Full text of DOI:10.1038/labinvest.3780438

0023-6837/02/8204-455$03.00/0
LABORATORY INVESTIGATION
Vol. 82, No. 4, p. 455, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
CD4؉ T-Lymphocytes Regulate Airway Remodeling
and Hyper-Reactivity in a Mouse Model of
Chronic Asthma
Paul S. Foster, Ming Yang, Cristan Herbert, and Rakesh K. Kumar
Department of Biochemistry and Molecular Biology (PSF, MY), John Curtin School of Medical Research, Australian
National University, Canberra; and Inflammation Research Unit (CH, RKK), Department of Pathology, University of
New South Wales, Sydney, Australia
SUMMARY: Asthma is an acute-on-chronic inflammatory disease of the airways, characterized by airflow obstruction and
hyper-reactivity of the airways to a variety of stimuli. Chronic asthma is associated with remodeling of the airway wall, which may
contribute to hyper-reactivity and fixed airflow obstruction. We used an improved mouse model of chronic asthma to investigate
the role of CD4ϩ T-lymphocytes in airway remodeling and hyper-reactivity. Animals functionally depleted of CD4ϩ T-lymphocytes
by repeated administration of a monoclonal antibody exhibited markedly decreased airway responsiveness. In addition, these
mice had greatly diminished subepithelial fibrosis, epithelial thickening, and mucous cell hyperplasia/metaplasia. Chronic
inflammation in the airway wall was moderately reduced, with a marked decrease in the accumulation of immunoglobulin-
synthesizing plasma cells. However, intraepithelial accumulation of eosinophils was not significantly inhibited and airway
epithelial expression of eotaxin was undiminished. This work provides the first experimental evidence that CD4ϩ T-lymphocytes
play a crucial role in the pathogenesis of the lesions of chronic asthma and lends support to the notion that functional inhibition
of these cells may be an important therapeutic target. (Lab Invest 2002, 82:455–462).
sthma is an acute-on-chronic inflammatory dis-
ease of the airways, clinically characterized by
1999). Increased numbers of activated CD4ϩ T cells
are recruited into the airways of asthmatics following
allergen challenge (Bentley et al, 1993) and T cell-
specific allergen peptides are able to trigger a late
asthmatic response (Haselden et al, 1999). Further-
more, treatment of severe asthmatics with a human-
ized monoclonal antibody to CD4ϩ T-lymphocytes
leads to marked improvement in peak expiratory flow
(Kon et al, 1998).
Studies in animal models of acute allergic broncho-
pulmonary inflammation provide additional support for
the pathogenetic role of CD4ϩ T-lymphocytes. Mice
lacking CD4ϩ T cells because of deficient ability to
express class II major histocompatibility complex an-
tigens fail to develop eosinophil-dominated inflamma-
tion or AHR following exposure to antigen (Brusselle et
al, 1994). Adoptive transfer of CD4ϩ T cells from
sensitized mice is associated with recruitment of eo-
sinophils and the development of AHR following anti-
gen challenge (Hogan et al, 1998a; Kaminuma et al,
1997). Conversely, depletion of CD4ϩ T-lymphocytes
by administration of antibody abrogates AHR (Gavett
et al, 1994; Hogan et al, 1998b; Mould et al, 2000).
Depletion of CD4ϩ T cells typically also inhibits eosin-
ophil recruitment (Garlisi et al, 1995; Gavett et al,
1994; Nakajima et al, 1992). However, both animal and
human studies indicate that there is no consistent
relationship between the accumulation of eosinophils
and the development of AHR (Crimi et al, 1998; Hessel
et al, 1997; Leckie et al, 2000; Mould et al, 2000; Nagai
et al, 1996; Wilder et al, 1999).
