AJP-Lung Articles in PresS. Published on June 5, 2002 as DOI 10.1152/ajplung.00061.2002
FINAL ACCEPTED VERSION LCMP-00061-2002.R1
Proteoglycan expression in bleomycin lung fibroblasts: Role of transforming growth factor-β1 and interferon-γ
Narayanan Venkatesan, PH.D1, Peter J. Roughley, PH.D2, Mara S. Ludwig, M.D.1
1
Meakins-Christie Laboratories, Royal Victoria Hospital and 2Genetics Unit, Shriners Hospital for Crippled Children, McGill University, Montreal, Quebec, Canada
Running title: TGF-β increases proteoglycan expression in fibrotic lung fibroblasts
Address for correspondence: Dr. Mara S. Ludwig Meakins-Christie Laboratories McGill University 3626 Ste. Urbain Street Montreal, QC Canada H2X 2P2 Tel: (514)-398-3864 Fax: (514)-398-7483 E-mail:
[email protected]
Copyright 2002 by the American Physiological Society.
1 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 ABSTRACT Bleomycin (BM)-induced pulmonary fibrosis involves excess production of proteoglycans (PGs). Because transforming growth factor-β1 (TGF-β1) promotes fibrosis, and interferon-γ (IFN-γ) inhibits it, we hypothesized that TGF-β1 treatment would upregulate PG production in fibrotic lung fibroblasts, and IFN-γ would abrogate this effect. Primary lung fibroblasts cultures were established from rats 14 days after intratracheal instillation of saline (CON) or BM (1.5 U). PGs were extracted and subject to Western blot analysis. Bleomycin-exposed lung fibroblasts (BLF) exhibited increased production of versican (VS), heparan sulfate proteoglycan (HSPG) and biglycan (BG) as compared to normal lung fibroblasts (NLF). Compared to NLF, BLF released significantly increased amounts of TGF-β1. TGF-β1 (5 ng/ml x 48 hr) upregulated PG expression in both BLF and NLF. Incubation of BLF with anti-TGF-β antibody (1, 5, 10 µg/ml) inhibited PG expression in a dose-dependent manner. Treatment of BLF with IFN-γ (500 U/ml/48 h) reduced VS, HSPG and BG expression. Furthermore, IFN-γ inhibited TGF-β1induced increases in PG expression by these fibroblasts. Activation of fibroblasts by TGF-β1 promotes abnormal deposition of PGs in fibrotic lungs; downregulation of TGF-β1 by IFN-γ may have potential therapeutic benefits in this disease. KEYWORDS: lung fibrosis; versican; heparan sulfate proteoglycan; biglycan
2 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 INTRODUCTION The lung extracellular matrix (ECM) consists of collagen and elastic fibers interspersed with structural glycoproteins and proteoglycans (PGs) (8). Proteoglycans, a heterogeneous group of macromolecules, consist of a core protein to which glycosaminoglycan (GAG) chains are covalently attached. PGs influence lung tissue mechanical properties, and through their interactions with various macromolecules, contribute to a variety of biological functions such as water balance, cell adhesion, cell migration and growth factor binding within the ECM (1, 20, 33). Versican (VS), the large aggregating chondroitin sulfate containing PG, forms macromolecular aggregates with hyaluronic acid in the lung interstitial matrix. In addition, perlecan, a heparan sulfate PG, and several small leucine-rich repeat PGs, i.e., biglycan, decorin, fibromodulin and lumican, have been identified in the lung tissue (11, 33, 43). Changes in ECM synthesis and degradation play a part not only in physiological processes such as development, growth and aging but also in wound healing, inflammation and fibrosis (33, 37). Bleomycin (BM)-induced pulmonary fibrosis, a well established animal model for the study of human pulmonary fibrosis, is an inflammatory interstitial lung disease characterized by excessive accumulation of fibroblasts and ECM molecules, including PGs, in the intraluminal and interstitial compartments of the lung (8, 40, 43). Evidence from human studies and animal models indicates that TGF-β1 plays a pivotal role in mediating pathophysiological changes in fibrotic diseases (37). TGF-β1 stimulates fibroblasts to synthesize large amounts of ECM proteins. TGF-β1 levels are upregulated in patients with cryptogenic fibrosing alveolitis and also in BM-induced lung fibrosis in rats (23). Fibroblasts participate in inflammatory responses and wound repair through their ability to release cytokines, secrete ECM proteins and through cell-cell interactions with other
3 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 inflammatory cells such as macrophages. At sites of injury and wound repair, fibroblasts have been shown to migrate from different anatomic sites and transform into a less proliferative but more contractile and collagen synthetic phenotype (14, 32). It has been reported that lung fibroblasts cultured from BM-induced fibrotic rat lungs produced more collagen than NLF in culture (30). Furthermore, Raghu et al., demonstrated that TGF-β1 stimulated collagen production and collagen mRNA levels in fibroblasts derived from normal and fibrotic human lungs (31). Whereas the effect of TGF-β1 on collagen expression in NLF and BLF has been relatively well defined, the pattern of synthesis of PGs by NLF and BLF and the effects of TGFβ1 on PG production have not been investigated. This is of particular interest because PGs are one of the first ECM components to be upregulated in the fibrotic process and may be critical in establishing the provisional matrix necessary for subsequent cell migration and proliferation (33). Moreover, an increase in TGF-β1, both at the messenger and protein levels, occurs well before any detectable increase in the expression of collagens in BM-induced pulmonary fibrosis (19). We have recently reported marked increases in both the large and small PGs in BMinduced pulmonary fibrosis in intact rats (12, 40). Based on these observations, we hypothesized that the upregulation of PGs in fibrotic lungs may be due to an increase in the biosynthetic activity of resident fibroblasts. Moreover, we postulated a role for TGF-β1 in this process, as has been shown in other organ systems (4, 22). The observation by Giri et al that anti-TGF-β antibodies and TGF-β-soluble receptors were effective in inhibiting bleomycin-induced lung fibrosis (16, 41) highlights TGF-β1 as a potential candidate for therapeutic intervention. Subsequent studies found decorin, a small PG and a natural inhibitor of TGF-β1, to ameliorate various fibrotic disorders including bleomycin-
