Modulation of cartilage destruction by select nonsteroidal antiinflammatory drugs.код для вставкиСкачать
257 MODULATION OF CARTILAGE DESTRUCTION BY SELECT NONSTEROIDAL ANTIINFLAMMATORY DRUGS In Vitro Effect on the Synthesis and Activity of Catabolism-Inducing Cytokines Produced by Osteoarthritic and Rheumatoid Synovial Tissue JEROME H. HERMAN, ALAN M. APPEL, and EVELYN V. HESS Non-enzymatic factors produced by synovial tissue can potentially mediate cartilage destruction by inducing the synthesis and release of matrix-degrading proteinases from chondrocytes. Pharmacologic control of this process is of potential clinical relevance. The in vitro effect of therapeutic concentrations of select nonsteroidal antiinflammatory drugs on the synthesis and activity of catabolism-inducing cytokines produced by 6-day explant cultures of osteoarthritic and rheumatoid synovial tissue was studied. Piroxicam regularly suppressed such factor synthesis by both types of tissue without significantly affecting total protein synthesis. This did not occur using sodium salicylate or indomethacin in osteoarthritis tissue cultures and was observed only occasionally in rheumatoid arthritis cultures. None of the nonsteroidal antiinflammatory drugs studied consistently blocked catabolism-inducing activity in osteoarthritis tissue, whereas piroxicam more consistently inhibited activity produced by rheumatoid arthritis tissue. Results suggest that the catabolisminducing factors produced by the 2 tissue sources may differ. From the Division of Immunology, Department of Internal Medicine, IJniversity of Cincinnati Medical Center, Cincinnati, Ohio. Supported in part by grants AM-16894 and AM-20615 from the NIH, an Arthritis Foundation Clinical Research Center grant, and a grant from Pfizer Laboratories. Jerome H. Herman, MD: Professor of Medicine, Department of Internal Medicine; Alan M. Appel, PhD: Research Assistant Professor, Department of Internal Medicine; Evelyn V. Hess, MD: Professor of Medicine and Director, Division of Immunology, Department of Internal Medicine. Address reprint requests to Jerome H. Herman, MD, Division of Immunology, ML 563, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, OH 45267. Submitted for publication February 24, 1986; accepted in revised form September 8, 1986. Arthritis and Rheumatism, Vol. 30, No. 3 (March 1987) Although nonsteroidal antiinflammatory drugs (NSAIDs) may effectively suppress articular inflammation, whether they are capable of modifying the natural course of cartilage destruction in joint disease is unknown. This possibility is suggested in experimental animal models of adjuvant arthritis (1-3), antigen-induced arthritis (4), type I1 collagen-induced arthritis (5-7), and osteoarthritis (OA) (8,9) in which the administration of select classes of NSAIDs has been associated with diminished pannus formation, cartilage erosion, and/or bone destruction. Although a given type of arthritis may have unique precipitating features, most forms of disease probably share common pathway mechanisms which induce and perpetuate cartilage destruction (10). One of the earliest manifestations in the incipient stage of cartilage involvement is a focal, or at times more diffuse, depletion of matrix proteoglycans. This appears to be primarily enzyme-mediated; however, the source of responsible proteinases remains unclear. The significance of enzymes evolving from synovial fluid must be questioned in that pathologic effusions contain proteinase inhibitors (1 1,12). Enzymes released in the immediate proximity of cartilage are probably of greater importance. They may originate in granulation tissue pannus, or as a consequence of phagocytosis or reversed endocytosis associated with immune complex deposition in the superficial collagenous layer of cartilage (13,14). Such sources of enzymes, however, would only provide a partial explanation for matrix destruction in that proteoglycan depletion may, at times, be observed in midzone and deeper regions of cartilage, which are anatomically divorced from events that occur in the superficial layer (15). HERMAN ET AL Recognizing that chondrocytes contain both acid and neutral proteinases (16,17), attention has focused in recent years on their potential role in mediating matrix depletion. Catabolin, a component(s) in a family of interleukin-1 (IL-1) molecules, is a product of synovial tissue which induces the synthesis and release of matrix-degrading proteinases from viable chondrocytes (18,191. IL-1 activity is produced by a variety of cell types, which include synovial fibroblasts, activated monocyte/macrophages, and monokine-dependent activated T lymphocytes (2CL23). IL-1 has been identified in pathologic synovial fluids (24,25). Its potential significance in the pathogenesis of cartilage destruction in rheumatoid arthritis (RA) and OA remains speculative. One cannot exclude the production of additional forms of chondrocyte catabolism-inducing activity by pathologic synovium. Based on their capacity to induce collagenase release from