A
episodic airflow obstruction and abnormally enhanced
reactivity to a variety of stimuli (Chung and Adcock,
2000). Chronic asthma is associated with a variety of
changes of airway wall remodeling, including subepi-
thelial and airway wall fibrosis, epithelial metaplasia
and increased smooth muscle mass (Hegele and
Hogg, 1996). Pathogenetic mechanisms remain in-
completely defined, but it has been suggested that
acute inflammation is primarily responsible for epi-
sodic bronchoconstriction, whereas chronic inflam-
mation and airway wall remodeling contribute to air-
way hyper-reactivity (AHR) and to the fixed airflow
obstruction that develops in chronic asthmatics
(Bousquet et al, 2000).
Studies in human asthmatics indicate that CD4ϩ
T-lymphocytes are likely to play a crucial role in the
development of asthma and AHR. Progressive accu-
mulation of activated CD4ϩ T cells in the lamina
propria of the airways is associated with development
of the clinical features of asthma in individuals who
previously exhibited asymptomatic AHR (Laprise et al,
Received November 27, 2001.
This work was supported by grants from the Human Frontiers Science
Program (to PSF) and the National Health and Medical Research Council
of Australia (to RKK).
Address reprint requests to: Dr. R. K. Kumar, Department of Pathology,
University of New South Wales, Sydney, Australia 2052. E-mail:
R.Kumar@unsw.edu.au
Laboratory Investigation • April 2002 • Volume 82 • Number 4 455

Foster et al
An important, and as yet unresolved, question is
Airway Inflammation
whether CD4ϩ T-lymphocytes play a significant role in
airway wall remodeling in asthma. This is of particular
interest because AHR in asthmatics may correlate
with the development of changes of remodeling, es-
pecially subepithelial fibrosis (Chetta et al, 1996;
Hoshino et al, 1998). Increasing subepithelial fibrosis
has also been associated with progression from
asymptomatic AHR to asthma (Laprise et al, 1999).
This issue cannot be satisfactorily addressed in stud-
ies using short-term inhalational exposure to antigen,
because the characteristic changes of remodeling fail
to develop in such experimental models.
We have previously shown that sensitized BALB/c
mice develop widespread multifocal accumulation of
lymphocytes, plasmacytoid lymphocytes, and other
chronic inflammatory cells in the lamina propria of the
trachea after long-term inhalation exposure to aero-
solized ovalbumin (Temelkovski et al, 1998). This
finding was reproduced in mice treated with the con-
trol monoclonal antibody ␤GL113. In comparison,
there was a statistically significant reduction in the
mean profile density of cells in the lamina propria in
mice treated with the anti-CD4 antibody GK1.5 (p Ͻ
0.05) (Fig. 1A).
We have described an improved model of chronic
asthmatic inflammation of the airways in mice (Te-
melkovski et al, 1998). This involves systemic sensiti-
zation of high IgE-responder BALB/c mice to ovalbu-
min and subsequent chronic inhalational challenge
with low levels of aerosolized antigen for 30 minutes/
day, 3 days/week for 6 weeks. This approach consis-
tently elicits airway lesions typical of chronic human
asthma, including acute and chronic inflammation in
the epithelial layer and lamina propria, together with
epithelial hypertrophy, mucous cell hyperplasia/meta-
plasia, and subepithelial fibrosis. In contrast to previ-
ously reported mouse models of asthma, in which the
inflammatory response is characterized by a high
density of lymphocytes and eosinophils surrounding
the airways and extending to the blood vessels (Cohn,
2001), there are fewer total inflammatory cells and no
evidence of pulmonary parenchymal inflammation.
However, numerous intraepithelial eosinophils are
seen in the upper airways. In addition, the mice exhibit
marked airway hyper-reactivity to an inhaled cholin-
ergic agonist, which can be attributed to abnormalities
of the airways rather than to parenchymal lesions.
Thus, the model replicates most features of the
chronic human disease (Kumar and Foster, 2001).
To test the hypothesis that CD4ϩ T-lymphocytes
play a crucial role in acute and chronic inflammation of
the airways, remodeling of the airway wall, and devel-
opment of airway hyper-reactivity in chronic asthma,
we used our improved experimental model to examine
these responses in mice depleted of CD4ϩ
T-lymphocytes by chronic administration of antibody.
