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EFFECTS O F INTERLEUKIN-6 ON THE METABOLISM OF CONNECTIVE TISSUE COMPONENTS IN RHEUMATOID SYNOVIAL FIBROBLASTS AKIRA ITO, YOSHIFUMI ITOH, YASUYUKI SASAGURI, MINORU MORIMATSU, and YO MORI Objective. High levels of interleukin-6 (IL-6) have been found in the synovial fluid of patients with rheumatoid arthritis (RA). We undertook the present study to investigate the role of IL-6 in this disease. Methods. We examined the effects of IL-6, in comparison with IL-1, on the biosynthesis of extracellular matrix macromolecules and of matrix-degrading proteinases in rheumatoid synovial fibroblasts. Results. In rheumatoid synovial fibroblasts, IL-6 by itself enhanced the production of plasminogen activator, its inhibitor, and tissue inhibitor of metalloproteinases, whereas it did not modulate the biosynthesis of precursor of matrix metalloproteinase 1 (proMMP-1) (tissue collagenase), proMMP-3 (stromelysin), or connective tissue components. However, IL- 1-induced production of proMMP-1 and proMMP-3 was preferentially augmented by IL-6. Conclusion. These results suggest that in RA, IL-6 may participate along with IL-1 in fine tuning of the catabolism of connective tissue components, by From the Department of Biochemistry, Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo, and the Second Department of Pathology, Kurume University School of Medicine, Kurume, Fukuoka, Japan. Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. Akira Ito, PhD: Department of Biochemistry, Tokyo College of Pharmacy; Yoshifumi Itoh. MS: Department of Biochemistry, Tokyo College of Pharmacy; Yasuyuki Sasaguri, MD. PhD: Second Department of Pathology, Kurume University School of Medicine; Minoru Morimatsu, MD, PhD: Second Department of Pathology, Kurume University School of Medicine; Yo Mori, PhD: Department of Biochemistry, Tokyo College of Pharmacy. Address reprint requests to Akira Ito, PhD, Department of Biochemistry, Tokyo College of Pharmacy, Horinouchi. Hachioji. Tokyo 192-03, Japan. Submitted for publication December 26, IWI; accepted in revised form June 8. 1992. Arthritis and Rheumatism, Vol. 35, No. 10 (October 1992)
modulating the balance between connective tissuedegrading enzymes and their inhibitors. Degradation of collagens and other matrix macromolecules in connective tissue is a common feature in rheumatoid arthritis (RA) ( I ) and osteoarthritis (2). Matrix metalloproteinases (MMPs) such as MMP- 1 (tissue collagenase; EC 3.4.24.7) and MMP-3 (stromelysin; EC 3.4.24.17) are thought to play a central role in cartilage destruction ( I ) . Cytokines such as interleukin-1 (IL-I) and tumor necrosis factor a (TNFa) are characterized as accelerators of such connective tissue destruction, since they stimulate the production of MMP-I and MMP-3 by connective tissue cells. IL-I and T N F a also induce IL-6 in various connective tissue cells, such as fibroblasts (3), synoviocytes (41, and chondrocytes ( 5 ) . The finding, by Houssiau et al (6), of elevated levels of IL-6 in the synovial fluid of patients with RA suggests that IL-6 may participate in the joint destruction in RA. However, its role in the disease is not fully understood. In the present study, we investigated the effects of IL-6 on the biosynthesis of connective tissue macromolecules such as glycosaminoglycans (GAGS) and collagens, and the production of MMPs, tissue inhibitor of metalloproteinases (TIMP), urokinase-type plasminogen activator (uPA), and PA inhibitor 1 (PAI-1) by human rheumatoid synovial fibroblasts.
MATERIALS AND METHODS Materials. The following reagents were obtained commercially: recombinant human IL-6 (rHuIL-6; R & D Systems, Minneapolis, MN), rabbit anti-human urokinase antibody (JCR Pharmaceuticals, Kobe, Japan), and goat anti-human PAI-I IgG (Biopool, Umea, Sweden). Recombi-
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Figure 1. Effects of human recombinant interleukin-6 (hrlL-6) and hrlL-I. separately or together. on the production of glycosaminoglycans (GAGS) by human rheumatoid synovial fibroblasts. A. Biosynthesis of total GAGs. Confluent sixth-passage synovial cells in 24-well plates were treated with hrlL-6 and/or hrIL-l in medium for I 2 hours, followed by labeling with 'H-glucosamine for a further 24 hours: GAGs in the medium and cell fractions were then extracted and purified (see Materials and Methods for details). Values are the mean and SD of 4 wells. B. Composition of GAGS in control samples and samples treated with hrlL-6 (50 ng/ml), hrlL-l (0.5 ng/ml), or hrlL-l (0.5 ng/ml) plus hrlL-6 (50 ng/ml). Results were calculated by paper chromatography of chondroitin AC-lyase- or ABC-lyasedigested GAGs. Relative composition is expressed as a percentage; values are the mean of triplicate determinations. HS = heparan sulfate; Ch-4S = chondroitin 4-sulfate: C h d S = chondroitin 6-sulfate; HA = hyaluronate.
