Time course of inflammatory and remodeling events in a murine
model of asthma: effect of steroid treatment
Alexandre
Trifilieff,
Ahmed
El-Hashim, and
Claude
Bertrand
Novartis Horsham Research Center, Horsham RH12 5AB, United Kingdom
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ABSTRACT |
The kinetics of
airway inflammation and remodeling processes following ovalbumin
aerosol challenge in sensitized BALB/c mice was studied. Mice were
exposed to either single or five ovalbumin challenges over 5 days. In
both protocols, time-dependent increases in bronchoalveolar lavage
(BAL) cellular fibronectin, neutrophils and eosinophils were observed.
The kinetics of these events were similar in both protocols; however,
the magnitude of the response was much greater following repeated
challenges. BAL protein levels and lymphocyte numbers were increased
only following repeated challenges, whereas interleukin (IL)-5 and IL-4
were increased in both protocols. Histological analysis revealed a
time-dependent increase in epithelial cell proliferation and in
mucus-producing epithelial cells. Proliferation of alveolar cells was
observed only following repeated challenges. Airway hyperreactivity was observed in both protocols but was much greater following repeated challenges. Pretreatment with dexamethasone fully inhibited the inflammatory response and airway hyperreactivity but only partially inhibited the remodeling process. These data suggest that
glucocorticoids, although potent anti-inflammatory agents, may not be
potent in reducing the lung remodeling process associated with asthma.
airway hyperreactivity; dexamethasone; eosinophils
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INTRODUCTION |
IT IS THOUGHT THAT CHRONIC
INFLAMMATION of the asthmatic airways is responsible for the
reversible airway obstruction and the nonspecific bronchial
hyperresponsiveness observed in these patients (5). In
addition to the inflammatory process, another regular feature of asthma
is a significant airway remodeling that leads to structural lung
changes. These changes include basement membrane thickening due to
collagen and fibronectin deposition (21), fibroblast
proliferation (2), airway smooth muscle thickening as a
result of both smooth muscle cell hyperplasia and hypertrophy
(11), and excessive production of mucus glycoproteins (24). All these modifications lead to the thickening of
asthmatic airway walls, which in turn could explain the
hyperresponsiveness observed in this disease (11, 27).
Although lung remodeling is a constant observation in chronic asthma
(23), very few studies have attempted to develop an animal
model to study this process (18, 19, 22). In this study,
we developed a murine model of lung inflammation using sensitized mice
and ovalbumin (OA) aerosol challenge, and we used this model to study
the airway hyperresponsiveness and the kinetics of lung inflammation
and remodeling, including inflammatory cell influx, interleukin (IL)-4,
and IL-5 levels, plasma leakage, cellular proliferation, cellular
fibronectin production, and mucus secretion. Moreover, we also studied
the effect of a glucocorticosteroid, dexamethasone, given 1 h
before each aerosol exposure on all these parameters.
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METHODS |
Experimental design.
Male BALB/c mice or C57BL/6 (25-30 g) were immunized
intraperitoneally with 10 µg of OA (grade V; Sigma, St. Louis, MO) in 0.2 ml of alum (Serva, Heidelberg, Germany) on days 0 and
14. On day 20, in some of the mice, ALZET
minipumps (model 2002; Charles River, St. Aullbin-les-Elbeuf, France)
filled with 5-bromo-2'-deoxyuridine (BrdU; 10 mg/ml; Sigma) were
implanted subcutaneously in the scapular region. The BrdU minipumps
lasted 2 wk and were replaced on day 34. Mice were
challenged with a nebulized solution of either OA (50 mg/ml of PBS) or
PBS alone for 20 min as described previously (4). One
group was challenged once on day 21 (acute protocol), and a
second group was challenged daily between days 21 and
25 (chronic protocol). At specified time points after the
last challenge, mice were killed by an injection of 0.2 ml ip of
pentobarbital sodium (60 mg/kg). Once deeply anaesthetized, mice were
used either for bronchoalveolar lavage (BAL; 5-6 mice) or for
tissue collection (3-4 mice).
