We assessed whether transforming growth factor- β (TGF- β ), a fibrogenic growth factor, may be involved in remodeling of asthma and chronic bronchitis; its expression was compared with that of epidermal growth factor (EGF) and granulocyte macrophage colony-stimulating factor (GM-CSF) in bronchial mucosal biopsies from 13 normal subjects, 24 asthmatics, and 19 patients with chronic bronchitis. TGF- β immunoreactivity was highly increased in epithelium and submucosa of those with bronchitis and to a lesser extent in asthmatics. By comparison, with normal subjects, EGF immunoreactivity was significantly increased in the epithelium of bronchitic subjects and submucosa of asthmatics, and, GM-CSF immunoreactivity was increased in both epithelial and submucosal cells of asthmatics and to a lesser extent in submucosa of bronchitics. A significant correlation was found between the number of epithelial or submucosal cells expressing TGF- β in both asthma and chronic bronchitis and basement membrane thickness and fibroblast number. No such correlation was found for EGF or GM-CSF. In situ hybridization for TGF- β 1 mRNA confirmed the results obtained by immunohistochemistry. By combining in situ hybridization and immunohistochemistry, it was found that eosinophils and fibroblasts were synthetizing TGF- β in asthma and bronchitis. These data suggest that TGF- β , but not EGF or GM-CSF, is involved in airways remodeling in asthma and chronic bronchitis.
Asthma is a chronic inflammatory disease associated with remodeling of the airways (1) as shown by subepithelial fibrosis (2) and by the activation of fibroblasts and myofibroblasts (3). In asthma, the increased thickness of the basement membrane appears to be an important and localized feature of fibrosis because collagen deposition does not appear to be present deeper in the submucosa (2). Chronic bronchitis is another chronic obstructive disease of the airways in which inflammation is a continuous process probably commencing with an increase in inflammatory cells (4) and followed by airway remodeling with extracellular matrix deposition on the airways wall (5). Tissue remodeling due to extracellular matrix production and fibroblasts proliferation is directly modulated by growth factors classified according to their biological properties into pro-fibrotic and non-fibrotic growth factors (6).
Transforming growth factor-β (TGF-β) is an important pro-fibrotic growth factor implicated in airway remodeling and pulmonary fibrosis (6-9). TGF-β can be generated by many cells, such as macrophages, epithelial cells, fibroblasts, and eosinophils (9-11).
Epidermal growth factor (EGF) is another growth factor that participates in repair processes targeted at the resurfacing of a wound with new epithelium (12). Although EGF may exert some fibrogenic properties (13), it is considered to be less fibrogenic than TGF-β (6).
Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor over-expressed in the bronchial mucosa of asthmatics, which is involved in eosinophil and macrophage accumulation in tissues (14, 15). Although there is some evidence showing that GM-CSF may have some fibrogenic properties, it is considered as a hematopoietic growth factor rather than a fibrogenic growth factor (6).
To evaluate the role of growth factors in the pathogenesis of airway remodeling, a study was undertaken to study the expression of a known fibrogenic growth factor (TGF-β), of a putative fibrogenic growth factor (EGF) and of a hematopoietic growth factor (GM-CSF) in the central airways of asthmatic and chronic bronchitis patients using immunohistochemistry. In addition, TGF-β expression was also evaluated in both central and peripheral airways of two non-smoking asthmatics and two chronic bronchitis patients undergoing lung resection for lung cancer. Furthermore, since in asthma subepithelial fibrosis is a characteristic histopathological feature of remodeling of central airways and fibroblast activation is considered a major causative factor (2), the immunoreactivity of TGF-β, EGF, and GM-CSF was compared with either the thickness of the basement membrane or the number of fibroblasts located beneath the basement membrane. Finally, since among the three growth factors only TGF-β was found to play a major role in the subepithelial fibrosis, we also performed in situ hybridization to detect mRNA for TGF-β as well as to characterize, by combining in situ hybridization and immunohistochemistry, the phenotype of submucosal cells positively staining for TGF-β. Since virtually all cells have receptors for TGF-β (16), we did not test their immunoreactivity.
