Direct in vivo evidence for mast cell

degranulation during allergen-induced

reactions in man

E. Gomez, M.Sc., 0. J. Corrado, M.R.C.P., D. L. Baldwin, F.R.C.S.,

A. R. Swanston, F.R.C.S., and R. J. Davies, M.D. London, England

There is little or no direct in vivo evidence in man to support the involvement of mast cell-
mediator release in the pathogenesis of immediate allergic reactions. We have pelformed a
within-subject controlled study to determine the changes that occur in nasal mast cells during
allergen-induced rhinitis. Twelve subjects with asymptomatic rhinitis were studied. Nasal
biopsy specimens were obtained from each subject after a control solution (isotonic saline, 0.9%
wlv) had been nebulized into one nostril and allergen sohtion (freeze-dried allergen extract
reconstituted with isotonic saline) into the other. The tissues obtained were @fixedin Carnoy’s
solution and stained with the a-naphthol AS-D chloroacetate esterase reaction (N AS-D CA ER).
Mast (cells were counted under light microscopy in the epi~helium and lamina propria, and the
integrity of each cell was assessed. No significant differences were found in the number of
epithelial or lamina propria mast cells in biopsy specimens obtained after saline or allergen
administration. However, the number of degranulated mas! cells after allergen provocation
(89%) was significantly greater than after instillation of control solution (15%) (p = 0.003).
Changes of mast cell degranulation after allergen provocc; tion were conjirmed by electron
microscopy. In six nonatopic, control subjects without rhinitis, there was no significant difference
between the percentages of degranulated mast cells after allergen provocation (25.8%) and
mstillation of saline (24.3%). This study provides direct in vivo evidence for allergen-induced
mast cell activation in man. (.I ALLERGYCLIN IMMJNOL 78:637-45, 1986.)

Allergic rhinitis is a worldwide problem affecting

between 2% and 20% of the population.’ The patho-
genesis of this common but important disease is be-
lieved to involve immediate type I allergic reactions.
The results of animal studies have provided consid-
erable evidence to support the involvement of the mast
cell in type I allergic reactions, and mast cell degran-
ulation and mediator release have been demonstrated
after allergen exposure.‘, ’ Furthermore, it has also
been demonstrated that mast cell-deficient mice ex-
press little or no passive cutaneous anaphylaxis.4, 5 It
is now increasingly recognized that mast cells from
different animal species and from different tissues
within the same species can display widely differing
functional characteristics.6-9 It would appear essential
to our understanding of allergic disease to seek con-
firmation that similar mechanisms occur during al-
lergic reactions in man.
From the Departmentof Respiratory Medicine, St. Bartholomew’s
Hospital, London, England.
Received for publication Jan. 28, 1985.
Accepted for publication March 18, 1986.
Reprint requests:R. J. Davies, M.D., Dept. of Respiratory Med-
icine. St. Bartholomew’s Hospital, London, ECl, U. K.
Abbreviations used
N AS-D CA ER: a-Napthol AS-D chloroacetate
esterase reaction
RHPF: Random high-power fields
GM: Geometric mean
EM: Electron microscopy
LM: Light microscopy
There is little or no direct in vivo evidence in hu-
mans demonstrating mast cell activation and degran-
ulation alter exposure to allergen. Most studies of type
I allergic reactions have relied on the measurement of
mediator:; such as histamine and neutrophil chemo-
tactic activity believed to have been released from
mast cells into peripheral blood after allergen prov-
ocation. “‘. I’ More recently, it has been possible to
measure these mediators in respiratory tract secre-
tions.“-15 However, such studies have produced con-
flicting results, and although histamine, neutrophil
chemotactic activity, and leukotrienes have been dem-
onstrated to increase in peripheral blood or secretions
during allergen-induced reactions, their source is not
limited to mast cells.‘“‘*
637
638 Gomez et al. J. ALLERGY CLIN. IMMUNOL.
OCTOBER 1986

TABLE I. Characteristics of the 12 subjects with asymptomatic rhinitis with extrinsic allergic rhinitis

Control solutioln Allergen extract

No. of No. of No. of No. of

Age mast cells degranulated mast cells degranulated
Subject (yr) Sex Allergen per 10 RHPF ma’st cells per 10 RHPF mast cells

33 M GP 0 0 2 2
.L 24 F DP I2 4 4
3 20 M GP 0 0 0
4 22 M GP 0 0 0
5 21 F GP 1 5 5
6 29 F GP 0 0 0
7 20 F GP 0 0 0
8 26 F CF 6 4 4
9 21 F GP 0 0 0
I0 25’ M GP IO I3 13
11 29 F GP 0 0 0
12 21 F GP 0 0 0
+ 0.58 +0.55
GM * SE 1.73 1.89
- 0.44 -0.43

GP = B, grass-pollen mix; DP = Dermatophagoides pteronyssinus; and CF = cat fur.