In another set of experiments, mice were treated with an injection of 3 mg/kg ip of water-soluble dexamethasone (Sigma) in PBS 1 h
before each challenge. Control mice received 0.1 ml of PBS. For BAL
cellular fibronectin and protein levels, BAL cellular content, and
total serum IgE, mice were killed 3 days after the last challenge. For
all the others parameters, mice were killed 7 days after the last challenge.
Assessment of BAL inflammatory cell infiltration.
After anesthesia, the trachea was cannulated, and BAL was performed by
injecting 0.3 ml of PBS, kept at room temperature, into the lung via
the trachea. The fluid was withdrawn and stored on ice. This procedure
was repeated four times. Total cell count was measured, and cytospin
preparation (Shandon Scientific, Cheshire, UK) was performed. Cells
were stained with Diff-Quik (Baxter Dade, Dudingen, Switzerland), and a
differential count of 200 cells was performed using standard
morphological criteria. The remaining BAL fluid was centrifuged (300 g for 10 min), and the supernatant was collected and stored
at
80°C for soluble mediator measurements.
BAL soluble mediator measurement.
Protein concentration was measured using the bicinchoninic acid
protein assay according to the manufacturer's instructions (Pierce,
Rockford, IL).
BAL cytokine levels [IL-5, IL-4, and interferon-
(IFN-
)] were
measured using commercially available kits (Endogen, Wolburn, MA). The
sensitivity of these assays was 15 pg/ml for IFN-
and 5 pg/ml for
IL-5 and IL-4.
To measure the BAL cellular fibronectin content, an ELISA procedure
modified from Rennard and colleagues (20) was used. Briefly, 96-well plates were coated overnight at 4°C with a solution of human cellular fibronectin (150 ng/ml; Sigma). BAL samples, at
appropriate dilution, were incubated overnight at 4°C with a mouse
anti-cellular fibronectin antibody (1:10,000; Sigma) and then
transferred to the fibronectin-coated wells. After the wells were
washed, antibodies that did not react with fibronectin in the BAL
samples were revealed by sequentially adding a biotinylated secondary
anti-mouse IgM (1:1,000; Sigma) and a streptavidin- horseradish
peroxidase complex (1:1,000; Amersham, Little Chalfont, UK). The
substrate 2,2-azino-bis(3-ethylbenzthiazole 6-sulfonic acid)diammonium
(Sigma) was then added for 5 min, the reaction was stopped with 10%
SDS, and the optical density was measured at 405 nm. Using these
procedures, the detection limit was 10 ng/ml. No signal was observed
when the plates were coated with collagen types I and IV or with laminin.
Determination of total serum IgE levels.
Following anesthesia, blood was taken from the aorta, the serum was
prepared, and the antibody titer was determined by ELISA as described
previously (15).
Determination of airway reactivity.
Airway reactivity was measured using barometric plethysmography and
whole body plethysmography (8). Twenty-four hours after the final challenge, unrestrained conscious mice were placed in a
plethysmographic chamber (Buxco Electronics, Sharon, CT), and respiratory parameters of each animal were measured in response to
increasing doses (0-0.3 M) of aerosolized methacholine dissolved in sterile PBS. The resistance was expressed as enhanced pause (Penh) according to the manufacturer's instructions.
Tissue preparation.
After anesthesia, the lungs were inflated through the trachea with 4%
buffered Formalin solution in PBS (pH 7.4) under a constant pressure of 150 mmH2O. After 2 h, the
lungs were removed from the thoracic cavity, cleared of nonlung tissue,
and immersed in 4% Formalin for 1 h. As a positive control for
BrdU incorporation and alcian blue-periodic acid-Schiff staining, a
section of gut was removed, perfused with 1 ml of 4% Formalin, and
immersed in fixative solution for 3 h. Lungs and gut were
routinely embedded in paraffin, and 4-µm sections were cut and
mounted on glass slides precoated with poly-L-lysine (Sigma).
BrdU and fibronectin immunostaining on lung sections.
