Human Asthmatic Epithelial Cells Stimulate Collagen Type III Production by Human Lung Myofibroblasts after Segmental Allergen Challenge | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Epithelial injury and subepithelial collagen deposition are characteristic of asthma. We hypothesized that epithelial cell proliferation increases after airway injury in asthmatics, that epithelial cells stimulate lung myofibroblast collagen production, and that both processes are modulated by allergen-recruited inflammatory cells. Epithelial cells obtained at baseline, 1 d, and 1 and 2 wk after endobronchial allergen challenge from asthmatics and nonasthmatics were placed in culture, with and without bronchoalveolar lavage cells obtained from the same segment. Epithelial cell proliferation and collagen synthesis by human lung myofibroblasts stimulated with culture medium from these epithelial cells were determined. Epithelial proliferation increased (108 ± 50% above baseline, p = 0.01 for d, and p = 0.004 for group × day interaction) 1 wk postchallenge in cells from asthmatics, but not from nonasthmatics, and required bronchoalveolar lavage cell coculture. Culture medium from epithelium harvested from asthmatics, but not from nonasthmatics, at 1 to 2 wk postchallenge stimulated collagen type III production 50% to 70% (p = 0.043 for clinical group, p = 0.012 for day, and p = 0.022 for group × day interaction), but not collagen type I. This effect was independent of an acute eosinophilic response. We conclude that epithelial cells from asthmatics, but not from nonasthmatics, are stimulated to proliferate after allergen challenge, and over 1 to 2 wk postchallenge, stimulate collagen type III synthesis by lung myofibroblasts. Epithelial cell proliferation appears dependent upon infiltrating inflammatory cells, but stimulation of collagen type III does not.
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Keywords: bronchial asthma; respiratory epithelium; fibroblasts; bronchial allergen challenge; endobronchial challenge tests
Airway epithelial damage and excess subepithelial collagen deposition are characteristic of asthma (1). Epithelial disruption may arise from the release of granule products and proteases by mast cells and infiltrating leukocytes, including eosinophils (4, 5), with subsequent degradation of extracellular matrix constituents (6), alterations in adherence proteins (7, 8), and/or disruptions in normal cycles of epithelial cell proliferation and apoptosis (9).
Regeneration of damaged epithelium involves recruitment, migration, and proliferation of epithelial cells from adjoining, undamaged areas (10, 11). Replacement of extracellular matrix involves synthesis and deposition of cross-linked collagen fibrils as well as fibronectin, tenascin, and other proteoglycans and glycoproteins by fibroblasts (12, 13). The thickened lamina reticularis observed in asthmatics contains increased amounts of collagen type III, type V, fibronectin, and tenascin, which is not seen in other inflammatory airway diseases (6, 14). This airway remodeling in asthma has been postulated to represent reactivation of the embryonal epithelial-mesenchymal trophic unit (18). Supporting this hypothesis, in vitro mechanical or chemical damage to bronchial epithelial cells causes increased proliferation of myofibroblasts co-cultured with epithelial cells (19).
We hypothesized that segmental antigen challenge of asthmatics initiates an inflammatory response with subsequent epithelial cell proliferation and modulation of myofibroblast collagen production. We have previously shown that resolution
of the acute inflammatory response and leukocyte infiltration
after segmental antigen challenge occurs over an extended period of recovery in asthmatics (i.e., 
2 wk) (20). Epithelial
cell proliferation and stimulation of extracellular matrix synthesis ex vivo have been examined over this same time period
after an in vivo inflammatory stimulus to determine the kinetics of airway repair and epithelial cell modulation of myofibroblast matrix synthesis. We report here both alterations in
epithelial cell proliferation and modulation of human lung myofibroblast collagen type III synthesis in asthmatics, but
not in nonasthmatic control subjects, 1 to 2 wk after allergen
challenge. A preliminary report of some of these findings has
been presented in abstract form (21).
