GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-beta receptors

Gang Chen1, Gary Grotendorst2, Thomas Eichholtz3, and Nasreen Khalil1

1 Department of Medicine, Vancouver Hospital and Health Sciences Centre, University of British Columbia, Vancouver, British Columbia V6Z 3Z6, Canada; 2 University of Miami School of Medicine, Miami, Florida 33136; and 3 GlaxoSmithKline, Stevenage, Herts SG1 2NY, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibrosis around the smooth muscle of asthmatic airway walls leads to irreversible airway obstruction. Bronchial epithelial cells release granulocyte/macrophage colony-stimulating factor (GM-CSF) in asthmatics and are in close proximity to airway smooth muscle cells (ASMC). The findings in this study demonstrate that GM-CSF induces confluent, prolonged, serum-deprived cultures of ASMC to increase expression of collagen I and fibronectin. GM-CSF also induced ASMC to increase the expression of transforming growth factor (TGF)-beta receptors type I, II, and III (Tbeta R-I, Tbeta R-II, Tbeta R-III), but had no detectable effect on the release of TGF-beta 1 by the same ASMC. The presence of GM-CSF also induced the association of TGF-beta 1 with Tbeta R-III, which enhances binding of TGF-beta 1 to Tbeta R-II. The induction of Tbeta Rs was parallel to the increased induction of phosphorylated Smad2 (pSmad2) and connective tissue growth factor (CTGF), indicative of TGF-beta -mediated connective tissue synthesis. Dexamethasone decreased GM-CSF-induced Tbeta R-I, Tbeta R-II, Tbeta R-III, pSmad2, CTGF, collagen I, and fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC to TGF-beta 1-mediated connective tissue expression by induction of Tbeta Rs, which is inhibited by corticosteroids.

airway remodeling; corticosteroids; irreversible airway obstruction; phosphorylated Smad2; connective tissue growth factor; granulocyte/macrophage colony-stimulating factor; transforming growth factor-beta ; airway smooth muscle cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A FUNCTIONAL ABNORMALITY in asthma is obstruction to airflow as a consequence of recruitment of inflammatory cells, mucus plugging, and airway wall thickening (17, 30). The airways are thickened by inflammatory cell infiltrates consisting of eosinophils, lymphocytes and mast cells, mucosal edema, and vasodilation (17, 30). Although many of these changes are reversible (30), airway obstruction in asthmatics may also be irreversible due to airway remodeling (30). Remodeling of airways occurs when myofibroblasts just beneath the bronchial epithelium proliferate and synthesize increased amounts of collagens I, III, and V and fibronectin (30). In addition, airway remodeling occurs from increases in collagens, elastins, fibronectin, laminin, hyaluronan, and versican around airway smooth muscle cells (ASMC) (27). ASMC may become hyperplastic and hypertrophied, which also contributes to structural changes, airway narrowing, and obstruction (27). On the basis of observations made on the pathogenesis of fibrosis at other sites, it is speculated that a number of cytokines and growth factors may be important in regulating the recruitment and proliferation of fibroblasts and connective tissue synthesis by fibroblasts, myofibroblasts, and ASMC (24, 33). The source of cytokines that regulate fibroblasts could be the structural cells themselves such as bronchial epithelial cells (BEC) and ASMC (3). Alternatively, the inflammatory cells recruited to the airways or plasma protein and platelets that leak into the airways could be additional sources of fibrogenic cytokines (3, 17).

Two cytokines of particular interest in the context of airway remodeling are granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta . GM-CSF is a 22-25-kDa homodimeric glycoprotein originally described to induce proliferation and differentiation of bone marrow cells (13, 15). GM-CSF is produced and released by a number of cells (13, 35), such as fibroblasts, keratinocytes, BEC, vascular cells, and ASMC (6, 7, 13, 25, 26, 35). With the use of immunohistochemistry and Northern analysis, the expression of GM-CSF is increased in the BEC of asthmatics compared with nonasthmatics (26). Isolated BEC from asthmatic airways release more GM-CSF than BEC from nonasthmatic controls (26). It is speculated that, in asthma, the increased presence of GM-CSF in the airways leads to survival of eosinophils, which have been demonstrated to have an important role in regulating a number of events in asthma (6, 7, 25, 26). Recently, an additional effect of GM-CSF has been described, demonstrating the induction of connective tissue synthesis (8, 31, 36). For example, in a rat model, subcutaneous instillation of GM-CSF led to fibroblast accumulation and stimulation of alpha -smooth muscle actin synthesis in myofibroblasts (31). In another rat model, intratracheal administration of an adenovirus containing the GM-CSF gene led to irreversible pulmonary fibrosis (36). The mechanism by which GM-CSF induces collagen synthesis is not well understood but could be due to induction of TGF-beta , a regulator of connective tissue synthesis (4, 24, 33). GM-CSF induced mRNA of TGF-beta 1 by vascular smooth muscle cells (29) and leiomyoma cells (10). GM-CSF also caused myometrial smooth muscle cells to release TGF-beta 1 (10).