nant human IL-la (2 x lo7 units/mg) was kindly supplied by Dainippon Pharmaceutical (Osaka, Japan). Other reagents used were those described in previous reports (7-9). Cell culture. Human rheumatoid synovial tissue obtained at arthroplasty from patients with definite or classic RA (10) were enzymatically digested, and adherent cell cultures were established. The synovial cells were maintained in Eagle's minimum essential medium (EMEM)-10% (volumeholume) fetal bovine serum until confluence was reached ( I ) . In most experiments, cells up to the seventh passage were plated in 24-well plates and maintained to confluence. To examine the production of proMMPs, TIMP, uPA, and PAI-I, the culture medium was changed to EMEM-0.2% (weight/volume) lactalbumin hydrolysate (LAH) after confluence. Harvested culture media were stored at -20°C until use. 3H-thymidine incorporation. Incorporation of 'Hthymidine into DNA was determined as described previously (8). Biosynthesis of GAGs and collagen. Labeling of confluent synovial cells with D-&'H-gIucosamine, extraction of labeled GAGs, digestion of GAGs with chondroitin AC and ABC lyases, and estimation of resulting unsaturated disaccharides were performed as described previously (9). To examine collagen biosynthesis, confluent cells were labeled with 'H-proline for 6 hours in EMEM-O.2% LAH-O.l mM ascorbate-0.5 pM @aminopropinnitrile. After the labeling, levels of collagen and noncollagenous protein were deter-
mined using purified bacterial collagenase as described previously (8). Labeling with ~-~~S-methionine, immunoprecipitation, and fluorography of proMMP-1, proMMP-3, and TIMP. Labeling of cells with L-3'S-methionine, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fluorography were carried out as described previously ( I I ) . Western blotting. Urokinase-type PA and PAI-I in culture media were analyzed by Western blotting, using their respective antibodies. SDS-PAGE using 10% (weight/ volume) acrylamide slub gel under reducing conditions, electrotransfer, and visualization of antigen were done as described previously (7).
RESULTS We first examined the effect of rHuIL-6 on 3H-thymidine incorporation into DNA. Recombinant human IL-6 (0.1 ng/ml, 10 ng/ml, and 20 ng/ml) and/or rHuIL-1 (0.5 ng/ml) did not show any effect on DNA synthesis by human synovial cells at either the growth or the stationary phase, indicating that neither cytokine influences cellular proliferation of rheumatoid synovial fibroblasts (results not shown). In contrast,
IL-6 IN RA SYNOVIAL FIBROBLASTS
rHuIL-l (0.5 ng/ml) augmented GAG production 2-fold, as compared with control cells (Figure IA). In addition, >75% of the GAG synthesized by synovial cells was found to be hyaluronate, and rHuIL-l accelerated its biosynthesis predominantly (Figure IB). Recombinant human IL-6, however, did not alter the levels or the composition of GAG in either control or IL-l-treated cells. Incorporation of 'H-proline into collagens and noncollagenous proteins in rheumatoid synovial fibroblasts was not influenced by rHuIL-6 and/or rHuIL-l (results not shown). The production of proMMP-l and proMMP-3 by untreated synovial cells was negligible (Figures 2A and B, lane I). Treatment of the cells with rHuIL-6 did not affect the production of proMMP-I or proMMP-3 (Figures 2A and B, lanes 2 4 , but rHuIL-l clearly promoted the production of both proMMPs (Figures 2A and B, lane 5). However, rHuIL-6 was found to further enhance IL-l-induced production of proMMPs, in a dose-dependent manner (Figures 2A and B, lanes 6-8): In cells treated with both rHuIL-6 (50 ng/ml) and rHuIL-l (0.5 ng/ml), production of proMMP- 1 and proMMP-3, respectively, was increased I .7-fold and 1 .Cfold, compared with cells treated with only rHuIL-I. Recombinant human IL- 1 increased the production of TIMP as well as of MMPs in rheumatoid synovial fibroblasts (Figure 2C, lane 5 ) . TIMP production was also increased, in a dose-dependent manner, by rHuIL-6 (Figure 2C, lanes 2 4 , with a maximal 2.4-fold stimulation at 50 ng/ml of rHuIL-6. Cotreatment of cells with both rHuIL-l and rHuIL-6 further enhanced the production of TIMP (Figure 2C, lanes 6-8), as was seen with proMMP-l and proMMP-3. Rheumatoid synovial cells constitutively produced a significant amount of uPA and its inhibitor PAI-I, and IL-6 by itself augmented the production of both uPA and PAI-1 in a dose-dependent manner (Figures 3A and B). However, in contrast to its stimulatory effect on the production of proMMP-1 and proMMP-3 (Figure 2), rHuIL-l did not modulate the production of uPA and PAI-I (results not shown).