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Subjects and Bronchoscopic Procedures
Twelve nonragweed allergic nonasthmatic subjects, 4 ragweed-allergic
nonasthmatics, and 18 ragweed-allergic asthmatics were enrolled in
this protocol approved by Jefferson Medical College Institutional Review Board. Each volunteer gave informed, written consent to screening and bronchoscopic procedures and was characterized as previously
described, including airway responses to methacholine and ragweed-antigen inhalation (early and late reactions) (20, 22). All subjects were
nonsmokers receiving no chronic medication; asthmatics used a 
-agonist on an as-needed basis. Subject demographic and pulmonary function data are listed in Table 1.
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The bronchoscopic protocol (Table 2), described previously in detail (20), included four bronchoscopies (on Days 1, 2, 9, and 16), two segmental antigen challenges (SAC) (Days 1 and 2) with 5 ml of short ragweed (Greer Laboratories, Lenoir, NC) at a concentration 100 times the amount needed to cause a 2+ intradermal skin reaction, (maximum of 0.05 µg/ml ragweed antigen E in some allergics and 0.5 µg/ml ragweed antigen E in some nonallergic control subjects), with sample collection (bronchoalveolar lavage [BAL] [150 ml] and two to three bronchial brush biopsies) at every bronchoscopy as outlined (Table 2). In some subjects (9 of 34), the Day 9 and Day 16 sampling of nonchallenged, control lung segments was eliminated so that each day's procedure involved lavage and brush biopsy of only one lung segment in these subjects.
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Cells from BAL fluid were pelleted (600 × g, 15 min) (22) and aliquots of 105 BAL cells were added to epithelial cell culture as indicated below.
Epithelial Cell Culture and In Vitro Proliferation Assay
Epithelial cells from the brushes were combined, divided into four equal aliquots (approximately 1 to 2 × 105 cells), and placed in Millicell-CM culture inserts (Millipore Corp., Bedford, MA) with serum-free medium (23). Two epithelial cell aliquots had 105 BAL cells placed in each outer well; and two aliquots had no BAL cells. Cells were incubated for 24 h (37° C, 5% CO2) before further experimentation. During the 24-h incubation, viable epithelial cells averaged 109 ± 13% of their initial number (determined by Cell Titer 96Aq assay; Promega, Madison, WI).
The culture medium from each 24-h epithelial cell incubation (± BAL cells) was harvested and stored at 
70° C until used for fibroblast
stimulation. Cell Titer 96Aq assay was performed at 24 and 96 h (one
aliquot with and one without BAL cell coculture each time), and proliferation, i.e., the increase in cell number relative to initial number,
calculated: (absorbance96 h absorbance24 h)/absorbance24 h × 100%.
The initial 24-h cell numbers varied between groups and over the four
time points (asthma [n = 16]: 1.30 ± 0.17, 2.08 ± 0.29, 1.12 ± 0.14, and
1.16 ± 0.16 × 105; nonasthma [n = 12]: 1.90 ± 0.31, 2.55 ± 0.34, 2.22 ± 0.58, and 1.34 ± 0.18 × 105 on Days 1, 2, 9, and 16, respectively) (p = 0.038, asthmatics versus nonasthmatics, and p < 0.001 for day, two-way
repeated measures ANOVA). In asthmatics, the cell number from the
Day 2 challenged segment was greater than from Day 1 (p = 0.008),
Day 9 (p = 0.002), and Day 16 (p < 0.001) (Tukey test) cell numbers, but cell numbers on Days 1, 9, and 16 did not differ from one another.
Fibroblast Cell Culture and Western Blot Analysis of Collagen Synthesis
Human lung fibroblasts, obtained from histologically normal areas of
lung resected for diagnostic or therapeutic purposes, were grown to
confluence (24). These cells, used at passages 3 to 6, exhibited a myofibroblast phenotype, i.e., expression of smooth muscle 
-actin. At
confluence, the medium was replaced with equal volumes of serum-free medium with 40 µg/ml ascorbic acid, and culture supernates from
epithelial cells (± BAL cells). After 48 h, supernates were harvested
from fibroblasts and frozen until analyzed.