TGF-beta is a multifunctional polypeptide (4, 24, 33) that is present as three isoforms in mammals, but TGF-beta 1 is the isoform most commonly associated with disorders characterized by inflammation and fibrosis (4). Signal transduction of TGF-beta 1 is mediated when TGF-beta 1 associates with TGF-beta receptor type II (Tbeta R-II), a 73-kDa serine/threonine receptor that is constitutively phosphorylated (5, 14). On binding TGF-beta 1, Tbeta R-II phosphorylates TGF-beta receptor type I (Tbeta R-I), a 53-kDa protein, leading to signal transduction mediated by a number of intracellular proteins that include the Smads (5, 14, 28). TGF-beta receptor type III (Tbeta R-III), or beta -glycan, is a 250-350-kDa proteoglycan that also binds TGF-beta 1 but has no definite signaling capability (5, 14). However, the association of TGF-beta with Tbeta R-III enhances the interaction of TGF-beta with Tbeta R-II and subsequent signal transduction (5, 14). With the use of immunohistochemistry, we have previously demonstrated that all isoforms of TGF-beta , Tbeta R-I, and Tbeta R-II, were expressed by ASMC of human and rat lungs (21, 23). We have also demonstrated that subconfluent bovine ASMC or mechanically wounded monolayers of ASMC release biologically active TGF-beta 1, that in an autocrine fashion, induces collagen I synthesis (12). Furthermore, the addition of TGF-beta 1 to ASMC also led to collagen synthesis in a dose-dependent fashion (12). Despite the fibrogenic effects of TGF-beta 1 on ASMC, previous studies using Northern blot analysis and immunohistochemistry have not demonstrated evidence of increased expression of TGF-beta 1 in ASMC of patients with asthma compared with nonasthmatic airways (2, 11, 32). Furthermore, there has been no correlation of expression of TGF-beta 1 with the severity of asthma and airway remodeling (11). Unlike our observations, these findings do not demonstrate the release of biologically active TGF-beta 1 in a model simulating ASMC injury (12). The release of active TGF-beta 1 is important in autocrine regulation of collagen synthesis by ASMC (12). In addition, the studies mentioned earlier do not provide any evidence on the regulation of Tbeta Rs, which might be equally important in TGF-beta 1-mediated effects.