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Figure 2. Effect of recombinant human interleukin-6 (rHulL-6) on rHulL-l-induced production of matrix metalloproteinases (MMPs) proMMP-l (A) and proMMP-3 (B),and tissue inhibitor of metalloproteinases (TIMP) (C), by human rheumatoid synovial fibroblasts. Confluent synovial cells were preincubated with rHulL-6 andor rHulL- I in medium for 24 hours, followed by labeling with '%-methionine in methionine-free medium containing cytokine(s1 for 3 hours. The harvested culture media were subjected to imrnunoprecipitation. followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography (see Materials and Methods for details). Bands corresponding to proMMPs or TIMP were excised and counted for radioactivity, and the respective amount of proMMPs or TIMP secreted into the media was then calculated in relation to the control. Lane I, Control. Lanes 2 4 , rHulL-6 (0.1. 20. and 50 ng/ml. respectively). Lane 5, rHulL-l (0.5 ng/ml). Lanes 6-8. rHulL-l (0.5 ng/ml) plus rHulL-6 (0.1. 20. and 50 ng/ml. respectively). Lane 9. rHulL-l (0.5 ng/ml) immunoprecipitated with nonimmune sheep I&.
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DISCUSSION This study demonstrated that neither rHuIL-6 nor rHuIL- 1 modulated 'H-thymidine incorporation by synovial cells at both the growth and the stationary phases, but rHuIL-I stimulated the biosynthesis of GAGS, predominantly hyaluronate production, whereas rHuIL-6 did not modulate basal or IL-Iinduced hyaluronate production in synovial fibroblasts. Neither cytokine altered collagen synthesis in
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Figure 3. Effect of recombinant human interleukin-6 (rHulL-6) on the production of urokinase-type plasminogen activator (u-PA) (A) and PA inhibitor I (PAI-I) (B) by human rheumatoid synovial fibroblasts. Confluent synovial cells were treated with rHulL-6 in medium for 2 days, after which the harvested culture media (1.5 ml) were mixed with 0.3 ml of 20% (weight/volume) trichloracetic acid. The precipitates were collected by centrifugation. dissolved in 25 pl of reducing sample buffer. and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Materials and Methods for details). Bands of u-PA were visualized by immunoblotting. The relative amount of u-PA or PAI-I was quantified by densitometric scanning, with control cells assigned a value of I .O. A. Lane I , Control. Lanes 2-5, rHulL-6 (0.I , I , 20. and 50 ng/ml. respectively). B, Lane 1 , Control. Lanes 2 4 , rHulL-6 (0.1. 20. and SO ng/ml, respectively).