The allergen exrract used for provocation, the total number, and the number of degralulated mast cells in the epithclium after administration
of the control solution and allergen extract are presented.

The direct study of the mechanism involved in al-
lergic asthma in man is limited by the relative inac-
cessibility of the lower airways. It is far easier to study
the mechanism underlying allergic rhinitis because
representative samples of nasal mucosa can be readily
obtained under local anaesthesia with minimal dis-
comfort to the patient. Wihl and Mygind” obtained
nasal biopsy specimens from allergic subjects before
and after nasal provocation with grass pollen and com-
pared ciliary structure and function and quantitative
changes in mast cells in the specimens obtained. In
contrast to a similar study performed in animals,“’ they
demonstrated no changes in mast cell numbers but did
not comment on any qualitative changes compatible
with mast cell degranulation. More recently, Kawa-
bori et al.” studied changes in the cellular content of
nasal secretions and biopsy specimens from three pa-
tients after allergen provocation. Specimens were
stained with toludine blue to facilitate examinations
under EM. This stain is taken up by mast and basophil
cells, both of which may be involved during allergic
reactions. Although the examination of tissues by EM
is particularly useful for demonstrating details of cel-
lular ultrastructure, only small areas of tissue can be
examined, making quantitation of cellular changes
difficult. The CY-NAS-D CA ER is a method of stain-
ing that enables mast cells to be distinguished from
basophils, since mast cell granules contain an ester-
ase capable of hydrolyzing the reaction between o-N
AS-D CA and hexazotized pararosaniline hydrochlo-
ride. “. 23Although neutrophil polymorphonuclear leu-
cocytes also contain this specific esterase, these cells
can be clearly distinguished by their characteristic nu-
clear morphology.“’ The purpose of this study was to
assess qualitative and quantitative changes in nasal
mast cells induced by allergen provocation.
MATERIAL AND METHODS
Subjects
We studied 12 subjects, eight female and four male, aged
between 20 and 33 years (mean age 24.3 years). All were
atopic, i.e , they demonstrated one or more positive skin
reactions to a battery of common inhalant allergens. All had
a history of rhinitis but no symptoms at the time of study.
The extrimic nature of their asymptomatic rhinitis was con-
firmed by nasal provocation with the relevant allergen ex-
tract. either B, grass-pollen mix, 2.5% w/v in IO subjects.
cat fur, 150% w/v in 1 subject, or Dermutophagoides pter-
onyssinus, I .2% w/v in 1 subject. The study was performed
between March and April 1984 when all the subjects were
asymptomatic and not receiving medication” Six subjects
(five male and one female) aged between 22 and 24 years
(mean age 22.8 years) who had no history of allergic disease
(asthma, rhinitis. or eczema) and negative skin prick tests
to four common inhalant allergens (including grass pollen)
acted as a control group of subjects.
Test solutions and method of administration
The allergen solutions used for nasal provocation were
freeze-dried allergen extracts (Bencard. Brentford. U. K.)
reconstitukd with isotonic saline, 0.9% w/v. isotonic saline
VOLUME 78
NUMBER 4. P4RT 1
In vivo evidence for mast cell degranulation 639

TABLE II

Control solution Allergen extract

No. of No. of % of No. of No. of % of

mast cells degranulated degranulated mast cells degranulated degranulated
Subject per 10 RHPF mast cells mast cells per 10 RHPF mast cells mast cells

1 76 15 20 47 4s 96
2 98 17 17 36 33 92
3 37 4 II 71 69 97
4 57 6 11 59 44 75
5 46 8 17 45 44 98
6 34 6 18 45 41 91
7 47 38 81 4s 40 x9
8 67 8 12 56 54 96
9 35 4 II 59 58 98
10 40 2 5 55 40 73
II 40 8 20 18 17 95
12 34 9 26 27 22 Xl
+5.1 + 1.9 +3.4 +5.* +4.7 + 2.7
GM 2 SE 47’9 7.81 ‘6’29 .44.3 31.7 89.7
-4.6 - 1.6 -2.8 -4.6 -4.2 -2.6

NS = statistically not significant.