Sections were deparaffinized for 20 min in xylene, dehydrated for 10 min in 100% ethanol, and then washed with PBS for 10 min. For BrdU
staining, slides were treated for 20 min with 0.2% trypsin (Zymed, San
Francisco, CA) at 37°C and washed under running tap water. After a 2 M HCl treatment for 30 min, sections were neutralized for 5 min in
sodium borate (0.1 M, pH 8.5) and washed in PBS. Endogenous peroxidase
activity was inhibited with 2% H2O2 in PBS for
30 min. After the blocking solution was applied (1% sheep serum in
PBS) for 15 min, sections were incubated with a rat anti-BrdU antibody
(1:50; abV Immune Response, Derry, UK) for 1 h, incubated with
biotinylated sheep anti-rat antibody (1:100; Amersham) for another
1 h, incubated with streptavidin-biotinylated horseradish
peroxidase complex (1:300; Amersham) for 30 min, incubated with
diaminobenzidine substrate for 10 min, and counterstained with Harris
hematoxylin. All slides were coded and counted blindly, under oil
immersion, using a ×400 magnification length. The bronchial epithelium
proliferation rate, previously shown to be a good index for the
measurement of lung cell proliferation (19)- was measured, as well as BrdU incorporation in alveolar cells as an index of the
changes occurring in the deep parenchyma. Care was taken to exclude all
the infiltrating inflammatory cells. In preliminary experiments,
airways were characterized according to the basement membrane
length and defined as large (>2 mm), medium (1-2 mm), or
small (<1 mm). BrdU-positive epithelial cells were expressed as a
percentage or as cells per millimeter of basement membrane. In either
case, no difference between the different airway sizes was observed.
Epithelial nuclear labeling index was expressed as a percentage of
BrdU-positive nuclei vs. total nuclei in at least 20 randomly chosen
airways. For alveolar cell proliferation, 1,000 cells were counted in
randomly chosen fields. Systemic distribution was confirmed by intense
BrdU staining in the gut of all animals.
Alcian blue-periodic acid-Schiff staining.
Sections were deparaffinized and immersed for 10 min in a
solution of alcian blue (1% in 3% acetic acid, pH 2.5). After a prolonged washing in running tap water, sections were treated with
0.5% periodic acid for 5 min, washed with several changes of distilled
water, placed in Schiff solution (Sigma) for 10 min, rinsed with
running tap water, and mounted without any counterstain. Section
analysis was performed in a blind fashion using a qualitative scoring
system (0-4), where 0 = no epithelial staining,
1 = slight epithelial staining, 2 = moderate epithelial
staining, 3 = heavy epithelial staining, and 4 = massive
epithelial staining.
Data analysis.
Data, expressed as means ± SE, were analyzed by ANOVA. A value of
P < 0.05 was taken as significant.
 |
RESULTS |
In preliminary experiments the response of both BALB/c and C57BL/6
mice to a single challenge of OA was compared. As shown in Table
1, C57BL/6 mice had significantly
decreased responses to OA compared with BALB/c mice for all the
inflammatory parameters examined, with the exception of the BAL
eosinophilia. More importantly, increased BAL fibronectin levels were
observed only in BALB/c mice. On the basis of these data, BALB/c mice
were chosen for study rather than C57BL/6 mice. The influence of the
number of challenges on the BAL eosinophilic influx by exposing
sensitized BALB/c mice to five challenges per week over 3 wk was
examined. Maximum response was obtained after five challenges (5.8 ± 0.4 × 105 eosinophils/ml, n = 5).
A diminished BAL eosinophilia was observed after 7 challenges (3.4 ± 0.3 × 105 eosinophils/ml,
n = 6) and disappeared after 12 challenges (0.1 ± 0.1 × 105 eosinophils/ml, n = 4).
Because allergen-induced lung inflammation wanes after 1 wk of allergen
exposure, the chronic protocol was established as five challenges.
BAL inflammatory cell counts.