synovial fibroblasts. Recent studies by Kandel et al (12) also showed that IL-6 did not alter the synthesis of proteoglycans and collagens in articular chondrocytes. It was reported that rHuIL-6 did not affect basal or IL- I-stimulated production of prostaglandin E, (PGE?)in human articular chondrocytes (12) or human synoviocytes (13). We have now confirmed this in the rheumatoid synovial cell system. Thus, it is unlikely that IL-6 participates in the hypertrophy of pannus tissue or the proinflammatory response of PGE, production in RA. Human recombinant IL-6 alone did not modulate the production of proMMP-l or proMMP-3, but it clearly augmented rHuIL- I-induced production of both proMMPs, indicating that IL-6 amplifies the signal transduction system of IL-I for proMMP production. Nevertheless, rHuIL-6 alone did enhance the production of TIMP in a dose-dependent manner in synovial cells, and additionally augmented 1L-Iinduced production of TIMP when cells were cotreated with both interleukins. These results are consistent with our previous findings in human skin and uterine cervical fibroblasts (14). Lotz and Guerne (13) reported that IL-6 could modulate the production of TIMP in human synoviocytes, but not basal or IL-I-stimulated collagenolytic activity. Thus, the action of IL-6 is likely dependent on the cell populations and experimental conditions used. Although we could
not quantify the exact amounts of MMP and TIMP induced by IL-6 and/or IL-I, it is probable that IL-6 participates in creating the imbalance between MMP and TIMP at sites of inflammation in RA and the eventual destruction of cartilage in vivo. It is well known that plasmin-PA participates directly and/or indirectly in the destruction of connective tissue components in RA, via activation of proMMP-3 (15-17) and direct destruction of proteoglycans (18). Recombinant human IL-6 stimulated human synovial cells to produce uPA in a dose-dependent manner. To our knowledge, this is the first evidence that IL-6 is one of the stimulators of uPA production. The appearance of PA activity at sites of inflammation is influenced by PA inhibitors, such as PAI-I (19). We observed that rHuIL-6 augmented PAI-I production by human rheumatoid synovial cells in a dose-dependent manner. Thus, IL-6 augmented the production of both uPA and its inhibitor. Although we could not evaluate the exact amounts of uPA and PAI-I in culture media, it is likely that IL-6 participates in the destruction of connective tissue components in RA by causing their imbalance. In fact, significantly high levels of uPA activity are found in the synovial fluid of patients with RA (20). In conclusion, we have demonstrated that IL-6 does not modulate PGE, production, synovial cell proliferation, or synthesis of GAGS and collagens, but
IL-6 IN RA SYNOVIAL FIBROBLASTS
d o e s stimulate the production of TIMP, uPA, and PAI-I, and augments the IL-I-stimulated production of MMP-1 and MMP-3, by human rheumatoid synovial cells. These results indicate that IL-6 may participate closely in the acceleration of catabolism of connective tissue components at sites of inflammation. Therefore, IL-6, together with I L - I , may be characterized as one of inflammatory mediators of joint destruction in RA.
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ACKNOWLEDGMENT We are grateful to Dr. H. Nagase (University of Kansas Medical Center, Kansas City, KS) for generously providing sheep anti-(human proMMP-I) and anti-(human MMP-3) antibodies and for critical reading of the manuscribt.
12.
REFERENCES I . Okada Y, Nagase H. Harris ED Jr: A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components: purification and characterization. J Biol Chem 261: 14245-14255, 1986 2. Dean DD, Martel-Pelletier J, Pelletier J-P, Howell DS. Woessner J F Jr: Evidence for metalloproteinase inhibitor imbalance in human osteoarthritic cartilage. J Clin Invest 84:678-685. 1989 3. Van Damme J, Cayphas S. Opdenakker G. Billiau A. van Snick J: Interleukin-I and poly-(rl).poly(rC) induce production of hybridoma growth factor by human fibroblasts. Eur J lmmunol 17:l-7, 1987 4. Guerne P-A, Zuraw BL, Vaughan JH, Carson DA, Lotz M: Synovium as a source of interleukin 6 in vitro: contribution to local and systemic manifestations of arthritis. J Clin Invest 83585-592, 1989 5. Guerne P-A. Carson DA, Lotz M: Interleukin 6 production by human articular chondrocytes: modulation of its synthesis by cytokines, growth factors, and hormones in vitro. J Immunol 144:49%505. 1990 6. Houssiau FA, Devogelaer J-P. van Damme J, Nagant de Deuxchaisnes C, van Snick J: Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 3 I :784788, 1988 7. Ito A, Nagase H: Evidence that matrix metalloproteinase 3 is an endogenous activator of procollagenase. Arch Biophys Biochem 267:211-216, 1988 8. Sakyo K, Ito A, Mori Y: Dehydroepiandrosterone sulfate stimulates collagenase synthesis without affecting the rate of collagen and noncollagen synthesis by rabbit