BEC, which express and release GM-CSF during episodes of asthma, are in close proximity to ASMC. It is, then, conceivable that GM-CSF previously demonstrated to induce TGF-beta 1 (10, 29) might regulate ASMC to synthesize and release TGF-beta 1, which, in turn, would induce collagen synthesis in the ASMC layer of bronchi. In this study, we demonstrate that GM-CSF induces collagen I and fibronectin expression by bovine confluent, prolonged, serum-deprived cultures of ASMC in a dose-dependent fashion. The presence of GM-CSF does not lead to detectable release of TGF-beta 1 by ASMC. However, the presence of GM-CSF in confluent, prolonged, serum-deprived cultures of ASMC increases the density of TGF-beta 1 on the ASMC and expression of Tbeta R-I, Tbeta R-II, and Tbeta R-III, as well as the association of TGF-beta 1 with Tbeta R-III. The presence of GM-CSF also induces TGF-beta -mediated signal transduction necessary for connective tissue synthesis. These findings, for the first time, demonstrate that regulation of Tbeta Rs on ASMC without evidence of release of TGF-beta 1 can lead to enhanced connective tissue expression that is mediated by TGF-beta 1.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. alpha -Modified Eagle's medium (alpha -MEM), fetal calf serum (FCS), and other cell culture reagents were purchased from GIBCO-BRL (Burlington, ON, Canada). Anti-Tbeta R-I and anti-Tbeta R-II antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To block the interaction of TGF-beta 1 with Tbeta R-II, anti-Tbeta R-II antibody from R&D Systems (Minneapolis, MN) was used. Anti-collagen type I and anti-fibronectin antibodies were from Cedarlane Laboratories (Hornby, ON, Canada). Anti-phosphorylated Smad2 antibody and anti-Tbeta R-III antibody were purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture. Bovine tracheas were obtained from the local slaughterhouse. An explanted culture of the smooth muscle tissue was established as described previously with some modification (12). Briefly, the associated fat and connective tissues were removed in cold Krebs-Ringer solution (in mM: 118 NaCl, 4.8 KCl, 2.5 CaCl2 2 H2O, 1.2 MgSO4, 25 NaCHO3, 1.15 KH2PO4, 5.6 dextrose, and 12.6 HEPES) with antibiotic-antimycotic reagents containing 100 U/ml of penicillin G, 100 µg/ml of streptomycin, 0.25 µg/ml of amphotericin B, and 0.25 µg/ml of sodium desoxycholate (GIBCO-BRL). The smooth muscle was then isolated, cut into 1-2-mm cubic size, and placed on culture dishes with a minimal volume of alpha -MEM supplemented with 10% FCS and antibiotic-antimycotic reagents. In an incubator at 37°C in a humidified atmosphere (5% CO2-balanced air), ASMC migrated from the tissue explants the following day. When cells were approaching confluence in some parts of the dish at 6-10 days after being explanted, the explants were removed, and the ASMC were passaged with 0.05% trypsin/0.53 mM EDTA. For the experiments, ASMC in passages 1-3 were grown to confluence in alpha -MEM with 10% FCS and antibiotic-antimycotic reagents and then changed to serum-free alpha -MEM containing antibiotics and ITS (10 µg/ml of insulin, 5.5 µg/ml of transferrin, and 5 ng/ml of sodium selenite, from Sigma) for 10 days before addition of GM-CSF with or without 10-4 M dexamethasone (SABEX, Boucherville, QC, Canada). It is of note that we have previously demonstrated that when ASMC are subconfluent in culture, they release biologically active TGF-beta 1 (12). In these conditions, the release of TGF-beta 1 results in the synthesis of collagen I (12). However, when ASMC were grown to confluence and kept in serum-free conditions for 10 days, there was no active TGF-beta 1 present in the conditioned media (CM) (12). Such conditions also had minimal or no collagen I synthesis (12) and were considered suitable to evaluate the effects of GM-CSF on the release of TGF-beta 1 or connective tissue synthesis by GM-CSF. Henceforth, when we refer to confluent monolayers of ASMC, the term refers to prolonged, serum-deprived, confluent ASMC.

Collection of CM. At various times after cells were incubated, the overlying CM were removed, stored in sterile, siliconized Eppendorf tubes in the presence of the protease inhibitors, including 5 µg/ml of leupeptin (Boehringer Mannheim), 5 µg/ml of aprotinin (Sigma), 5 µg/ml of pepstatin A (Sigma), and 1 mM PMSF (Sigma), and frozen at -86°C until ready for use.

TGF-beta 1 receptor assay by flow cytometry. Twenty-four hours after being treated with GM-CSF or normal saline, ASMC were removed from the culture dishes using a nonenzymatic cell disassociation solution (Sigma) and then stained with Fluorokine Cytokine Flow Cytometry Reagents (R&D Systems) as instructed by the manufacturer. Briefly, each sample in both the control and the GM-CSF group was divided into two aliquots of 1 × 105 cells. The first aliquot was incubated at 4°C for 1 h with biotinylated recombinant human (rh)TGF-beta 1, which binds to the cell predominantly via specific cell surface receptors. The cells were then incubated with avidin-fluorescein, which attaches to the cell-bound biotinylated TGF-beta 1, for 30 min at 4°C in the dark. The other aliquot, which served as the negative staining control, was stained exactly as the first aliquot except biotinylated negative control reagent was used instead of biotinylated rhTGF-beta 1. The cells were washed twice with RDF1 buffer to remove unreacted avidin-fluorescein and resuspended in RDF1 buffer for final flow cytometric analysis using Coulter Epics XL-MCL Flow Cytometer and Expo 32 software. Each sample was normalized by its individual negative staining control before obtaining a mean and SE and applying statistical analysis. The specificity of the reaction was confirmed using anti-TGF-beta 1 antibody.