Twenty-four asthmatic subjects (18–64 yr, 36.7 ± 12.7 yr) were studied. Asthma was defined according to the criteria of the American Thoracic Society (17, 18). The clinical severity of asthma was assessed according to the score of Aas. None of the subjects participating in this study was a current smoker, and none had smoked during the previous 2 yr. No subject had any bronchial or respiratory tract infection during the month preceding the test.
Nineteen chronic bronchitis patients (35–71 yr, 57.3 ± 9.6 yr) were studied. Chronic bronchitis was defined according to the criteria of the American Thoracic Society (17, 19). Chronic obstructive pulmonary disease (COPD) was defined as a disorder characterized by abnormal results for expiratory flow tests, and, although there is no definitive cut-off limit, only patients with FEV1 capacities of less than 70% of the predicted value were considered to have COPD (19, 20). Smoking habits were carefully checked, and a minimum of 30-pack years smoked was required (30–150 pack-years) for inclusion in the chronic bronchitis group.
Patients with asthma or chronic bronchitis were excluded from the study if they had had a severe exacerbation or respiratory tract infection during the month preceding the study. Inhaled corticosteroids or oral corticosteroids had been withdrawn for at least 2 mo prior to the commencement of the study and use of nedocromil sodium or cromoglycate had been stopped for at least two weeks. No patient was under long-acting β2-agonist or under oral theophylline. Short-acting β2-agonist were stopped for 12 h.
Thirteen healthy subjects (18–68 yr; 46.3 ± 18.0 yr) were the control group. They were non-allergic, had never suffered from asthma, and none of them was a smoker. No member of the control group had suffered from any respiratory tract infection during the month preceding the test.
The study was performed after informed consent was obtained from the participants and approval of the Ethics Committee of the Hospital was obtained.
Biopsies were recovered using fiberoptic bronchoscopy performed as previously described in subsegmental bronchi (18). Specimens were fixed in 3.7% weight/volume formaldehyde and embedded in paraffin blocks. Four-micrometer sections were deparaffined and rehydrated before hematoxylin and eosin staining or immunohistochemistry. Slides were incubated with the monoclonal antibody against TGF-β1, 2, 3 at a dilution of 1:30 (Genzyme, Cambridge, MA) (21), the polyclonal antibody against-EGF (Z-12) at a dilution of 1:30 (Santa Cruz Biotechnology, CA) (23), the fibroblast specific monoclonal antibody against prolyl-4 hydroxylase (clone 585; Dako, Glostrup, Denmark) (24, 25) at a dilution of 1:50, and the monoclonal antibody against eosinophil cationic protein (EG2) at a dilution of 1:5 (Kabi Pharmacia, Uppsala, Sweden) (26). The reaction was revealed using the APAAP method (Dako LSAB®; Dako). The specificity of each immunostaining process was assessed using the recombinant growth factors (Genzyme) at a concentration fourfold greater than that of the antibodies, and no positive staining was observed after the neutralization by immunoprecipitation.
For the overall length of each biopsy, the number of positively stained epithelial cells was quantified using an eyepiece graticule per unit length (mm) of basement membrane. In addition, in order to avoid any influence of the epithelial cell counting due to differences in the height of the epithelium, to hyperplasia or to metaplasia, we also expressed the immunoreactivity of bronchial epithelial cells as percentage of positive stained cells. The results were similar, and, in the paper, the results were expressed as a number of positive epithelial cells per length of basement membrane. On the other hand, the number of positively stained cells beneath the basement membrane was quantified per unit area (mm2) of submucosa. The thickness of the basement membrane was assessed in the sections stained with hematoxylin and eosine. The results are expressed as the medians and 25–75% percentiles of the total measurements (at least four measurements were needed to calculate averages).