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uterine cervical fibroblasts. Biol Reprod 36:277-28 I , I987 Katsura M. Ito A, Hirakawa S. Mori Y: Human recombinant interleukin I increases biosynthesis of collagenase and hyaluronic acid in cultured human chorionic cells. FEBS Lett 244:315-318, 1989 Ropes MW, Bennett GA, Cobb S, Jacox R , Jessar RA: 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 9:175-176. 1958 Ito A. Sato T. Ojima Y, Chen L-C, Nagase H, Mori Y: Calmodulin differentially modulates the interleukin 1induced biosynthesis of tissue inhibitor of metalloproteinases (TIMP) and matrix metalloproteinases in human uterine cervical fibroblasts. J Biol Chem 266: 1359813601, 1991 Kandel RA. Petelycky M. Dinarello CA, Minden M. Pritzker KPH. Kruz TF: Comparison effect of interleukin 6 and interleukin 1 on collagenase and proteoglycan production by chondrocytes. J Rheumatol 17:953-957. I990 Lotz M. Guerne P-A: Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-l/erythroid potentiating activity (TIMP-I/EPA). J Biol Chem 266: 20 17-2020, I99 I Sato T, I t 0 A, Mori Y: Interleukin 6 enhances the production of tissue inhibitor of metalloproteinases (TIMP) but not matrix metalloproteinases by human fibroblasts. Biochem Biophys Res Cornmun 170:824 829. 1990 Nagase H, Enghild JJ, Suzuki K, Salvesen G: Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and 4aminophenylmercuric acetate. Biochemistry 2957835789. 1990 Suzuki K, Enghild JJ, Morodomi T, Salvesen G, Nagase H: Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 29: 10261-10270, 1990 He C. Wilhelm SM. Pentland AP. Marmer BL, Grant GA, Eisen AZ, Goldberg GI: Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc Natl Acad Sci U S A 86:2632-2636, 1989 Lack CH. Rogers HJ: Action of plasmin on cartilage. Nature 182:948-949, 1958 Pfeilschifter J. Erdmann J, Schmidt W, Naumann A, Minne HW. Ziegler R: Differential regulation of plasminogen activator and plasminogen activator inhibitor by osteotropic factors in primary culture of mature osteoblast precursors. Endocrinology 126:703-71 I , 1990 Mochan E, Uhr J: Elevation in synovial fluid plasminogen activator in patients with rheumatoid arthritis. J lmmunol 11:123-128. 1983
EFFECTS O F INTERLEUKIN-6 ON THE METABOLISM OF CONNECTIVE TISSUE COMPONENTS IN RHEUMATOID SYNOVIAL FIBROBLASTS AKIRA ITO, YOSHIFUMI ITOH, YASUYUKI SASAGURI, MINORU MORIMATSU, and YO MORI Objective. High levels of interleukin-6 (IL-6) have been found in the synovial fluid of patients with rheumatoid arthritis (RA). We undertook the present study to investigate the role of IL-6 in this disease. Methods. We examined the effects of IL-6, in comparison with IL-1, on the biosynthesis of extracellular matrix macromolecules and of matrix-degrading proteinases in rheumatoid synovial fibroblasts. Results. In rheumatoid synovial fibroblasts, IL-6 by itself enhanced the production of plasminogen activator, its inhibitor, and tissue inhibitor of metalloproteinases, whereas it did not modulate the biosynthesis of precursor of matrix metalloproteinase 1 (proMMP-1) (tissue collagenase), proMMP-3 (stromelysin), or connective tissue components. However, IL- 1-induced production of proMMP-1 and proMMP-3 was preferentially augmented by IL-6. Conclusion. These results suggest that in RA, IL-6 may participate along with IL-1 in fine tuning of the catabolism of connective tissue components, by From the Department of Biochemistry, Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo, and the Second Department of Pathology, Kurume University School of Medicine, Kurume, Fukuoka, Japan. Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. Akira Ito, PhD: Department of Biochemistry, Tokyo College of Pharmacy; Yoshifumi Itoh. MS: Department of Biochemistry, Tokyo College of Pharmacy; Yasuyuki Sasaguri, MD. PhD: Second Department of Pathology, Kurume University School of Medicine; Minoru Morimatsu, MD, PhD: Second Department of Pathology, Kurume University School of Medicine; Yo Mori, PhD: Department of Biochemistry, Tokyo College of Pharmacy. Address reprint requests to Akira Ito, PhD, Department of Biochemistry, Tokyo College of Pharmacy, Horinouchi. Hachioji. Tokyo 192-03, Japan. Submitted for publication December 26, IWI; accepted in revised form June 8. 1992. Arthritis and Rheumatism, Vol. 35, No. 10 (October 1992)
modulating the balance between connective tissuedegrading enzymes and their inhibitors. Degradation of collagens and other matrix macromolecules in connective tissue is a common feature in rheumatoid arthritis (RA) ( I ) and osteoarthritis (2). Matrix metalloproteinases (MMPs) such as MMP- 1 (tissue collagenase; EC 3.4.24.7) and MMP-3 (stromelysin; EC 3.4.24.17) are thought to play a central role in cartilage destruction ( I ) . Cytokines such as interleukin-1 (IL-I) and tumor necrosis factor a (TNFa) are characterized as accelerators of such connective tissue destruction, since they stimulate the production of MMP-I and MMP-3 by connective tissue cells. IL-I and T N F a also induce IL-6 in various connective tissue cells, such as fibroblasts (3), synoviocytes (41, and chondrocytes ( 5 ) . The finding, by Houssiau et al (6), of elevated levels of IL-6 in the synovial fluid of patients with RA suggests that IL-6 may participate in the joint destruction in RA. However, its role in the disease is not fully understood. In the present study, we investigated the effects of IL-6 on the biosynthesis of connective tissue macromolecules such as glycosaminoglycans (GAGS) and collagens, and the production of MMPs, tissue inhibitor of metalloproteinases (TIMP), urokinase-type plasminogen activator (uPA), and PA inhibitor 1 (PAI-1) by human rheumatoid synovial fibroblasts.