Samples of fibroblast culture medium were separated on polyacrylamide gels (25), either without further processing or after complete pepsin digestion (1 mg/ml pepsin [P-7012; Sigma Chemical, St Louis, MO] 2.9 µl of glacial acetic acid/100 µl at 30° C for 30 min followed by 15 h at 4° C; twice dried, and reconstituted with H2O) (26). Gel transfers to nitrocellulose were probed with primary antibody (either 1/ 3,000 dilution of monoclonal antihuman collagen type III [Accurate Chemical & Scientific Co., Westbury, NY], or 1/ 5,000 dilution of polyclonal antihuman procollagen type III [Chemicon International, Temecula, CA]), biotin-labeled secondary antibody, streptavidin-horseradish peroxidase complex, and developed with enhanced chemiluminescent (ECL) substrate (Amersham Pharmacia Biotech, Les Ulis, France). Nine of 10 membranes in each group (one blot in each group was lost) were stripped and reprobed with a 1/ 1,000 dilution of monoclonal type I collagen antibody (Accurate Chemical & Scientific Co.).
Collagen band densities were quantitated by a densitometry (1D Image Analysis Software, v 3.0.1; Eastman Kodak, Rochester, NY), and expressed relative to the level from fibroblasts stimulated by control segment (Day 1) epithelial cell supernates (cultured without BAL cells) within each subject. The raw collagen III band density from Day 1 asthmatic samples averaged 54,980 ± 11,930 units, and was comparable to 67,050 ± 10,550 units from Day 1 nonasthmatic samples (p = 0.458).
ELISA Assay for Transforming Growth Factor 1
Total transforming growth factor 1 (TGF1) isotype was measured by
ELISA in epithelial cell supernates after activation with acid pH according to manufacturer's protocol (R & D Systems, Minneapolis, MN). Levels (pg/ml) were normalized to cell number and expressed as pg/104 cells.
Statistical Analysis
Data were tested for normal distribution and equal variances by Sigmastat (version 2.0). Some data, when indicated, were transformed (usually by log10) to meet criteria for normal distribution and/or equal variance. Data of normal distribution were examined by one- or two-way repeated measures analysis of variance, and post hoc pairwise comparisons by Tukey test at individual time points, or by t test as appropriate (Sigmastat or Systat 5). Otherwise, data were analyzed by nonparametric tests such as Kruskal-Wallis one-way ANOVA on ranks, Friedman's repeated measures ANOVA on ranks, or Dunn's method for posthoc pairwise comparison (Sigmastat). A p < 0.05 was considered significant and a 0.05 < p < 0.10, a trend. If analysis of epithelial cell proliferation or fibroblast collagen synthesis did not differ based on subjects grouped according to one dependent variable, subjects were regrouped according to other variables for further analysis.
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Subject Characteristics
As noted above, Table 1 lists subject demographics, pulmonary function data, and BAL fluid return. The asthmatic group consisted of 6 subjects who had a single (early) bronchoconstrictive response to whole-lung ragweed challenge, and 10 subjects with dual (early and late) responses; 2 asthmatics were not characterized by whole-lung ragweed challenge. Epithelial cell proliferation and collagen type III stimulation were not different between asthmatics with a single versus those with a dual response to allergen; therefore, all asthmatics were grouped together.
The four allergic nonasthmatic subjects (rhinitics), had dual responses to ragweed inhalation. As we have previously reported (22), and as shown in Table 3, ragweed-allergic subjects who display a dual airway response after whole-lung ragweed challenge, but are not asthmatic (i.e., rhinitic) also display a marked airway (BAL) eosinophilia after SAC. Subjects with only an early response after airway ragweed challenge show a markedly attenuated BAL eosinophilia after SAC (shown for asthmatics in Table 3). The two nonasthmatic groups (allergic nonasthmatic and nonallergic nonasthmatic subjects) did not differ from each other in terms of epithelial cell proliferation and collagen III stimulation and were combined as "nonasthmatics" for comparison to asthmatics.