synovial cells and dermal fibroblasts, macrophage-derived tumor necrosis factor (26) and products of mast cells (27) have such a potential. The present study was conducted to assess the effect of select classes of NSAIDs on the synthesis and activity of catabolism-inducing factors produced by RA and OA syrlovial tissue. MATERIALS AND METHODS Synovial tissue culture. Synovium samples were obtained during total hip or knee replacement from 16 patients who fulfilled American Rheumatism Association diagnostic criteria for classic RA (28). Samples were also taken from 15 patients diagnosed as having primary OA, based on the absence of predisposing secondary factors and the presence of radiologic evidence of disease. A 72-hour NSAID washout period was instituted prior to surgery. This time period was selected arbitrarily, since data assessing synovial tissue levels of various classes of NSAIDs after their discontinuation are unavailable. The administration of steroids, hydroxychloroquine, chrysotherapy, penicillamine, o r cytotoxic drugs was not interrupted in the RA group. Tissue was immediately processed under aseptic conditions. Representative portions were submitted for routine histologic analysis. The synovium was dissected free of fat and adherent fibrous tissue and was minced into 2-3-mm segments. Contaminating blood and synovial fluid were removed by repetitive washing over a 4-hour period in Hanks' balanced salt solution, pH 7.4, augmented with penicillin (100 units/ml) and streptomycin (100 pg/rnl). Tissue was maintained in Eagle's minimum essential medium (EMEM; 10 ml medium per gram of tissue) supplemented with 2 mM L-glutamine, 25 mM HEPES buffer, antibiotics, and 15% a2-macroglobulin-depleted, decomplemented newborn calf serum. Six-day cultures were established at 37"C, in an atmosphere of 5% C 0 2 and 95% air, in the presence and absence of piroxicam, 5 pg/ml (lot no. 9603-262-2m; Pfizer, Groton, CT), indomethacin, 1.5 &ml, or sodium salicylate, 160 &ml. Like medium was exchanged at 72 hours, and that derived from the 2 harvest periods was pooled. In further experiments to assess the effect of NSAIDs on the activity of catabolism-inducing factors, drugs were added to synovium conditioned medium (SCM) or control medium at the time of harvest. The concentrations of NSAIDs used corresponded to therapeutic plasma levels achieved in humans after oral drug administration. Each drug was solubilized in 0.93M sodium carbonate buffer, pH 9.0, and was subsequently diluted with fresh medium to achieve the desired concentration. Assay for catabolism-inducingactivity. SCM and control media (media not exposed to synovial tissue, but maintained in the presence or absence of NSAIDs, or media to which "SAIDs were added at culture termination) were diluted 1:l with fresh medium and exposed to bovine weight-bearing articular cartilage discs (4 x 1 mm) which were prelabeled with 35S04 as described previously (22). Aliquots of cartilage were repetitively frozen and thawed to destroy chondrocytes, and were heated to 60°C for 1 hour to inactivate indigenous enzymes. This nonviable substrate was used to measure proteoglycan-degrading enzyme activity in SCM, whereas viable cartilage provided an index of catabolism-inducing activity as well. Lipopolysaccharide (25 pg/ml) was used in assays as a positive control because of its ability to induce chondrocyte proteinase release (29). Cultures were maintained at 37"C, in 5% COz and 95% air for 6 days. Analogous fresh medium was replaced at 72 hours. The labeled sulfate present in combined culture supernatants and explant washes and that remaining in acid-hydrolyzed explants (6N HCl, lOO"C, 1 hour) was determined by scintillation counting. Catabolism-inducing activity was expressed as the percentage of 3sS04 released from viable cartilage minus that released from nonviable tissue. Release from nonviable tissue was negligible (mean 5 SEM 0.37 4 0.28%). Assays were performed in replicates of 5. The significance of differences between 35S04release occurring in SCM derived from NSAID-treated and untreated cultures and that in corresponding control media was determined by 2-tailed t-test. Effect of piroxicam on synovial tissue protein synthesis. Six-day OA and RA synovial tissue cultures of comparable wet weight were established in leucine-free EMEM shpplemented as described above, in the presence and absence of 5 pg/ml of piroxicam. Cultures were continuously pulsed with 2 pCi/ml of L[4,5-3H]-leucine (New England Nuclear, Boston, MA). Medium and label were exchanged at 3 days. Culture supernatants were dialyzed free of unincorporated label, and an aliquot was counted. Tissue was resuspended in phosphate buffered saline, pH 7.4, and homogenized (Polytron; Brinkmann Instruments, Westbury, NY). An aliquot was removed for protein determination according to the method of Lowry et a1 (30). The clarified homogenate was precipitated with trichloroacetic acid (TCA) in a final concentration of 5%. The precipitated protein was solubilized in 1N NaOH, and an aliquot was counted. Newly synthesized protein was expressed as the counts per minute derived from culture supernatants and the tissue homogenate per gram of tissue protein content. 