Our results establish a crucial role for CD4ϩ T cells in
airway wall remodeling and the development of AHR.
Whereas eosinophils are rarely identifiable within
the tracheal epithelium of normal BALB/c mice, sen-
sitized BALB/c mice treated with the control monoclo-
nal antibody ␤GL113 exhibited recruitment of signifi-
Results
Long-term administration of anti-CD4 and control
monoclonal antibodies had no apparent deleterious
effects upon the experimental animals. At the end of
the period of treatment, there was a slight decrease in
the mean weight of the anti-CD4-treated group rela-
tive to the control group (18.36 Ϯ 0.32 g versus 19.07
Ϯ 0.24 g, n ϭ 16, p Ͻ 0.05). There was no evidence of
pulmonary infection or other significant abnormalities
in the anti-CD4-treated group at autopsy.
Figure 1.
Inflammation in the airways in sensitized, chronically-exposed mice treated
with ␤GL113 control monoclonal antibody or GK1.5 monoclonal antibody to
CD4. A, Inflammation in the airway wall, as assessed by quantifying the
numerical density of cells in the lamina propria of the trachea. B, Numerical
density of intraepithelial eosinophils.
456 Laboratory Investigation • April 2002 • Volume 82 • Number 4

CD4ϩ T Cells in Chronic Asthma
cant numbers of eosinophils, comparable with that
previously described in untreated animals. Accumula-
tion of eosinophils was unaffected in mice treated with
the anti-CD4 antibody GK1.5 (Fig. 1B).
Airway Wall Remodeling
As has previously been demonstrated in untreated
animals, a prominent subepithelial reticulin-stained
zone was apparent in sensitized mice exposed to
antigen and treated with control monoclonal antibody.
The mean thickness of this zone was significantly
decreased in mice treated with anti-CD4 antibody (p Ͻ
0.001) (Fig. 2A) and was equivalent to that in naïve
mice. In parallel, treatment with anti-CD4 prevented
the development of thickening of the airway epithelium
that was observed in mice treated with control mono-
clonal antibody (p Ͻ 0.001) (Fig. 2B).
Mucus-secreting goblet cells, which are virtually
absent in the intrapulmonary airways of naïve mice,
were strikingly increased in sensitized exposed mice
treated with the control antibody. These animals had a
median grade of 3.5, whereas mice treated with anti-
CD4 exhibited significantly decreased mucous cell
hyperplasia/metaplasia, with a median grade of 1.0 (p
Ͻ 0.05) (Fig. 2C).
Airway Responsiveness
As reported previously for sensitized, chronically-
exposed BALB/c mice, animals treated with the con-
trol monoclonal antibody exhibited a left-shifted dose-
response curve and increased maximal reactivity,
characteristic of airway hyper-reactivity (Fig. 3). In
contrast, animals treated with anti-CD4 exhibited
markedly decreased airway responsiveness, compa-
rable with that of naïve mice. The difference in en-
hanced Pause (Penh) was statistically significant at all
methacholine concentrations tested.
Immunohistochemistry
Consistent with our previously reported observations
in sensitized, chronically-exposed BALB/c mice (Ku-
mar et al, 2000), there were numerous cells immuno-
reactive for cytoplasmic Ig in the lamina propria of the
airways in animals treated with the control monoclonal
antibody (Fig. 4A). Immunostaining for IgG subclass
revealed the presence of a corresponding number of
IgG1-positive cells (Fig. 4B) and no evidence of IgG2a-
positive cells (Fig. 4C). In contrast, Ig-positive cells
were virtually absent in mice treated with anti-CD4
antibody. The profile density of these cells was re-
duced from 3.79 Ϯ 0.95 to 0.19 Ϯ 0.07 cells/100 ␮m
basement membrane (p Ͻ 0.01).
There was no evidence of diminished expression of
growth factors of the epidermal growth factor (EGF)
family by airway epithelial cells in animals chronically
treated with anti-CD4. For TGF-␣, the median grade
for intensity of immunoreactivity of the epithelium was
2.0 in mice treated with the control antibody and 2.5 in
mice treated with anti-CD4, whereas for amphiregulin
Figure 2.
