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REGULATION OF SYNOVIAL CELL GROWTH Coexpression of Transforming Growth Factor f3 and Basic Fibroblast Growth Factor by Cultured Synovial Cells DAVID H. GODDARD, SCOTT L. GROSSMAN, WILLIAM V. WILLIAMS, DAVID B. WEINER, JANET L. GROSS, KRISTI EIDSVOOG, and JAMES R. DASCH
Objective. To demonstrate expression of transforming growth factor f3 (TGFf3) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. Methods. Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGFf3 and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGFf3 bioactivity was determined by MvlLu assay. Presented in part at the 55th Annual Meeting of the American College of Rheumatology, Boston, MA, November 1991. From the Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Winthrop-University Hospital, Mineola, New York, and Temple University School of Medicine, Philadelphia, the Division of Rheumatology, University of Pennsylvania, and The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania; The Du Pont Merck Pharmaceutical Company, Wilmington, Delaware; and the Department ofImmunology, Celtrix Laboratories, Palo Alto, California. Supported in part by a grant from the Eastern Pennsylvania Chapter, Arthritis Foundation. David H. Goddard, MD, MRCP: Division of Rheumatology, Immunology, and Allergy, Winthrop-University Hospital and Temple University School of Medicine; Scott L. Grossman, PhD: Temple University School of Medicine; William V. Williams, MD: Division of Rheumatology, University of Pennsylvania; David B. Weiner, PhD: The Wistar Institute of Anatomy and Biology; Janet L. Gross, PhD: The Du Pont Merck Pharmaceutical Company; Kristi Eidsvoog, PhD: The Du Pont Merck Pharmaceutical Company; James R. Dasch, PhD: Department of Immunology, Celtrix Laboratories. Address reprint requests to David H. Goddard, MD, MRCP, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Winthrop-University Hospital, 222 Station Plaza North, Suite 430, Mineola, NY 11501. Submitted for publication December 17,1991; accepted in revised form July 8, 1992. Arthritis and Rheumatism, Vol. 35, No. 11 (November 1992)
Results. Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGFf3 and bFGF. Western blot analysis identified a single TGFf3 band (MW -25 kd) in each of the cell Iysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW ~ 18.0 kd) in normal synovial cell Iysates and 4 bFGF bands (MW -18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell Iysates. RA and normal synovial cells expressed mRNA transcripts encoding TGFf31 but not TGFf32' and FGF·2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb IDII.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 p,g/ml) had no effect on cell growth. Conclusion. RA and normal synovial cells cultured in serum-free medium express TGFf31 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGFf31 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown. Rheumatoid arthritis (RA), a disease of unknown etiology, is characterized by excessive growth of the synovial membrane, causing inflammatory destruction of cartilage and bone. In this disease, the synovium displays characteristics similar to a localized malignancy. Cultured RA synovial cells exhibit several characteristics of neoplastic and virally transformed cells, e.g., a tendency to grow as disorganized monolayers and to form foci, the ability to form
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TGFf3 AND bFGF IN SYNOVIAL CELLS
colonies in soft agar, the presence of many multinucleated cells, and tumorigenicity in nude mice (1-4). Accumulating data indicate that polypeptide growth factors and cytokines play an important role in regulating normal as well as neoplastic cell growth (5-10). Furthermore, the observations that the protein products of several viral oncogenes show close homology with polypeptide growth factors encoded by their human homologs suggest a possible link between certain viruses and excessive cell growth (11-14). Similarities between rheumatoid synovitis and neoplasia have led to speculation that polypeptide growth factors may playa role in causing synovial proliferation. This speculation is strengthened by the identification of several growth factors and cytokines in both the intact rheumatoid synovium and in synovial fluid (15-18). In this regard, the identification of transforming growth factord (TGFI3) and basic fibroblast growth factor (bFGF) in RA synovial fluid is of particular interest (18,19). TGFI3, a homodimeric peptide (MW ~25 kd) with a ubiquitous distribution and a broad range of target tissues, is present both in the intact synovium and, in latent and activated forms, in RA synovial fluid (17,18). When injected into the normal joints of Lewis rats, this growth factor causes an inflammatory synovitis characterized by synovial cell proliferation, new blood vessel formation, and fibrosis (20). Basic fibroblast growth factor, also found in RA synovial fluid (16), is synthesized in situ by a variety of different types of cells, including several tumor cell lines (for review, see ref. 21). It is both a potent mitogen and an angiogenic factor, stimulating growth in many different types of cells, including mesenchymal cells, endothelial cells, and various tumor cell lines (21). We have previously observed that long-term cultures of RA, but not osteoarthritis (OA), synovial cells proliferate in serum-free medium (22). Moreover, we have shown that the addition of neutralizing monoclonal antibodies (MAb) to TGFI3 causes a dosedependent reduction in RA synovial cell growth (22). This led us to speculate that RA synovial cell growth in vitro is regulated by endogenous TGFI3. Moreover, the recent identification of endogenous bFGF in synoviocytes in culture (19) suggests that RA synovial cell growth in vitro may be regulated by both growth factors. To further explore these possibilities, we undertook studies in which TGFI3, and bFGF were identified in synovial cells cultured in serum-free medium. We also measured cell growth in serum-free medium containing increasing amounts of neutralizing
MAbs to TGFI3 (MAb 1011.16) or bFGF (MAb OG2). Whereas the addition of MAb 1011.16 to the medium caused a dose-dependent reduction in RA synovial cell growth, the addition of MAb OG2 had no effect on cell proliferation. These findings indicate that endogenous TGFI3, but not endogenous bFGF, has a direct regulatory effect on RA synovial cell growth in vitro.