TGF-beta 1 assay by ELISA. TGF-beta 1 is secreted in a biologically latent form due to a noncovalent association of the latency associated peptide-1 (LAP-1) with the NH2-terminal 25-kDa portion of the protein. In this form, TGF-beta 1 is called latent TGF-beta 1 (LTGF-beta 1). To convert LTGF-beta 1 to a biologically active TGF-beta 1, the LAP-1 must be removed, which can be achieved by acidification of the CM. The current assay detects TGF-beta 1 only in its active conformation. Each sample was divided into two aliquots in which one aliquot of CM had a neutral pH containing biologically active TGF-beta 1. To detect total TGF-beta 1 in each sample, the other aliquot of CM was acidified by 1 N HCl for 10 min, neutralized with 1.2 N NaOH/0.5 M HEPES, and used in the assay as described. DuoSet TGF-beta 1 ELISA kit (R&D Systems) was used to determine TGF-beta 1 in neutral CM (representing active TGF-beta 1) or CM that were acidified and subsequently neutralized (representing total TGF-beta 1) according to the manufacturer's instructions. Briefly, anti-TGF-beta 1 capture antibody was coated onto a 96-well microplate (Costar) overnight at room temperature. The antibody was diluted to a working concentration of 2 µg/ml, and 100 µl of the preparation were added to each well. After being washed with wash buffer and blocked with block buffer, 100 µl of sample or the standard, rhTGF-beta 1, were added and incubated for 2 h at room temperature. The plate was washed before adding 100 µl of the detection antibody (biotinylated chicken anti-human TGF-beta 1). The TGF-beta 1 binding was colored by streptavidin-horseradish peroxidase, and the optical density was read using a microplate reader at 450 nm with correction at 540 nm.

Western blot and immune detection. After collection of the CM, ASMC were washed with phosphate-buffered saline and then detached by trypsinization. Cell numbers were determined using a hemocytometer. Whole cell protein was extracted on ice with triple-detergent lysis buffer (50 mM Tris · HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 5 mg/ml of sodium deoxycholate) in the presence of the protease inhibitors (as above). In addition to these inhibitors, the samples used for detection of phosphorylated Smad2 (pSmad2) were extracted in the presence of NaF (1 mM) and Na3VO4 (1 mM), potent phosphatase inhibitors. Protein concentration was calculated using the Bradford method with Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). Protein extracts were separated by SDS-PAGE [gel concentrations were 8% for collagen I, fibronectin, and Tbeta R-III; 10% for Tbeta R-II and pSmad2; and 12% for Tbeta R-I and connective tissue growth factor (CTGF)] as per Laemmli's method and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using CAPS transfer buffer (25 mM CAPS, pH 10, 20% methanol) (12). The equal loading of proteins was confirmed before immunoblotting using Ponceau S staining solution (Sigma). Briefly, after transfer of the protein from SDS-PAGE, the PVDF membrane was immersed in Ponceau S staining solution for 5 min and rinsed, and the protein bands were visualized. Once equal loading was established, the membrane was prepared for immunoblotting. After blocking with 5% milk in Tris-buffered saline containing 0.05% Tween 20 overnight at 4°C, the membrane was incubated with primary antibody at various dilutions. The dilutions were: for collagen I, 1:3,000; fibronectin, 1:3,000; Tbeta R-I, 1:500; Tbeta R-II, 1:500; Tbeta R-III, 1:600; CTGF, 1:500; and pSmad2, 1:500 for 1 h at room temperature or overnight at 4°C. This was followed by incubating the blots with a secondary antibody (Santa Cruz) at a dilution of 1:8,000 for 1 h at room temperature. The proteins on the membrane were then immunodetected by the ECL system (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. Relative absorbance of the resultant bands was determined using the Quantity One imaging system (Bio-Rad) normalized with data of untreated control and expressed as fold of control.

Immunoprecipitation of TGF-beta 1-associated proteins. One hundred micrograms of total cell lysate prepared as described above were precleared for 1 h at 4°C with 0.25 µg of normal rabbit IgG and 20 µl of Protein A/G plus-Agarose (Santa Cruz). The precleared extract was incubated with 1 µg of rabbit anti-human TGF-beta 1 polyclonal IgG (Santa Cruz) at 4°C for 1 h, then 20 µl of Protein A/G plus-Agarose was added and incubated at 4°C on a rocker platform overnight. The pellet was washed with triple-detergent lysis buffer four times and then resuspended in 40 µl of nonreducing sample buffer. After being boiled for 3 min, 20 µl of the sample were loaded on 8% polyacrylamide gel, and electrophoresis and immunoblotting were done as described above. Beads, both unused and precleared, were mixed with the sample buffer and served as negative control.