The total number and the number and percentage of degranulated mast cells in the lamina propria after administration of the control solution
and allergen extract and the values of significance are presented

alone was used as the control solution. Allergen and control
solutions were admmistered to separate nostrils by nebuliz-
ing 100 PI of each solution with a modified air spray (Hum-
brol, Hull, II. K.).
Study design
An identical protocol was followed for subjects with
asymptomatic rhinitis and nonatopic subjects without rhi-
nitis. Fifteen minutes after nebulizing the control solutions
to one nostril, two sprays of local anesthetic, lidocaine (Xy-
locaine: Astra Pharmaceutical Products, Inc., Worcester,
Mass., 10% w/v) were delivered from a metered-dose aero-
sol. Five minutes later a nasal biopsy specimen was obtained
1 cm from the anterior edge of the inferior turbinate with
cup forceps. After a further 5 minutes, allergen was ad-
ministered to the opposite nostril. In subjects with asymp-
tomatic rhinitis, nasal provocation with allergen produced
symptoms ot rhinitis (nasal itching, sneezing, rhinorrhea,
and nasal obstruction) and a visible change in appearance
of the inferior turbinate that became pale and edematous.
None of the nonatopic subjects without rhinitis who were
tested with grass-pollen extract developed any of these fea-
tures. After 20 minutes, a biopsy specimen was obtained
from this nostril as previously described. This time interval
was chosen to coincide with the maximal change in allergen-
induced nasal obstruction measured by rhinomanometry.”
A biopsy of the nose earlier in the time course of the allergic
reaction is hindered by the presence of sneezing. In four
subjects with asymptomatic rhinitis (Nos. 9 to I2 in Tables
I and II). biopsy specimens were obtained after any residual
allergen solution had been washed from the nasal mucosa
by nebulizing three separate 200 pJ aliquots of isotonic
saline into this nostril. This was to ensure that any changes
observed in nasal mast cells were due to allergen deposition
on the nasal epithelium rather than excess solution reacting
with the tissue after biopsy. In one subject with asymptom-
atic rhinitis (No. I), an identical biopsy procedure was
repeated itt a later date to obtain tissue for examination
by EM.
Preparation of nasal tissue
LM. Biopsy specimens were immediately placed in iden-
tical glass bottles containing Camoy’s fixative (absolute al-
cohol, 60 ml; chloroform. 30 ml; glacial acetic acid, 10 ml)
and coded to ensure that the tissues were examined blind
by one of the investigators (E. G.) who was unaware of the
results of nasal provocation and biopsy. The biopsy speci-
mens were fixed for 3 hours, then removed and placed in
an automatic tissue processor (Histokinette, Reichert-Jung,
Slough, U. K.), embedded in wax, and sectioned at 5 km
thickness by use of a rotary microtome (Reichert-Jung). The
sections w’zre rehydrated and stained with the aN AS-D CA
ER. The staining solution was prepared as follows: 20 mg
of pararosaniline (Sigma Chemical Co. Ltd.. Poole. U. K.)
was dissolved in 0.5 ml of 2 mol/L of hydrochloric acid.
Hexazotization was performed by allowing the pararosan-
iline solution to react with 0.5 ml of freshly prepared
4% sodium nitrite solution (BDH Chemicals. Ltd., Poole,
U. K.) for I minute. This was then added to 46 ml of Verona1
acetate buffer (pH 6.8) to lower the pH to 6.3. To this
640 Gomez et al. J. ALLERGY CLIN. IMMUNOL.
OCTOBER 1966

FIG. 1. A, Section of the lamina propria of a subject with asymptomatic rhinitis, demonstrating
intact mast cells after the instillation of the control solution (isotonic saline). (Original magni-
fication x400.1 6, Section of the lamina propria of the same subject demonstrating mast cell
degranulation after allergen administration. (Original magnii’ication x400.)
FIG. 2. A, High-power view of Fig. 1, A. (Original magnificati’on x 1000.) B, High-power view of
Fig. 1, B. (Original magnification x 1000.)