Lung specimens resected for cancer were processed by inflating them with a cryoembedding medium diluted 1:1 in saline (OCT; Miles, Elkhart, IN) and freezing them in liquid nitrogen according to a method previously described (27). Samples were cut randomly in three distinct sites corresponding to segmental, sub-segmental, and peripheral airways. The samples were then frozen at −80° C until they were analyzed. Immunoreactivities for TGF-β was expressed as intensity of staining that was graded from − to +++ as compared with the immunoreactivity of three positive controls (lung specimens obtained from subjects suffering from lung fibrosis) included in each experiment.
TGF-β1 cDNA probe (R&D System, Abingdon, UK) was labeled using digoxigenin-11-dUTP (Boehringer Manheim, Manheim, Germany) by a random primed labeling method (Boehringer Manheim), and unincorporated nucleotides were removed by ethanol precipitation. Digoxigenin-labeled probe was stored at −20° C in TE (0.01 M Tris-Cl pH 7.6, 0.001 M EDTA) at a concentration of 15 μg/ml.
In situ hybridization was performed on 4% paraformaldehyde-fixed frozen sections (5 μm thick) obtained from 15 asthmatic and 15 chronic bronchitis patients by bronchoscopy as previously described (18) using the method proposed by Lawrence and coworkers (28). Hybridized probe was detected using a polyclonal serum anti-digitoxigenin-AP-conjugated (Boehringer Mannheim) at a final concentration of 1:4000 in TMA (0.1 Tris-HCL, 0.15 MgCl2, 10 mg/ml Bovine Serum Albumin), in a humid chamber at room temperature for 15 min (29). Colour development was achieved by adding a freshly prepared substrate solution consisting of 0.175 mg of X-phosphate-5-bromo-4-chloro-3-indolphosphate (BCIP) (Boehringer Mannheim) and 0.37 mg of NBT (nitroblue tetrazolium) salt (Boehringer Mannheim) per 1 ml of alkaline-substrate buffer to the slides of 30–120 min at room temperature. To ensure that results were not due to methodological artefacts, control slides were prepared in a similar fashion during the same experimental series either without the TGF-β1 hybridization probe or using the sense probe or omitting the polyclonal serum anti-digitoxigenin-AP-conjugated and after digestion using RNAse (Sigma Chemical Company, St. Louis, MO) (20 μg/ml) for 30 min at 37° C in a humidified chamber.
Sections obtained from 15 asthmatic and 15 chronic bronchitis patients were used for both immunohistochemistry and in situ hybridization. A slightly different technique was therefore required for immunohistochemistry in which cryostat sections were used. In order to identify the cells expressing mRNA, slides were fixed in 4% paraformaldehyde for 10 min and stained with monoclonal antibodies against prolyl 4-hydroxylase (fibroblast) or EG2, as previously described, before performing the in situ hybridization technique. In order to avoid RNAse contamination, all solutions used for the APAAP technique were made in 0.1% diethyl pyrocarbonate (DEPC)-treated distilled water. These sections were not counterstained.
Nonparametric tests were used. For multiple correlations, the Bonferroni's correction was used. Results are given as medians and 25–75% percentiles.
Fourteen patients had mild asthma, seven had moderate asthma, and three had severe asthma. FEV1 values ranged from 40 to 100% of predicted values. Thirteen patients were allergic. In patients with chronic bronchitis, FEV1 values ranged from 56 to 90% of predicted values. Seven patients were considered as having COPD.
Satisfactory biopsies were obtained from all controls. The sections obtained from three asthmatics could only be used for staining with TGF-β. In one patient with chronic bronchitis, the sample was represented by the epithelium layer and the basal membrane thereby precluding the staining of submucosa (Table 1).