4 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 induced lung fibrosis (15). Interferon- γ (IFN-γ), a major effector cytokine produced by activated T-lymphocytes and natural killer cells, has immunomodulatory, as well as antiviral and antiproliferative properties (3). It modulates the metabolism of connective tissue cells, inhibiting fibroblast proliferation, and the production of collagenous and non-collagenous ECM proteins (9). Gurujeyalakshmi and Giri (17) reported that IFN-γ downregulates TGF-β1 and collagen gene expression in the bleomycin-model of lung fibrosis. IFN-γ has also been shown to inhibit the growth of fibroblast cultures and collagen synthesis derived from normal and fibrotic human lungs (27). However, the role of IFN-γ in providing protection against increases in PG production by bleomycin lung fibroblasts remains poorly defined. Therefore, we compared PG production by NLF and BLF. We also investigated the modulating effect of TGF-β1 on PG production, and determined whether endogenous TGF-βinduced increases in PG production would be abrogated by the addition of neutralizing antiTGF-β antibodies. Finally, the anti-fibrotic effect of IFN-γ on TGF-β1-induced increases in proteoglycan expression was studied.
MATERIALS AND METHODS Chemicals Reagents were obtained from the following sources: bleomycin (Blenoxane) from Bristol-Myers Squibb, Princeton, NJ, U.S.A.; agarose from Park Scientific Limited, Northampton, U.K. acrylamide, 6-aminohexanoic acid, benzamidine hydrochloride, chondroitinase ABC, EDTA, Nethylmaleimide (NEM), guanidine hydrochloride (GuHCl), N,N’-methylene-bisacrylamide, N,N,N’N’-tetramethyethylenediamine, L-glutamine, phenylmethylsulfonyl fluoride (PMSF), sodium acetate, Trizma base, Tween 20, and monoclonal antibodies anti-vimentin and anti-alpha
5 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 smooth muscle actin from Sigma Chemicals Co., Oakville, ON, Canada; monoclonal antibodies 12C5 and C17 from Developmental Studies Hybridoma Bank, Iowa City, IA, U.S.A.; hyper film, hybond ECL nitrocellulose membrane, molecular weight standards, ECL Western blotting detection reagents, and streptavidin-biotinylated HRP complex from Amersham Pharmacia Biotech Inc., QC, Canada; collagenase type I, DNase, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), fungizone, interferon-gamma, penicillin-streptomycin, phosphate-buffered saline (PBS), recombinant human transforming growth factor-β1 (TGF-β1), and trypsin from GIBCO, Burlington, ON, Canada; pan-specific TGF-β antibody and TGF-β1 elisa kit from R&D systems Inc., MN, U.S.A.
Experimental design Pulmonary fibrosis was induced in male Sprague-Dawley rats (weight: 275 to 350 g) by a single intratracheal instillation of 1.5 U of BM in 0.3 ml of saline. Control rats received an equal volume of saline. Control and BM rats were killed at 14 days by exanguination under phenobarbitol anesthesia according to standard ethical procedures. Using sterile techniques the lungs were perfused until they were pale with sterile phosphate-buffered saline (PBS) via the right ventricle. The lungs were removed and trimmed of extraneous tissues, bronchial and vascular structures.
Primary rat lung fibroblast culture Cultures of rat lung fibroblasts were established by enzymatic dissociation of finely minced lung tissues according to the standard protocol of Phan et al, (30) with slight modifications. Tissue fragments (after mincing into 2-4 mm pieces) were digested in DMEM containing trypsin (2.5
6 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 mg/ml), collagenase (1 mg/ml), and DNase I (2 mg/ml) and incubated with gentle stirring at 37oC in 5% CO2:95% O2 in humidified air for 30 min. Digestion was carried out for three cycles of 30 min each, with removal of medium and free cells after each cycle, and addition of fresh medium for each successive cycle. Medium removed at the completion of each cycle was mixed with an equal volume of DMEM. Pooled liberated cells, collected and separated from undigested tissue and debris, were filtered through sterile gauze and centrifuged to pellet cells. The pelleted cells were washed twice with PBS and DMEM containing 10% fetal bovine serum (FBS), and finally suspended in this media. The cells were then incubated in a 5% CO2 incubator at 37oC. After 24-48 h, unattached cells were removed by washing and fresh medium added. After another 48 h, cells were confluent and harvested by trypsinization with 0.25% trypsin-EDTA for 3-5 min at 37oC, and then counted and split 1:3. Fibroblasts were maintained in DMEM + 10% FBS, containing
2 mM L-glutamine, 0.37 g of sodium bicarbonate/100 ml, 200 Units of
penicillin/ml, 200 µg/ml of streptomycin sulfate and 2.5 µg/ml of fungizone. Cells were passaged every 3-5 days and by the fourth passage they were homogeneous monolayers (morphologically consistent with fibroblast-like cells, phase contrast light microscopy). Experiments were carried out with fibroblasts between 4th and 6th passages.