As shown in Fig. 1, both the
acute and chronic OA challenges induced neutrophil and eosinophil
infiltration into the BAL. In the acute protocol, neutrophils were
apparent at 6 h postchallenge, peaked at day 1, and
resolved by day 3. The BAL eosinophilia was delayed,
appearing on day 1, peaking at day 3, and lasting
through to day 14. In the chronic protocol, a significant
BAL neutrophilia was observed from the first time point studied up to
the 6-h time point. BAL eosinophils were already present at the first
time point studied. Thereafter, the kinetics of cell infiltration and resolution were similar to those observed in the acute protocol. However, eosinophilia was much more pronounced in the chronic model
compared with the acutely challenged animals (e.g., a 4- to 5-fold
increase at day 3). BAL lymphocytes were increased only following chronic challenge, and a significant increase was observed from day 1 until day 14. In both protocols, no
change was observed in the number of macrophages (data not shown). In
the acute protocol, no increase in the BAL protein content was
observed, whereas in the chronic protocol, protein content increased
from day 1, peaked at day 3, and resolved by
day 7 (Fig. 2). An increase in
BAL T helper cell type 2 (Th2) cytokines (IL-4 and IL-5) was observed in both protocols as early as 0 and 6 h for the chronic and acute protocols, respectively. By day 3, no more Th2 cytokines
were detectable (Fig. 3). In both
protocols, no IFN-
was detected in the BAL (data not shown). At
day 1 postchallenge in both protocols, no significant
increase in BAL inflammatory cell infiltration, protein, or cytokine
levels were observed in sensitized mice challenged with PBS (data not
shown).

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Fig. 1.
Time course of bronchoalveolar lavage (BAL) cellular
infiltration following acute and chronic challenges. Sensitized mice
were challenged once (acute, A) or challenged daily for 5 consecutive days (chronic, B) with ovalbumin (OA) and then
killed at specified time points. No significant variation in the number
of macrophages was observed in either protocol. Data shown are from 2 different experiments, each including 5-6 mice per group, and are
expressed as means ± SE. *Significance (P < 0.05) was determined between OA-challenged mice and nonchallenged mice
(before).
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Fig. 2.
Time course of BAL protein levels following acute and chronic
challenges. Sensitized mice were challenged once (acute, A)
or challenged daily for 5 consecutive days (chronic, B) with
OA and then killed at specified time points. Data shown are from 2 different experiments, each including 5-6 mice per group, and are
expressed as means ± SE. *Significance (P < 0.05) was determined between OA-challenged mice and nonchallenged mice
(before).
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Fig. 3.
Time course of BAL cytokine levels following acute (A
and C) and chronic (B and D)
challenges. Sensitized mice were challenged once (acute) or challenged
daily for 5 consecutive days (chronic) with OA and then killed at
specified time points. Data shown are from 2 different experiments,
each including 5-6 mice per group, and are expressed as
means ± SE. *Significance (P < 0.05) was
determined between OA-challenged mice and nonchallenged mice
(before). UD, undetectable.
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Total serum IgE.
The immunization procedure induced a time-dependent increase in the
level of total serum IgE, which peaked at day 9 following the initial sensitization. Following the boost, on day 14, a
more rapid and dramatic increase in total serum IgE was observed (Fig. 4). No further increase was induced by a
single OA challenge (Fig. 5). However,
repeated OA challenges induced a further significant increase of total
serum IgE that started 1 day following the last challenge and peaked at
3 days. Total serum IgE levels had begun to return to basal
values after 14 and 21 days for the acute and chronic protocols,
respectively (Fig. 5).

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Fig. 4.
Time course of total serum IgE levels during the
sensitization period. Mice were sensitized via the intraperitoneal
route on days 0 and 14. Data shown are from 1 experiment including 6 mice per time point and are expressed as
means ± SE. *Significance (P < 0.05) was
determined between sensitized mice and nonsensitized mice (day
0).
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Fig. 5.
Time course of total serum IgE level following acute and chronic
challenges. Sensitized mice were challenged once (acute, A)
or challenged daily for 5 consecutive days (chronic, B) with
OA and then killed at specified time points. Data shown are from 2 different experiments, each including 5-6 mice per group, and are
expressed as means ± SE. *Significance (P < 0.05) was determined between OA-challenged mice and nonchallenged mice
(before).
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Lung remodeling.
BAL cellular fibronectin content was measured as a marker of
extracellular matrix component production. Using either protocol, a
similar time-dependent BAL cellular fibronectin increase was observed.
However, the level of BAL cellular fibronectin was much more elevated
in the chronic protocol than in the acute protocol (Fig.
6).

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Fig. 6.
Time course study of BAL cellular fibronectin level following acute
and chronic challenges. Sensitized mice were challenged once (acute,
A) or challenged daily for 5 consecutive days (chronic,
B) with OA and then killed at specified time points. Data
shown are from 2 different experiments, each including 5-6 mice
per group, and are expressed as means ± SE. *Significance
(P < 0.05) was determined between OA-challenged mice
and nonchallenged mice (before).
