Induction of PDGF receptor-alpha in rat myofibroblasts during pulmonary fibrogenesis in vivo

James C. Bonner1, Pamela M. Lindroos1, Annette B. Rice1, Cindy R. Moomaw2, and Daniel L. Morgan3

Laboratories of 1 Pulmonary Pathobiology, 2 Experimental Pathology, and 3 Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. Induction of the PDGF receptor-alpha (PDGF-Ralpha ) in vitro enhances PDGF-induced mitogenesis and chemotaxis. Thus we investigated whether the PDGF-Ralpha is induced in vivo during pulmonary fibrogenesis using a vanadium pentoxide (V2O5) model of lung injury. PDGF-Ralpha mRNA expression was induced 24 h postinstillation. PDGF-Rbeta mRNA was constitutively expressed and did not increase. Western blotting showed upregulation of PDGF-Ralpha protein by 48 h, and immunohistochemical analysis localized PDGF-Ralpha primarily in mesenchymal cells residing within fibrotic lesions. Upregulation of PDGF-Ralpha in vivo preceded mesenchymal cell hyperplasia (3-7 days) and collagen deposition by day 15. Supernatants from alveolar macrophages treated with V2O5 in vitro released upregulatory activity for PDGF-Ralpha on cultured lung myofibroblasts, and this activity was blocked by the interleukin-1-receptor antagonist. These data suggest that interleukin-1beta -mediated induction of PDGF-Ralpha in vivo is important to lung myofibroblast hyperplasia during fibrogenesis.

lung fibrosis; vanadium pentoxide; platelet-derived growth factor-receptor system; interleukin-1; transforming growth factor-beta 1

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE PROLIFERATION of fibroblasts in the lung is a key component of lung fibrosis. The disease may be initiated by exposure to a variety of factors including chemotherapeutic drugs such as bleomycin (27, 30), man-made fibers such as asbestos (4, 5), and metals such as vanadium and cadmium (10, 25). Myofibroblast hyperplasia is an early event in the fibrotic process and generally precedes extracellular matrix deposition (21). The factors that initiate and perpetuate the myofibroblast growth response during fibrosis have not been fully clarified. However, several growth-promoting cytokines are increased within bronchoalveolar lavage fluid during the early stages of pulmonary fibrogenesis, and these agents include platelet-derived growth factor (PDGF), interleukin-1beta (IL-1beta ), tumor necrosis factor-alpha , transforming growth factor-alpha (TGF-alpha ), and insulin-like growth factor-1 (for a review see Ref. 16).

PDGF is a major mitogen and chemoattractant for cells of mesenchymal origin, including fibroblasts, myofibroblasts, and smooth muscle cells (reviewed in Ref. 13). Several studies have emphasized the importance of PDGF in fibroproliferative diseases such as idiopathic pulmonary fibrosis (1), obliterative bronchiolitis (14), and atherosclerosis (26) in humans. Mesenchymal cells produce PDGF-AA, whereas macrophages secrete primarily PDGF-AB and PDGF-BB (6). PDGF isoforms stimulate cell replication and chemotaxis by interacting with cell-surface receptor subtypes termed PDGF receptor-alpha (PDGF-Ralpha ) and PDGF-Rbeta (28). PDGF binding initiates PDGF-receptor dimerization that results in autophosphorylation of tyrosine residues within the intracellular domain of the receptor (13). PDGF-AA binds to PDGF-Ralpha and does not bind to PDGF-Rbeta (28). The expression of PDGF-Ralpha on adult fibroblasts is relatively low compared with the normally abundant PDGF-Rbeta , yet PDGF-Ralpha is highly expressed during development (23). A recent study (7) with PDGF-A-deficient mice demonstrated that PDGF-AA and its receptor (PDGF-Ralpha ) are essential for the development of myofibroblasts in the lung, and surviving offspring develop a fatal emphysema due to reduced elastin deposition. PDGF-Ralpha is necessary for maximal mitogenic and chemotactic responses to PDGF (8, 20, 24, 29), and this is likely due to unique signaling events mediated via the heterodimeric alpha beta -receptor complex compared with the beta beta -receptor complex (29).