Phenotypic characterization of lung fibroblasts Immunofluorescent staining was used to characterize the phenotype of isolated lung fibroblasts. Lung fibroblasts were grown to subconfluence on glass coverslips, and fixed with ice-cold acetone for 20 min at –20oC. The cells were then treated with 70% ethanol for 5 min at –20oC. The cells were washed in sterile PBS and incubated with mouse monoclonal antibodies for vimentin (a fibroblast marker) and α-smooth muscle actin (α-SMA) for 1 h at room temperature.
7 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 The cells were again washed in PBS and reincubated with fluorescent goat anti-mouse IgG (Molecular Probes, Eugene, OR, U.S.A.) for 45 min at room temperature. After washing in PBS, the coverslips were sealed with crystal mount and the fluorescence pattern was examined by confocal microscopy. Negative controls were processed similarly, except incubation with the primary antibody was omitted.
Determination of cell proliferation and cell viability Fibroblast proliferation was assayed by direct cell counting. Fibroblasts were seeded at a density of 1 x 105 cells in 25-cm2 culture flasks in DMEM + 0.1% FBS and the rates of growth were measured with or without TGF-β1 at 24, 48 and 72 h. Cells were trypsinized and placed in a hemocytometer for actual cell counting using a microscope. Experiments were performed in triplicate with cells obtained from saline and BM-exposed rat lungs. Cell viability was determined by trypan blue exclusion test.
Characterization of PGs synthesized by fibroblasts in culture Fibroblasts were grown to confluence, and then serum deprived (0.1% FBS) for 24 h before stimulation with TGF-β1 (5 ng/ml), anti-TGF-β antibody (1, 5, 10 µg/ml), IFN-γ (5, 50, 500 U/ml) or TGF-β1+IFN-γ (5, 50, 500 U/ml) for 48 hr. The amount of TGF-β1 used in the present study was based on a previous study on collagen production using fibroblasts derived from fibrotic human lung (31). At the end of the experimental period, PGs were fractionated into cell layer and medium compartments. The cell layer was rinsed three times with PBS and extracted with ice cold 4 M GuHCl/50 mM sodium acetate, pH 5.8/1% Triton X-100 containing proteinase inhibitor cocktail: 100 mM 6-aminohexanoic acid/10mM EDTA/5 mM benzamidine
8 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 hydrochloride/10 mM NEM/0.1 mM PMSF at 4oC overnight. The PG extracts were then centrifuged at 15,000 rpm for 30 min, and the supernatants were dialyzed exhaustively against 50 mM Tris-HCl, pH 8.0 containing proteinase inhibitors and distilled water, concentrated and then protein content estimated. Cell culture media were dialyzed as described above and then protein content measured (BioRad protein assay).
Composite agarose-PAGE and Western blotting of versican and HSPG Electrophoretic separation of large PGs was performed in a composite gel (0.6% agarose:1.2% polyacrylamide) as described previously (40). Samples were run into the gel at 60 V and then separated at 160 V until the bromophenol blue marker migrated to 3 cm. Separated PGs were electrophoretically transferred and probed with antibodies to VS or large basement membrane heparan sulfate proteoglycans (HSPGs). After electrophoresis, separated PGs were transferred to nitrocellulose membranes, using a BioRad (Mississauga, ON, Canada) blotter apparatus (20 V overnight at 4°C). After blocking, membranes were probed with monoclonal antibodies 12C5 (1:2,000) or C17 (1:1,000), to detect VS or large basement membrane HSPGs , respectively, in Tris-buffered saline with Tween (TBST) (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. After washing with TBST, membranes were incubated with a biotinylated rabbit antimouse secondary antibody (1:2,500) for 1 h at RT, washed again with TBST, and then incubated in streptavidin-biotinylated HRP complex (1:3,000) for 45 min at RT. After washing of membranes, antibody binding was visualized through enhanced chemiluminescence (ECL) detection.
9 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 SDS-PAGE and Western blotting of biglycan Specific polyclonal antipeptide IgG that has been previously shown to interact with biglycan in human cartilage extracts (34) was used to identify the expression of biglycan protein expressed by lung fibroblasts. Proteoglycan extracts were digested with chondroitinase ABC (0.1 U/ml at 37°C for 4 h), run in a 10% SDS-PAGE gel, and then analyzed by immunoblotting. After electrophoresis, the separated proteins were electrophoretically transferred to nitrocellulose membranes and blocked for 1 h at 20oC. After blocking, membranes were washed with TBST and then incubated with primary antibodies for 1 h at room temperature to detect biglycan (1:500 dilution) core protein. After washing with TBST, membranes were incubated with a 1:1,000 dilution of biotin-labeled swine anti-rabbit secondary antibody for 1 h at 20oC. After further washing with TBST, membranes were incubated in streptavidin-biotinylated HRP complex (1:5,000) for 45 min at RT, washed once again, and then visualized by ECL detection.
Quantification of immunoblots Densitometric analysis of both the large and small PGs was accomplished with an image analyzer software (Fluorchem, Alpha Innotech Corporation, San Leandro, CA, U.S.A.) which measures the sum of all the pixel values after background correction. The mean values of three experiments are presented.