Statistical analysis. The results were expressed as means ± SE. All P values (two-tailed) were based on Wilcoxon's signed rank test. Results were considered statistically significant when P < 0.05.


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ABSTRACT
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MATERIALS AND METHODS
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GM-CSF regulation of connective tissue expression and release of TGF-beta 1 by ASMC. We previously demonstrated that addition of TGF-beta 1 to monolayers of ASMC induced collagen I synthesis in a dose-dependent fashion (12). Similar to TGF-beta 1, the presence of GM-CSF also induced collagen I synthesis in a dose-dependent manner (Fig. 1, A and B). In addition, TGF-beta 1 increased the expression of fibronectin by a greater magnitude than GM-CSF at an equivalent amount of 0.05 and 0.1 ng/ml. We next determined whether the increase in connective tissue protein by ASMC treated with GM-CSF was associated with an increased release of TGF-beta 1 by the ASMC. The presence of GM-CSF at a number of concentrations and for a number of lengths of incubation time had no effect on the secretion of TGF-beta 1 in the active or latent form by confluent ASMC in culture (Fig. 2, A-C).


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Fig. 1.   Induction of collagen I and fibronectin by granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta 1. Confluent, prolonged, serum-deprived cultures of airway smooth muscle cells (ASMC) were cultured in the presence of various concentrations of GM-CSF or TGF-beta 1 for 24 h before protein extraction, electrophoresis, and immunoblotting, followed by measuring the relative absorbance of the resultant bands. A: collagen I and fibronectin increased in a dose-dependent fashion after the addition of GM-CSF. B: collagen I and fibronectin had a similar tendency to increase in a dose-dependent fashion after the addition of TGF-beta 1. *P < 0.05, **P < 0.01 compared with ASMC cultured in the absence of GM-CSF (A) and TGF-beta 1 (B). The data presented are from 6 (A) and 3 (B) different experiments.



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Fig. 2.   Release of TGF-beta 1 from confluent ASMC. A: prolonged, serum-deprived cultures of confluent ASMC were incubated with various concentrations of GM-CSF for 24 h, and the conditioned media (CM) were collected and used to measure the active and total TGF-beta 1 activity in each sample using ELISA. The quantities of active and total TGF-beta 1 were not significantly different compared with control conditions in which no GM-CSF was present. B: ASMC in the absence or presence of GM-CSF generated small quantities of active TGF-beta 1 that increased slightly with increase in time interval. ASMC in the absence or presence of GM-CSF generated increasing quantities of total TGF-beta 1 with time in culture detected after acidification and neutralization of CM. There was not significant difference in active TGF-beta 1 or total TGF-beta 1 at each interval between the untreated and GM-CSF-treated ASMC. C: percentage of active TGF-beta 1 in the absence of GM-CSF was not statistically different compared with that in the presence of GM-CSF at each time interval. The data presented are from 9 (A) and 4 (B) different experiments.

