities of
H202. Hydrogen peroxide does not have the bi-
ological reactivity of the oxygen radicals, 02-
and OH-. However, in combination with MPO
released from the PMN primary granules, and a
halide ion (usually chloride) present in plasma,
the powerful oxidant HOCI is formed (Klebanoff,
1988). This oxidant forms the basis for com-
mercial bleach and attacks a wide range of bio-
logical substrates. The MPO-H202-halide system
kills bacteria, inactivates bacterial toxins, and
kills tumor cells and numerous other host cells
(Klebanoff, 1988). The MPO system is also re-
sponsible for the inactivation of chemotactic pep-
tides, causing a localization of the PMN at the
site of primary granule release. In addition to the
direct toxicity of the MPO system on host cells,
HOC1 has a profound indirect potentiating effect
on the local destruction of extracellular tissues
by proteolytic enzymes (Weiss, 1989). Studies
into the role of reactive oxygen species in the
pathogenesis of periodontal destruction have not
been performed. However, indirect evidence has
been gained from experimental models of per-
iodontitis using drugs known to affect the pro-
duction of oxygen radicals by human PMN. Us-
ing the squirrel monkey model of experimental
periodontitis, it has been demonstrated that the
systemic administration of gold sodium thioma-
late (GST) dramatically decreases the extent of
periodontal destruction during ligature-induced
periodontitis (Novak et al., 1984). GST is a po-
tent antiinflammatory drug known to inhibit ox-
ygen radical production by human PMN at the
level of protein kinase c (Parente et al., 1989).
The studies in the squirrel monkey model clearly
demonstrated that a reduction in number and
function of PMN, brought about by an inhibition
of oxygen radical production, could result in de-
creased periodontal tissue destruction. The extent
of the decrease in destruction of the periodontal
tissues is clearly demonstrated in Figure 1. Fol-
lowing 2 weeks of ligature placement, little to
no destruction of the supporting alveolar bone is
observed around the ligated teeth of the gold-
treated animals when compared with the exten-
sive loss of alveolar support in the non-gold-
receiving animals. Similar differences were ob-
served for the levels of connective tissue attach-
ment in these animals. An analysis of the cellular
content of the periodontal tissues revealed a dra-
matic decrease in the numbers of PMN present
(Polson et al., 1984). Since protein kinase c ac-
tivity is essential for chemotaxis and respiratory
burst activity, it is possible that GST not only
diminished oxygen radical production, but also
the chemotaxis of PMN to the site of infection.
It has been postulated previously that diminished
chemotaxis of PMN to a site of infection may
predipose the host to less protection and greater
tissue destruction. Although this is certainly true
in leukocyte adhesion deficiency and other ge-
netic or induced disorders of PMN function that
lead to a near absence of PMN at infected sites
(reviewed by Van Dyke and Hopps, 1990), these
conditions are also characterized by generalized
life-threatening infections that are not observed
in most patients with periodontitis. Support for
the proinflammatory and tissue destructive role
of PMN has been accumulated from several ear-
lier animal studies (for review see Taichman et
al., 1984). What is not clearly understood, at this
time, are the conditions necessary to change a
protective PMN response into a destructive one
that initiates pathophysiologic changes.
The extensive array of proteolytic enzymes
neatly packaged within the primary and second-
ary PMN granules are potential mediators of tis-
sue destruction if released into the extracellular
environment. However, to prevent unwanted de-
struction of host tissues, plasma and extracellular
fluids contain high levels of proteinase inhibitors,
such as a,-proteinase inhibitor, a-2-M, and se-
cretory leukoproteinase inhibitor (Travis and Sal-
49
FIGURE 1. The effect of systemic gold salts on ligature-induced periodontitis in the squirrel monkey. Buccal view
of the defleshed maxilla following 2 weeks of ligature placement. (A) Extensive destruction of the alveolar bone in
animals not receiving systemic gold salts. The 3-0 silk ligatures are located at the cementoenamel junction, and
30 to 50% of the supporting alveolar bone has been destroyed. Arrows mark the level of crestal alveolar bone. (B)
No bone destruction was observed in the animals that received gold salts prior to and during the induction of
experimental periodontitis. The crestal alveolar bone is seen at its normal location, approximately 1 mm apical to
the cementoenamel junction.