|Controls||Asthma||Chronic Bronchitis||Statistical Analysis|
|Number of subjects||13||24||19|
|TGF-beta|| 0 0–5.8|| 4 0–31||4940–71||0.015||0.0001||0.006|
|GM-CSF|| 3.5 0–6.5||5029.5–86.7||10 0–21||0.0003||NS||0.0003|
|EGF|| 0 0–6.2||11 0–51.7||22.5 5–36.5||NS||0.0025||NS|
|TGF-beta|| 0 0–0||31.5 3–81.5||69.538–102||0.007||0.0002||NS|
|GM-CSF|| 0 0–1||8427.2–128|| 4 0–25||0.0001||0.0005||NS|
|EGF|| 2 0–7||14 0–50.2|| 8 0–76||0.01||NS||NS|
|Vimentin positive cells†|| 7 1.5–9.5||20.5 6–57.5||8641–145||NS||0.0006||0.0001|
|Basement membrane, μm|| 4 3–7.2|| 7.9 4.5–17.25|| 5.8 4–8.4||0.005||0.01||0.01|
TGF-β was rarely expressed in epithelial cells from controls. In comparison with the controls, there was a small but significant increase (p < 0.015, Mann-Whitney U test) in the number of cells expressing TGF-β in asthmatics and a great and highly significant increase (p < 0.0001, Mann-Whitney U test) in chronic bronchitis patients (Figures 1 and 2 and Table 1). In asthmatics, positive staining was observed on ciliated and basal bronchial epithelial cells. The intensity and extent of immunostaining was not homogeneous within the same sample being mainly localized where the epithelial layer was partially desquamated. In chronic bronchitis patients, positive staining was mainly observed on basal bronchial epithelial cells, and the number of positive cells and the intensity of the staining was greater in metaplastic epithelium.
EGF was rarely expressed in epithelial cells from controls. The number of positive epithelial cells was not significantly greater in asthmatics. On the other hand, EGF expression was significantly increased in chronic bronchitis patients in comparison with the controls (p < 0.0025, Mann-Whitney U test) (Figures 1 and 2 and Table 1). The expression of EGF on epithelial cells was not significantly different between asthmatic and chronic bronchitis patients. In asthmatics, the intensity and extent of EGF immunostaining was not homogeneous within the same sample and was mainly localized where the epithelial layer was partially desquamated. In chronic bronchitis patients, EGF staining was also observed on ciliated and basal bronchial epithelial cells and in the metaplastic epithelium.
GM-CSF was minimally expressed on epithelial cells from controls and chronic bronchitis patients. In asthma GM-CSF was expressed at a higher level in both epithelial and submucosal cells than in control or chronic bronchitis subjects (p < 0.0003, Mann-Whitney U test) (Figures 1 and 2 and Table 1).
In the submucosa, the number of cells expressing TGF-β was low in controls and significantly greater in asthmatic or chronic bronchitis patients (p < 0.001 in asthma, p < 0.0002 in chronic bronchitis, Mann-Whitney U test) (Figures 1 and 2 and Table 1). In asthma and chronic bronchitis, the TGF-β immunostaining was localized on cells infiltrating the submucosa, on smooth muscle cells, and on mucous glands and endothelial cells. Moreover, positive immunostaining for TGF-β was localized in subepithelial collagen zone of the basement membrane and in the extracellular matrix.
EGF-positive cells were lower in control subjects than in asthmatic or chronic bronchitis patients. EGF immunoreactivity was mainly localized on cells infiltrating the submucosa, on smooth muscle and mucous glands.
GM-CSF-positive cells were lower in controls and chronic bronchitis patients than in asthmatic subjects (p < 0.0001 and p < 0.0005, Mann-Whitney U test). The staining for GM-CSF was localized in submucosal cells as well as in smooth muscle cells.
Epithelium. TGF-β immunoreactivity was found to be similar in the epithelium of segmental, sub-segmental bronchi and of bronchioles both in asthmatic and chronic bronchitis patients. The staining was localized in both ciliated and basal epithelial cells (figure not shown).
Sub-mucosa. TGF-β immunostaining was localized on smooth muscle cells, on cells of mucous glands and on endothelial cells. A positive immunostaining for TGF-β was found in subepithelial collagen zone of the basement membrane and, in some biopsies, in the extracellular matrix. Several cells infiltrating the sub-mucosa were positively stained and there was no difference between segmental, sub-segmental bronchi and bronchioles (figure not shown).