buffered solution, 10 mg of a-N AS-D CA (Sigma Chemical
Co., Ltd.) dissolved in 1 ml of N,N-dimethylformamide
(Sigma Chemical Co.. Ltd.) was added.
The tissue sections were incubated in this solution for 30
minutes at room temperature, washed in running tap water,
counterstained with Mayer’s hematoxylin (Raymond A.
Lamb, London, U. K.) for 7 minutes, dehydrated, cleared
in xylene, and mounted in dibutyl pthalate xylene. With
this stain, mast cell granules appear red (Fig. I), whereas
their nuclei are blue black in color.z4
EM. Biopsy specimens were fixed within 30 seconds of
their excision in 3% glutaraldehyde buffer with 0.1 mol/L
of sodium phosphate (pH 7.6) for between 2 and 4 hours
and then postfixed in 2% osmic acid buffered with 0.2
mol/L of cacodylate (SO: 50 by volume). The tissues were
subsequently dehydrated through a graded series of aqueous
alcohol solutions, absolute alcohol, and propylene oxide
before embedding in TAAB 812 resin (TAAB Laboratories
Ltd., Reading, IJ. K.). Sections were cut with an LKB
ultratome (LKB Instruments Ltd., Surrey, U. K.). After
examining thick sections stained with toluidine blue for
metachromasia. thin sections were cut and stained with
uranyl acel:ate and counterstained with Reynold’s lead
citrate.
Examination of tissue sections
LM. Tissue sections were examined with an Olympus
BH-2 light nicroscope (Gallenkamp, London, U. K.), eye-
piece X 10, objective X 40) incorporating a square graticule
in the eyeplece. The section was moved from left to right
10 high-power fields (each separated by an interval equal
to the width of the graticule) in the epithelium and the lamina
propria. Mast cell counts included those cells lying within
the confines of the square and those touching the upper and
left hand sides but not those touching the other two sides,
thus avoiding counting the same cell twice. The mean mast
cell count for the 10 high-power fields in the epithelium and
lamina propria was determined. Mast cell degranulation
was assessed with criteria similar to those reported by
Dvorak et al.‘h for toluidine blue stained sections of the
human gastrointestinal tract; however, the a-N AS-D CA
ER enabled us to detect additionally extrusion of mast cell
VOLUME 78
NUMBER 4, P4RT 1
granules (Table III). Examples of intact and degranulated
mast cells are illustrated in Fig. I, A and B, and Fig. 2,
A and B.
EM. The tissue sections were examined with a Philips
300 transmission electron microscope (Pye Unicorn Ltd.,
Cambridge. U. K.) at an accelerating voltage of 100 kV.
Statistical analyses
Differences in the numberandproportion of degranulated
mast cells after administration of allergen and control so-
lution were comparedwith Wilcoxon matched-pairssigned-
rankstest. and differencesbetweenthe numberof mastcells
in the tissues of subjects with asymptomatic rhinitis and
nonatopic subjects without rhinitis were comparedwith the
Mann-Whitney U test.
RESULTS
The use of cup biopsy forceps provided specimens
of roughly equal size, approximately 4 mm’. The pro-
cedure was well tolerated by all subjects and associ-
ated with only mmimal discomfort and bleeding. Bi-
opsy specimens included the superficial epithelium
and the deeper lamina propria in all subjects. The
lamina propria could be subdivided into a superficial
subepithelial layer lying immediately below the epi-
thelium and a deeper glandular layer.
The number of mast cells present in both groups in
the epithelium was small compared to the number
observed in the lamina propria. For the asymptomatic
subjects with rhinitis, this ranged from 0 to 12110
RHPF. (GM + SE 1.73 I:::) after instillation of the
control solution, and 0 to 13 (GM c SE 1.89 +i::)