vesen, 1983; Weiss, 1989). Recent studies have
demonstrated that the oxygen metabolites de-
scribed above, especially the MPO system, are
able to locally inactivate proteinase inhibitors
(Weiss, 1989; Janoff, 1985). In the event that
PMN primary granule contents should be re-
leased into the extracellular environment in com-
bination with activation of the respiratory burst,
it is clear that extensive localized tissue destruc-
tion can occur through an inactivation of the host's
protease inhibitors. In addition to the inactivation
of the inhibitors, it has been demonstrated that
oxygen metabolites, including hypochlorous acid
(HOCl) generated by the MPO system and OH-,
activate PMN collagenase and gelatinase, which
are normally released in an inactive form (Weiss,
1989; Saari et al., 1990). It is therefore clear that
the extracellular release of oxygen radicals, com-
bined with the release of proteolytic enzymes,
may be responsible for the initiation of tissue
destruction. Situations leading to an exacerbation
of respiratory burst activity, enhancement of PMN
infiltration, and proteolytic enzyme release would
be expected to result in enhanced tissue destruc-
tion. It has been proposed that pathophysiologic
changes may occur that can lead to periodontal
destruction if these events occur within the per-
iodontal tissues instead of in the gingival crevice
(Taichman et al., 1984).
D. Extracellular Release of Oxygen
Metabolites and Proteases
As we have described previously, the pro-
duction of superoxide radical is the result of ac-
tivation of the NADPH oxidase, with the reduced
oxygen product being formed on the outer surface
of the PMN plasma membrane. During phago-
cytosis of particulate material, such as bacteria,
the external surface of the PMN plasma mem-
brane becomes the internal surface of the phag-
ocytic vesicle. Under these conditions, super-
oxide is released into the phagosome, where it
can interact with granule contents, such as MPO,
to effect bacterial killing (Weissmann, 1978).
Activation of the respiratory burst, in response
to opsonized or unopsonized particulates, is pre-
ceded by a lag time of several minutes following
ligand-receptor binding. During this time, inter-
nalization of the bound particulate material con-
tinues and may be complete before significant
NADPH oxidase activity is evident. Previous
studies have demonstrated that some loss of con-
tents from the phagocytic vesicle can be mea-
sured prior to its closure and this has been termed
regurgitation while feeding (Weissmann et al.,
1972). However, significant superoxide produc-
tion can be measured extracellularly by pretreat-

50
ment of PMN with the fungal metabolite cyto-
chalasin B, which prevents the closure of the
phagocytic vesicle by disruption of cytoskeletal
components (Hartwig and Stossel, 1976). During
the ingestion process, primary granules fuse with
the phagocytic vesicle. The phagocytic vesicle
then contains a battery of proteolytic enzymes,
which can cause tissue destruction, and MPO,
which can combine with H202 and a halide to
produce HOCI. Should conditions exist that pre-
vent the closure of the phagocyte vesicle, the
otherwise contained battery of proteases and
powerful oxidants are released to effect their
damage on extracellular tissues. Such conditions
have been demonstrated in vitro and appear to
exist in vivo. Conditions under which PMN en-
counter immobilized immune complexes or im-
munoglobulin aggregates (Henson et al., 1988),
where PMN are presented with either an over-
whelming exposure to particulate stimuli (i.e.,
the bacteria in the gingival crevice) or an un-
phagocytosable surface, have resulted in the ex-
tracellular release of oxygen metabolites and pro-
teolytic enzymes (Weissmann, 1978). This
process has been termed frustrated phagocytosis
(Henson et al., 1988) and results in the degra-
dation of extracellular tissues.
The phagocytosis by PMN of bacteria col-
onizing the gingival crevice does not appear to
be the primary mechanism for controlling the
accumulation of microorganisms in the crevice
or their dissemination into the gingival tissues.