Changes of remodeling in sensitized, chronically-exposed mice treated with
monoclonal antibodies. A, Subepithelial fibrosis, as assessed by measuring the
thickness of the reticulin-stained zone. B, Hypertrophy of the tracheal
epithelium. C, Mucous cell hyperplasia/metaplasia in the intrapulmonary
airways.
Laboratory Investigation • April 2002 • Volume 82 • Number 4 457

Foster et al
Figure 3.
Airway reactivity of sensitized, chronically-exposed mice, assessed by change
in enhanced Pause (Penh) in response to increasing concentrations of
aerosolized ␤-methacholine. Significant differences between mice treated with
␤GL113 control monoclonal antibody or GK1.5 monoclonal antibody to CD4
are shown as ** ϭ p Ͻ0.01, *** ϭ p Ͻ0.001.
the corresponding figures were 2.0 and 3.0. These
differences were not statistically significant.
We have previously shown that in chronically chal-
lenged mice, expression of eotaxin by airway epithelial
cells is maximal 3 hours after inhalational exposure
(Kumar et al, 2002b). Airway epithelium was strongly
immunoreactive for eotaxin both in mice treated with
control antibody and in those treated with anti-CD4.
Indeed, expression of eotaxin seemed to be moder-
ately elevated in the latter animals, with median
grades of 2.0 and 3.0, respectively (p Ͻ 0.05).
Discussion
In this investigation, we have demonstrated the feasi-
bility and effectiveness of long-term administration of
an anti-CD4 antibody to study the role of these T cells
in an experimental model of chronic asthma. In mice
that received injections of anti-CD4 over a period of 6
weeks, there was no evidence of illness, and although
there was a small decrease in mean weight compared
with mice that received the control antibody, there
was no evidence of pulmonary infection or other
significant abnormalities at autopsy.
Figure 4.
A, Many cells strongly immunoreactive for cytoplasmic immunoglobulin were
present in the lamina propria of the trachea of sensitized, chronically-exposed
mice treated with ␤GL113 control monoclonal antibody. B, Immunostaining for
cytoplasmic IgG1 demonstrated numerous immunoreactive cells (some indi-
cated by arrows). C, In contrast, virtually no cells immunoreactive for
cytoplasmic IgG2a could be identified in a consecutive section. Immunoper-
oxidase, counterstained with hematoxylin (A) or methyl green (B and C).
That the anti-CD4 antibody was effective in blocking
CD4 T cell function was clearly demonstrated by the
abrogation of the local humoral immune response in
the airway wall in the treated animals. We have previ-
ously shown that long-term inhalational exposure of
sensitized mice to antigen is associated with accumu-
lation of numerous IgG-producing plasma cells in the
lamina propria of the airways (Kumar et al, 2000). In
the present study, this finding was replicated in mice
treated with control antibody, while staining for IgG
subclasses revealed that the plasma cells were essen-
tially all IgG1-positive and IgG2a-negative, which is
consistent with a Th2 pattern of response (Finkelman
et al, 1990). In contrast, the response was abrogated
in mice treated with anti-CD4, presumably as a con-
sequence of the lack of helper T cell function for
antibody production.