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Both acute and chronic OA challenge induced a significant increase in
the number of BrdU-positive epithelial cells compared with PBS
challenge (Fig. 7A). This
effect was rapid in onset as evident from the significant proliferation
observed at the first time point studied (3 days after the last
challenge). During the acute protocol, OA-specific cellular
proliferation increased until day 14 and then appeared to
plateau at day 21. In contrast, during the chronic protocol,
the antigen-induced cellular proliferation was much more pronounced on
the 3rd day following the last challenge but did not show any further
increase (Fig. 7A).

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Fig. 7.
Time course of epithelial cell (A) and alveolar cell
(B) proliferation after acute and chronic challenges.
Sensitized mice were challenged with PBS or OA and killed at specified
time points. Percentage of 5-bromo-2'-deoxyuridine (BrdU)-positive
nuclei vs. total nuclei was counted in at least 20 airways for
epithelial cells. For alveolar cells, 1,000 cells were counted in
randomly chosen fields. Data shown are from 2 different experiments,
each including 3-4 mice per group, and are expressed as means ± SE. *Significance (P < 0.05) was determined between
OA and PBS challenged mice at the same time point.
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When compared with PBS challenge, the OA challenge did not increase
alveolar cell proliferation in the acute protocol (Fig. 7B).
In contrast, significant OA-specific alveolar cell proliferation was
observed in the chronic protocol. As observed for the epithelial cells,
this effect was rapid in onset (3 days after the last challenge) and
plateaued from day 7 (Fig. 7B).
In PBS-challenged mice, either no or very few alcian blue-periodic
acid-Schiff positive epithelial cells were observed. However, in
contrast, OA provocation induced a dramatic change in the secretory phenotype of the epithelium in both protocols (Fig.
8). This secretory phenotype was mainly
observed at the level of the large bronchi. A semiquantitative analysis
of the epithelial mucus secretory phenotype showed that it was maximal
at the first time point studied (3 days after the last challenge) and
thereafter decreased through to day 21. The mucin secretory
phenotype was more intense in the chronic protocol but was more
prolonged in the acute protocol (Fig. 9).

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Fig. 8.
Alcian blue-periodic acid-Schiff (PAS) staining of
representative lung sections at day 7 after chronic
challenge. A: PBS-challenged mice. B:
OA-challenged mice. Similar pattern of staining was observed after
acute challenge. Magnification: ×200.
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Fig. 9.
Time course of alcian blue-PAS staining on lung section after acute
(A) or chronic (B) challenges. Sensitized mice
were challenged with PBS or OA and killed at specified time points. A
semiquantitative estimation was done with a score from 0 (no epithelial
staining) to 4 (strong epithelial staining). Data shown are from 2 different experiments, each including 3-4 mice per group, and are
expressed as means ± SE. *Significance (P < 0.05) was determined between OA-challenged mice and PBS-challenged
mice.
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Measurement of airway reactivity.
Animals acutely challenged with OA showed a significant increased
Penh in response to increasing doses of methacholine
compared with PBS-challenged animals. However, no significant
difference was observed at the highest dose of methacholine (0.3 M).
The increased Penh to the dose response of methacholine
observed following chronic challenge was much more pronounced.
Moreover, this hyperreactivity was also present at the highest dose of
methacholine. Dexamethasone (3 mg/kg ip) fully inhibited the
hyperreactivity seen in the acute and chronic protocols (Fig.
10).

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Fig. 10.
Measurement of airway hyperreactivity to methacholine
after acute (A) and chronic (B) challenges.
Sensitized mice were nontreated and challenged with PBS (baseline),
treated with dexamethasone (3 mg/kg ip) and challenged with OA, or
treated with PBS (0.1 ml ip) and challenged with OA. Airway
hyperreactivity was measured 24 h after the last challenge by
exposing the animals to increasing concentrations of aerosolized
methacholine. Data shown are from 2 different experiments, each
including 6-7 mice per group, and are expressed as means ± SE. *P < 0.05 vs. untreated/PBS-challenged mice.