Measurement of TGF-β1 in cell-conditioned medium The concentration of TGF-β1 released by NLF and BLF was determined using a human TGF-β1 enzyme-linked immunosorbent assay kit (R&D Systems) that detects rat TGF-β1 protein. Briefly, NLF and BLF were plated and grown to confluency in complete medium containing
10 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 10% FBS. Then these cells were washed in PBS and incubated in a serum-free medium containing 0.2 mg/ml bovine serum albumin. The medium was changed every 4 hours for a period of 24 h. After 24 h, the conditioned media were separated from cells and centrifuged. The cell-free conditioned media were assayed for TGF-β1 after acidification and neutralization according to the manufacturer’s protocol. All assays were done in duplicate wells. The assay detects only the active form of TGF-β1, and the results, normalized to equal number of cells, were expressed as ng/ml/106 cells.
Statistical analysis All data are presented as the mean ± SD of three observations. Student's unpaired t-test was used to analyze the statistical significance of the differences between the results of NLF and BLF, and that of cells treated with or without TGF-β1. One-way analysis of variance (ANOVA) with post hoc Bonferroni correction was used to determine the significance of dose-response studies with neutralizing anti-TGF-β antibody and IFN-γ. Statistical analyses were performed using GraphPad Prism software (version 3.0).
RESULTS Characterization of lung fibroblasts Immunofluorescent studies on cultured cells revealed that both NLF (Fig. 1A) and BLF (Fig. 1B) were strongly positive for vimentin (a fibroblast marker). There was no systematic difference between NLF and BLF in the staining pattern of vimentin. We also stained the isolated fibroblasts with α-SMA (a smooth muscle marker). The results demonstrated that NLF (Fig. 1C)
11 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 expressed a weak signal for α-SMA, whereas BLF (Fig. 1D) contained more cells positive for α-SMA. Growth rate and cell viability In a previous study, we observed a maximal increase in lung PGs at 14 days post-BM treatment (40): lung fibroblasts were isolated at this time point in the current study. The growth rate of BLF did not differ significantly from NLF (data not shown). Since TGF-β1 is a potent regulator of growth, cell proliferation was also measured in the presence of exogenously added TGF-β1. The results revealed no change in proliferation rate indicating TGF-β1 did not affect growth rates in either NLF or BLF. We were also interested in determining whether TGF-β1 or anti-TGF-β antibody had any cytotoxic effects on these fibroblasts. To this end, we used the trypan blue exclusion method to assess cell viability. Our results indicated that neither TGF-β1 nor antiTGF-β antibody caused detachment of cells from the culture flask. More than 95% of cells were attached to the culture flask and excluded trypan blue stain.
BLF express increased amounts of PGs at the protein levels in vitro Similar results were obtained for both the cell layer and medium compartments with regard to PG expression, and therefore in the present study, we present the data obtained from the cell layer. Our data indicate that NLF and BLF differ in their ability to produce PGs in vitro. BLF demonstrated a 7.3-fold increase in the in vitro expression of VS (Fig. 2B) in the cell layer in comparison with NLF (Figure 2A). Two immunoreactive bands were detected on composite gels for VS, suggesting that the protein has two isoforms perhaps due to alternative splicing of the
12 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 versican gene. We observed a 3.6-fold increase of HSPG in the cell layer of BLF (Fig. 3B) as compared with NLF (Fig. 3A). Finally, BLF (Fig. 4B) produced larger amounts (3.5-fold increase) of cell-associated BG in comparison with NLF (Fig. 4A). BLF secrete higher amounts of TGF-β1 protein To investigate the hypothesis that enhanced expression of PGs in BLF may be due to increased expression of TGF-β1, we measured the levels of TGF-β1 released in serum-free cellconditioned media of NLF and BLF. Our results demonstrated that there was a significant increase (3.4-fold increase) in TGF-β1 protein secreted by BLF (Fig 5) as compared to NLF. TGF-β1 upregulates PG expression in NLF and BLF in vitro After exposure to TGF-β1 (5 ng/ml) there was a 2-fold increase in VS in the cell layer of BLF (Fig. 1B). BLF exposed to TGF-β1 exhibited a 3.8–fold increase of HSPG in the cell layer (Fig. 3B) and a 3.2-fold increase in the cell-associated BG (Fig. 4B). Similarly, NLF increased the expression of PGs at the protein level in response to TGF-β1 stimulation. A 4.4-fold increase in the expression of VS in the cell layer was observed for NLF treated with TGF-β1 (Fig. 2A). NLF treated with TGF-β1 demonstrated a 5.5-fold increase for cell-associated HSPG (Fig. 3A). Similarly, a 3.6-fold increase in the expression of BG in the cell layer was noted for NLF treated with TGF-β (Fig. 4A).