The most striking feature of this study was that in
comparison with mice treated with control antibody,
there was marked suppression of airway wall remod-
eling in mice treated with anti-CD4. This was evident
both with respect to collagen deposition in the sub-
epithelial region and epithelial hypertrophy. Subepi-
thelial fibrosis is a distinctive lesion of human asthma
that is well reproduced in our chronic inhalation expo-
sure model (Temelkovski et al, 1998) but is not ame-
nable to study in animal models of acute allergic
bronchopulmonary inflammation. Our investigation is
the first to demonstrate the crucial role of the T
cell-mediated response in regulating airway wall re-
modeling in chronic asthma. Furthermore, these find-
ings are consistent with our earlier work using exper-
imental models of pulmonary fibrosis, which
demonstrated a role for T cells in regulation of the
458 Laboratory Investigation • April 2002 • Volume 82 • Number 4

CD4ϩ T Cells in Chronic Asthma
production of macrophage-derived growth factors for
fibroblasts (Kumar, 1989; Li et al, 1992), including
growth factors of the epidermal growth factor receptor
(EGFR) ligand family (Kumar et al, 1993). Recent
studies have emphasized the potential role of EGFR
ligands in airway wall remodeling in human asthma
(Davies et al, 1999; Puddicombe et al, 2000), particu-
larly with respect to epithelial injury and repair. There-
fore, we investigated whether there was any diminu-
tion of epithelial expression of TGF-␣ or amphiregulin
in the airways of mice treated with anti-CD4, because
these are the major EGFR ligands produced by airway
epithelial cells (Tsao et al, 1996). However, we were
unable to demonstrate any differences compared with
animals treated with control antibody, which might
indicate that other growth factors could be more
important in the induction of epithelial hypertrophy.
Nevertheless, our observations do not exclude a role
for EGFR ligands produced by macrophages (Madtes
et al, 1988), by eosinophils (Brach et al, 1994), or by T
cells themselves (Blotnik et al, 1994) in the pathogen-
esis of subepithelial fibrosis.
Another significant finding to emerge from the
present study was that CD4ϩ T cells played a crucial
role in the development of AHR. This result was not
unexpected, because the role of CD4ϩ T cells in AHR
has previously been demonstrated in a variety of
short-term, high-level exposure models of allergic
bronchopulmonary inflammation (Gavett et al, 1994;
Hogan et al, 1998a, 1998b; Mould et al, 2000). How-
ever, this work provides the first experimental evi-
dence that CD4ϩ T cells regulate AHR in chronic
asthma. This finding is entirely consistent with data
from long-term follow-up studies in human asthmatics
(Laprise et al, 1999).
An interesting question in this context is whether
there is a pathogenetic relationship between the sup-
pression of remodeling and the decrease in airway
reactivity following treatment with anti-CD4 in this
model of chronic asthma. Human studies reveal an
association between severity of remodeling and AHR
in asthma (Chetta et al, 1996; Hoshino et al, 1998), as
well as a correlation between increasing subepithelial
fibrosis and progression from asymptomatic AHR to
asthma (Laprise et al, 1999). However, our previous
studies using this experimental model suggest that
remodeling and development of AHR can be uncou-
pled in IL-5-deficient animals, which develop signifi-
cant airway wall remodeling but not AHR (Foster et al,
2000), as well as in IL-13-deficient animals, which do
not exhibit remodeling but continue to be hyper-
reactive to methacholine (Kumar et al, 2002a).
With respect to the inflammatory changes in the
airway walls, we wish to emphasize that because these
animals do not develop a significant alveolitis, relatively
few inflammatory cells are present in bronchoalveolar
lavage fluid and lavage is thus an inappropriate method
of quantifying inflammation in this model. We, therefore,
employed morphometric quantification of eosinophils
within the airway epithelium and of chronic inflammatory
cells within the lamina propria. In mice treated with
anti-CD4, there was a diminution in the number of
chronic inflammatory cells in the airway wall, as demon-
strated by morphometric quantification of nuclear pro-
files. This was possibly related to the absence of the
local humoral immune response and the lack of accu-
mulation of plasma cells (Kumar et al, 2000). Unexpect-
edly, however, there was no diminution in the intraepi-
thelial recruitment of eosinophils in anti-CD4-treated
mice. In experimental studies using short-term, high-
level challenge models of allergic bronchopulmonary
inflammation, recruitment of eosinophils was shown to
be abrogated by administration of the GK 1.5 antibody
against CD4ϩ T cells (Gavett et al, 1994), and the T cell
dependence of eosinophil recruitment was shown to be
related to induction of eotaxin. However, in our study
using chronic low-dose challenge, expression of eotaxin
by airway epithelium was undiminished in mice treated
with anti-CD4 antibody. We have recently demonstrated
up-regulation of the induction of eotaxin and other eo-
sinophil chemoattractants in this model as compared
with short-term, high-level exposure models (Kumar et
al, 2002b), and we speculate that this may account for
the lack of effect of administration of anti-CD4 on eosin-
ophil recruitment.