GM-CSF regulation of Tbeta Rs, pSmad2, and CTGF. To study the changes in TGF-beta signal transduction, we first determined the expression of Tbeta Rs using flow cytometry and Western analysis. Flow cytometry revealed that the presence of GM-CSF in cultures of ASMC significantly increased the intensity of fluorescence from labeled TGF-beta 1, reflecting an increased density of TGF-beta 1 receptor on the ASMC compared with untreated ASMC (Fig. 3A, P < 0.05). In addition, the presence of GM-CSF increased the expression of Tbeta R-I, Tbeta R-II, and Tbeta R-III compared with untreated control ASMC as detected by Western analysis (Fig. 3B). Next, we determined whether there was an increase in Tbeta R-III associated with TGF-beta 1 by immunoprecipitating ASMC proteins using TGF-beta 1 antibody followed by Western analysis using anti-Tbeta R-III or anti-TGF-beta 1 antibody. ASMC protein extracts immunoprecipitated with TGF-beta 1 all expressed the same quantity of TGF-beta 1 (Fig. 3C, bottom blots). ASMC cultured alone had a minor quantity of Tbeta R-III when immunoblotted with anti-Tbeta R-III antibody (Fig. 3C, top blots). However, in the presence of GM-CSF, the quantity of Tbeta R-III was markedly increased (Fig. 3C, top blots), indicating that in the presence of GM-CSF, there is an increase in the number of Tbeta R-III associated with TGF-beta 1. We next determined whether GM-CSF also increased TGF-beta -mediated downstream signal transduction. An index of target cell response to TGF-beta is the phosphorylation of the COOH-terminal SSXS motif of Smad2 and -3 that mediates intracellular signals of the TGF-beta superfamily (28). ASMC cultured with GM-CSF had induction of pSmad2 compared with untreated controls (Fig. 3D). Induction of connective tissue proteins by TGF-beta is mediated by CTGF, a 33-38-kDa cysteine-rich protein (16). To confirm that connective tissue synthesis was mediated by TGF-beta 1, the protein from ASMC was immunoblotted with anti-CTGF antibodies. ASMC cultured with GM-CSF had an induction of CTGF (Fig. 3D). To further confirm that the GM-CSF-induced above effects are via Tbeta Rs, GM-CSF-treated ASMC were cultured with or without Tbeta R-II antibody, which interrupts the TGF-beta 1/Tbeta R-II interaction. The increased pSmad2 by GM-CSF was inhibited in the presence of 20 µg/ml of Tbeta R-II antibody (Fig. 3E). It is of note that in the presence of GM-CSF, the magnitude of increases in the expression of Tbeta Rs, collagen I, fibronectin, pSmad2, and CTGF is modest. However, the results are highly reproducible. Furthermore, there is a statistically significant difference in the expression of all these proteins when ASMC treated with GM-CSF are compared with ASMC receiving no treatment.


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Fig. 3.   GM-CSF regulation of TGF-beta receptors, phosphorylated Smad2 (pSmad2), and connective tissue growth factor (CTGF). A: after 24-h treatment of prolonged, serum-deprived cultures of confluent ASMC with normal saline (lines a and b) or 5 ng/ml of GM-CSF (lines c and d), the cells were stained with biotinylated recombinant human TGF-beta 1 (lines b and d) or biotinylated negative control reagent (lines a and c), followed by incubation with avidin-FITC and flow cytometric analysis. Inset: geometric mean of the fluorescence intensity from each sample was normalized by its individual negative staining control. The normalized values representing relative density of TGF-beta receptor were calculated for a mean, SE, and statistical significance using Wilcoxon's signed rank test. The density of TGF-beta receptor increased significantly after being treated with GM-CSF compared with control receiving no GM-CSF (*P < 0.05). The data are from 6 separate experiments. B: proteins from confluent cultures of ASMC without treatment () are control conditions. ASMC treated with 5 ng/ml of GM-CSF in the absence () or presence (gray bars) of dexamethasone (Dex; 10-4 M) were used for separation by SDS-PAGE before immunoblotting with antibodies to TGF-beta receptors Tbeta R-I, Tbeta R-II, or Tbeta R-III. All culture conditions were maintained for 24 h before collection of cells for Western analysis. There was induction of Tbeta R-I, Tbeta R-II, and Tbeta R-III in the presence of GM-CSF. Treatment with GM-CSF + Dex demonstrated a decrease in expression of Tbeta R-I, Tbeta R-II, and Tbeta R-III compared with GM-CSF treatment alone. The data are from 3-6 separate experiments. C: protein from ASMC lysate immunoprecipitated with TGF-beta 1 antibody was electrophoresed and immunoblotted with anti-Tbeta R-III antibody or anti-TGF-beta 1 antibody. For each condition, the quantity of TGF-beta 1 detected by Western analysis was the same (bottom blots). ASMC cultured in the absence of GM-CSF (control conditions) had barely detectable Tbeta R-III. ASMC incubated with GM-CSF had a marked increase in Tbeta R-III associated with TGF-beta 1. The presence of Dex had no effect on the increased association of Tbeta R-III with TGF-beta 1. The data are from 5 separate experiments. D: cells used for controls received no treatment. There was induction of pSmad2 and CTGF in the presence of GM-CSF, which was inhibited by the presence of Dex. The data are from 6-8 separate experiments. E: ASMC that received no treatment were used as conditions for controls. ASMC cultured in the presence of GM-CSF and an antibody to Tbeta R-II that interrupts association of TGF-beta 1 with Tbeta R-II had decreased pSmad2 expression compared with ASMC cultured with GM-CSF alone. The data are from 5 separate experiments. *P < 0.05, **P < 0.01 compared with control. black-lozenge P < 0.05 compared with GM-CSF treatment.