Neutralizing antibody to TGF-β decreases PG expression in fibroblasts There was a dose-dependent decrease in PG expression in response to neutralizing antibody. Whereas the lowest concentration (1 µg/ml) of anti-TGF-β antibody had no inhibitory effect on PG expression, incubation with 5 and 10 µg/ml significantly modulated PG production. As
13 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 shown in Figure 1B, anti-TGF-β antibody at 5 and 10 µg/ml inhibited 48.9% and 86.7% of VS expression, respectively, in the cell layer, obtained for BLF. Anti-TGF-β antibody treatment resulted in a 57.4% and 80.8% inhibition of HSPG expression in the cell layer (Fig. 3B) at 5 and 10 µg/ml, respectively, in BLF. Anti-TGF-β antibody treatment led to a 40.9% and 62.6% inhibition of BG in the cell layer at 5 and 10 µg/ml (Fig. 4B), respectively, for BLF. We also investigated whether NLF responds to the antifibrotic effects of anti-TGF-β antibody. Neutralizing antibody significantly reduced the basal levels of PG expression in NLF. Quantification of Western blots of cell-associated PGs showed that anti-TGF-β antibody at 10 µg/ml inhibited 53.6% of VS expression in NLF (Fig. 2A). Similarly, NLF treated with 10 µg/ml demonstrated a significant decrease in HSPG production (54.5% inhibition for cell layer PGs; Fig. 3A). Treatment of NLF with 10 µg/ml of anti-TGF-β antibody resulted in a 54% inhibition in the expression of cell-associated BG (Fig. 4A). These results clearly indicate that inhibition of endogenous TGF-β activity results in downregulation of PGs in both NLF and BLF.
IFN-γ reduces PG expression in fibroblasts To determine whether IFN-γ can suppress PG protein expression in BLF, we used three different concentrations of IFN-γ (5, 50 or 500 U/ml). We found that the two lower concentrations (5 and 50 U/ml) had no significant effect in reducing PG production, whereas IFN-γ at 500 U/ml markedly decreased PG expression. A 64%, 63% and 62% inhibition of VS (Fig. 2B), HSPG (Fig. 3B) and BG (Fig. 4B) expression, respectively, was observed in the cell layer of BLF treated with 500 U/ml of IFN-γ. We were also interested to determine if IFN-γ treatment would block the TGF-β1-induced increases in PG expression by fibroblasts. Whereas the two lower
14 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 concentrations (5 or 50 U/ml) of IFN-γ resulted in no inhibitory effect on PG expression, IFN-γ treatment at a dose of 500 U/ml resulted in a 74%, 61%, and 76% inhibition of VS (Fig. 2A), HSPG (Fig. 3A), and BG (Fig. 4A), respectively, in NLF treated with TGF-β1. Similarly, IFN-γ treatment resulted in a 75%, 78%, and 67%, inhibition of VS (Fig. 2B), HSPG (Fig. 3B), and BG (Fig. 4B), expression, respectively, in BLF treated with TGF-β1.
DISCUSSION The most significant findings of the present study are (i) BLF produced larger amounts of all classes of PGs than NLF, (ii) BLF secreted increased amounts of TGF-β1 protein than NLF, (iii) exposure of fibroblasts to exogenous TGF-β1 stimulated these cells to produce more PGs than untreated cells (iv) anti-TGF-β antibody inhibited the levels of PGs expressed by these fibroblasts in a dose-dependent fashion, and (v) IFN-γ attenuated the TGF-β1-induced increases in PG expression. In the present study, we have analyzed PG expression at the protein level. Proteoglycan expression at the mRNA level would have provided additional information, however, the presence of mRNA does not necessarily predict protein production. A previous study on cytokine regulation of PG expression in lung fibroblasts has reported that changes in mRNA levels for the various PGs were not consistent with changes in protein production (38).
We questioned whether bleomycin induced phenotypic changes in lung fibroblasts. Previous studies have demonstrated the emergence of myofibroblasts as the predominant cell type involved in the increased deposition of ECM proteins and enhanced contractility of lung tissue in this disease process (13, 24, 26, 44). Our findings are in agreement with these studies, in so far as the bleomycin fibroblasts showed positive staining for both vimentin and α-SMA and
15 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 hence a phenotype more consistent with that of the myofibroblast. Whether this specific cell type produces relatively increased amounts of PGs upon cytokine stimulation warrants investigation. The importance of the marked upregulation of PGs by BLF, and the significant response to TGF-β1, are relevant to events in the development of pulmonary fibrosis. Increased VS expression may be of importance in providing the provisional matrix for cell migration and cell proliferation (2, 33). This provisional matrix may be required for subsequent collagen deposition (2). In this regard, VS is one of the early response ECM components to be upregulated during the development of BM-induced pulmonary fibrosis (2). Given the observation that HSPG binds to growth factors, and regulates cell growth and cell-matrix interactions (21), its upregulation by BLF could influence many cellular functions pertinent to fibrosis. Since the core protein of BG has been shown to bind to collagens and fibronectin, it may modulate cell adhesion, cell migration and collagen fiber assembly during fibrogenic processes (35). Further, in a recent study from this laboratory, biglycan expression was correlated with biomechanical changes in lung tissue behavior (12). Hence these molecules may also be important in determining the mechanical changes observed in this disease process.