It has been observed that the continuous efflux
of PMN from the subepithelial vascular plexus
into the gingival crevice acts to form a biological
barrier between bacteria and the crevicular epi-
thelium (Novak et al., 1984). Since numerous
studies have demonstrated that potential perio-
dontal pathogens are able to activate human PMN
and to initiate the release of oxygen radicals and
granule contents (Passo et al., 1982; Taichman
et al., 1984; Mangan et al., 1989), it appears
that frustrated phagocytosis occurs within the
crevice. This extracellular release of microbicidal
agents may be the primary mechanism for con-
trolling the subgingival microflora. The high lev-
els of PMN granule contents isolated from gin-
gival crevicular fluid support this hypothesis.
Primary granule enzymes, such as beta glucu-
ronidase, are normally retained within the PMN
and are discharged into the phagosome during
phagocytosis. During frustrated phagocytosis,
both primary and secondary granule contents are
released into the extracellular space and can be
recovered in gingival fluid.
The PMN contains a wide variety of enzymes
packaged in granules that differ morphologically,
histochemically, developmentally, and in their
granule content (Table 1). There is considerable
evidence that the secretion of granule contents is
a regulated phenomenon (Boxer and Smolen,
1988; Henson et al., 1988). Primary granules
function intracellularly, being released into the
phagocytic vesicle following ingestion of partic-
ulate material. The secondary and tertiary gran-
ules, and the so-called replenishsomes, are re-
leased extracellularly and are important in
replenishing membrane receptors, enhancing the
respiratory burst, amplifying the PMN response,
and regulating granulopoiesis (Boxer and Smo-
len, 1988). These secretory granules also contain
large quantities of collagenase and gelatinase,
both of which are released in an inactive form
but which can be activated locally by oxygen
metabolites. Therefore, it is clear that the extra-
cellular release of lysosomal enzymes, combined
with the activation of the respiratory burst, has
the potential to lead to extensive, localized tissue
destruction. When attempting to use granule con-
tents as a marker of active tissue destruction,
several factors must be taken into consideration.
The distinct difference between primary granules
and the other granule types is that the former are
normally retained intracellularly. Secondary
granules (that contain collagenase) are "secre-
tory" granules. This means that they are released
extracellularly upon activation of the cells. This
usually occurs during chemotaxis from the vas-
cular compartment to the tissue compartment and,
therefore, will occur naturally during the normal
physiologic response of PMN to inflammatory
stimuli. The quantity of collagenase retrievable
at sites of inflammation may be directly propor-
tional to the numbers of PMN present and not
their metabolic state. It is therefore not surprising
that the level of collagenase activity in crevicular
fluid fluctuates with the level of tissue inflam-
mation (Kowashi et al., 1979) but is not predic-
tive of active alveolar bone loss in humans (Bir-
kedal-Hansen et al., 1989). In contrast, the
51
presence in GCF of markers for the release of
PMN primary granules signifies the extracellular
release of a battery of proteolytic, tissue-destruc-
tive enzymes. Combined with the concomitant
release of oxygen radicals capable of activating
the extracellular pool of inactive collagenase and
gelatinase, a potent armamentarium of tissue-de-
grading weapons is unleashed. As noted previ-
ously, markers of primary granule release in GCF,
such as P-glucuronidase (Lamster, 1989) and
elastase (Palcanis et al., 1990), have been as-
sociated with active loss of attachment along the
root surface.