Effects of dexamethasone on GM-CSF-induced expression of Tbeta Rs, pSmad2, CTGF, and connective tissue proteins. Corticosteroids are used as standard therapy in the treatment of asthma and have been demonstrated to inhibit collagen I synthesis (18, 31, 36). There were no significant differences in the expression of collagen I, fibronectin, pSmad2, CTGF, Tbeta R-I, Tbeta R-II, and Tbeta R-III between the extracts from ASMC treated with dexamethasone (10-4 M) and with normal saline (Table 1). However, the presence of dexamethasone inhibited collagen I and fibronectin expression induced by GM-CSF (Fig. 4, A and B). Because the induction of connective tissue synthesis by GM-CSF was demonstrated to be on the basis of increase in Tbeta Rs, it was next determined whether the presence of dexamethasone altered Tbeta Rs. The presence of dexamethasone inhibited the increased expression of Tbeta R-I, Tbeta R-II, and Tbeta R-III (Fig. 3B) as well as pSmad2 and CTGF (Fig. 3D). These findings demonstrate that dexamethasone inhibits the expression of GM-CSF-mediated induction of Tbeta Rs and TGF-beta 1-mediated signal transduction, important for collagen synthesis. The presence of dexamethasone did not have an effect on GM-CSF-mediated increase of association of TGF-beta 1 with Tbeta R-III (Fig. 3C, top band). The use of corticosteroids in asthmatics can vary from being used chronically or at various time intervals after the onset of an exacerbation of asthma (9). To simulate these possibilities, in some instances, ASMC were pretreated with dexamethasone by 1.5 h before the addition of GM-CSF. In other instances, dexamethasone was added concomitantly or at a number of intervals after the addition of GM-CSF (Fig. 4, A and B). The GM-CSF-induced increases in collagen I and fibronectin were inhibited by dexamethasone added at all time intervals (Fig. 4, A and B).

                              
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Table 1.   Effects of dexamethasone on expression of collagen I, fibronectin, pSmad2, CTGF, Tbeta R-I, -II, and -III



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Fig. 4.   Dex regulation of collagen I and fibronectin by GM-CSF stimulated ASMC. With the use of prolonged, serum-deprived confluent cultures of ASMC, GM-CSF induced increases in expression of collagen I (A) and fibronectin (B). Dex was added 1.5 h before GM-CSF (-1.5; lane 3), concomitantly with GM-CSF (0; lane 4), or 2 (lane 5), 4 (lane 6), and 6 (lane 7) h after addition of GM-CSF. Each culture condition was maintained for 24 h before collection of protein for Western analysis. The data presented are from 9 (A) and 4 (B) different experiments. *P < 0.05 compared with control. black-lozenge P < 0.05, black-lozenge black-lozenge P < 0.01 compared with GM-CSF treatment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although a vast spectrum of cytokines is induced and released in asthma, some cytokines have been reported to be fibrogenic, such as TGF-beta and GM-CSF (3, 8, 17, 25, 27, 30, 31, 36). However, this is the first observation to demonstrate that GM-CSF increased induction of TGF-beta receptors Tbeta R-I, Tbeta R-II, and Tbeta R-III, as well as downstream signal transduction, as evidenced by increases in pSmad2 and CTGF. The connective tissue effects of GM-CSF mediated by TGF-beta 1 occurred without a detectable release of biologically active TGF-beta 1. These findings are highly significant to our understanding of the biology of TGF-beta 1. Because TGF-beta 1 is secreted as a biologically latent protein, the most important mechanism in the regulation of the effects of TGF-beta 1 is the conversion of latent TGF-beta to its biologically active form (19, 37). However, the current findings demonstrate that an increase in the receptors to TGF-beta can be equally important in regulating the effects of TGF-beta 1 in the pathogenesis of fibrosis (22, 23). In the context of airway remodeling, previous observations did not report differences in expression of TGF-beta 1 in airways of asthmatics and normal controls (2, 11, 32). However, the findings of others do not take into account that biologically active TGF-beta 1 may be released during ASMC injury (12) or that there may be induction of Tbeta Rs by cytokines present in asthmatic airways (11, 32). A cytokine reported to be released by BEC during an episode of asthma is GM-CSF (26). On the basis of current findings, the release of GM-CSF by bronchial epithelium, which is in direct contact with ASMC, could then induce expression of Tbeta R-I, Tbeta R-II, and Tbeta R-III. Of equal importance is the observation that GM-CSF increased the association of TGF-beta 1 with Tbeta R-III. When TGF-beta 1 is associated with Tbeta R-III, the Tbeta R-III presents TGF-beta 1 to the signal-transducing complex composed of Tbeta R-I and Tbeta R-II (5). It has been demonstrated that binding of TGF-beta to Tbeta R-III enhances Smad2 phosphorylation, an indication of TGF-beta -mediated signaling (14). Collectively, the findings demonstrate that enhanced association of TGF-beta 1 to ASMC leads to induction of connective tissue synthesis. This then suggests that in patients with asthma, the presence of GM-CSF can induce fibrotic effects via a TGF-beta 1 pathway by increasing the expression of Tbeta Rs, despite the lack of apparent increase in TGF-beta 1 expression (2). Alternatively, it is possible in the current model that GM-CSF induced the release of active TGF-beta 1, but the quantity was so low that it was not detectable by the assay used. However, since there is an increase in Tbeta R-I, Tbeta R-II, and Tbeta R-III on ASMC, these cells are likely to be more responsive to the effects of TGF-beta 1 in the CM even if the increase in the quantity is not detectable.