Upregulation of the PDGF-receptor system has been proposed as a mechanism of mesenchymal cell hyperplasia in fibroproliferative diseases other than lung fibrosis. Animal models of disease associated with induction of PDGF-Rbeta include liver fibrosis (31) and atherosclerosis (26). In contrast, induction of PDGF-Ralpha has been reported in vascular hypertension in rats (17). In vitro, we have reported that PDGF-Ralpha on rat lung myofibroblasts is upregulated by IL-1beta (20), basic fibroblast growth factor (2), lipopolysaccharide (8), and asbestos fibers (5). Induction of PDGF-Ralpha by asbestos fibers (5) could be due to surface-bound endotoxin (8). TGF-beta 1 downregulates PDGF-Ralpha and suppresses PDGF-mediated responses (3). Alveolar macrophages stimulated with fibrogenic particles in vitro release an upregulatory factor(s) for PDGF-Ralpha on lung myofibroblasts, and this activity is due mainly to IL-1beta (19). Induction of the PDGF-Ralpha subtype in vitro enhances PDGF-mediated mitogenesis and chemotaxis of lung myofibroblasts (8, 20, 24).

The induction of PDGF-Ralpha has not been studied in vivo during the progression of pulmonary fibrogenesis. We investigated possible alterations of the PDGF-receptor system in a rat model of pulmonary fibrosis induced by vanadium pentoxide (V2O5). Occupational exposure to V2O5 is common in petrochemical industries (18), and vanadium-containing emission dusts released from oil-fired, electricity-generating plants contribute to environmental exposure in humans (9). We previously reported that a V2O5-containing emission dust caused PDGF-Ralpha upregulation 24 h postinstillation in rats (19). V2O5 elicited a fibrogenic response as measured by trichrome staining within 15 days after instillation. Upregulation of PDGF-Ralpha in vivo peaked during the early proliferative phase (24-48 h) after lung injury but returned to control levels before collagen deposition within fibrotic lesions. Macrophages treated in culture with V2O5 released a factor(s) that upregulated PDGF-Ralpha on myofibroblasts in vitro, and the majority of this upregulatory activity was blocked by the IL-1-receptor antagonist protein (IRAP). We postulate that induction of PDGF-Ralpha through an IL-1beta -dependent mechanism could be important to mesenchymal cell hyperplasia in vivo during lung fibrogenesis.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Reagents. V2O5 was purchased from Aldrich Chemical (Milwaukee, WI). Human recombinant PDGF-AA, rabbit anti-murine PDGF-Ralpha , and rabbit anti-human PDGF-Rbeta were purchased from Upstate Biotechnology (Lake Placid, NY). 125I-PDGF-AA was purchased from Biomedical Technologies (Stoughton, MA). Human recombinant IL-1beta and human recombinant IRAP were purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS; heat inactivated) and bovine serum albumin (BSA; Cohn fraction V) were purchased from Sigma Chemical (St. Louis, MO). The human PDGF-Rbeta cDNA probe was kindly provided by Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden), and the rat PDGF-Ralpha cDNA probe was the kind gift of Dr. Yutaka Kitami (Ehime University, Ehime, Japan).

Intratracheal instillation. Sprague-Dawley rats (Charles River) weighing ~200 g were instilled intratracheally with 200 µl of sterile saline or 2 mg/kg (0.4 mg/rat) of V2O5 suspended in saline. This protocol was similar to a previously published intratracheal instillation procedure (10). V2O5 suspensions were vortexed thoroughly, then bath sonicated for 30 min at 25°C before instillation. At 24, 48, and 72 h and 6 and 15 days postinstillation, the animals (three saline instilled and five V2O5 instilled per time point) were overdosed with an intraperitoneal injection of Nembutal, and the lungs were removed en bloc. The left lung was instilled with buffered Formalin in phosphate-buffered saline (PBS), pH 7.2; the trachea was tied off; and the lungs were immersed in Formalin overnight. After fixation, the lung tissues were embedded in paraffin. Four-micrometer-thick sections were mounted and stained with hematoxylin and eosin, Masson's trichrome for collagen, and Verhoeff's stain for elastin and PDGF-Ralpha (see Immunohistochemistry). The right lobes of the lung were homogenized with a motor-driven tissue grinder in 10 ml of TRI reagent (Molecular Research Center, Cincinnati, OH). Total RNA was isolated according to the manufacturer's specifications, except that the aqueous phase from the first chloroform separation was extracted an additional time with TRI reagent to inhibit endogenous ribonuclease activity. The protein fraction from the TRI reagent procedure was separated according to the manufacturer's specifications, frozen at -80°C, and processed for Western blot analysis of PDGF receptors as described in Western blot analysis. In the present study, we did not use a nonfibrogenic particle of similar particle diameter to V2O5. However, we previously reported that rats exposed to iron particles (>93% iron and a mean diameter of 3 µm) developed no acute inflammation or fibrosis compared with fibrogenic chrysotile asbestos fibers (4).