In conclusion, our studies in this model of chronic
asthma have facilitated investigation of the pathogen-
esis of changes in the airway wall, which simply could
not be carried out with conventional short-term mod-
els of allergic bronchopulmonary inflammation. They
have also highlighted some striking differences be-
tween the pattern of inflammation observed in our
model and in short-term, high-level exposure models.
The results emphasize the critically important role of
CD4ϩ T-lymphocytes in asthma and lend support to
the notion that functional inhibition of these cells may
be an important therapeutic target (Kon and Kay,
1999).
Methods
Animals
Specific pathogen-free female BALB/c mice were ob-
tained from the Biological Resources Centre, Univer-
sity of New South Wales, Sydney, Australia. Animals
were aged approximately 8 weeks at the commence-
ment of experimental studies. They were maintained in
a laminar flow holding unit (Gelman Sciences, Sydney,
Australia) and housed in autoclaved cages on auto-
claved bedding in an air-conditioned room on a 12
hour light/dark cycle. Irradiated food and acidified
water were provided ad libitum throughout. All exper-
imental procedures complied with the requirements of
the Animal Care and Ethics Committee of the Univer-
sity of New South Wales (ref. no. 98/29).
Sensitization, Antibody Administration, and
Inhalational Exposure
This was performed as previously described (Temelk-
ovski et al, 1998), except that, to ensure reproducible
induction of an antiovalbumin IgE response, mice
were sensitized by an intraperitoneal injection of 50 ␮g
of alum-precipitated chicken egg ovalbumin (Grade V,
Laboratory Investigation • April 2002 • Volume 82 • Number 4 459

Foster et al
Ն98% pure; Sigma, St. Louis, Missouri; unless other-
wise specified, all chemicals were obtained from this
source) 21 days and 7 days (booster injection) before
inhalational exposure. Experimental groups com-
prised of 7 to 9 immunized and boosted animals were
administered weekly intraperitoneal injections of 0.75
mg of ammonium sulfate-purified anti-CD4 monoclo-
nal antibody GK1.5 or isotype control monoclonal
antibody ␤GL113 (Hogan et al, 1998b), beginning 3
days before inhalation exposures were commenced.
Mice were exposed to aerosolized ovalbumin for 30
minutes/day on 3 days/week for 6 weeks in a whole-
body inhalation exposure system (Unifab Corporation,
Kalamazoo, Michigan). During the exposure, the ani-
mals were held in wire flow-through cage racks and
filtered air was drawn through the 0.5 m3 inhalation
chamber at a flow rate of 250 l/minutes. A solution of
2.5% ovalbumin in normal saline was aerosolized by
delivery of compressed air to a sidestream jet nebu-
lizer (Trimed, Sydney, Australia) and injected into the
airstream entering the chamber. The concentration
and size distribution of particles within the breathing
zone of the mice were continuously and cumulatively
monitored using a light scatter device (PCAM-TX;
PPM, Knoxville, Tennessee). Particle concentration
was maintained in the range of 10 to 20 mg/m3 by
controlling the airflow into the nebulizer.
incubation for 30 minutes with a complex of strepta-
vidin (Boehringer Mannheim, Sydney, Australia) and
biotinylated horseradish peroxidase in PBS-1% BSA.
Diaminobenzidine was used as the substrate and
sections were lightly counterstained with hematoxylin
for 30 seconds.
To assess relative production of IgG1 and IgG2a by
the Ig-producing cells, immunostaining was per-
formed using rabbit antibodies specific for each of
these immunoglobulin subclasses (Zymed, San Fran-
cisco, California) (5 ␮g/ml). Antigen retrieval and
blocking of nonspecific binding were performed as
above. Following incubation with the primary anti-
body, detection was by sequential 30-minute incuba-
tions with a 1:50 dilution of swine anti-rabbit immuno-
globulins, followed by a 1:100 dilution of rabbit
peroxidase-antiperoxidase complex (Dako).