During the development of pulmonary fibrosis, fibroblasts are exposed to a number of inflammatory mediators, including eicosanoids, immune complexes, serum components, inflammatory growth and differentiation factors and cytokines, many of which have the potential to influence fibroblast function (8). In addition, resident fibroblasts participate in the inflammatory response with other inflammatory cells such as macrophages, resulting in fibroblast chemotaxis to sites of injury and excessive ECM synthesis and deposition (14). As a
16 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 result, fibroblasts attain a different metabolic phenotype with increased biosynthetic activity as evidenced by increased ECM gene expression (32). In our study, PG production and secretion were significantly increased in BLF compared to NLF. Our results are consistent with the studies of Phan et al., (30) who reported that the BLF secreted increased amounts of collagen. Bleomycin has been reported to increase the synthesis of acidic glycosaminoglycans in cultured fibroblasts derived from carrageenin granuloma (29). Our data are also in agreement with findings of increased fibroblast PG production in other types of fibrotic disorders: human granulation tissue fibroblasts have been shown to produce increased amounts of PGs compared to human gingival fibroblasts (18). Enhanced ECM synthesis and deposition could be attributed to an increase in fibroblast proliferation and/or synthetic capacity. However, there was no significant difference in growth rates, as measured by in vitro cell proliferation, between NLF and BLF. Similar results were reported by Phan et al., (30) who also showed no change in growth rates of NLF and BLF. Our results are also supported by the studies of Wegrowski et al., (42) who reported that fibroblasts derived from patients with primary hypertrophic osteopathy synthesized more PG with no change in cell proliferation. Thus, the observed increases in PG production by BLF can be explained by either increased synthesis or decreased degradation. Our findings indicate an enhancement in PG biosynthetic activity by fibroblasts isolated from BM-exposed rat lungs compared to saline-exposed rat lungs. Fibroblasts may be “primed” by BM to increase PG production. This corroborates our previous in vivo findings of marked upregulation of these PGs in the lung tissue of BM-induced fibrosis in rats (12, 40). Enhanced secretion of PGs by fibroblasts may represent a mechanism to explain increased ECM deposition in this model.
17 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 TGF-β1 is believed to be a critical mediator involved in the fibrotic response through its ability to regulate ECM production (37). Normally, TGF-β1 is expressed in bronchiolar epithelial cells and interstitial fibroblasts. However, following tissue injury and inflammation. TGF-β1 is highly expressed in macrophages and mesenchymal, endothelial and mesothelial cells of the lung (23). TGF-β1 is increased in biopsies from fibrotic lungs (23). Elevations in TGF-β1 mRNA and protein content precede the increased expression of collagens in BM-induced pulmonary fibrosis (19). BM has also been shown to modulate the expression of TGF-β1 in rat lung fibroblasts (6). These observations indicate that TGF-β1 contributes to the general lung fibrotic process. We questioned whether this cytokine would affect production of PGs by fibroblasts in the bleomycin model. Indeed, in the current study, TGF-β1 increased the expression of PGs in both NLF and BLF. These observations are consistent with those demonstrated in previous in vitro studies using other tissue specific fibroblasts. Dermal fibroblasts exposed to TGF-β1 increased their PG levels: expression of VS, HSPG and BG was upregulated (22). In general the response to TGF-β1 stimulation by both NLF and BLF was similar. Interestingly, it was reported previously that fibrotic human lung fibroblasts and normal lung fibroblasts exhibited a similar level of increase in collagen production in response to TGFβ1 treatment (31). TGF-β1-induced PG production was not coupled to enhanced cell proliferation. The biological activity of TGF-β1 depends on cell type and culture conditions. Hence, in our experimental conditions, the proliferative effect of TGF-β1 may not have been evident. Our findings are in accordance with the studies of Schonherr et al., (36) who reported that TGF-β1 increased the synthesis of a large versican-like chondroitin sulfate PG by arterial smooth muscle
18 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 cells without changing smooth muscle growth rate. The present results suggest that the stimulation of PG production by TGF-β1 can occur independently of effects on cell proliferation
The marked upregulation of PGs by BLF may be due to an increase in TGF-β1 expression by these cells in response to BM-induced lung injury. Accordingly, we compared the amount of TGF-β1 protein released by NLF and BLF. The results indicated that BLF secreted higher amounts of TGF-β1 protein than that of NLF, supporting our hypothesis that increased expression of PGs in BLF is due to an increase in the expression of TGF-β1. Increased expression of TGF-β1 mRNA was observed in BM-induced pulmonary fibrosis in rats (43). Fibroblasts isolated from fibrotic human gingiva have been demonstrated to produce more TGFβ1 in vitro than normal fibroblasts, which in turn can stimulate PG production (39). We investigated the putative autocrine role of TGF-β1 in the over production of PGs in this model using neutralizing antibody to TGF-β isoforms. The present findings revealed that the elevated production of PGs by BLF was significantly inhibited by anti-TGF-β antibody in a dosedependent manner. At the highest dose of anti- TGF-β antibody, PG production by BLF was similar to the basal levels produced by NLF, that is, the enhanced PG synthesis was totally abrogated. Inhibition by neutralizing antibody to TGF-β of PG production to near normal levels has also been demonstrated in rat fibrotic glomeruli (5). In addition, antibody to TGF-β reduced the levels of fibronectin, laminin and chondroitin sulfate proteoglycan in injured rat brain (25). The results of these studies suggest that increased production of TGF-β play a key role in the pathogenesis of fibrogenic diseases, including BM-induced lung fibrosis. Further, the significant
19 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 reduction in PG production by NLF with anti-TGF-β antibody treatment suggests that the constitutive production of PGs in these fibroblasts is also under autocrine control by TGF-β.