Isolation of pulmonary myofibroblasts. Early-passage rat lung myofibroblasts from male Sprague-Dawley rats were isolated and characterized as described previously (8). Cell isolates at passage 1 or 2 were plated onto 3-aminopropyltriethoxysilane-coated glass chamber slides and grown to confluence, then fixed briefly in ice-cold acetone. Fixed cells were then subjected to overnight incubation with a murine monoclonal antibody to the antigen of interest, followed by a biotinylated horse anti-mouse antibody, avidin-biotin immunoperoxidase, and 3,3'-diaminobenzidine chromogen (all from Vector Laboratories, Burlingame, CA). An irrelevant monoclonal antibody (anti-5-bromo-2'-deoxyuridine; Becton-Dickinson, San Jose, CA) at an equivalent immunoglobulin G concentration served as a control for nonspecific immunoreactivity. Concentrations of primary antibodies were set by titration on appropriate rat-derived positive control cells. Cells stained positively for vimentin and alpha -smooth muscle actin and negatively for factor VIII, desmin, and rat leukocyte common antigen (OX 1). In addition, examination of glutaraldehyde-fixed cell pellets by transmission electron microscopy showed ultrastructural features consistent with a myofibroblast phenotype (abundant intermediate filaments and rough endoplasmic reticulum and lack of Weibel-Palade bodies characteristic of endothelial cells). Cells were grown to confluence in 10% FBS-Dulbecco's modified Eagle's medium (DMEM) before being seeded for the assays described in Western blot analysis and 125I-PDGF-AA binding assay.

Isolation of rat alveolar macrophages. Alveolar macrophages from male Sprague-Dawley rats were obtained by bronchoalveolar lavage as previously described (6). Macrophages (30-35 × 106) that were >95% viable (as determined by trypan blue exclusion) were suspended in serum-free DMEM (0.25% BSA) and cultured in 175-cm2 flasks coated with poly(2-hydroxyethyl methacrylate) (Sigma Chemical) that prevents macrophage attachment to the tissue culture surface and activation via adherence. After 1 h of equilibration in culture at 37°C in 5% CO2, the macrophages were treated with 0.01, 0.1, or 1 µg/cm2 (0.3-30 µM) of V2O5. After 24 h, the culture medium was centrifuged at 1,500 revolutions/min to pellet the macrophages, and the supernatant was filtered (0.45 µm) and stored at -20°C. These concentrations of V2O5 did not significantly affect cell viability as determined by trypan blue exclusion.

Northern blot analysis. Total lung RNA isolated with TRI reagent (see Intratracheal instillation) was electrophoresed (20 µg/lane) in 1% agarose-2 M formaldehyde gels and capillary transferred onto Immobilon S membranes (Millipore, Bedford, MA). RNA from the in vitro experiments was hybridized to a 32P-labeled cDNA probe for the PDGF-Ralpha or PDGF-Rbeta subunit. [alpha -32P]dCTP (Amersham, Arlington Heights, IL) was used to label the cDNA with a Prime-It II random-primer labeling kit (Stratagene, La Jolla, CA). The autoradiographic signal was visualized with a phosphorimaging system.

Western blot analysis. Total lung protein collected after in vivo V2O5 instillation (see Intratracheal instillation) was separated from RNA and DNA according to the manufacturer's directions and mixed 1:4 with lysis buffer A [20 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml of pepstatin, leupeptin, and aprotinin]. The mixture was centrifuged at 100,000 g for 30 min (Beckman TLA.3 fixed-angle rotor), and the pellet was resuspended in 0.25 ml of lysis buffer B (lysis buffer A supplemented with 1% Triton X-100) and probe sonicated for 30 s. After a second centrifugation at 100,000 g for 30 min, the supernatant (membrane fraction) was removed and stored at -80°C. In another set of experiments in which the cell lysates were harvested in vitro, rat lung myofibroblasts were grown to confluence in 75-cm2 flasks and rendered quiescent for 24 h in serum-free defined medium (SFDM). The SFDM consisted of Ham's F-12 medium with CaCl2 and 0.25% BSA and was supplemented with an insulin-transferrin-selenium mixture (Boehringer Mannheim). Myofibroblasts were treated with 0.25× macrophage-conditioned medium (MØCM; see Isolation of rat alveolar macrophages) collected from macrophages treated with V2O5. Some cultures received V2O5 alone (0.01-1 µg/cm2) to test the direct effect of this metal on myofibroblast PDGF-Ralpha expression. IL-1beta (2 ng/ml) was used as a positive control for PDGF-Ralpha upregulation (20). Parallel flasks of all agents were set up to test the effect of an IRAP on PDGF-receptor expression. After incubation for 24 h at 37°C, the cells were washed with PBS, and 250 ml of lysis buffer B were added to cover the surface of the attached cells for 20 min. Lysates were stored at -80°C. Twenty microliters of each sample were mixed with sample buffer [0.5 M Tris · HCl, pH 6.8, 10% sodium dodecyl sulfate (SDS), 0.1% bromphenol blue, and 20% glycerol], boiled for 5 min, and sonicated for 30 s before electrophoresis in a 2-15% Tris-glycine SDS-polyacrylamide gel (Integrated Separation Systems, Hyde Park, MA) for 2 h at 130 V and 30 mA. The proteins were transferred to nitrocellulose membrane (Hybond, Amersham, Arlington Heights, IL). The membrane was blocked with 3% milk-PBS for 1 h before overnight incubation at 4°C with a 1:500 dilution of rabbit anti-mouse PDGF-Ralpha or PDGF-Rbeta antibody (UBI, Lake Placid, NY). After being washed three times with PBS-Tween, a 1:2,000 dilution of secondary horseradish peroxidase-conjugated swine anti-rabbit antibody (Dako, Carpinteria, CA) was added for 1.5 h. An enhanced chemiluminescence luminol kit (Amersham, Arlington Heights, IL) was used for detection of bound secondary antibody.