after administration of allergen (Table I). Epithelial
mast cells in the nonatopic subjects without rhinitis
were found in only two of the biopsy specimens ex-
amined in which two and three epithelial mast cells
were observed. Both of these were in postsaline bi-
opsy specimens, and all the cells were intact. In sub-
jects with asymptomatic rhinitis, allergen provocation
led to the degranulation of all the mast cells present
in the epithelium, although, because of the small num-
bers. it was impossible to perform statistical analyses
of these qualitative changes when compared to those
changes produced by administration of the control
solution. In contrast, many more mast cells were ob-
served in the lamina propria, most being located in
the subepithelial layer. In subjects with asymptomatic
rhinitis, the number of mast cells per 10 RHPF present
in the lamina propria after instillation of the control
solution ranged from 34 to 98 (GM 2 SE 47.7 12::)
and did not differ significantly from the number of
mast cells present after the administration of allergen
ranging from 18 to 71 (GM 5 SE 44.3 T:.$. How-
ever, many more mast cells were degranulated after
allergen provocation when the number was compared
In vivo evidence for mast cell degranulation 641
TABLE III. The criteria used to assess the
state of mast cell granulation
Granulated cells Degranulated cells
Stain bright red Stain pink
Clearly defined with Loss of outline with interruptions
intact cell mem- in cell membrane
bran<:
Granules within cell Extrusion of granules outside cell
membrane membrane
to the number after instillation of the control solution
(p = 0.003), the percentage of degranulated mast
cells after allergen provocation being substantially
greater ihan after administration of the control solution
in all but one subject as illustrated in Table II. Exactly
the same pattern of results was obtained in the four
subjects (Nos. 9 to 12) in whom any residual allergen
had been washed off the nasal epithelium.
The number of mast cells in the lamina propria of
the nonatopic subjects without rhinitis ranged from 16
to 36 per IO RHPF (GM + SE 21.56 I:,$) in post-
saline biopsy specimens and 12 to 39 (GM t SE
24.34 Z f ii) in biopsy specimens taken after the ad-
ministration of allergen. These results are presented
in Table IV. No significant differences were found
between either the total number or percentage of de-
granulated mast cells in paired biopsy specimens. Sub-
jects wil:h asymptomatic rhinitis had a significantly
greater rumber of mast cells present in the nasal lam-
ina propria than did the nonatopic subjects without
rhinitis (p < 0.02).
In the biopsy specimens obtained from subject 1
(Table 1) that were examined by EM, no mast cells
were found in the epithelium of either the postsaline
or postallergen specimens. Mast cells were clearly
identified in the lamina propria and exhibited diverse
morphologic shapes, although many were elongated,
and a few of these cells had small surface projections
arising from the cell membranes. The granules ranged
from being homogenous and electron dense to being
uniforml:y particulate, whereas others exhibited char-
acteristic whorled and scrolled patterns (Fig. 3, A).
In the postallergen biopsy specimens, many of the
mast cells demonstrated stages of degranulation. In
some, the granules exhibited swelling, a loss in elec-
tron density (Fig. 3, A), and vacuolation. In others,
the fusion of the perigranular membrane and cyto-
plasmic rnembrane were observed (Fig. 3. B).
DISCUSSION
There is little in vivo evidence in man to support
the current concept of the involvement of mast cell
642 Gomez et al. J. ALLERGY CLIN. IMMUNOL.
OCTOBER 1986

