Abstract
The histopathology of bronchial asthma is associated with structural changes within the airways, including subepithelial fibrosis, as well as chronic eosinophilic inflammation. The mechanisms responsible for this tissue remodeling, and in particular the role of inflammatory cells, remain to be established. Transforming growth factor-β (TGF-β) is a potent profibrotic cytokine which may contribute to the thickening of the reticular lamina by the deposition of collagen fibers. To investigate the molecular mechanisms underlying these structural changes, we have investigated the expression of TGF-β1 mRNA and immunoreactivity within the bronchial mucosa of mild to severe asthmatic individuals and normal control subjects using the techniques of in situ hybridization and immunocytochemistry. As eosinophils are prominent within the asthmatic airway and are known to synthesize pro-inflammatory cytokines, the presence of TGF-β1 mRNA and immunoreactive protein in eosinophils was also examined. Asthmatic individuals exhibited a greater expression of TGF-β1 mRNA and immunoreactivity in the airways submucosa than normal control subjects (P < 0.05), and these increases were directly related to the severity of the disorder. The extent of airways fibrosis, as detected histochemically, was also increased in asthmatics compared with normal control subjects (P < 0.005). In asthmatic subjects, the presence of subepithelial fibrosis was associated with the severity of the disease and correlated with the decline in forced expiratory volume in 1 s (r2 = 0.78; P < 0.05). Within the asthmatic airways, EG2-positive eosinophils represented the major source of TGF-β1 mRNA and immunoreactivity. These results provide evidence that TGF-β1 may play a role in the fibrotic changes occurring within asthmatic airways and that activated eosinophils are a major source of this cyto-kine.
Pathologic features associated with asthma include subepithelial fibrosis, epithelial desquamation, smooth muscle hypertrophy/hyperplasia, and an eosinophilic inflammatory cell infiltration (1-3). While initially described in postmortem studies, these structural alterations can be observed even in mild and newly diagnosed asthmatics (4, 5). Because any thickening of the airway wall will profoundly increase the maximal degree of airway narrowing when the smooth muscle constricts, it has been proposed that architectural changes similar to those observed in asthmatics contribute to the development of chronic airways hyperresponsiveness and the progressive deterioration in lung function over time (6, 7). While many studies have focused on the mechanisms underlying the acute presentations of bronchial asthma, the factors responsible for the more chronic structural changes within the lungs remain to be elucidated. Several reports have suggested that thickening of the reticular lamina results from interstitial collagen and fibronectin deposition (4, 5). Although myofibroblasts are recognized as being the cell type responsible for subepithelial collagen deposition in these individuals (8), the mechanisms contributing to the onset of airways fibrosis in asthma and the role of inflammatory cells, in particular eosinophils, remain to be established.
Transforming growth factor-beta (TGF-β) is a potent profibrotic cytokine which stimulates fibroblasts to promote the synthesis and secretion of many proteins of the extracellular matrix (9, 10). In addition, TGF-β is an immunomodulatory cytokine and a potent chemoattractant for several cell types including monocytes (11), fibroblasts (12), and mast cells (13). Due to its actions in promoting growth and repair, TGF-β is thought to play an important role at sites of wound healing and tissue remodeling. However, the role of TGF-β in contributing to the structural alterations evident in bronchial asthma remains to be established.
Mammalian cells synthesize three isoforms of TGF-β, each encoded by its own gene, which appear to be differentially regulated (14). TGF-β1 was the first isoform of TGF-β to be purified and is the most extensively characterized to date. As such, the biologic functions reported for TGF-β are generally those of TGF-β1 (14). In lung diseases associated with fibrosis, the cellular source of TGF-β1 includes lymphocytes, macrophages, and epithelial cells (15, 16). Recently, several reports have demonstrated that eosinophils infiltrating nasal polyps represent a major source of TGF-β1 (17, 18). Furthermore, expression of TGF-β1 is evident in circulating eosinophils from hypereosinophilic individuals (19). Since bronchial asthma is intimately associated with the recruitment and activation of eosinophils within the airways, the expression of TGF-β1 by these cells may link chronic eosinophilic inflammation to the fibroblast activation and characteristic deposition of collagen fibers seen in this disease (4, 5).