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Proliferation of Bronchial Epithelium after Allergic Inflammation
Epithelial cells from each time point were assessed for proliferation. To examine interactions of epithelial cells and BAL cells, via soluble mediators, epithelial cells were cocultured with BAL cells under conditions in which cells were physically separated but soluble factors were accessible to both cell populations, and compared to epithelial cells without coculture. There was no significant change in epithelial cell proliferation without BAL cell coculture in either asthmatics or nonasthmatics (Figure 1A). In contrast, epithelial cell proliferation with BAL cell coculture was greater in the asthmatic group than in the nonasthmatic group, and this effect was most marked on Day 9 (108 ± 50% increase, Figure 1B, p = 0.01 for day and p = 0.004 for clinical group × day interaction). In post hoc analyses, differences between asthmatics and nonasthmatics were significant on Days 9 and 16 (p = 0.046 and 0.004, respectively, Tukey test). Thus, at 1 and 2 wk after challenge, epithelial cells from allergic asthmatics were stimulated by autologous BAL cell coculture to proliferate more than epithelial cells from nonasthmatics.
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Cell Titer, % of
baseline) in vitro without
(A) and with (B) BAL cell
coculture from control (Day
1) and allergen-challenged
lung segments (Days 2, 9, and 16) in asthmatics
(n = 15) and in nonasthmatics (n = 10). The data
were analyzed by a two-way repeated measures
ANOVA on transformed
data (log10 [
, asthma; 
, nonasthma.
Stimulation of Lung Myofibroblast Collagen III Synthesis, but Not Collagen I Synthesis, by Asthmatic Epithelial Cell Supernates
Because there was a stimulatory effect of BAL cells on epithelial cell proliferation, we examined culture supernates from epithelial cells both with and without BAL cell coculture for stimulation of lung fibroblast collagen synthesis (see Figure 2). Analysis of collagen production, both type III and type I, by fibroblasts revealed no effect of BAL cell coculture with epithelial cells. Therefore, collagen stimulation data from epithelial samples with and without BAL cell coculture were averaged, so that assays, in effect, were performed in duplicate.
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Initial experiments to assess lung fibroblast synthesis of collagen type III induced by epithelial cells from asthmatics (n = 8) and nonasthmatics (n = 6, which included three nonallergic, nonasthmatics plus three allergic nonasthmatic subjects) examined samples that were not pepsin-digested (Figure 3). The sum of the two type III bands, procollagen and processed collagen (i.e., terminal peptides removed), both identified by the monoclonal antihuman collagen III antibody and by the polyclonal antihuman collagen III antibody, were analyzed. As shown in Figure 3, increased collagen type III synthesis was observed from lung fibroblasts stimulated with asthmatic epithelial cell supernates but not with nonasthmatic epithelial cell supernates (p = 0.011).
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These data were consistent with data from another approach to collagen type III quantitation (i.e., quantitation of pepsin-digested samples), which separates collagen types III and I based on different electrophoretic mobilities of the processed collagen molecules. The stimulation of collagen type III by epithelial supernates from 10 asthmatic subjects compared with epithelial supernates from 10 nonallergic, nonasthmatics is shown in Figure 4. Collagen type III release from lung fibroblasts was again significantly increased in asthmatics versus nonasthmatics (p = 0.043 for clinical group) with significant effects for day (p = 0.012) and group × day interaction (p = 0.022). A post hoc analysis of these data revealed significantly enhanced collagen III release from fibroblasts stimulated with epithelial cell supernates from asthmatics compared with nonasthmatic epithelial cell supernates on Days 9 and 16 (p < 0.03, Tukey test).
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Ragweed asthmatic subjects can vary markedly in their response to SAC, with some developing a marked airway eosinophilia, and others developing a minimal airway eosinophilia; such divergent responses correspond, respectively, to the presence or absence of a dual (early and late) airway response to whole-lung ragweed inhalation (Table 3). Asthmatics who developed a single (early) reaction to ragweed inhalation increased fibroblast collagen III synthesis stimulated by epithelial cell supernates equal to asthmatics who developed a dual (early and late) airway reaction to ragweed inhalation (Figure 5). Thus, the fibrogenic stimulus from asthmatic epithelium is not dependent upon, and does not reflect, the magnitude of inflammation, as measured in terms of eosinophil influx.
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, single (n = 4); 
, dual (n = 4).