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Fig. 1.   A: temporal expression of platelet-derived growth factor receptor-alpha (PDGF-Ralpha ) mRNA in vivo after instillation of vanadium pentoxide (V2O5; +) into rats. -, Saline instillation. Northern blot analysis of total lung RNA was performed as described in METHODS, and autoradiographic signals were normalized to 18S ethidium bromide rRNA. Each lane represents a single rat. dy, Day. B: quantitive densitometry showing that PDGF-Ralpha gene expression was induced maximally (8- to 10-fold) 24 h after instillation of V2O5 (hatched bars), and mRNA expression returned to saline (open bars) control levels by 72 h postinstillation. Data are representative of 3-5 rats for either saline or V2O5 instillation at each time point (see Fig. 2 for variability among animals within a group).


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Fig. 2.   Relative mRNA expression of PDGF-Ralpha and PDGF-Rbeta in vivo 24 h after instillation of V2O5. A: total lung RNA from 3 rats each from saline (lanes 1-3) and V2O5 (lanes 4-6) groups was analyzed for PDGF-receptor gene expression. B: densitometry confirmed that PDGF-Ralpha mRNA (solid bars) was strongly increased after V2O5 instillation compared with saline control lungs in all rats (see Fig. 1 for time course of expression). In contrast, PDGF-Rbeta (hatched bars) was constitutively expressed, and level of this receptor did not change after lung injury. Densitometry data are means ± SE and were normalized against 18S rRNA.

Immunohistochemistry. Lung tissue was fixed overnight in 10% neutral buffered Formalin. Testes from 6-day-old rats were used as a positive control tissue for PDGF-Ralpha expression because the connective tissue surrounding the seminiferous tubules stains intensely for this receptor (11). Immunohistochemistry was performed with the avidin-biotin-peroxidase method. Tissue sections (6 µm) were dehydrated through a series of graded alcohol solutions to 1× automation buffer (AB) consisting of 5% NaCl and 2% HCl (Biomeda, Foster City, CA). Endogenous peroxidase was blocked in 3% (vol/vol) H2O2 for 15 min. After a 1× AB wash, the slides were immersed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven (750 W) for 5 min. This procedure was repeated two times at 1-min intervals to add fresh citrate buffer. After cooling for 15 min, the slides were rinsed in distilled H2O and incubated in 1× AB for 5 min. Sections were blocked with normal goat serum (Vector Laboratories) for 20 min at room temperature, then incubated for 1 h at room temperature with a rabbit anti-murine PDGF-Ralpha antibody (UBI) diluted 1:100 in 1% BSA. Sections were washed two times with AB and then incubated for 30 min with a 1:400 dilution of biotinylated secondary goat anti-rabbit immunoglobulin G (Vector Laboratories). The slides were washed again and incubated with the Elite Avidin-Biotin Complex (Vector Laboratories) for 30 min. Visualization of the antibody complex was done with a diaminobenzidine tablet (10 mg; Sigma Chemical) dissolved in 20 ml of 1× AB containing 12 µl of 30% H2O2 for 6 min in the dark. The slides were then rinsed in running tap water, counterstained with Harris hematoxylin (Harelco, Gibbstown, NJ), dehydrated through a series of graded alcohols to xylene, and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). Some paraffin-embedded sections were stained for elastin (Verhoeff's stain) or desmin as a smooth muscle cell phenotypic marker or vimentin (Clone LN6, Accurate Antibodies, Westbury, NY) as a marker of fibroblast phenotype. Mature collagen was detected by Masson's trichrome stain.