TABLE IV. Characteristics of the six nonatopic subjects without rhinitis

-__
Control solution Allergen extract

No. of No. of % Of No. of No. of % of

Age mast cells degranulated degranulated mast cells degranulated degranulated
Subject (yr) Sex per 10 RHPF mast cells mast cells per 10 RHPF mast cells mast cells

I :!4 F 36 II 30.5 24 4 16.6

2 22 M 16 5 31 I? 8 66
3 Z!3 M 34 8 23.5 39 II 28.2
4 :!2 M 15 3 20 I6 2 12.5
5 >!3 M I9 4 21.1 IX 5 27.1
h 23 M I8 4 22.2 22 6 21.3
+3.63 +6.73
GM f SE 21.58 +3’67 24.34 + “95 20.36 - 3.08 25.76
--3.13 -1.81 -5.34

The number and percentage of degranulated mast cells in the lamina propria after administration of the control solution and allergen extract
are presented.

reactions in allergic respiratory disease. In this con-
trolled study we have demonstrated that allergen does
indeed cause mast cell degranulation, not only in the
epithelium but also in the deeper lamina propria.
These changes confirmed by EM are a specific re-
sponse to allergen since they do not occur in nonatopic
subjects without rhinitis. We found no significant al-
teration in mast cell numbers after allergen challenge,
although subjects with asymptomatic rhinitis have a
significantly greater number of mast cells in nasal
tissue than nonatopic subjects without rhinitis. Wihl
and Mygind’” performed a controlled study of 28 sub-
jects with hay fever, obtaining nasal biopsy specimens
before and after allergen challenge. As in this study,
they found no changes in mast cell numbers 30 min-
utes after allergen provocation. However, unlike in
this study, they did not comment on the state of mast
cell granulation.
The existence of two distinct subpopulations of
mast cells, termed “mucosal” and “connective tis-
sue” types, each with different characteristics, was
first recognized in the intestinal mucosa of rats.” Mast
cell heterogeneity also occurs in the human intestinal
mucosa,” and there is accumulating evidence to sug-
gest that “mucosal” type mast cells are found in the
lower respiratory tract of man.29% 3” Our preliminary
studies suggest that mast cells of this type occur in
the nasal mucosa,” and this has recently been con-
firmed by Okuda et al.” Wihl and Mygind” do not
provide details regarding the number of mast cells that
they observed in the nasal epithelium, but the number
of mast cells that they found in the lamina propria
was equivalent to between 2 and 43 per 10 RHPF
(mean 13). This difference might in part be explained
*Gomcz E. Con-ado 01, Baldwin DL, Davies RJ: In preparation.
by the fact that these workers used glutaraldehyde to
fix their tissues, which has been demonstrated to effect
the staining properties of mast cells of the rat intestinal
mucosa.*” Even though we found few mast cells in
the nasal epithelium in comparison to the lamina pro-
pria, the numbers present were much greater than
those observed by Pipkom” who again fixed tissues
in glutaraldehyde and found a total of only two mast
cells in the epithelium in all the biopsy specimens
stained from 14 subjects. The differences between
these studies in mast cell numbers could similarly be
explained by the use of glutaraldehyde as a tissue
fixative. .We used Camoy’s solution that is recom-
mended as one of the fixatives of choice for the study
of “mucosal” type mast cells.”
A number of investigators have demonstrated sig-
nificant d: fferences between the numbers of basophils
and mast cells present in the nasal secretions, smears,
and scrapings of individuals with asymptomatic rhi-
nitis compared to nonatopic subjects without rhini-
tis. *‘. “. 34Although in this study mast cells were found
more frequently in the epithelium in biopsy specimens
taken from subjects with asymptomatic rhinitis com-
pared to nonatopic subjects without rhinitis, it was
impossible to make meaningful comparisons between
the two groups since in many biopsy specimens. no
mast cells were observed in the epithelium. Contrary
to previous reports ,j’. j4 we found that the number of
mast cells. present in the lamina propria of subjects
with asymptomatic rhinitis was significantly greater
than in that of nonatopic subjects without rhinitis. This
finding is of considerable importance because it would
suggest that allergic disease may be a stimulus to the
increased production of mast cells” and that their con-
tinued proliferation may perhaps be dependent on a
T cell-derived lymphokine.36 Such a conclusion
VOLUME 78 IT vivo evidence for mast cell degranulation 643
NUMBER 4, PART 1

FIG. 3. A, Electron micrograph of a postallergen biopsy specimen. One mast cell granule exhibits
characteristic whorled and scrolled patterns (P), whereas the other two (0) exhibit swelling
and a loss in electron density. (Original magnification x 140,000.) B, Electron micrograph of a
different view of the same biopsy specimen demonstrating a direct connection between a
swollen, electron-lucent cytoplasmic granule and the extra cellular space ($). (Original mag-
nification x 5000.)