Interferon-gamma (IFN-γ), a lymphokine produced by activated T-lymphocytes, macrophages and natural killer cells, has been shown to modulate the metabolism of connective tissue cells. Numerous studies have demonstrated that IFN-γ has a regulatory role on collagen accumulation by inhibiting the synthesis of types I and III collagen, and abrogating the stimulatory effect of TGF-β1 (17). Additionally, IFN-γ has been shown to inhibit the growth of fibroblasts and collagen synthesis from normal and fibrotic human lungs (27). Likewise, inhibitory effects of IFN-γ on aggrecan and decorin core protein gene expression in cultured human chondrocytes have been reported (10). In addition, IFN-γ was reported to inhibit experimental renal fibrosis (28). We also examined whether IFN-γ would interefere with the increased PG expression by fibrotic lung fibroblasts and fibroblasts treated with TGF-β1 as well. Our results indicate that IFN-γ treatment led to a marked decrease in the production of PGs by BLF. Because of the importance of TGF-β1 in the regulation of ECM production, the ability of IFN-γ to decrease the production of PGs by fibroblasts exposed to TGF-β1 may have beneficial effects in controlling the excessive PG accumulation in the fibrotic lung. It is possible that IFN-γ could block the abnormal deposition of PGs in the fibrotic lung by altering TGF-β1 gene expression and/or TGF-β signaling. Consistent with this hypothesis, it was reported that in the bleomycin-induced model of pulmonary fibrosis, exogenous IFN-γ downregulates the transcription of the gene for TGF-β1 (17), which in turn could modulate the turnover of ECM proteins, including PGs. In contrast, Chen et al, (7) reported that IFN-γ -/- mice exhibited
20 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 reduced inflammation and fibrosis when treated with bleomycin, suggesting that IFN-γ may cause inflammation early in the development of bleomycin fibrosis, but its continuous presence either endogenously or administered exogenously may have a net antifibrotic effect. An exciting study by Ziesche et al, (45) demonstrated the potency of IFN-γ for the treatment of lung fibrosis in humans, revealing an increase in total lung capacity and improved arterial oxygenation that ran parallel with decreases in mRNA levels of TGF-β and connective tissue growth factor.
In conclusion, our study has demonstrated that rat lung fibroblasts in culture are capable of synthesizing various PGs that are normally expressed in lung tissue. Following BM-induced lung injury, these fibroblasts respond with an increased expression of PG; TGF-β1 further activates these cells. Since anti-TGF-β antibody reduced the levels of PGs expressed by these fibroblasts to near normal levels, our observations indicate an autocrine role for TGF-β1. Inhibition of endogenous TGF-β activity may therefore be of importance in controlling the excessive deposition of ECM components in pulmonary fibrosis. We have also demonstrated that IFN-γ suppresses the excessive production of PGs by fibrotic lung fibroblasts, and fibroblasts exposed to TGF-β1. This observation supports a potential therapeutic role for the IFN-γ via modulation of the abnormal deposition of PGs observed during pulmonary fibrosis.
ACKNOWLEDGMENTS The monoclonal antibodies 12C5, developed by Dr. R. Asher, and C17 developed by Dr. J. R. Sanes, were obtained from the Developmental Studies Hybridoma Bank of the University of Iowa Department of Biological Sciences, Iowa City, IA. We wish to thank Drs. Barbara Tolloczko and Vasanthi Govindaraju for their help in immunofluorescence studies.
21 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 Dr. Venkatesan was a recipient of a fellowship from Canadian Lung Association/ and the Canadian Institutes of Health Research. Supported by the J.T. Costello Memorial Research Fund and the Canadian Institutes of Health Research.
22 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 REFERENCES
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24 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 18.Hakkinen L, Westermarck J, Kahari VM and Larjava H. Human granulation-tissue fibroblasts show enhanced proteoglycan gene expression and altered response to TGF-β1. J Dent Res 75: 1767-1778, 1996. 19. Hoyt DG and Lazo JS. Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice. J Pharmacol Exp Ther 246: 765-771, 1988. 20. Iozzo RV. Matrix proteoglycans: From molecular design to cellular function. Ann Rev Biochem 67: 609-652, 1998. 21. Iozzo RV and Murdoch AD. Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J 10: 598-614, 1996. 22. Kahari VM, Larjava H and Uitto J. Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-β1 upregulates biglycan (PG), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture. J Biol Chem 266: 10608-10615, 1991. 23. Khalil N and Greenberg AH. The role of TGF-β in pulmonary fibrosis. Ciba Found Symp 157: 194-207, 1991. 24. Kuhn C and McDonald JA. The roles of the myofibroblast in idiopathic pulmonary fibrosis. Am J Pathol 138: 1257-1265, 1991. 25. Logan A, Green J, Hunter A, Jackson R and Berry M. Inhibition of glial scarring in the injured rat brain by a recombinant human monoclonal antibody to transforming growth factor-β2. Eur J Neurosci 11: 2367-2374, 1999.
25 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 26. Mitchell J, Woodcock-Mitchell J, Reynolds S, Low R, Leslie K, Adler K, Gabbiani G and Skalli O. α-Smooth muscle actin in parenchymal cells of bleomycin-injured rat lung. Lab Invest 60: 643-650, 1989. 27. Narayanan AS, Whithey J, Souza A and Raghu G. Effects of γ-interferon on collagen synthesis by normal and fibrotic human lung fibroblasts. Chest 101: 1326-1331, 1992. 28. Oldroyd SD, Thomas GL, Gabbiani G and El Nahas AM. Interferon-gamma inhibits experimental renal fibrosis. Kidney Int 56: 2116-2127, 1999. 29. Otsuka K, Murota S and Mori Y. Stimulatory effect of bleomycin on the synthesis of acid glycosaminoglycans in cultured fibroblasts derived from a rat carrageenin granuloma. Biochim Biophys Acta 444: 359-368, 1976. 30. Phan SH, Varani J and Smith D. Rat lung collagen metabolism in bleomycin-induced pulmonary fibrosis. J Clin Invest 76: 241-247, 1985. 31. Raghu G, Masta S, Meyers D and Narayanan AS. Collagen synthesis by normal and fibrotic human lung fibroblasts and the effect of transforming growth factor-β. Am Rev Respir Dis 140: 95-100, 1989. 32. Regan MC, Kiek SJ, Wasserkrug HL and Barbul A. The wound environment as a regulator of fibroblast phenotype. J Surg Res 50: 442-448, 1991. 33. Roberts CR, Wight TN and Hascall VC. Proteoglycans. 1997. In The Lung: Scientific Foundations (2nd ed.). R.G. Crystal, J.B. West, P.J. Barnes, and E.R. Weibel, editors. Lippincott-Raven Publishers, Philadelphia. 757-767. 34. Roughley PJ, White RJ, Magny MC, Liu J, Pearce RH and Mort JS. Non-proteoglycan forms of biglycan increase with age in human articular cartilage. Biochem J 295: 421-426, 1993.