125I-PDGF-AA binding assay. Rat lung myofibroblasts were seeded in 24-well plates and grown to confluence in 10% FBS-DMEM, then rendered quiescent in SFDM for 24 h. The cells were then treated with fresh SFDM supplemented with 0.25× MØCM (from control or V2O5-stimulated macrophages), V2O5 alone (0.01-1 µg/cm2), or IL-1beta (2 ng/ml) in the absence or presence of 2 µg/ml of IRAP. After 24 h, the cultures were chilled to 4°C, rinsed in ice-cold binding buffer (Ham's F-12 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, CaCl2, and 0.25% BSA), and incubated with 2 ng/ml of 125I-PDGF-AA (specific activity 125 µCi/µg) in the absence or presence of 400 ng/ml of PDGF-AA (nonspecific binding) for 3 h at 4°C on an oscillating platform. Cells were then rinsed three times in ice-cold binding buffer and solubilized (1% Triton X, 0.1% BSA, and 0.1 N NaOH; 1 ml/well), and cell-associated radioactivity was measured in a gamma counter.

    RESULTS
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Abstract
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Methods
Results
Discussion
References

PDGF-Ralpha mRNA expression is upregulated in vivo after instillation with V2O5. Total lung mRNA was analyzed for PDGF-receptor genes at several time points (24, 48, and 72 h and 6 and 15 days) after the intratracheal instillation of V2O5. PDGF-Ralpha expression in the whole lung was low and barely detectable by Northern blot analysis. PDGF-Ralpha mRNA expression was markedly increased 24 h after V2O5 instillation (8- to 10-fold above the saline control lung), but this transient induction returned to control levels of expression by 72 h postinstillation (Fig. 1). No significant change in PDGF-Ralpha mRNA expression was observed in rats that were instilled with saline alone. In contrast to PDGF-Ralpha expression, PDGF-Rbeta was highly expressed constitutively, and instillation of V2O5 did not change the expression of this receptor subtype (Fig. 2).

In vivo expression of PDGF-Ralpha protein is increased after V2O5 instillation and is localized in early fibrotic lesions. Western blotting of total lung protein homogenates was performed with antibodies specific to either PDGF-Ralpha or PDGF-Rbeta . Total lung PDGF-Ralpha protein was strongly upregulated 24 h postinstillation of V2O5 and was maximally expressed at 48 h (Fig. 3). PDGF-Rbeta protein levels were constitutively high, and the levels did not significantly change after V2O5 instillation. Immunohistochemical analysis of the PDGF-Ralpha was performed on paraffin-embedded sections of V2O5-instilled rat lung with the same rabbit anti-murine PDGF-Ralpha antibody that was employed in the Western blotting procedure. In agreement with the Western blot analysis, PDGF-Ralpha protein was detected 24 and 48 h postinstillation of V2O5. PDGF-Ralpha was visualized (brown staining) in inflammatory foci that were characterized by inflammatory cell infiltration (primarily macrophages, neutrophils, and lymphocytes) and thickened foci composed of epithelial and mesenchymal cells (Fig. 4). Weak PDGF-Ralpha staining was observed 6 days postinstillation of V2O5 in proliferative lesions characterized by myofibroblast hyperplasia. As a negative control, the staining procedure was also performed with normal rabbit serum. Testes from 6-day-old rats were used in the immunohistochemical procedure as a positive control because PDGF-Ralpha expression has been shown to be high during development of the mesenchyme surrounding the semiferous tubules of the testes (11). In agreement with this earlier study, we observed strong PDGF-Ralpha staining in the connective tissue surrounding the seminiferous tubules of the testes but no staining within Sertoli cells (Fig. 4).


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Fig. 3.   Western blot analysis of PDGF-Ralpha in vivo after instillation of V2O5. A: temporal upregulation of PDGF-Ralpha protein was observed, with no change in PDGF-Rbeta . Membrane fractions of whole lung protein (20 µg/lane) were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and blotted with antibodies specific to either PDGF-Ralpha or PDGF-Rbeta . B: densitometry of PDGF-receptor signals showing maximal induction of PDGF-Ralpha protein 48 h postinstillation of V2O5 (hatched bars). Open bars, saline group. Data are from a single rat per treatment and are representative of 3-5 rats at each time point for each group.