The aims of the present study were to examine the expression of TGF-β1 messenger ribonucleic acid (mRNA) and immunoreactivity within the airways of mild to severe asthmatics compared with control individuals, and to determine the potential role of eosinophils in contributing to the synthesis of this cytokine in bronchial asthma. Furthermore, we investigated the association between TGF-β1 mRNA expression, the extent of subepithelial fibrosis, and baseline lung function, using a combination of histochemical and molecular biologic techniques.
The technique of fiberoptic bronchoscopy with bronchial biopsies in asthmatic volunteers and normal control subjects has been described elsewhere in detail (20). To study the expression of TGF-β1 mRNA and immunoreactivity in asthmatics compared with normal controls, 28 subjects were recruited. These included 18 atopic asthmatics and 10 nonatopic normal controls, whose clinical data is shown in Table 1. The subjects were recruited from the Asthma Clinic at the Montréal General Hospital and Montréal Chest Institute (Montréal, PQ, Canada), and the General Clinical Research Center at the National Jewish Center for Immunology and Respiratory Medicine (Denver, CO). All patients fulfilled the ATS criteria for asthma (21), had typical clinical symptoms, documented airways reversibility, and increased airways responsiveness to methacholine (PC20 < 8 mg/ml). Informed consent, approved by the Montréal Chest Institute, the Montréal General Hospital, and the National Jewish Center Institutional Review Board, was obtained from all patients before entry into this study. Of the 18 asthmatic individuals, 6 were classified as severe asthmatics with forced expiratory volume in 1 s (FEV1) values less than 60% predicted and required the regular use of inhaled steroids. Six asthmatics had frequent symptoms which were controlled by regular use of β2-agonists or inhaled steroids with FEV1 values between 60 and 80% predicted, and were classified as moderately severe. The remainder (n = 6) were mild asthmatics who required only occasional β2-agonists, had FEV1 values greater than 80% predicted, and had been asymptomatic for at least 1 wk before fiberoptic bronchoscopy. None of the asthmatics had taken oral corticosteroids in the preceding 12 wk and all patients were atopic as defined by a positive skin-prick test to one or more aeroallergens. The normal control subjects had negative skin-prick tests to aeroallergens. All of the subjects were nonsmokers and none had any other serious lung disease. Two to five endoscopic bronchial biopsies were obtained from each subject. Biopsies from asthmatics and normal control subjects were fixed immediately in 4% paraformaldehyde for 2 h, washed in 15% phosphate-buffered saline-sucrose, blocked, and sectioned on a cryostat at −20°C. Sections (10 μm-thick) were then baked in an oven at 37°C and stored at −80°C until further use.
| Study Participants | n | FEV1 ± SEM(% predicted) | Male/FemaleRatio | Age(yr) | ||||
|---|---|---|---|---|---|---|---|---|
| Normal subjects | 10 | 107.6± 2.9 | 7:3 | 26.4 ± 1.4 | ||||
| Mild asthmatics | 6 | 89 ± 2.2 | 3:3 | 32.7 ± 4.8 | ||||
| Moderate asthmatics | 6 | 73.8 ± 2.1 | 4:2 | 36.0 ± 3.4 | ||||
| Severe asthmatics | 6 | 45.0 ± 2.4 | 4:2 | 31.0 ± 3.0 |
A radiolabeled complementary RNA probe coding for TGF-β1 mRNA was prepared from complementary DNA (cDNA) as described previously (22). The probe was constructed to be specific for the TGF-β1 isoform. In brief, cDNA was inserted into PGEM vectors, linearized, and transcribed in vitro in the presence of 35S-uridine triphosphate and either SP6 or T7 polymerases. Antisense (complementary to mRNA) and sense probes (identical to mRNA) were prepared.