The use of corticosteroids in many asthmatics has been demonstrated to relieve acute airway obstruction, and, when used chronically, corticosteroids inhibit irreversible airway obstruction attributed to increased connective tissue deposition (30, 32). The findings in the current study demonstrate that when dexamethasone is present concomitantly with GM-CSF, there is a reduction of Tbeta R-I, Tbeta R-II, Tbeta R-III, collagen I, and fibronectin expression. Dexamethasone did not affect the increase in the association of TGF-beta 1 with Tbeta R-III induced by GM-CSF. However, in the same culture conditions, pSmad2 and CTGF, both indicators of TGF-beta -mediated effects, were decreased. The later findings strongly suggest that the effect of corticosteroids on connective tissue synthesis is the result of inhibiting GM-CSF regulation of Tbeta R-I and Tbeta R-II, despite a lack of effect on the association of TGF-beta 1 with Tbeta R-III.

The current findings demonstrate a unique interaction between two totally different cytokines, GM-CSF and TGF-beta 1, commonly found at sites of injury and fibrosis (8, 13, 19, 20, 21, 23, 25, 29, 31, 33, 36, 37). For example, in models of pulmonary fibrosis induced by the antineoplastic antibiotic bleomycin, there is increase of both GM-CSF (1, 36) and TGF-beta 1 (20, 37). Furthermore, after intratracheal administration of an adenovirus carrying the gene for GM-CSF to rats, there was an increase in TGF-beta 1 in the bronchoalveolar lavage fluid (12). In those instances where both GM-CSF and TGF-beta 1 are increased, the induction of Tbeta Rs by GM-CSF could lead to a synergistic effect of GM-CSF and TGF-beta 1 on cells for connective tissue synthesis.

In conclusion, we have demonstrated that GM-CSF, a prevalent cytokine released by BEC in asthma, stimulates confluent, prolonged, serum-deprived culture of ASMC to synthesize connective tissue proteins by induction of Tbeta Rs type I, II, and III and association of TGF-beta 1 with Tbeta R-III. This effect of GM-CSF on ASMC is reversible by corticosteroids, confirming the importance of using corticosteroids in asthma to prevent irreversible airway obstruction.


    ACKNOWLEDGEMENTS

We thank Valerie Romanchuk for preparation of the manuscript.


    FOOTNOTES

The work presented in this paper was supported by a grant from the Canadian Institute of Health Research-Pharmaceutical Medical Association of Canada, GlaxoSmithKline.

Address for reprint requests and other correspondence: N. Khalil, Division of Respiratory Medicine, Dept. of Medicine, Univ. of British Columbia, Jack Bell Research Centre, Rm. 350, 2660 Oak St., Vancouver, BC V6H 3Z6, Canada (E-mail: nkhalil{at}interchange.ubc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 6, 2002;10.1152/ajplung.00091.2002

Received 26 March 2002; accepted in final form 26 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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