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Fig. 4.   Immunohistochemistry of PDGF-Ralpha during inflammatory and proliferative phases of V2O5-induced pulmonary fibrosis. Paraffin-embedded sections of rat lungs instilled with V2O5 were stained with a rabbit anti-mouse antibody specific to PDGF-Ralpha (A-D, left) or with normal rabbit serum as a control (A-D, right). A: 24 h postinstillation. B: 48 h postinstillation. C: 6 days postinstillation. D: 6-day-old rat testis stained as a positive control for PDGF-Ralpha (note strong brown staining in connective tissue surrounding seminiferous tubules). PDGF-Ralpha staining (brown) in lung sections was observed 24 and 48 h postinstillation, with little or no staining observed at 6 days (light blue staining).

End-stage fibrotic lesions containing collagen and elastin do not express PDGF-Ralpha . In the V2O5 model of pulmonary fibrosis, we observed inflammatory cell infiltration at 24-48 h, followed by a mesenchymal cell proliferative stage from 72 h to 6 days. Collagen staining (blue trichrome stain) was not evident in fibrotic lesions between 24 h and 6 days but was intense at day 15 postinstillation of V2O5 (Fig. 5). PDGF-Ralpha expression within fibrotic lesions had declined by day 6 (Fig. 4), and PDGF-Ralpha was not detectable by immunohistochemistry within lesions at day 15 (data not shown). Myofibroblasts are the principal source of elastin in the lung parenchyma (7). Fibrotic lesions stained postive for elastin, which is a smooth muscle cell marker (Fig. 5). Mesenchymal cells within the lesions also stained postively for vimentin (a phenotypic marker of fibroblasts) and desmin (a marker of smooth muscle cells) (data not shown). Taken together, these data indicated that myofibroblasts were present within fibrotic lesions. Thus induction of PDGF-Ralpha was confined to the inflammatory and early proliferative phases of lung fibrosis and preceded deposition of mature collagen.


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Fig. 5.   Progression of fibrotic lesions containing collagen and elastin after V2O5 instillation. A-D: Masson's trichrome, ×100. E: Masson's trichrome, ×400. F: Verhoeff's stain for elastin, ×400. Inflammatory foci containing abundant leukocytes were apparent 24 h post-V2O5 instillation (A). Proliferative lesions at 48 h (B) and 6 days (C) did not stain positively for collagen (lack of blue staining). At day 15 post-V2O5, numerous fibrotic lesions staining for collagen (blue trichrome stain; arrows) were present (D). High magnification shows blue-staining collagen fibrils (E, arrows) and red-staining elastin filaments (F, arrows) within lesions.

V2O5 upregulates PDGF-Ralpha on cultured rat lung myofibroblasts through a macrophage-dependent pathway involving IL-1beta . To address the mechanism whereby V2O5 causes upregulation of PDGF-Ralpha in vivo, we investigated the possibility that 1) V2O5 might directly induce PDGF-Ralpha on cultured lung myofibroblasts or 2) V2O5 induction of PDGF-Ralpha on myofibroblasts could require the presence of alveolar macrophage-derived inflammatory mediators. Therefore, confluent cultures of rat lung myofibroblasts were treated in vitro directly with V2O5 or with MØCM from macrophages that had been stimulated with V2O5 for 24 h. As a positive control for PDGF-Ralpha upregulation, some myofibroblasts were stimulated with IL-1beta (2 ng/ml). Parallel cultures of myofibroblasts were coincubated with IRAP. V2O5 alone did not significantly increase PDGF-Ralpha expression as determined by Western blot analysis (Fig. 6). However, V2O5 stimulated macrophages to release a factor(s) that strongly upregulated PDGF-Ralpha on myofibroblasts, and the majority of this activity (>75%) was blocked by IRAP (Fig. 6). IRAP alone did not cause any change in PDGF-Ralpha expression (data not shown). PDGF-Rbeta expression was not changed by any of these treatments. Similar experiments were performed with an 125I-PDGF-AA binding assay to more accurately measure upregulation of the cell-surface PDGF-AA binding site (i.e., PDGF-Ralpha ; Fig. 7). These data confirmed the Western blotting experiments in Fig. 6 and showed that V2O5 stimulated alveolar macrophages to release a factor(s) that strongly upregulated the number of 125I-PDGF-AA binding sites on cultured myofibroblasts (seven- to eightfold increase in upregulatory activity between control MØCM and MØCM from V2O5 treatment). IRAP blocked this upregulatory activity by >70%.