E. PMN "Priming" and its Potential Role
in Periodontal Tissue Destruction
The physiologic evaluation of PMN from the
peripheral blood of patients with acute bacterial
infection and bacteremia revealed that the cells
were hyperactive when compared with PMN of
healthy controls (McCall et al., 1973; Simms et
al., 1989; Barbous et al., 1980). It was realized
that components of Gram-negative bacterial cell
walls, such as lipopolysaccharide (LPS), are able
to enhance the oxidative burst of human PMN in
response to secondary stimulation without them-
selves initiating any measurable activity of the
NADPH oxidase (Kaku et al., 1983; Guthrie et
al., 1984; Forehand et al., 1989). This obser-
vation could be repeated using synthetic ana-
logues of a bacterial product, the N-formylated
oligopeptides (Schiffman et al., 1975) at con-
centrations that are chemotactic for PMN but did
not activate the respiratory burst (Van Epps and
Garcia, 1980; English et al., 1981; Bender et al.,
1983; Dewald and Baggiolini, 1985). The en-
hanced response of PMN to stimulation, induced
by their prior exposure to agents that do not, in
themselves, initiate the response, has been termed
priming. Although initially observed for FMLP
and LPS, PMN priming has since been demon-
strated using a variety of PMN stimuli at substi-
mulatory concentrations (McPhail et al., 1984),
and more recently using recombinant cytokines
(Steinbeck and Roth, 1989). Cytokines are poor
stimulants of the PMN oxidative metabolic burst
when the PMN are in suspension, but may have
a more profound effect when PMN are adherent
52
to biological surfaces (Nathan, 1987). When used
as priming agents at substimulatory concentra-
tions, recombinant cytokines, such as granulo-
cyte-macrophage colony stimulating factor, IL-
11 , and TNF-a, have been shown to dramatically
enhance the oxidative burst of PMN in response
to secondary stimulation (Steinbeck and Roth,
1989). In contrast, TNF-a and the interferons
gamma and alpha inhibit chemotaxis. In the case
of TNF-(a, the inhibition of PMN chemotaxis in
response to FMLP appears to be due to a change
in the affinity, but not the number, of FMLP
receptors present on the cell surface (Atkinson et
al., 1988).
The significance of these reactions may be
apparent when considering the acute "bursts" of
inflammation observed during infection with
Gram-negative organisms (Movat et al., 1987).
Gram-negative organisms contain LPS in their
outer membranes, and this may be released dur-
ing infection. It has been demonstrated that LPS
elicits TNF production (Beutler et al., 1985). The
priming of PMN by LPS and TNF results in an
enhanced oxidative burst and enhanced degran-
ulation (Steinbeck and Roth, 1989; Henson et al.,
1988). Since priming by these stimuli also results
in an inhibition of PMN chemotaxis, it is clear
that tissue-destructive proteases and products of
oxidative metabolism may be released within the
tissues or microvasculature, resulting in exten-
sive tissue damage, microvascular injury, and
enhanced vasopermeability (Movat et al., 1987).
For these reasons, an enhancement of the PMN
oxidative metabolic burst and associated degran-
ulation of tissue-destructive proteases, combined
with a chemotactic immobilization of the PMN
within the tissues, have been implicated in the
tissue injury associated with inflammation (Hen-
son and Johnston, 1987; Weiss, 1989; Malech
and Gallin, 1987).
Since Gram-negative microorganisms pre-
dominate in the subgingival environment, and
bacteremia and endotoxemia are commonly ob-
served even during normal oral hygiene proce-
dures, it is distinctly possible that priming of
PMN in the periodontal environment occurs. Re-
cent studies have demonstrated that PMN isolated
from patients with rapidly progressive periodon-
titis do, in fact, demonstrate an exaggerated or
hyperactive response, producing considerably
greater quantities of oxygen radicals than PMN
from healthy controls (Shapira et al., 1991).
Priming of PMN with LPS has also been shown
to mediate damage to periodontal ligament fi-
broblasts (Deguchi et al., 1990), supporting the
concept that the conversion of a physiologic PMN
response to a pathologic response may be initi-
ated by PMN priming that results in a hyperactive
PMN response.
F. Conclusions
Studies of GCF chemistry have suggested the
importance of an exuberant PMN response to
subgingival plaque in the active phases of per-
iodontal destruction. Furthermore, the accumu-
lated data regarding the functional status of PMN
at sites of infection and inflammation suggest that
tissue destruction associated with an influx of
PMN is the result of PMN hyperreactivity. In
contrast, both quantitative and qualitative PMN
defects may be associated with some rapidly pro-
gressive forms of periodontal disease. These dif-
fering conclusions do not create a dilemma, but
rather may represent opposite ends of a balance
that is no longer in equilibrium. Future studies
should focus on defining this equilibrium, and
identifying shifts in the equilibrium that occur
with different periodontal disease states and that
may be characterized by changes in GCF chem-
istry. This information may then be applied to
development of strategies for diagnosing and
treating the periodontal diseases.
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