26 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 35. Ruoslahti E and Yamaguchi Y. Proteoglycans as modulators of growth factor activities. Cell 64: 867-869, 1991. 36. Schonherr E, Jarvelainen HT, Sandell LJ and Wight TN. Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem 266: 1764017647, 1991. 37. Sime PJ and O’Reilly KMA. Fibrosis of the lung and other tissues: New concepts in pathogenesis and treatment. Clin Immunol 99:308-319, 2001. 38. Tiedemann K, Malmstrom A and Westergren-Thorsson G. Cytokine regulation of proteoglycan production in fibroblasts: separate and synergistic effects. Matrix Biol 15: 469478, 1996. 39. Tipton DA and Dabbous MKh. Autocrine transforming growth factor β stimulation of extracellular matrix production by fibroblasts from fibrotic human gingiva. J Periodontol 69: 609-619, 1998. 40. Venkatesan N, Ebihara T, Roughley PJ and Ludwig MS. Alterations in large and small proteoglycans in bleomycin-induced pulmonary fibrosis in rats. Am J Respir Crit Care Med 161: 2066-2073, 2000. 41. Wang Q, Wang Y, Hyde DM, Gotwals PJ, Koteliansky VE, Ryan ST and Giri SN. Reduction of bleomycin induced lung fibrosis by transforming growth factor beta soluble receptors in hamsters. Thorax 54: 805-812, 1999. 42. Wegrowski Y, Gillery P, Serpier H, Georges N, Combemale P, Kalis B and Maquart FX. Alteration of matrix macromolecule synthesis by fibroblasts from a patient with pachydermoperiostosis. J Invest Dermatol 106: 70-74, 1996.
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28 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 FIGURE LEGENDS
Figure 1. Immunofluorescence staining of vimentin and α-smooth muscle actin in lung fibroblasts. Isolated lung fibroblasts were cultured on glass coverslips and fixed with acetone before staining for vimentin and α-smooth muscle actin as described under “Materials and Methods”. Both NLF (A) and BLF (B) demonstrated positive staining for vimentin. α-Smooth muscle actin expression was increased in BLF (D) compared to NLF (C).
Figure 2. Immunoblot analysis and quantification of versican expression in the cell layer of NLF (A) and BLF (B). Fibroblasts derived from saline or bleomycin-exposed lungs were exposed to TGF-β1 (5 ng/ml), or neutralizing anti-TGF-β antibody (1, 5, or 10 µg/ml), or IFN-γ (500 U) or TGF-β1+IFN-γ for 48 h and then extracted for proteoglycans. Extracted proteoglycans were run on composite gels and probed with 12C5 antibody to detect versican epitope as described under “Materials and Methods.” Data are mean ± S.D. of three independent observations.
Figure 3. Immunoblot analysis and quantification of heparan sulfate proteoglycan expression in the cell layer of NLF (A) and BLF (B). Fibroblasts isolated from saline or bleomycin-treated rats were treated with or without TGF-β1 (5 ng/ml), or neutralizing anti-TGF-β antibody (1, 5, or 10 µg/ml) or IFN-γ (500 U) or TGF-β1+IFN-γ for 48 h. Proteoglycans isolated from the cell layer was run on a agarose-polyacrylamide gel and then probed with C17 antibody to detect heparan sulfate proteoglycan epitope as described under “Materials and Methods.” Data are mean ± S.D. of three separate experiments.
29 FINAL ACCEPTED VERSION LCMP-00061-2002.R1 Figure 4. Immunoblot analysis and quantification of cell-associated biglycan in NLF (A) and BLF (B) treated with or without TGF-β1 (5 ng/ml), or neutralizing anti-TGF-β antibody (1, 5, or 10 µg/ml) or IFN-γ (500 U) or TGF-β1+IFN-γ for 48 h. Cell layer proteoglycans were separted by SDS-PAGE electrophoresis on 10% gels and probed with a specific polyclonal antipeptide IgG for biglycan core protein as described under “Materials and Methods.” Values are mean ± S.D. of three separate experiments.
Figure 5. Measurement of TGF-β1 release in cell-conditioned media of NLF and BLF. After confluency, NLF and BLF were cultured in serum-free media for a period of 24 h and the conditioned media was analyzed for bioactive TGF-β1 protein as described under “Materials and Methods”. Compared to NLF, BLF secreted significantly increased amounts of TGF-β1. Values are mean ± S.D. of three separate experiments.
FIG. 1
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D
Versican (Density Units x 106) 3
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1
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