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Effect of dexamethasone on inflammatory events and lung remodeling.
At day 3 postchallenge, in both protocols, dexamethasone (3 mg/kg ip) fully inhibited the inflammatory parameters found to be
increased (BAL eosinophil and neutrophil numbers in the acute protocol;
BAL eosinophil and lymphocyte numbers, BAL protein levels, and total
serum IgE in the chronic protocol). Other cell types were not affected
by this treatment (Table 2). In the same
way, at day 1 postchallenge, BAL IL-4 and IL-5
levels were also fully inhibited in both protocols (Table
3). In contrast to the inflammatory parameters, cellular fibronectin content was only partially inhibited (Table 2).
At day 7 postchallenge, dexamethasone significantly reduced
the allergen-induced epithelial cell proliferation in both protocols. However, it did not fully reverse this effect (Fig.
11). Similar results were obtained for
proliferation of alveolar cells in the chronic protocol (Fig. 11). No
allergen-specific alveolar cell proliferation was observed during the
acute protocol, and dexamethasone had no effect on the basal
proliferation of these cells (Fig. 11). The epithelial mucus secretory
phenotypes induced by OA challenge were also attenuated, but
not completely abrogated, in the acute and chronic protocols (data not
shown).

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Fig. 11.
Effect of dexamethasone on proliferation of epithelial
(A) and alveolar (B) cells. One hour before
each challenge, mice were treated intraperitoneally with 0.1 ml of PBS
(open bars) or 3 mg/kg of dexamethasone (hatched bars). PBS-challenged
mice (crosshatched bars) were not treated. Seven days after the last
challenge, mice were killed, and BrdU-positive cells were counted. Data
shown are from 4 mice per group and expressed as means ± SE.
*P < 0.05.
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DISCUSSION |
There is widespread evidence to support an important role for
airway wall remodeling in chronic asthma patients (23).
However, probably because of the lack of experimental tools, the
mechanisms leading to this phenomenon are still not fully elucidated.
In the present study, we have characterized an allergen-driven murine model of lung inflammation and have shown that the airway inflammation is associated with some of the remodeling features typically seen in
asthmatics. Nonmurine antigen-driven models have been used to model
this feature of human asthma (18, 19, 22). However, the
increasing number of reagents capable of probing the murine immune
system and its genetic variants may be helpful to unravel the events
leading to airway remodeling.
We report an allergen-driven murine model of lung inflammation that
simulates many of the characteristic features of human asthma. On
sensitization and aerosol challenge, the mice developed an inflammatory
cell infiltration that became more pronounced with repeated aerosol
exposure to the allergen. In the acute protocol, the inflammatory cells
present in the BAL were mainly neutrophils and eosinophils, whereas an
influx of lymphocytes was observed only following chronic challenge.
Consistent with this lung eosinophilic inflammation, a similar increase
in BAL Th2 cytokines was observed in both protocols. Although repeated
challenges clearly increased the intensity of the lung inflammatory
cell infiltration, they also induced new inflammatory processes
compared with the acute allergen exposure. In addition to the
lymphocytic BAL infiltration, an allergen-induced plasma leakage as
measured by BAL protein levels was observed only after repeated
exposure. Similarly, an increase in total serum IgE over the
sensitization level following aerosol exposure of the allergen was
evident only in the chronic protocol. Overall, the pattern of the
inflammation obtained in the chronic protocol was closer to what is
observed in human asthma (5).
In both protocols, the allergen challenge induced an increased airway
responsiveness to methacholine when compared with PBS-challenged animals. However, in the acute protocol, the mice were only
hypersensitive to methacholine (no difference was observed at the
highest dose of methacholine). In contrast, in the chronic protocol,
mice were both hypersensitive and hyperreactive to methacholine, and
the magnitude of the response was much higher when compared with
the acute protocol. Although the mechanisms of airway hyperreactivity in human asthma are not fully understood, many studies have tried to
address this problem using murine models of asthma. Both IgE (7) and T cells (9) have been implicated as
major contributors to airway hyperreactivity in these models. In our
model, the BAL lymphocytic influx and increase in total serum IgE,
observed only following repeated challenge, could at least partially
explain the increase in severity of the hyperreactivity observed in the chronic protocol. In addition to the immunological component, lung
structural changes have also been suggested to play a role in airway
hyperreactivity (6, 18). Accordingly, in our model, the
parenchymal cell proliferation and the plasma leakage, observed only in
the chronic protocol, may also play a role in the increased hyperreactivity.