Collagen type I synthesis by fibroblasts was subsequently examined for nine asthmatics and nine nonasthmatic subjects (Figure 6). In contrast to collagen III, collagen type I production was not significantly increased by fibroblasts stimulated with either asthmatic or nonasthmatic epithelial cell supernates.
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The Day 9 brush biopsies of a previously unchallenged and unsampled segment allowed us to determine whether the localized SAC produced a generalized alteration in epithelial cell function in areas of the lung that did not directly receive SAC. As previously shown (Figures 3-5), epithelial cells from an antigen-challenged segment obtained 1 wk after SAC from asthmatics, but not from nonasthmatics, released fibrogenic factor(s) that stimulated collagen III production (asthma, 207 ± 34 versus nonasthma, 96 ± 8% of baseline [= 100%]; two-way repeated measures ANOVA, p = 0.0016, for clinical group). Comparison of epithelial cells from the unchallenged segments from asthmatics versus nonasthmatics also showed a significant difference (asthma, 144 ± 14 versus nonasthma, 97 ± 15; p = 0.04, t test).
Role of Inflammatory versus Mechanical Injury in Asthmatic Epithelial Cell Supernate Stimulation of Myofibroblast Fibrogenic Responses
Although the major stimulus examined in this human model involves an IgE-mediated inflammatory event (i.e., segmental antigen challenge), bronchial brush biopsies were performed, which permitted us to also examine the effect of a mechanical injury to the airway epithelium. In eight asthmatic subjects, epithelium was obtained on Day 16 from both the baseline, unchallenged lung segment first sampled on Day 1, in addition to the antigen-challenged lung segment first sampled on Day 2. The resampling of the challenged segment after 2 wk (Day 16) provided epithelium that received both mechanical injury from bronchial brushing and immunologic injury from allergen challenge, and resampling of the unchallenged segment provided epithelium that received only mechanical injury. Stimulation of collagen III synthesis by asthmatic epithelium from these two lung segments did not differ (resampled challenged segment epithelium: 154 ± 30 versus resampled unchallenged segment epithelium: 173 ± 21; p = 0.209, paired t test), indicating that at this late time point, the immunologic injury did not contribute an additional stimulus for epithelial cells to release fibrogenic factor(s) other than that caused by mechanical injury alone.
Release of TGF1 from Epithelial Cells
To determine if the principal fibrogenic stimulus deriving from
asthmatic epithelium was TGF1, epithelial cell culture supernates from the initial 24 h, either with BAL cell coculture or without, were assayed by ELISA. TGF1 levels were normalized to
cell number at 24 h. Again, there was no difference in levels
from epithelial cells with or without BAL cell coculture, so
these were averaged for each subject at each time point. In Figure 7 the level of total TGF1 (both active and latent) was significantly greater in the supernates from asthmatic epithelial
cells than from nonasthmatic epithelial cells at all time points
(p = 0.032). There was no further increased release, however,
on Days 9 and 16 to correspond with the increased stimulus
for collagen type III synthesis observed at these time points.
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Subepithelial collagen deposition is one of the cardinal histologic features of the asthmatic airway (6, 27). Repair of injury after an inflammatory response to allergen is thought to underlie the remodeling of the airways observed in allergic asthma (12). We have developed a novel, safe bronchoscopic protocol that permits sampling at multiple time points to assess the repair process, and to reconstruct cellular interactions ex vivo that may modulate certain components of this process. In the present report we have focused on airway epithelial proliferation necessary for covering denuded basement membrane, and on collagen synthesis necessary for replacing extracellular matrix as two separate, but possibly interrelated, components of repair.
We have shown (1) increased proliferation of epithelial cells cocultured with autologous BAL cells from asthmatic, but not nonasthmatic subjects, after segmental allergen challenge, and (2) increased production of collagen III, but not collagen I, from human lung fibroblasts exposed to factors from the epithelial cells of asthmatics, but not those of nonasthmatics, after challenge. This histologic phenotype (increase in collagen III with no alteration in collagen I) is characteristic of the asthmatic subepithelium (6, 14). The time course of these two epithelial activities in asthmatics is prolonged and appears to coincide: both peak proliferation and fibrogenic stimulus occurred at 1 to 2 wk after segmental allergen challenge. However, epithelial cell proliferation was increased only with autologous BAL cell coculture, whereas BAL cell coculture had no effect on epithelial cell release of fibrogenic factor(s). This finding indicates independent execution of these epithelial cell activities after allergen-induced inflammation.