would be supported by in vitro studies because rodent
mast cells grown in culture appear to be dependent
on T cell-derived growth factor(s) for proliferation
and maintainence.37 The exact nature of the growth
factor(s) invoked is still being investigated, but the
putative lymphokine interleukin-3 appears a likely
candidate.38 39
Recent studies by Okuda et a1.33suggested that ba-
sophils present at or close to the epithelial surface may
play a major role in the genesis of allergic reactions
in the nose. Support for this concept came from a
direct EM study on the nasal mucous membrane after
allergen provocation. Although these investigators ob-
served changes of mast cell degranulation in the ep-
ithelial and subepithelial layers, this was not observed
deeper in the lamina propria. In biopsy specimens
obtained from one subject after allergen provocation,
we have observed similar ultrastructural changes of
mast cell degranulation after allergen challenge. There
are difficulties in the interpretation of some of the
results of the investigation by Kawabori et al.*’ They
studied biopsy specimens of patients with perennial
rhinitis for EM before undergoing turbinectomy for
persistent symptoms. Under these circumstances the
investigators may well have been examining mast cells
altered by continuing disease. Furthermore, although
EM allows detailed examination of cellular ultrastruc-
ture, it is difficult to examine the whole specimen to
obtain an accurate quantitative analysis. Finally, no
comment is made in the article whether or not the
644 Gomez et al.
biopsies were randomized and the specimens were
examined in a blind manner.
It is possible to explain our findings of mast cell
degranulation throughout the nasal mucous membrane
within 20 minutes of exposure to allergen in several
ways. Inhaled allergens mav react initiallv with sen-
I Y d I
sitized basophils or mast cells at or close to the nasal
epithelium. Subsequent mediator release may result
in the opening of tight intraepithelial junctions, al-
lowing penetration of allergen into the deeper lamina
propria where further interaction with sensitized mast
cells may occur.4o Possibly, as suggested by Newson
et al.“’ from studies on the rat intestines, released
mediators may stimulate nerve endings initiating re-
flex-mediated mast cell degranulation throughout the
nasal mucosa, but evidence that such mechanisms may
occur in the human nose is awaited.
We have provided direct evidence for allergen-in-
duced mast cell activation in the respiratory tract of
man. Mast cell degranulation occurs in the epithelium
and lamina propria. To our knowledge this is the first
time that this has been conclusively demonstrated.
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T cell reactivity to penicillin: Phenotypic

analysis of in vitro activated cell subsets

Marianne Koponen, Ph.D., Werner J. Pichler, M.D., and

Alain L. de Week, M.D. Bern, Switzerland

Patients with penicillin allergy demonstrate a T cell prolyerative response after in vitro
stimulation with penicillin G (Pen G) and other /I-lactam antibiotics. To understand better
penicillin-allergic reactions, T cell subset stimulation with Pen G was studied and compared with
other soluble (tetanus toxoid and puri$ed protein derivitive [PPD]) and membrane-bound viral
(injuenza A and Epstein-Barr viruses) antigens. A double jluorescence method for flow cytometry
was used to evaluate the activated cells simultaneously by pyronin Y staining of RNA and by
indirect immunofluorescence of cell surface T4, T8, or Leu 8 antigens. The antigens used
stimulated mainly the T4’ subset(>90%), whereas the nu.nber of activated T8 cells was slightly
increased only in Pen G- and influenza A-triggered cultures (5% to 15%). Leu 8 antigen was
used to analyze more precisely the activated T4’ cells. Pen G and injuenza A and Epstein-
Barr viruses stimulated both T4’, Leu 8’ (>.50% of activated cells, inducers for suppressor
cells), and T4’, Leu 8- (helpers for B cells) subsets, whereas PPD activated mainly T4’,
Leu 8 subpopulations. These results indicate that penicillin-allergic patients with skin symptoms
demonstrate a T cell subset stimulation that resembles more the reaction versus viral antigens
(membrane incorporated) than to soluble antigens like PPD. These results suggest that Pen G is
presented to T cells like viral proteins and might thus cause allergic reactions resembling skin
symptoms observed in viral diseases. (J ALLERGYCLINIMMUNOL 78.645-52, 1986.)

Allergic reactions to p-lactam antibiotics can cause

clinically different symptoms, such as anaphylaxis, Abbreviations used
pruritus, exanthema, vasculitis, interstitial nephritis, EB V: Epstein-Barr virus
hemolytic anemia, etc.’ Although the anaphylactic re- FITC: Fluorescein isothiocyanate
PBL: Peripheral blood lymphocytes
PPD: Purified protein derivative
From the Institute of Clinical Immunology, Inselspital, Bern, Switz- PY: Pyronin Y
erland. SI: Stimulation index
Supported by Swiss National Foundation Grant No. 3-543-0.83. ‘IT: Tetanustoxoid
Received for publication Oct. 11, 1985.
HLA: Human leukocyte antigens
Accepted for publication March 19, 1986.
Reprint requests: W. J. Pichler, M.D., Institute of Clinical Im-
Pen G: Penicillin G
munology. Inselspital, 3010 Bern, Switzerland.

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