In situ hybridization was performed as previously described (23, 24). Briefly, cryostat sections from biopsies were permeabilized with Triton X-100 and proteinase K solution (1 μg/ml) in 0.1 M Tris containing 50 mM EDTA for 20 min at 37°C. The samples were subsequently incubated with 0.1 M triethanolamine and 0.5% acetic anhydride for 20 min at 37°C in order to prevent the nonspecific binding of the 35S-labeled complementary RNA (cRNA) probes. Prehybridization of the samples was carried out in 50% formamide in 2X standard saline citrate (SSC) for 15 min at 37°C. Hybridization was carried out using a hybridization mixture containing the appropriate sense or antisense probes. Posthybridization involved high-stringency washing of the samples in decreasing concentrations of SSC at 42°C. In order to remove any unbound RNA probes, the samples were washed with RNase solution for 20 min at 42°C. The samples were then dehydrated with increasing concentrations of ethanol, and air-dried. Following this, the samples were dipped in Amersham LM-2 emulsion (Oakville, Ontario, Canada) and exposed for a period of 10 days. The autoradiographs were then developed in Kodak D-19 developer (Eastman Kodak, Toronto, Canada), fixed, and counterstained with hematoxylin. As negative controls, tissue sections were hybridized with the sense probe and/or pretreated with RNase A, then hybridized to antisense probes.
To ascertain the expression of TGF-β1 mRNA by activated eosinophils, we simultaneously applied radiolabeled in situ hybridization (ISH) with EG2-immunoreactivity as previously described in detail elsewhere (25). Briefly, cryostat sections (5 μm) of frozen bronchial biopsies were cut and the sections were immunostained with an anti-EG2 monoclonal antibody directed against the cleaved form of eosinophil cationic protein (ECP) (Pharmacia, Uppsala, Sweden). Following visualization of the positive immunostaining using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique, the sections underwent a modified ISH for TGF-β1 mRNA.
TGF-β1 immunoreactivity was detected in 5-μm tissue sections by the use of a TGF-β1 specific polyclonal antibody (AB-101-NA; R&D Systems, Minneapolis, MN). Briefly, endogenous peroxidase activity in cryostat sections of nasal biopsies was blocked using 1% H2O2 (plus 0.02% sodium azide in Tris-buffered saline [TBS]) for 30 min. Immunocytochemistry using the avidin-biotin-peroxidase complex method was then performed as previously described (26). For negative control preparations, the primary antibody was replaced by either nonspecific rabbit immunoglobulin or TBS.
To confirm the phenotype of TGF-β1 immunoreactive positive cells, we undertook double sequential immunocyto-chemistry as previously described (27). Briefly, endogenous peroxidase activity in cryostat sections of bronchial biopsies was blocked using 1% H2O2 (plus 0.02% sodium azide in TBS) for 30 min. A mixture of primary antibodies was then applied consisting of the polyclonal anti-TGF-β1 antibody used to detect TGF-β1 immunoreactivity, and the appropriate monoclonal antibody to determine the presence of eosinophils (EG2+). After incubating with the appropriate secondary antibodies, a tertiary layer of streptavidin peroxidase and murine APAAP conjugate was then applied. Sections were developed sequentially in Fast Red (the APAAP substrate) and diaminobenzidine (a peroxide substrate). TGF-β1 immunoreactive cells stained brown, the eosinophils stained red, and eosinophils expressing eotaxin-immunoreactivity stained a reddish-brown. In all immunochemical studies the appropriate negative controls were included, which included TBS alone, omission of the primary antibodies, and the use of an irrelevant mouse or rabbit IgG antibody.
For the detection of collagen fibers, sections of bronchial biopsies were stained with van Gieson's stain. Using this technique, the collagen fibers could be identified by their characteristic red staining.
Slides were coded and positive cells counted blindly using ×100 magnification with an eyepiece graticule. Hybridization signals between cytokine mRNA and cRNA probes were localized as dense collections of silver grains in photographic emulsion overlying individual cells. Positively staining cells were counted to a depth of 0.45 mm below the basement membrane (BM) and the results were expressed as the mean number of positive cells per mm BM. The severity of airway fibrosis was quantitated on a scale of 1 to 4 according to the extent of fibrosis within the lamina propria, with a score of 4 representing extensive fibrosis within the airways which extended from the BM to the smooth-muscle layer. The within observer coefficient of variation for repeated measures was less than 5%.