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Fig. 6.   A: Western blotting of PDGF receptors, demonstrating that V2O5 upregulates PDGF-Ralpha on rat lung myofibroblasts in vitro through a macrophage-dependent pathway involving interleukin (IL)-1beta . B: densitometry of PDGF-Ralpha . Rat alveolar macrophages were lavaged from untreated control rats and incubated in absence or presence of 1 µg/cm2 of V2O5 for 24 h. Macrophage-conditioned medium (MØCM) was added to confluent cultures of rat lung myofibroblasts. IL-1beta was tested as a positive inducer of PDGF-Ralpha . Parallel cultures of myofibroblasts were incubated with IL-1-receptor antagonist protein (IRAP; hatched bars). Open bars, control. V2O5 stimulated macrophages to release a factor(s) that upregulated PDGF-Ralpha , and the majority of this activity (>75%) was blocked by IRAP. V2O5 alone did not affect PDGF-Ralpha expression, and PDGF-Rbeta was not changed by any treatment.


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Fig. 7.   Specific binding of 125I-PDGF-AA to rat lung myofibroblasts in vitro after 24-h stimulation with MØCM from alveolar macrophages stimulated with V2O5. Conditions for IL-1beta and MØCM treatment of cultured myofibroblasts were the same as in Fig. 6. Binding assay was performed as described in METHODS. Data are means ± SE. Experiment was repeated 3 times, and triplicate determinations were performed for total and nonspecific binding in each experiment. cpm, Counts/min. V2O5 stimulated a 7- to 8-fold increase in upregulatory activity for PDGF-Ralpha on myofibroblasts, and IRAP blocked >70% of this activity, * P < 0.01. IRAP completely blocked IL-1beta -induced upregulation of PDGF-Ralpha .

    DISCUSSION
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Discussion
References

The hyperplastic growth of myofibroblasts is a key event in the progression of pulmonary fibrosis. In this study, we have shown that PDGF-Ralpha , but not PDGF-Rbeta , is induced during the inflammatory and early proliferative stages of fibrogenesis in rats instilled with V2O5. Because the expression of PDGF-Ralpha is required for maximal PDGF-stimulated chemotaxis and mitogenesis of myofibroblasts in vitro (8, 20, 24), our findings suggest that induction of PDGF-Ralpha in vivo is a mechanism that contributes to enhanced myofibroblast proliferation in the fibrotic lung. Furthermore, we showed that V2O5 stimulated cultured alveolar macrophages to release a factor(s) that upregulated the PDGF-Ralpha on lung myofibroblasts in vitro and that the majority of this activity was due to IL-1beta . PDGF-Ralpha induction appears to be a novel mechanism of lung fibroblast hyperplasia during pulmonary fibrogenesis.

Previous studies from our laboratory (19, 20) have shown that IL-1beta is a potent inducer of PDGF-Ralpha on myofibroblasts in vitro. Other investigators have demonstrated with rat models that IL-1beta gene expression in vivo occurs within 4 h after the instillation of vanadium sulfate (9) and that increased protein levels of IL-1 in lavage fluid are maximal within 24 h after the inhalation of asbestos fibers (12) or the instillation of bleomycin (15). In humans, alveolar macrophages from patients with idiopathic pulmonary fibrosis or individuals exposed to asbestos release enhanced levels of IL-1beta (33). Induction of PDGF-Ralpha gene expression by IL-1beta occurs 4 h after treatment of lung myofibroblasts in vitro, and cell-surface PDGF-Ralpha appears within 12 h (20). Thus the temporal expression of IL-1beta in vivo during fibrosis induced by fibrogenic agents, including vanadium, suggests that this proinflammatory cytokine is a likely candidate for turning on PDGF-Ralpha after lung injury. In the present study, we showed that V2O5 stimulates macrophages to release a factor(s) that upregulates PDGF-Ralpha on lung myofibroblasts in culture and that this activity is due mainly to IL-1beta . V2O5 did not directly stimulate PDGF-Ralpha , and, therefore, IL-1beta may be the major inducer of PDGF-Ralpha in vivo after lung injury.

We did not measure the induction of PDGF in the V2O5 model of fibrosis. However, the temporal expression of PDGF in rats has been investigated after the instillation of bleomycin (30). In these studies, the induction of PDGF occurs within 2-3 days after lung injury. This temporal induction of PDGF coincides with the upregulation of PDGF-Ralpha in the present study. Thus it appears that PDGF and the inducible PDGF-Ralpha are coregulated during the progression of fibrosis. It is important to keep in mind that upregulation of PDGF-Ralpha has a significance beyond simply increasing the overall number of PDGF receptors at the cell surface of mesenchymal cells. Indeed, even a relatively small increase in cell-surface PDGF-Ralpha relative to PDGF-Rbeta (1:10 ratio) renders cells severalfold more responsive to PDGF (19, 20). Indeed, Seifert et al. (29) have shown that maximal mitogenic responses to PDGF isoforms require the presence of PDGF-Ralpha in combination with PDGF-Rbeta and that the PDGF heterodimeric alpha beta -receptor complex is a more potent inducer of Swiss 3T3 fibroblast proliferation compared with the PDGF beta beta -receptor complex. Another study from our laboratory (24) emphasized that maximal chemotaxis of lung myofibroblasts requires the presence of PDGF-Ralpha . During fibrosis, it is now apparent that two changes in the PDGF system occur: 1) induction of PDGF-A and -B chain genes (30) and 2) a coordinated upregulation of the normally suppressed PDGF-Ralpha gene (reported in the present study).