MATERIALS AND METHODS Materials. The following reagents were obtained commercially: recombinant human IL-6 (rHuIL-6; R & D Systems, Minneapolis, MN), rabbit anti-human urokinase antibody (JCR Pharmaceuticals, Kobe, Japan), and goat anti-human PAI-I IgG (Biopool, Umea, Sweden). Recombi-
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Figure 1. Effects of human recombinant interleukin-6 (hrlL-6) and hrlL-I. separately or together. on the production of glycosaminoglycans (GAGS) by human rheumatoid synovial fibroblasts. A. Biosynthesis of total GAGs. Confluent sixth-passage synovial cells in 24-well plates were treated with hrlL-6 and/or hrIL-l in medium for I 2 hours, followed by labeling with 'H-glucosamine for a further 24 hours: GAGs in the medium and cell fractions were then extracted and purified (see Materials and Methods for details). Values are the mean and SD of 4 wells. B. Composition of GAGS in control samples and samples treated with hrlL-6 (50 ng/ml), hrlL-l (0.5 ng/ml), or hrlL-l (0.5 ng/ml) plus hrlL-6 (50 ng/ml). Results were calculated by paper chromatography of chondroitin AC-lyase- or ABC-lyasedigested GAGs. Relative composition is expressed as a percentage; values are the mean of triplicate determinations. HS = heparan sulfate; Ch-4S = chondroitin 4-sulfate: C h d S = chondroitin 6-sulfate; HA = hyaluronate.
nant human IL-la (2 x lo7 units/mg) was kindly supplied by Dainippon Pharmaceutical (Osaka, Japan). Other reagents used were those described in previous reports (7-9). Cell culture. Human rheumatoid synovial tissue obtained at arthroplasty from patients with definite or classic RA (10) were enzymatically digested, and adherent cell cultures were established. The synovial cells were maintained in Eagle's minimum essential medium (EMEM)-10% (volumeholume) fetal bovine serum until confluence was reached ( I ) . In most experiments, cells up to the seventh passage were plated in 24-well plates and maintained to confluence. To examine the production of proMMPs, TIMP, uPA, and PAI-I, the culture medium was changed to EMEM-0.2% (weight/volume) lactalbumin hydrolysate (LAH) after confluence. Harvested culture media were stored at -20°C until use. 3H-thymidine incorporation. Incorporation of 'Hthymidine into DNA was determined as described previously (8). Biosynthesis of GAGs and collagen. Labeling of confluent synovial cells with D-&'H-gIucosamine, extraction of labeled GAGs, digestion of GAGs with chondroitin AC and ABC lyases, and estimation of resulting unsaturated disaccharides were performed as described previously (9). To examine collagen biosynthesis, confluent cells were labeled with 'H-proline for 6 hours in EMEM-O.2% LAH-O.l mM ascorbate-0.5 pM @aminopropinnitrile. After the labeling, levels of collagen and noncollagenous protein were deter-
mined using purified bacterial collagenase as described previously (8). Labeling with ~-~~S-methionine, immunoprecipitation, and fluorography of proMMP-1, proMMP-3, and TIMP. Labeling of cells with L-3'S-methionine, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fluorography were carried out as described previously ( I I ) . Western blotting. Urokinase-type PA and PAI-I in culture media were analyzed by Western blotting, using their respective antibodies. SDS-PAGE using 10% (weight/ volume) acrylamide slub gel under reducing conditions, electrotransfer, and visualization of antigen were done as described previously (7).