The numbers of cells expressing TGF-β1 in mild, moderate, and severe asthmatic airways and normal control subjects were compared using a nonparametric Kruskal-Wal-lis analysis of variance. Statistically significant differences between groups were subsequently analyzed using a Mann-Whitney U test. Correlation coefficients were calculated from Pearson's moment coefficient and were corrected for multiple comparisons by the use of Bonferroni's correction factor (Systat v5.0, v6.1; SPSS Inc., Chicago, IL). Results were considered statistically significant for P < 0.05.
Hybridization between the labeled cRNA probes and mRNA encoding TGF-β1 was demonstrated by specific deposits of silver grains in the photographic emulsion overlying the tissue sections. No positive hybridization signals were observed when the sense probe was used, nor when the tissues were pretreated with RNase. TGF-β1 mRNA was detected in bronchial biopsies from control and asthmatic individuals (Figure 1c). The expression of this cytokine was significantly increased in severe asthmatics (Figure 2; mean mRNA positive cells/mm BM ± SEM; 18.5 ± 3.1; n = 6) compared with moderate asthmatics (mean ± SEM; 10.8 ± 1.3; n = 6; P < 0.05), mild asthmatics (mean ± SEM; 7.8 ± 1.5; n = 6; P < 0.01), and normal control subjects (mean ± SEM; 3.5 ± 0.8; n = 10; P < 0.001). As a group, asthmatics (mean ± SEM; 12.4 ± 1.6; n = 18) had significantly greater TGF-β1 mRNA expression than did normal individuals (Figure 2; P < 0.05). The expression of TGF-β1 mRNA was also increased in moderate asthmatics compared with normal control subjects (Figure 2; P < 0.05).
Fig. 1. Immunostaining for EG2-positive cells in mild (a) and severe (b) asthmatic bronchial biopsies, and in situ hybridization detecting TGF-β1 mRNA-positive cells in a moderate (c) and a severe asthmatic individual (d). Insert in panel (d) shows a TGF-β1 mRNA-positive cell, indicated by the silver grains, associated with EG2+-immunoreactivity (pink staining). Immunocytochemistry for TGF-β1 immunoreactive protein in a moderate asthmatic (e) and double immunostaining showing the presence of TGF-β1 immunoreactivity in EG2-positive eosinophils (f ). Hematoxylin and eosin staining of a bronchial biopsy section from a severe asthmatic (g), and representative examples of subepithelial fibrosis as detected by van Gieson's staining in mild (h), moderate (j), and severe (k) asthmatic individuals.
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Fig. 2. TGF-β1 mRNA expression and EG2-positive cells in mild to severe asthmatics, and normal control subjects. There were increased numbers of cells expressing TGF-β mRNA and staining specifically for EG2 in airways from severe (***P < 0.001) and moderate (**P < 0.02) asthmatics compared with normal control subjects.
[More] [Minimize]The numbers of cells expressing immunoreactivity for TGF-β1 were also enumerated in bronchial biopsies from the severe, moderate, and mild asthmatic individuals, as well as the normal control subjects. Compared with the normal individuals (mean ± SEM; 5.2 ± 1.0; n = 5), there was a significant increase in TGF-β1 immunoreactive cells in mild (mean ± SEM; 9.2 ± 1.6; n = 5; P < 0.05), moderate (mean ± SEM; 12.25 ± 1.0; n = 4; P < 0.05), and severe asthmatics (mean ± SEM; 18.8 ± 4.9; n = 5; P < 0.01). There were no significant differences in TGF-β1 immunoreactivity between the groups of asthmatics classified according to disease severity (P > 0.05).
Cells exhibiting positive immunoreactivity for ECP were detected by the presence of discrete red staining within the airway submucosa (Figures 1a and 1b). Numbers of EG2-positive cells were increased in severe asthmatics (Figure 2; mean EG2-positive cells/mm BM ± SEM; 15.1 ± 1.5; n = 6) compared with moderate asthmatics (mean ± SEM; 8.8 ± 0.9; n = 6; P < 0.05), mild asthmatics (mean ± SEM; 5.3 ± 1.0; n = 6; P < 0.01), and normal control subjects (mean ± SEM; 0.4 ± 0.2; n = 10; P < 0.001). Moderate and mild asthmatics also had a greater degree of airway eosinophilia than normal controls (Figure 2; P < 0.001).