Several factors previously identified as stimulating lung
epithelial cells to proliferate include mast cell tryptase (31), granulins/epithelins (32), neuregulins/heregulins (33), or other
epidermal growth factor receptor ligands such as TGF or amphiregulin (34, 35), keratinocyte growth factor/FGF-7 (36, 37), and hepatocyte growth factor/scatter factor (HGF) (38, 39), or a heparin-binding variant of HGF (40). Of these factors, the tryptase from mast cells, HGF from alveolar macrophages (41), granulins from leukocytes, or possibly a heparin-binding epidermal growth-factor-like growth factor from
T lymphocytes (42) are perhaps more likely to be proliferative
stimuli derived from BAL cells in coculture with the epithelial
cells, rather than FGF-7, which predominantly comes from fibroblasts (36). Because many of these factors reportedly require serum (36), act in autocrine as well as paracrine manner, induce or
enhance other factors (34, 35, 43), it is possible that the proliferative stimulus may also originate partly from epithelial cells, e.g.
epithelins, as well as from the BAL cells in the asthmatic subjects.
Although epithelial cell stimulation of fibroblast collagen type III synthesis was not modulated by BAL cells ex vivo, there was greater stimulus from epithelial cells from the challenged segment than from the unchallenged segment in asthmatics after 1 wk. Therefore, inflammation in vivo enhances the asthmatic epithelial cell stimulation of collagen III synthesis.
Our studies also found no difference in collagen III stimulation between asthmatics with a dual airway response to allergen and asthmatics with only a single response, despite a markedly different airway eosinophilia in these two groups. These observations lend further support to the conclusion that it is the epithelial response of asthmatics to inflammatory or other injury, rather than the magnitude of the response by infiltrating inflammatory cells such as eosinophils, which drives the excess collagen deposition. Analogous to this concept of excess collagen deposition induced by an asthmatic epithelial response to injury instead of the inflammatory cell influx, very little difference in collagen III stimulation occurred between allergic nonasthmatic subjects and nonallergic nonasthmatic subjects, despite increased eosinophil influx in the allergic nonasthmatic group.
The number of subjects in the allergic nonasthmatic group, and in the dual and single-response asthmatic groups (n = 4 for each) were small. Therefore, the null hypothesis of no difference could have been accepted when, in fact, it should have been rejected, particularly in light of variability in response to challenge. However, the maximum differences in both epithelial proliferation and collagen III stimulation between the allergic nonasthmatic group and the nonallergic, nonasthmatic group were 3- to 4-fold less compared with their differences versus the asthmatic group after challenge. These large differences between the asthmatic and two nonasthmatic groups (allergic and nonallergic nonasthmatics) permitted us to observe significant differences between the asthmatic and nonasthmatic subjects.
In our model, both epithelial proliferation and fibrogenic stimulation occurred over an extended time frame. Previous reports have suggested that isolated mechanical injury to epithelium results in replacement of epithelial cells within minutes to hours and completely differentiated epithelium within 5 d (10, 11, 44). In contrast, our allergen-induced inflammatory injury is spread over a much longer time frame (20), and repair, therefore, presumably becomes prolonged as well. By Day 16, in asthmatics we found no difference in the increased stimulation of collagen type III synthesis from the resampled Day 1 unchallenged lung segment (mechanical injury only) compared with the resampled Day 2 antigen-challenged lung segment (inflammatory plus mechanical injury). This suggests that by 2 wk after allergen challenge, the inflammatory stimulus to collagen synthesis may be subsiding and the increased stimulus to collagen synthesis is mainly due to mechanical injury.