One of the characteristics of the lung remodeling in human asthma is
basement membrane thickening due to extracellular matrix protein
deposition (21). In our model, we do not have evidence for
deposition of extracellular matrix in the lung. However, the increased cellular fibronectin levels observed in the BAL could be the
first step of a cascade, eventually leading to extracellular matrix
protein deposition in the tissue. Indeed, using a similar model, a
previous study has suggested that subepithelial fibrosis is only
apparent after 4-6 wk of allergen exposure (25).
Another aspect of the lung remodeling is the proliferation of various cellular types that have been reported in both asthmatic individuals (2, 10) and animal models (18, 19, 22). Our
data clearly show that allergen challenge induced epithelial cell
proliferation in both protocols. It has to be noted that alveolar cell
proliferation was observed only in the chronic protocol; however, the
relevance of this observation is not clear, since alveolar cell
hyperplasia was never described in human asthma. Although smooth muscle
hyperplasia and epithelial desquamation are characteristic features of
human asthma (23), such a phenomenon was not evident in
the present study. However, all these features may be related to the
severity and the chronicity of the disease (12), and,
despite the chronic allergen challenges, we still may have induced an
acute and mild inflammatory response in our model.
It has been suggested that airway smooth muscle thickening may be the
most important determinant of airway responsiveness alterations
(14). However, an increase in airway submucosal area
(11) or an increase in adventitial thickness
(16) could also exaggerate airway narrowing. Our data have
demonstrated, using the chronic protocol, a significant increase in
BrdU incorporation in the alveolar cells that may account for an
increased thickness of the alveolar wall. This, in turn, may decrease
the elastic load of the parenchyma on smooth muscle, eventually
resulting in airway obstruction (16). This concept is
further supported by the fact that the airway hyperreactivity observed
following repeated challenges was much more pronounced compared with
the acute protocol.
Mucus hypersecretion (24) is also thought to
contribute to the structural changes occurring in asthmatic lungs.
Excessive production of mucus glycoproteins may lead to a decrease in
airway caliber, airway obstruction, and progressive respiratory
insufficiency. However, very few studies have attempted to understand
the mechanism(s) responsible for this increase in mucus production.
Whether the increase in mucus-producing cells observed in the present
study is related to proliferation of secretory cells or to
differentiation of other epithelial cells to a secretory type remains
to be determined. However, the high proliferative rate observed in the
epithelium following allergen challenge, plus the fact that secretory
cells are known to be able to divide (1), may favor of the
first hypothesis.
Steroids are the most effective class of drugs to inhibit the
inflammatory reaction in asthma (3), but the question of whether they are also able to inhibit the airway remodeling in human
asthma is still controversial (13, 17, 26). In this study,
intraperitoneal administration of dexamethasone (3 mg/kg) before each
challenge fully inhibited the inflammatory reaction and the airway
hyperreactivity but only partially affected the remodeling process in
both protocols. These data suggest that steroids may be at least partly
effective in reducing the airway remodeling seem in asthmatic patients.
Although we were unable to demonstrate a complete picture of the
asthmatic airway remodeling, most probably due to the fact that this
process is related to the chronicity of the disease, we believe that
the allergic murine models described in the present study may be useful
to study the initial events leading to this process. Further studies
using these models combined with genetically modified mice and/or
specific receptor antagonists may prove useful in determining the link
between the allergic airway response and tissue remodeling in diseases
such as asthma.
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ACKNOWLEDGEMENTS |
Part of this work was performed at Novartis (Basel, Switzerland)
with the technical assistance of Antje Holle, Marinette Erard, Isabelle
Bruckhardt, and Junko Tsuyuki.
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FOOTNOTES |
Present address of C. Bertrand: Institut de Recherche Jouveinal/Parke
Davis, 94265 Fresnes, France.
Address for reprint requests and other correspondence: A. Trifilieff, Novartis Horsham Research Center, Wimblehurst Rd.,
Horsham RH12 5AB, UK.
The costs of publication of this
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