Histochemically, the extent of airways fibrosis could be visualized by the use of van Gieson's stain, which results in a characteristic red staining of the subepithelial collagen layer (Figures 1e through 1g). Individuals with severe asthma had the greatest degree of airway fibrosis (Figure 3; mean score ± SEM; 3.1 ± 0.2; n = 6) compared with moderate asthmatics (mean score ± SEM; 2.2 ± 0.2; n = 6; P < 0.05), mild asthmatics (mean score ± SEM; 1.4 ± 0.1; n = 6; P < 0.005) and normal control subjects (mean score ± SEM; 0.4 ± 0.2; n = 10; P < 0.001). Significantly increased airway fibrosis was observed even in moderate and mild asthmatics compared with normal control subjects (Figure 3; P < 0.005).
Fig. 3. The extent of subepithelial airways fibrosis in mild to severe asthmatics, and normal control subjects. There was a significant increase in airways fibrosis in severe (***P < 0.001), moderate (**P < 0.001), and mild (*P < 0.005) asthmatics compared with normal control subjects.
[More] [Minimize]When examining the numbers of TGF-β1 mRNA-positive cells with respect to the numbers of EG2-positive cells and extent of airways fibrosis in asthmatic individuals, positive correlations could be observed (TGF-β1 mRNA and EG2-positive cells, r2 = 0.86; n = 18; TGF-β1 mRNA and airways fibrosis, r2 = 0.70; n = 18; P < 0.05). These observed increases in subepithelial fibrosis and EG2-positive cells could also be correlated to baseline FEV1 values in the asthmatic individuals (Figures 4a and 4b). There were also significant correlations between the FEV1 value and the extent of subepithelial fibrosis (r2 = 0.78; P < 0.05) and EG2-positive cells (r2 = 0.84; P < 0.05).
Fig. 4. Correlations between (a) FEV1 and subepithelial fibrosis, and (b) FEV1 and EG2-positive cells in mild to severe asthmatics. There were significant correlations between the FEV1 and the degree of subepithelial fibrosis (P < 0.001) and the numbers of EG2-positive cells (P < 0.001).
[More] [Minimize]In phenotyping the cells expressing mRNA for TGF-β1, combined immunocytochemistry and ISH revealed that approximately 65% of the TGF-β1 mRNA-positive cells were cells expressing immunoreactivity for the cleaved form of ECP. The other 35% of the TGF-β1 mRNA and immunoreactive cells exhibited a phenotype consistent with macrophages and fibroblasts. Only approximately 75% of the EG2+ cells expressed TGF-β1 mRNA.
Since subepithelial fibrosis resulting from the deposition of collagen beneath the BM has been reported in mild and newly diagnosed asthmatics (4, 5), we hypothesized that the mRNA expression of the potent profibrotic cytokine TGF-β1 would be markedly upregulated in bronchial biopsies from patients with more severe asthma, and that the increased expression of TGF-β1 would be associated with the degree of airway fibrosis. The results of this study demonstrate that TGF-β1 mRNA is upregulated within the airways of asthmatic individuals and that the expression of this cytokine correlates with the degree of subepithelial fibrosis. We also show that the major source of this cytokine mRNA is activated eosinophils.
Upregulation of TGF-β1 mRNA and its association with airways fibrosis is a novel observation in the pathogenesis of bronchial asthma. To date, there has been only one previous study examining the expression of TGF-β1 in bronchial asthma (28). Using Northern blot analysis, these investigators found no difference between asthmatic subjects and individuals with chronic obstructive pulmonary disease or smokers with no fixed airway obstruction. The discrepancies between these results and our own may lie in the severity of the asthmatic subjects examined. Indeed, we found that expression of TGF-β1 mRNA was greatest in the most severe asthmatics, with no significant differences noted between mild asthmatics and the normal control subjects (Figure 2). Other possible explanations include the use of appropriate control individuals, and/or the inability of the Northern blot technique to detect local increases in TGF-β1 mRNA.