MATERIALS AND METHODS Source of materials and synovial samples. MAb ID11.16 was provided by one of the authors (JRD) and is a neutralizing antibody recognizing TGFI3, and TGF132 in Western blot analysis (23). MAb DG2 and DE6 were provided by one of the authors (JLG) and are neutralizing antibodies recognizing bFGF in Western blot analysis (24). All other antibodies were purchased as needed. TGFI3 and bFGF were purchased from R&D Systems (Minneapolis, MN). Recombinant human bFGF (rHubFGF) was generously provided by Synergen (Boulder, CO). CHAPS detergent, protease inhibitors, and crude collagenase were purchased from Sigma (St. Louis, MO). RPMI 1640, Dulbecco's modified essential medium, Hanks' balanced salt solution, nonessential amino acids, streptomycin, penicillin, and fungazone were all purchased from Flow Laboratories (Mcl.ean, VA). Fetal bovine serum was purchased from Hyclone (Logan, UT), bovine serum albumin (BSA) from Miles (Naperville, IL), and trypsin/EDTA from Gibco (Grand Island, NY). All tissue culture plastics were purchased from Falcon (Oxnard, CAl. Normal dermal fibroblasts and mink lung epithelial cells were obtained from American Type Culture Collection (Rockville, MD). Synovial samples were obtained, with the patients' informed consent, at the time of joint replacement surgery in 4 patients with RA (25). Synovium was also obtained, with the informed consent of the next of kin, from I patient with irreversible brain death from whom organs were being removed for transplantation. This patient was
REGULATION OF SYNOVIAL CELL GROWTH Coexpression of Transforming Growth Factor f3 and Basic Fibroblast Growth Factor by Cultured Synovial Cells DAVID H. GODDARD, SCOTT L. GROSSMAN, WILLIAM V. WILLIAMS, DAVID B. WEINER, JANET L. GROSS, KRISTI EIDSVOOG, and JAMES R. DASCH
Objective. To demonstrate expression of transforming growth factor f3 (TGFf3) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. Methods. Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGFf3 and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGFf3 bioactivity was determined by MvlLu assay. Presented in part at the 55th Annual Meeting of the American College of Rheumatology, Boston, MA, November 1991. From the Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Winthrop-University Hospital, Mineola, New York, and Temple University School of Medicine, Philadelphia, the Division of Rheumatology, University of Pennsylvania, and The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania; The Du Pont Merck Pharmaceutical Company, Wilmington, Delaware; and the Department ofImmunology, Celtrix Laboratories, Palo Alto, California. Supported in part by a grant from the Eastern Pennsylvania Chapter, Arthritis Foundation. David H. Goddard, MD, MRCP: Division of Rheumatology, Immunology, and Allergy, Winthrop-University Hospital and Temple University School of Medicine; Scott L. Grossman, PhD: Temple University School of Medicine; William V. Williams, MD: Division of Rheumatology, University of Pennsylvania; David B. Weiner, PhD: The Wistar Institute of Anatomy and Biology; Janet L. Gross, PhD: The Du Pont Merck Pharmaceutical Company; Kristi Eidsvoog, PhD: The Du Pont Merck Pharmaceutical Company; James R. Dasch, PhD: Department of Immunology, Celtrix Laboratories. Address reprint requests to David H. Goddard, MD, MRCP, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Winthrop-University Hospital, 222 Station Plaza North, Suite 430, Mineola, NY 11501. Submitted for publication December 17,1991; accepted in revised form July 8, 1992. Arthritis and Rheumatism, Vol. 35, No. 11 (November 1992)
Results. Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGFf3 and bFGF. Western blot analysis identified a single TGFf3 band (MW -25 kd) in each of the cell Iysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW ~ 18.0 kd) in normal synovial cell Iysates and 4 bFGF bands (MW -18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell Iysates. RA and normal synovial cells expressed mRNA transcripts encoding TGFf31 but not TGFf32' and FGF·2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb IDII.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 p,g/ml) had no effect on cell growth. Conclusion. RA and normal synovial cells cultured in serum-free medium express TGFf31 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGFf31 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown. Rheumatoid arthritis (RA), a disease of unknown etiology, is characterized by excessive growth of the synovial membrane, causing inflammatory destruction of cartilage and bone. In this disease, the synovium displays characteristics similar to a localized malignancy. Cultured RA synovial cells exhibit several characteristics of neoplastic and virally transformed cells, e.g., a tendency to grow as disorganized monolayers and to form foci, the ability to form