The stimulation of collagen type III, but not of collagen type I, is consistent with earlier reports of the thickened subepithelial basal lamina in asthmatics having increased amounts of type III, but not type I (14). A recent study of guinea pig epithelial cell-fibroblast coculture found that both procollagen types I and III mRNAs increased 4 d after mechanical injury to the epithelium, and decreased to baseline by 8 d (44). Transient expression of collagen I mRNA may have been missed between our Day 2 and Day 9 samples, may not have generated sufficient additional extracellular protein to be detected, or may represent a difference in cytokine production and stimulation for mechanical versus immune injury to the airways.
In vivo "activated" fibroblasts, or myofibroblasts, increase
in numbers in the subepithelium of subjects with mild atopic asthma after allergen but not diluent challenge (45). TGF, insulin-like growth factor (IGF-1), platelet-derived growth
factor (PDGF), and interleukin 11 (IL-11), all known to stimulate fibroblast collagen synthesis but differing in proliferative
effects, are produced by airway epithelium (17, 46). Both
TGF1 (49) and fibronectin (50) become elevated in BAL
fluid 24 to 48 h after segmental allergen challenge in asthmatics, but not in saline-challenged segments, or in control subjects. In an in vitro model of mechanical or chemical damage
to epithelial cells, increased proliferation of myofibroblasts
cocultured with epithelial cells was due to the combined effects
of basic fibroblast growth factor, IGF-1, PDGF, and endothelin 1 (19). However, the epithelial cells were simian virus-40
transformed, rather than primary epithelial cells from asthmatics or control subjects, injury was brief (10 min or less),
and there was no examination of effects beyond 48 h. In another series of experiments, bovine lung epithelial cell-fibroblast coculture revealed an antiproliferative effect on fibroblasts caused by TGF, but no effect caused by epidermal growth
factor (EGF), PDGF, IGF-1, tumor necrosis factor alpha (TNF-)
or fibronectin (51). Our data show an elevated release of total
TGF1 from epithelial cells of asthmatics compared with nonasthmatics, but without further increase to correspond to stimulation of collagen III on Days 9 and 16. However, the proportion of active TGF1 (below the ELISA minimum) to latent
may differ over these time points with increased release of
matrix proteases (6), or other fibrogenic factors may contribute to the increased collagen III synthesis. Nevertheless, the
potential of asthmatic epithelium to release greater amounts
of active TGF1 than nonasthmatic epithelium suggests that
epithelial cells may play an important role in inducing the greater
numbers of myofibroblasts observed in asthmatics (45) through
release of this growth factor (44).
In summary, the time course of certain elements important in airway repair after allergen-induced inflammation has been examined in allergic asthmatics, allergic nonasthmatics, and nonallergic nonasthmatics. Our observations demonstrate the important part the airway epithelium plays in airway remodeling after allergen- and mechanical-injury-induced airway damage in allergic asthmatics. Identification of the specific epithelial cell-derived soluble factor or factors that induce excess collagen III production, and the time frame during which they are operating, may permit targeting the responsible elements that play a key role in the subepithelial fibrosis characteristic of asthma.
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Correspondence and requests for reprints should be addressed to Annette T. Hastie, Ph.D., Department of Medicine, Division of Critical Care, Pulmonary, Allergic & Immunologic Diseases, Jefferson Medical College of Thomas Jefferson University, 805 College Building, 1025 Walnut Street, Philadelphia, PA 19107. E-mail: Annette.T.Hastie{at}mail.tju.edu
(Received in original form January 17, 2001 and accepted in revised form October 18, 2001).
Dr. Kraft was supported by Grant GM08562 from the National Institutes of Health.Dr. Zangrilli is the recipient of Clinical Investigator Award HL03663 from the National Institutes of Health.
Acknowledgments: The writers gratefully acknowledge the useful discussions and technical suggestions provided by Dr. Arturo Diaz (now a Fellow at U. of Michigan, Ann Arbor, MI) and Dr. George Dodge (now at A. I. Dupont Hospital for Children, Wilmington, DE). They also thank their volunteer subjects, who made this work possible, and the entire Jefferson Medical College Airways Inflammation Research Group for their assistance with these studies.
Supported in part by Grants ES05721, HL67663 and AI24509 from the National Institutes of Health.
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References |
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