Using the ISH techniques, it is possible to determine the presence of transcribed mRNA only for TGF-β1. Thus, from the results of this study, we are unable to determine whether this mRNA is being translated into the active protein. Indeed, even if translation were taking place, we still could not be certain that the TGF-β1 was secreted in a biologically active form (29). However, several lines of evidence do suggest that eosinophil-derived TGF-β1 mRNA is transcribed into the active protein. Previous immunocytochemical and ISH studies examining TGF-β1 in eosinophils have confirmed the presence of active cytokine secretion (17). We have also conducted preliminary studies which show that supernatants from activated eosinophils are able to induce the synthesis of collagen types I, II, III, and IV in a fibroblastic cell line (unpublished observations). Furthermore, our own data show that there is a positive correlation between the extent of subepithelial fibrosis and the number of cells positive for TGF-β1 mRNA.
In order to examine asthmatic individuals with a wide range of disease severity, we used subjects who were on regular inhaled steroids. It may be argued that the use of these agents could have influenced the data obtained in the moderate and severe asthmatics. The ability of corticosteroids to modulate TGF-β1 mRNA expression has not been systematically examined to date. However, there are reports that steroids do not directly influence TGF-β1 mRNA production (30, 31). Indeed, steroids may indirectly reduce TGF-β1 mRNA production within the lungs of asthmatic individuals by attenuating eosinophil recruitment (32). The low FEV1 values and increased numbers of EG2-positive cells in our moderate and severe asthmatics would suggest that these individuals were not being well controlled by these agents. Alternatively, it would suggest that inhaled steroids have a limited ability to resolve tissue eosinophilia compared with oral steroids. In addition, significant amounts of TGF-β1 mRNA were present in cells not staining for EG2 and may be coming from cells already resident within the lung, such as epithelial cells, macrophages, lymphocytes, and fibroblasts themselves (15, 16). The lack of effect of steroids on fibrosis formation obviously has important clinical considerations and may explain why even aggressive steroid therapy fails to restore normal airways responsiveness (33, 34).
There has been a relative paucity of data examining the molecular mechanisms underlying the structural abnormalities observed in bronchial asthma. Structural changes within the airways of asthmatic individuals may contribute to the development of persistent airways hyperresponsiveness (6) and chronic alterations in airflow obstruction (7), although the mechanisms by which these alterations occur is unclear. It has been proposed that collagen types I and III and fibronectin deposition are involved in the thickening of the reticular layer (4, 5) and that this is accompanied by a change in the phenotype of fibroblasts to resemble a contractile cell (8). However, the inflammatory stimulus which induces collagen synthesis from fibroblasts and/or myofibroblasts remains to be elucidated. Our findings suggest that TGF-β1 released locally from beneath the BM may contribute both to the development of airways fibrosis and the subsequent decline in lung function.
Our colocalization studies show that 65% of the TGF-β1 mRNA-positive cells were activated eosinophils. These cells were located within the reticular lamina (Figure 1) and therefore were appropriately situated to generate inflammatory stimuli capable of inducing collagen synthesis. The production by eosinophils of profibrotic agents such as TGF-β1 is not novel and it has been shown previously that these cells are a potential source of platelet-derived growth factor B-chain in bronchial asthma (35). In addition to its role as a potent profibrotic agent within the airways, the local secretion of TGF-β1 by eosinophils is known to inhibit eosinophil survival and function (36). The activation of this pathway may be one autoregulatory mechanism which limits the numbers of tissue-dwelling cells in chronic eosinophilic diseases. Evidence to support such a role of TGF-β1 in bronchial asthma remains to be established.
In summary, we have demonstrated that TGF-β1 mRNA is upregulated in bronchial asthma and is associated with the degree of airways fibrosis. The finding that eosinophils are a potential major source of this cytokine within the reticular lamina links chronic allergic inflammation with structural remodeling and the decline in FEV1 observed in this disease. Novel therapeutic agents which prevent TGF-β1 production may be of use in the restoration of normal lung function in asthmatic individuals.