PDGF-Ralpha expression was turned off before the appearance of mature collagen within fibrotic lesions as determined by trichrome staining. This may be due to the expression of TGF-beta 1, which stimulates collagen expression and also serves to downregulate PDGF-Ralpha on lung fibroblasts (3). We did not measure TGF-beta 1 levels in the present study, but another study (27) has shown that TGF-beta 1 expression occurs within 5-7 days during fibrogenesis caused by the instillation of bleomycin. Previous work from our laboratory (3) showed that TGF-beta 1 acts at the transcriptional level to suppress PDGF-Ralpha gene expression and that TGF-beta 1 inhibits lung fibroblast proliferation, at least in part, by suppression of PDGF-Ralpha . Thus TGF-beta 1 has multifunctional roles in the fibroproliferative process: inhibition of fibroblast proliferation in concert with activation of genes encoding extracellular matrix proteins such as collagen. Other investigators (32) have shown that development of interstitial lung disease in rats requires expression of both PDGF and TGF-beta 1. This could be explained by the fact that PDGF is important to the early proliferative phase of fibrosis, whereas TGF-beta 1 drives the matrix deposition phase of the disease process.

The data presented in the present study, taken together with earlier in vitro observations of PDGF-Ralpha upregulation (8, 20), suggest that induction of this receptor is important to mesenchymal cell hyperplasia in vivo during lung fibrogenesis. Induction of PDGF-Ralpha in vitro has been demonstrated on cultured human lung fibroblasts in response to thrombin (22), human bronchial smooth muscle cells stimulated with basic fibroblast growth factor (2), and rat lung myofibroblasts treated with IL-1beta (20). However, induction of PDGF-Rbeta rather than PDGF-Ralpha appears to be important to some other proliferative diseases. During liver fibrosis, adipocytes differentiate into myofibroblasts, and this is accompanied by the appearance of PDGF-Rbeta (31). During atherosclerosis, PDGF-Rbeta is upregulated with no apparent change in PDGF-Ralpha (26). Thus, although induction of PDGF-Ralpha appears to be important to the progression of lung fibrosis, it may not occur in all fibroproliferative disease states.

Elastin staining indicated that alveolar myofibroblasts (contractile interstitial cells) were present within fibrotic lesions during V2O5-induced fibrogenesis. Alveolar myofibroblasts constitute the major source of elastin fibers in the lung parenchyma (7), and fibrotic lesions stained positively for both elastin and collagen (Fig. 5). Furthermore, our early-passage isolates of rat lung mesenchymal cells that possessed inducible PDGF-Ralpha stained positively for alpha -smooth muscle actin and vimentin and contained abundant intermediate filaments as determined by ultrastructural analysis (8). Thus these cells are most likely a myofibroblast phenotype and represent the target cell that we observed in vivo within fibrotic lesions.

In summary, we have shown that induction of PDGF-Ralpha occurs in vivo during the early events associated with myofibroblast proliferation after vanadium-induced lung injury. This is the first report of PDGF-Ralpha induction in vivo during the multistep progression of fibrogenesis. Expression of PDGF-Rbeta remained unchanged during fibrogenesis. Induction of PDGF-Ralpha is due, in part, to a macrophage-dependent pathway involving IL-1beta . Because PDGF-Ralpha expression is necessary for maximal PDGF-stimulated mitogenic and chemotactic responses in vitro, the present study suggests that upregulation of PDGF-Ralpha in vivo is a novel mechanism of myofibroblast hyperplasia that contributes to the development of fibrotic lesions after lung injury.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Robert Maronpot and Julie Foley for providing technical support in platelet-derived growth factor-receptor-alpha immunohistochemistry and lung pathology. We thank Dr. Darryl Zeldin and Dr. Robert Maronpot for comments during the preparation of the manuscript. The technical assistance of Herman Price in performing intratracheal instillations is greatly appreciated.

    FOOTNOTES

Address for reprint requests: J. C. Bonner, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709.

Received 4 June 1997; accepted in final form 16 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 274(1):L72-L80