Transcripts for TGF-β1 were found on the same cells expressing this protein in biopsies from asthmatic and chronic bronchitis patients (Figure 3). The median number of epithelial cells expressing the transcript for TGF-β1 was 17.5 (25–75% percentiles: 10-35) per mm of basement membrane in asthma and 65 (50–80) in chronic bronchitis. The median number of submucosal cells expressing the transcript for TGF-β1 was 50 (30–55) per mm2 in asthma and 80 (60–142) in chronic bronchitis. Control slides carried out by omitting TGF-β1 probe, sense probe and/or biotin or by using RNase digestion gave no cellular hybridization signal (Figure 3). Combining immunohistochemistry and in situ hybridization, it was found that in bronchial biopsies from 15 asthmatic and 15 chronic bronchitis patients, transcripts for TGF-β1 were found in eosinophils (Figure 4) and fibroblasts. The median number of TGF-β positive eosinophils per mm2 in the submucosa ranged from 40 (20–40: median and 25-75% percentiles) in asthmatics to 15 (10–45) in chronic bronchitis patients. The median number of TGF-β positive fibroblasts per mm2 in the submucosa ranged from 10 (3–18: median and 25–75% percentiles) in asthmatics to 70 (52–100) in chronic bronchitis patients.
The number of fibroblasts was not significantly greater in asthmatics than in control subjects and was greatly and significantly increased in chronic bronchitis compared to control or asthmatic subjects (p < 0.001 and p < 0.005, Mann-Whitney U test). In both asthmatic and chronic bronchitis patients, the number of fibroblasts significantly correlated with the thickness of the basement membrane (Figure 5).
In asthmatic and chronic bronchitis patients, the number of epithelial and sub-mucosal cells expressing TGF-β was significantly correlated with the overall thickness of the basement membrane (Figure 6). However, the median size of the basement membrane was significantly greater in asthma than in chronic bronchitis (p < 0.01, Mann-Whitney U test), whereas the median number of TGF-β (p < 0.006, Mann-Whitney U test) or fibroblasts (p < 0.001, Mann-Whitney U test) was significantly greater in chronic bronchitis (Table 1). There was a significant correlation between the number of fibroblasts in the submucosa and the number of cells expressing TGF-β in asthma (p < 0.01, Spearman rank correlation) or chronic bronchitis (p < 0.01, Spearman rank correlation).
There was no significant correlation between the numbers of cells expressing EGF or GM-CSF and the thickness of the basement membrane or the number of fibroblasts.
This study shows that by comparison with normal subjects: (1) the expression of TGF-β is significantly increased in chronic bronchitis patients and to a lesser extent in asthmatics; (2) EGF immunoreactivity is significantly increased in epithelial cells from chronic bronchitis patients and in submucosal cells of asthmatics; (3) GM-CSF immunoreactivity is significantly increased in both epithelial and submucosal cells in asthmatics. In asthma and chronic bronchitis, the increased expression of TGF-β is significantly correlated with the thickness of the basal membrane and the number of fibroblasts. Moreover, in bronchial biopsies from asthmatic and chronic bronchitis patients, the expression of TGF-β is localized in structural cells such as epithelial cells and fibroblasts as well as in recruited cells such as eosinophils.
Asthma and chronic bronchitis are chronic inflammatory diseases of the airways associated with remodeling due to the deposition of connective tissue in both asthma. Since growth factors are able to modulate the proliferation of fibroblasts and the synthesis of extracellular matrix components, we put forward the hypothesis that these cytokines may play a role in the pathogenesis of subepithelial fibrosis associated with chronic airway inflammation. We first evaluated the spontaneous expression of three growth factors, such as TGF-β, EGF, and GM-CSF, depending on their different potential fibrotic ability to affect tissue remodeling processes. Our results indicate that, although EGF immunoreactivity is significantly increased in bronchial epithelial cells of chronic bronchitis patients and in submucosal cells of asthmatics, it is not significantly correlated with either the thickness of the basement membrane or the number of fibroblasts. Moreover, our results confirm that there is a strong GM-CSF expression in bronchial biopsies obtained from asthmatics but fail to show any relationship with the thickness of the basement membrane or the number of fibroblasts. In this regard, since at the time of the biopsy patients were not suffering from acute exacerbations of their diseases, one might speculate that the contribution to airway remodeling of EGF and/or GM-CSF is greater during exacerbations and may be reduced by the time that the disease process has settled down and the patients can be biopsied.