The authors thank the Glaxo Institute for Molecular Biology for their generous gift of human cDNA for TGF-β1. Dr. Eleanor Minshall is a recipient of a Canadian Lung Association/Medical Research Council Fellowship. The authors also to thank Ms. Elsa Schotman and Ms. Zivart Yasruel for their technical assistance. This work was supported by MRC Canada and in part by NIH grants HL 36577 and RR 00051.
| 1. | Dunnill M. S.The pathology of asthma, with special reference to changes in the bronchial mucosa. J. Clin. Pathol.1319602733 |
| 2. | Jeffery P. K.Comparative morphology of the airways in asthma and chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med.1501994S6S13 |
| 3. | Laitinen L. A., Laitinen A., Haahtela T.Airway mucosal inflammation even in patients with newly diagnosed asthma. Am. Rev. Respir. Dis.1471993697704 |
| 4. | Roche W. R., Beasley R., Williams J. H., Holgate S. T.Sub-epithelial fibrosis in the bronchi of asthmatics. Lancet11989520524 |
| 5. | Jeffery P. K., Wardlaw A. J., Nelson F. C., Collins J. V., Kay A. B.Bronchial biopsies in asthma. An ultra-structural, quantitative study and correlation with hyperreactivity. Am. Rev. Respir. Dis.140198917451753 |
| 6. | James A. L., Pare P. D., Hogg J. C.The mechanisms of airway narrowing in asthma. Am. Rev. Respir. Dis.1391989242246 |
| 7. | Van Schayck C. P., Dompeling E., Van Herwaarden C. L. A., Wever A. M. J., Van Weel C.Interacting effects of atopy and bronchial hyperrepsonsiveness on the annual decline in lung function and the exacerbation rate in asthma. Am. Rev. Respir. Dis.144199112971301 |
| 8. | Brewster C. E. P., Howarth P. H., Djukanovic R., Wilson J., Holgate S. T., Roche W. R.Myofibroblasts and sub-epithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol.31990507511 |
| 9. | Raghu C., Masta S., Meyers D., Narayanan A. S.Collagen synthesis by normal and fibrotic lung fibroblasts and the effect of transforming growth factor-β. Am. Rev. Respir. Dis140198995100 |
| 10. | Raghow, R. 1991. Role of transforming growth factor-β in repair and fibrosis. Chest 99(3S):61S–65S. |
| 11. | Wahl S. M., Hunt D. A., Wakefield L. M., McCartney-Francis N., Wahl L. M., Roberts A. B., Sporn M. B.Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc. Natl. Acad. Sci. USA84198757885792 |
| 12. | Postlethwaite A. E., Keski-Oja J., Moses H. L., Kang A. H.Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor-β. J. Exp. Med.1651987251256 |
| 13. | Gruber B. L., Marchese M. J., Kew R. R.Transforming growth factor-β mediates mast cell chemotaxis. J. Immunol.152199458605867 |
| 14. | Roberts, A. B., and M. B. Sporn. 1990. The transforming growth factor-betas. In Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology. M. B. Sporn and A. B. Roberts, editors. Springer-Verlag, New York. 419–472. |
| 15. | Khalil N., O'Connor R. N., Flanders K. C., Unruh H.TGF-β1, but not TGF-β2 or TGF-β3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am. J. Respir. Cell Mol. Biol.141996131138 |
| 16. | Zhang K., Flanders K. C., Phan S. H.Cellular localisation of TGF-β expression in bleomycin-induced pulmonary fibrosis. Am. J. Pathol.1471995352361 |
| 17. | Ohno I., Lea R. G., Flanders K. C., Clark D. A., Banwatt D., Dolovich J., Denberg J., Harley C. B., Gauldie J., Jordana M.Eosinophils in chronically inflamed human upper airway tissues express transforming growth factor β1 gene (TGFβ1). J. Clin. Invest.89199216621668 |
| 18. | Elovic A., Wong D. T. W., Weller P. F., Matossian K., Galli S. J.Expression of transforming growth factors-α and β1 messenger RNA and product by eosinophils in nasal polyps. J. Allergy Clin. Immunol.931994864869 |
| 19. | Wong D. T. W., Elovic A., Matossian K., Nagura N., McBride J., Chou M. Y., Gordon J. R., Rand T. H., Galli S. J., Weller P. F.Eosinophils from patients with blood eosinophilia express transforming growth factor β1. Blood78199127022707 |