Expression by airway epithelium of growth factors
of the EGF family and of eotaxin were also assessed
by immunostaining. Expression of EGF family ligands
was demonstrated using rabbit polyclonal antibodies
to transforming growth factor-␣ (TGF-␣) (Santa Cruz
Biotechnology, Santa Cruz, California, sc-9043) (10
␮g/ml) and amphiregulin (NeoMarkers, Fremont, Cal-
ifornia, Ab-1) (20 ␮g/ml), and the procedure was as
described above. Eotaxin expression was demon-
strated using a goat polyclonal antibody to a 19-amino
acid peptide corresponding to an epitope at the car-
boxy terminus of mouse eotaxin (Santa Cruz, sc-6182)
(2.5 ␮g/ml). The procedure was similar except that
blocking was with rabbit serum and detection was
with a 1:200 dilution of rabbit anti-goat immunoglobu-
lins, followed by a 1:100 dilution of goat peroxidase-
antiperoxidase complex (Dako).
Histopathology
Tissues for histopathologic examination were col-
lected 18 hours after the last inhalational exposure
and fixed in 10% buffered formalin overnight. Tissues
for immunostaining were collected 3 hours after the
last exposure. The longitudinally-oriented trachea and
a horizontal slice from the mid-zone of the single-
lobed left lung were embedded in paraffin. Ribbons of
2 to 3 sections, cut at 5 ␮m, were stained with
hematoxylin and eosin for assessment of inflammatory
changes in sections of the longitudinally-oriented tra-
chea, with Gordon & Sweet’s reticulin stain for assess-
ment of collagen deposition, or with Alcian blue-PAS
for enumeration of mucin secreting cells in the in-
trapulmonary conducting airways.
Morphometry
The validity and reliability of the morphometric tech-
niques we employed for quantifying inflammation and
remodeling have been established in previous reports
(Foster et al, 2000; Temelkovski et al, 1998). To
enumerate inflammatory cells and Ig-secreting cells,
images were captured with a Spot Cooled Color
Digital camera (Diagnostic Instruments, Sterling
Heights, Michigan), which was calibrated with a refer-
ence measurement slide. Nuclear profiles in hematox-
ylin and eosin-stained sections, or numbers of cells
exhibiting cytoplasmic immunoreactivity for immuno-
globulins, were counted in every second consecutive
microscopic field in the lamina propria and the length
of the epithelial basement membrane was measured
by tracing using the Spot software. The data were
used to calculate the mean number of cells per 100
␮m of epithelial basement membrane in the lamina
propria for individual animals.
Immunohistochemistry
To identify Ig-producing cells within the airway wall,
immunostaining was performed using a capillary ac-
tion staining system as previously described (Kumar et
al, 1989). Sections of formalin-fixed, paraffin-
embedded tissue were subjected to antigen retrieval
by boiling in 0.01M citrate buffer (pH 6.0) for 10
minutes in a microwave. Sections were washed with
PBS containing 0.1% Triton X-100 and nonspecific
binding was blocked with 50% normal goat serum for
10 minutes at 37° C. They were then incubated for 30
minutes at 37° C with an appropriate concentration of
a biotinylated rabbit antibody to mouse immunoglobu-
lins (Dako, Sydney, Australia). Sections were washed
four times with PBS-Triton after this and each subse-
quent incubation. Bound antibody was detected by
In parallel, the full length of the epithelial surface was
examined using a ϫ100 oil immersion objective to enu-
merate intraepithelial eosinophils, which were readily
identified on the basis of their strongly staining cytoplas-
mic granules. The data were expressed as number of
cells per millimeter of epithelial basement membrane for
individual animals. The thickness of the epithelial layer
460 Laboratory Investigation • April 2002 • Volume 82 • Number 4

CD4ϩ T Cells in Chronic Asthma
was also measured in these sections and the mean
thickness for each animal was calculated.
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