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TGFf3 AND bFGF IN SYNOVIAL CELLS
colonies in soft agar, the presence of many multinucleated cells, and tumorigenicity in nude mice (1-4). Accumulating data indicate that polypeptide growth factors and cytokines play an important role in regulating normal as well as neoplastic cell growth (5-10). Furthermore, the observations that the protein products of several viral oncogenes show close homology with polypeptide growth factors encoded by their human homologs suggest a possible link between certain viruses and excessive cell growth (11-14). Similarities between rheumatoid synovitis and neoplasia have led to speculation that polypeptide growth factors may playa role in causing synovial proliferation. This speculation is strengthened by the identification of several growth factors and cytokines in both the intact rheumatoid synovium and in synovial fluid (15-18). In this regard, the identification of transforming growth factord (TGFI3) and basic fibroblast growth factor (bFGF) in RA synovial fluid is of particular interest (18,19). TGFI3, a homodimeric peptide (MW ~25 kd) with a ubiquitous distribution and a broad range of target tissues, is present both in the intact synovium and, in latent and activated forms, in RA synovial fluid (17,18). When injected into the normal joints of Lewis rats, this growth factor causes an inflammatory synovitis characterized by synovial cell proliferation, new blood vessel formation, and fibrosis (20). Basic fibroblast growth factor, also found in RA synovial fluid (16), is synthesized in situ by a variety of different types of cells, including several tumor cell lines (for review, see ref. 21). It is both a potent mitogen and an angiogenic factor, stimulating growth in many different types of cells, including mesenchymal cells, endothelial cells, and various tumor cell lines (21). We have previously observed that long-term cultures of RA, but not osteoarthritis (OA), synovial cells proliferate in serum-free medium (22). Moreover, we have shown that the addition of neutralizing monoclonal antibodies (MAb) to TGFI3 causes a dosedependent reduction in RA synovial cell growth (22). This led us to speculate that RA synovial cell growth in vitro is regulated by endogenous TGFI3. Moreover, the recent identification of endogenous bFGF in synoviocytes in culture (19) suggests that RA synovial cell growth in vitro may be regulated by both growth factors. To further explore these possibilities, we undertook studies in which TGFI3, and bFGF were identified in synovial cells cultured in serum-free medium. We also measured cell growth in serum-free medium containing increasing amounts of neutralizing
MAbs to TGFI3 (MAb 1011.16) or bFGF (MAb OG2). Whereas the addition of MAb 1011.16 to the medium caused a dose-dependent reduction in RA synovial cell growth, the addition of MAb OG2 had no effect on cell proliferation. These findings indicate that endogenous TGFI3, but not endogenous bFGF, has a direct regulatory effect on RA synovial cell growth in vitro.
MATERIALS AND METHODS Source of materials and synovial samples. MAb ID11.16 was provided by one of the authors (JRD) and is a neutralizing antibody recognizing TGFI3, and TGF132 in Western blot analysis (23). MAb DG2 and DE6 were provided by one of the authors (JLG) and are neutralizing antibodies recognizing bFGF in Western blot analysis (24). All other antibodies were purchased as needed. TGFI3 and bFGF were purchased from R&D Systems (Minneapolis, MN). Recombinant human bFGF (rHubFGF) was generously provided by Synergen (Boulder, CO). CHAPS detergent, protease inhibitors, and crude collagenase were purchased from Sigma (St. Louis, MO). RPMI 1640, Dulbecco's modified essential medium, Hanks' balanced salt solution, nonessential amino acids, streptomycin, penicillin, and fungazone were all purchased from Flow Laboratories (Mcl.ean, VA). Fetal bovine serum was purchased from Hyclone (Logan, UT), bovine serum albumin (BSA) from Miles (Naperville, IL), and trypsin/EDTA from Gibco (Grand Island, NY). All tissue culture plastics were purchased from Falcon (Oxnard, CAl. Normal dermal fibroblasts and mink lung epithelial cells were obtained from American Type Culture Collection (Rockville, MD). Synovial samples were obtained, with the patients' informed consent, at the time of joint replacement surgery in 4 patients with RA (25). Synovium was also obtained, with the informed consent of the next of kin, from I patient with irreversible brain death from whom organs were being removed for transplantation. This patient was