| 20. | Azzawi M., Bradley B., Jeffery P. K., Frew A. J., Wardlaw A. J., Assoufi B., Collins J. V., Durham S. R., Knowles G. K., Kay A. B.Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am. Rev. Respir. Dis.142199014071413 |
| 21. | American Thoracic SocietyStandards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am. Rev. Respir. Dis.1361987225244 |
| 22. | Hamid Q., Wharton J., Terenghi G., Hassall C. J. S., Aimi J., Taylor K. M., Nakazato H., Dixon J. E., Burnstock G., Polak J. M.Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage. Proc. Natl. Acad. Sci. USA84198767606764 |
| 23. | Tsicopoulous A., Hamid Q., Varney V., Ying S., Moqbel R., Durham S. R., Kay A. B.Preferential messenger RNA expression of Th1-type cells (IFNγ+, IL-2+) in classical delayed-type (tuberculin) hypersensitivity reactions in human skin. J Immunol148199220582061 |
| 24. | Ying S., Durham S. R., Barkans J., Masuyama K., Jacobson M., Rak S., Lowhagen O., Moqbel R., Kay A. B., Hamid Q.T cells are the principle source of interleukin-5 mRNA in allergen-induced rhinitis. Am. J. Respir. Cell Mol. Biol.91993356360 |
| 25. | Hamid Q., Barkans J., Meng Q., Abrams J., Kay A. B., Moqbel R.Human eosinophils synthesize and secrete interleukin-6, in vitro. Blood80199214961501 |
| 26. | Giad A., Michel R. P., Stewart D. J., Sheppard M., Corrin B., Hamid Q.Expression of endothelin-1 in lungs of patients with cryptogenic fibrosing alveolitis. Lancet341199315501554 |
| 27. | Moqbel R., Ying Y., Barkans J., Newman T. M., Kimmitt P., Wakelin M., Taborda-Barata L., Meng Q., Corrigan C. J., Durham S. R., Kay A. B.Identification of messenger RNA for IL-4 in human eosinophils with granular localization and release of the translated product. J. Immunol155199549394947 |
| 28. | Aubert J.-D., Dalal B. I., Bai T. R., Roberts C. R., Hayashi S., Hogg J. C.Transforming growth factor β1 gene expression in human airways. Thorax491994225232 |
| 29. | Miyazono K., Yuki K., Takaku F., Wernstedt C., Kanzaki T., Olofsson A., Hellman U., Heldin C. H.Latent forms of TGF-beta: structure and biology. Ann. N.Y. Acad. Sci.59319905158 |
| 30. | Khalil N., Whitman C., Zuo L., Danielpour D., Greenberg A.Regulation of alveolar macrophage transforming growth factor-beta secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat. J. Clin. Invest.92199318121818 |
| 31. | Ayanlar-Batuman O., Ferrero A. P., Diaz A., Jimenez S. A.Regulation of transforming growth factor-beta 1 gene expression by glucocorticoids in normal human T lymphocytes. J. Clin. Invest.88199115741580 |
| 32. | Jeffery P. K., Godfrey R. W., Adelroth E., Nelson F., Rogers A., Johansson S.-A.Effects of treatment on airway inflammation and thickening of basement membrane reticular collagen layer. Am. Rev. Respir. Dis.1451992890899 |
| 33. | Lundgren R., Soderberg M., Horstedt P., Stenling R.Morphological studies of bronchial mucosal biopsies from asthmatics before and after ten years of treatment with inhaled steroids. Eur. Respir. J.11988863889 |
| 34. | Kerrebijn K. F., van Essen-Zandvliet E. E. M., Neijens H. J.Effect of long-term treatment with inhaled corticosteroids and beta-agonists on the bronchial responsiveness in children with asthma. J. Allergy Clin. Immunol.791991653659 |
| 35. | Ohno I., Nitta Y., Yamauchi K., Hoshi H., Honma M., Woolley K., O'Byrne P., Dolovich J., Jordana M., Tamura G., Tanno Y., Shirato K.Eosinophils as a potential source of platelet-derived growth factor B-chain in nasal polyposis and bronchial asthma. Am. J. Respir. Cell Mol. Biol131995639647 |
| 36. | Alam R., Forsythe P., Stafford S., Fukuda Y.Transforming growth factor β abrogates the effects of hematopoietins on eosinophils and induces their apoptosis. J. Exp. Med.179199410411045 |