RESULTS We first examined the effect of rHuIL-6 on 3H-thymidine incorporation into DNA. Recombinant human IL-6 (0.1 ng/ml, 10 ng/ml, and 20 ng/ml) and/or rHuIL-1 (0.5 ng/ml) did not show any effect on DNA synthesis by human synovial cells at either the growth or the stationary phase, indicating that neither cytokine influences cellular proliferation of rheumatoid synovial fibroblasts (results not shown). In contrast,
IL-6 IN RA SYNOVIAL FIBROBLASTS
rHuIL-l (0.5 ng/ml) augmented GAG production 2-fold, as compared with control cells (Figure IA). In addition, >75% of the GAG synthesized by synovial cells was found to be hyaluronate, and rHuIL-l accelerated its biosynthesis predominantly (Figure IB). Recombinant human IL-6, however, did not alter the levels or the composition of GAG in either control or IL-l-treated cells. Incorporation of 'H-proline into collagens and noncollagenous proteins in rheumatoid synovial fibroblasts was not influenced by rHuIL-6 and/or rHuIL-l (results not shown). The production of proMMP-l and proMMP-3 by untreated synovial cells was negligible (Figures 2A and B, lane I). Treatment of the cells with rHuIL-6 did not affect the production of proMMP-I or proMMP-3 (Figures 2A and B, lanes 2 4 , but rHuIL-l clearly promoted the production of both proMMPs (Figures 2A and B, lane 5). However, rHuIL-6 was found to further enhance IL-l-induced production of proMMPs, in a dose-dependent manner (Figures 2A and B, lanes 6-8): In cells treated with both rHuIL-6 (50 ng/ml) and rHuIL-l (0.5 ng/ml), production of proMMP- 1 and proMMP-3, respectively, was increased I .7-fold and 1 .Cfold, compared with cells treated with only rHuIL-I. Recombinant human IL- 1 increased the production of TIMP as well as of MMPs in rheumatoid synovial fibroblasts (Figure 2C, lane 5 ) . TIMP production was also increased, in a dose-dependent manner, by rHuIL-6 (Figure 2C, lanes 2 4 , with a maximal 2.4-fold stimulation at 50 ng/ml of rHuIL-6. Cotreatment of cells with both rHuIL-l and rHuIL-6 further enhanced the production of TIMP (Figure 2C, lanes 6-8), as was seen with proMMP-l and proMMP-3. Rheumatoid synovial cells constitutively produced a significant amount of uPA and its inhibitor PAI-I, and IL-6 by itself augmented the production of both uPA and PAI-1 in a dose-dependent manner (Figures 3A and B). However, in contrast to its stimulatory effect on the production of proMMP-1 and proMMP-3 (Figure 2), rHuIL-l did not modulate the production of uPA and PAI-I (results not shown).
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Figure 2. Effect of recombinant human interleukin-6 (rHulL-6) on rHulL-l-induced production of matrix metalloproteinases (MMPs) proMMP-l (A) and proMMP-3 (B),and tissue inhibitor of metalloproteinases (TIMP) (C), by human rheumatoid synovial fibroblasts. Confluent synovial cells were preincubated with rHulL-6 andor rHulL- I in medium for 24 hours, followed by labeling with '%-methionine in methionine-free medium containing cytokine(s1 for 3 hours. The harvested culture media were subjected to imrnunoprecipitation. followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography (see Materials and Methods for details). Bands corresponding to proMMPs or TIMP were excised and counted for radioactivity, and the respective amount of proMMPs or TIMP secreted into the media was then calculated in relation to the control. Lane I, Control. Lanes 2 4 , rHulL-6 (0.1. 20. and 50 ng/ml. respectively). Lane 5, rHulL-l (0.5 ng/ml). Lanes 6-8. rHulL-l (0.5 ng/ml) plus rHulL-6 (0.1. 20. and 50 ng/ml. respectively). Lane 9. rHulL-l (0.5 ng/ml) immunoprecipitated with nonimmune sheep I&.