Since one of the most critical steps in remodeling processes is the proliferation of fibroblasts (6), and, since in central airways the increased thickness of the basement membrane has been attributed to collagen deposition induced by fibroblast activation (2, 3), we then compared the spontaneous expression of TGF-β, EGF, and GM-CSF with the number of fibroblasts and with the thickness of the basement membrane measured in bronchial biopsies obtained from both asthmatic and chronic bronchitis patients. Among the three growth factors studied, only TGF-β immunoreactivity was significantly correlated with the number of fibroblasts and with the thickness of the basement membrane. These data extended previous findings regarding the ability of TGF-β to stimulate fibroblast proliferation and collagen synthesis (6) and, to our knowledge, show for the first time that TGF-β is associated with subepithelial fibrosis in asthma. Because the basement membrane was found to be significantly thicker in asthmatic than in chronic bronchitis patients, whereas TGF-β- or fibroblasts were found to be in greater number in chronic bronchitis, it might be argued that: (1) the mechanisms regulating collagen deposition beneath the basement membrane in both diseases differs; (2) other fibrogenic factors may be involved in asthma and act in a synergistic manner with TGF-β; or (3) collagen turnover and degradation may differ in asthma and chronic bronchitis. In addition to its ability to regulate the turnover of the extracellular matrix components and the activation of stromal cells, TGF-β is also capable of enhancing the expression of desmosomal proteins in bronchial epithelial cells (31) as well as their squamous differentiation (32). Therefore, the increased TGF-β immunoreactivity in chronic bronchitis may play a major role in the pathogenesis of epithelial metaplasia characterizing this disease. However, since epithelial metaplasia is not a common feature of asthma, it may be suggested that either the amount of TGF-β needed for inducing a metaplastic epithelium is greater than that released in asthma or, alternatively, there are other metaplastic factors which are specifically involved in chronic bronchitis. In this study, we have also shown that bronchial epithelial cells, eosinophils and fibroblasts are three cellular sources of TGF-β (33, 34).
The results of our study differs with those of Aubert and associates (27) who found that TGF-β1 mRNA and protein were expressed at a similar level in control subjects, asthmatics and COPD patients. However, the discrepancies between the two studies can be attributed to several factors: (1) we used bronchial biopsies obtained by bronchoscopy whereas Aubert and coworkers used resected lung specimens; (2) we carefully avoided smokers in the control and asthmatic groups whereas many, if not most, subjects tested in the other study were smokers; (3) in the asthmatic group, none of the subjects had had corticosteroids of any form for at least the month previous to the study whereas all patients for whom the treatment regimen was known in Aubert's study had recently received corticosteroids; and (4) the antibodies used differed. Moreover, in this study, the clinical characterization of the patients was checked carefully. Since overlaps may exist between asthma and chronic bronchitis (19), we attempted to distinguish between asthmatic and chronic bronchitis patients by avoiding the inclusion of patients who did not present with a well defined and characteristic form of the disease. Thus, the results of our study cannot be directly compared with those of Aubert and colleagues and suggests that smoking may up regulate TGF-β expression.
In conclusion, this study demonstrates that TGF-β may play an important role in the pathogenesis of fibrotic alterations associated with asthma or chronic bronchitis and suggests the involvement of epithelial cells, eosinophils, and fibroblasts in the cellular network underlying airway remodeling in chronic obstructive inflammatory diseases of the airways.
Supported by INSERM (France) and CNR (Italy).
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