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DISCUSSION This study demonstrated that neither rHuIL-6 nor rHuIL- 1 modulated 'H-thymidine incorporation by synovial cells at both the growth and the stationary phases, but rHuIL-I stimulated the biosynthesis of GAGS, predominantly hyaluronate production, whereas rHuIL-6 did not modulate basal or IL-Iinduced hyaluronate production in synovial fibroblasts. Neither cytokine altered collagen synthesis in
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Figure 3. Effect of recombinant human interleukin-6 (rHulL-6) on the production of urokinase-type plasminogen activator (u-PA) (A) and PA inhibitor I (PAI-I) (B) by human rheumatoid synovial fibroblasts. Confluent synovial cells were treated with rHulL-6 in medium for 2 days, after which the harvested culture media (1.5 ml) were mixed with 0.3 ml of 20% (weight/volume) trichloracetic acid. The precipitates were collected by centrifugation. dissolved in 25 pl of reducing sample buffer. and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Materials and Methods for details). Bands of u-PA were visualized by immunoblotting. The relative amount of u-PA or PAI-I was quantified by densitometric scanning, with control cells assigned a value of I .O. A. Lane I , Control. Lanes 2-5, rHulL-6 (0.I , I , 20. and 50 ng/ml. respectively). B, Lane 1 , Control. Lanes 2 4 , rHulL-6 (0.1. 20. and SO ng/ml, respectively).
synovial fibroblasts. Recent studies by Kandel et al (12) also showed that IL-6 did not alter the synthesis of proteoglycans and collagens in articular chondrocytes. It was reported that rHuIL-6 did not affect basal or IL- I-stimulated production of prostaglandin E, (PGE?)in human articular chondrocytes (12) or human synoviocytes (13). We have now confirmed this in the rheumatoid synovial cell system. Thus, it is unlikely that IL-6 participates in the hypertrophy of pannus tissue or the proinflammatory response of PGE, production in RA. Human recombinant IL-6 alone did not modulate the production of proMMP-l or proMMP-3, but it clearly augmented rHuIL- I-induced production of both proMMPs, indicating that IL-6 amplifies the signal transduction system of IL-I for proMMP production. Nevertheless, rHuIL-6 alone did enhance the production of TIMP in a dose-dependent manner in synovial cells, and additionally augmented 1L-Iinduced production of TIMP when cells were cotreated with both interleukins. These results are consistent with our previous findings in human skin and uterine cervical fibroblasts (14). Lotz and Guerne (13) reported that IL-6 could modulate the production of TIMP in human synoviocytes, but not basal or IL-I-stimulated collagenolytic activity. Thus, the action of IL-6 is likely dependent on the cell populations and experimental conditions used. Although we could
not quantify the exact amounts of MMP and TIMP induced by IL-6 and/or IL-I, it is probable that IL-6 participates in creating the imbalance between MMP and TIMP at sites of inflammation in RA and the eventual destruction of cartilage in vivo. It is well known that plasmin-PA participates directly and/or indirectly in the destruction of connective tissue components in RA, via activation of proMMP-3 (15-17) and direct destruction of proteoglycans (18). Recombinant human IL-6 stimulated human synovial cells to produce uPA in a dose-dependent manner. To our knowledge, this is the first evidence that IL-6 is one of the stimulators of uPA production. The appearance of PA activity at sites of inflammation is influenced by PA inhibitors, such as PAI-I (19). We observed that rHuIL-6 augmented PAI-I production by human rheumatoid synovial cells in a dose-dependent manner. Thus, IL-6 augmented the production of both uPA and its inhibitor. Although we could not evaluate the exact amounts of uPA and PAI-I in culture media, it is likely that IL-6 participates in the destruction of connective tissue components in RA by causing their imbalance. In fact, significantly high levels of uPA activity are found in the synovial fluid of patients with RA (20). In conclusion, we have demonstrated that IL-6 does not modulate PGE, production, synovial cell proliferation, or synthesis of GAGS and collagens, but
IL-6 IN RA SYNOVIAL FIBROBLASTS
d o e s stimulate the production of TIMP, uPA, and PAI-I, and augments the IL-I-stimulated production of MMP-1 and MMP-3, by human rheumatoid synovial cells. These results indicate that IL-6 may participate closely in the acceleration of catabolism of connective tissue components at sites of inflammation. Therefore, IL-6, together with I L - I , may be characterized as one of inflammatory mediators of joint destruction in RA.
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ACKNOWLEDGMENT We are grateful to Dr. H. Nagase (University of Kansas Medical Center, Kansas City, KS) for generously providing sheep anti-(human proMMP-I) and anti-(human MMP-3) antibodies and for critical reading of the manuscribt.
12.
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