259 CARTILAGE DESTRUCTION BY NSAID catabolism-inducing factor was analyzed in 12 cultures. As shown in Figure 2A, piroxicam appeared to suppress factor synthesis in 11 of the 12 assays. The postculture addition of drug to SCM blocked activity in only 3 of these experiments. In contrast, in 6 assays, neither indomethacin nor sodium salicylate affected factor synthesis (Figures 2B and C). When these drugs were added to SCM, catabolism-inducing activity was blocked in 1 experiment in each group. Rheumatoid arthritis synovium. Sixteen specimens that produced catabolism-inducing activity were studied. Piroxicam appeared to suppress factor synthesis in 11 of 12 experiments, whereas postculture addition of drug had a blocking effect in 8 of 11 studies (Figure 3A). Indomethacin suppressed factor synthesis and blocked activity in 2 of 7 experiments (Figure 3B), whereas comparable effects were observed in only 1 of 6 assays using sodium salicylate (Figure 3C). Effect of piroxicam on synovial tissue protein synthesis. Because piroxicam, in contrast to the other NSAIDs studied, appeared to consistently suppress catabolism-inducing factor synthesis, experiments RESULTS Effect of NSAIDs on cartilage catabolism. NSAIDs alone had no effect on cartilage proteoglycan catabolism as reflected by release of labeled sulfate from viable cartilage during the 6-day culture period. Representative experiments are shown in Figure 1. Results using NSAID-containing media cultured for 6-day periods (medium exchanged at 3 days) or media to which NSAIDs were freshly added at the time of catabolic assay were comparable. The percentage of spontaneously released 35S04 differed between batches of cartilage used. The mean percentage k SEM of label release, contrasting, respectively, naive cultures to NSAID-exposed cartilage, was as follows: 2.98 versus 30.3 ? 2.89; indopiroxicam, 31.01 methacin, 31.63 4.1 versus 30.88 f. 4.1; Na salicylate, 31.7 ? 3.9 versus 30.9 f. 3.95. Osteoarthritic synovium. Fifteen OA specimens producing catabolism-inducing activity were studied. The limited quantity of synovial tissue available per specimen restricted the number of experiments that would permit simultaneous drug analysis using each of the 3 classes of NSAIDs investigated. The in vitro effect of piroxicam on the synthesis and activity of * * 60 lndornethactn (1.5 UQlml) 50 m -Wm 40 $ 40 30 d m 2 $ a+ 30 0 ’ # 20 20 10 5 10 I 2 3 4 5 6 7 9 8 10 I 2 7 8 3 4 5 6 7 8 9 1 0 60 50 W f 40 -m0 Kc 30 0’ $ 20 ip 10 5 1 2 3 4 5 6 9 10 Figure 1. The effect of nonsteroidal antiinflammatory drugs (NSAIDs) on the release of ’%04 from viable bovine articular cartilage explants in 6-day cultures. Results represent the mean SEM of 5 replicate assays * per experiment. Comparable results were obtained whether NSAIDs were maintained in culture for 6 days (comparable with synovial tissue cultures) prior to their addition to 3sS04-prelabeled cartilage or added freshly to explants. Prior to assay, control and NSAID-containingmedia were diluted 1 : 1 with fresh medium. 260 HERMAN ET AL A P m 35 r 30 - E < 30 i 0. Indomethacm 25 . 10- Y) SCM Alone SCM Generated m Presence ol Drug t - 15 - 10 - POS-CUI1YrB Drug Addition to SCM of 5 - # 01 I SCM Alone SCY Qmerated k R..me of Drug POSt-CURUe AddRlon 01 Drug to SCY Figure 2. The effect of nonsteroidal antiinflammatory drugs (NSAIDs) on the synthesis (synoviumconditioned medium [SCM] generated in presence of drug) and activity (postculture addition of drug to SCM) of the catabolism-inducingfactor produced in 6-day explant cultures of osteoarthritic synovial tissue. Each joined series of data points represents the mean percentage of '304release (assays performed in replicates of 5 ) greater than corresponding control values (media with or without NSAID) derived from independent tissue specimens. Closed circles represent results that are significantly different from the effect of SCM alone. were performed to exclude a drug toxic effect; total protein synthesis by synovial tissue cultured in the presence and absence of the drug was assessed. As shown in Table 1, piroxicam had no suppressive effect on the incorporation of 'H-leucine into TCAprecipitable counts in either 6-day OA or RA synovial tissue cultures, which suggests a selectivity of effect on catabolism-inducing factor production. The percentage of 3H-leucine-incorporated counts released into culture supernatants was comparable between drug-exposed (34.75 2 3.05%) and untreated (34.8 t 4.3%) OA and RA specimens (Table 1). In one experiment using RA synovial tissue, a small, but statistically significant, increase in 3H-leucine incorporation was observed. DISCUSSION Explant cultures of idiopathic OA and RA synovial tissue have been shown to produce catabolism-inducing activity, as measured by release of 35S04-labeledproteoglycans from viable articular cartilage substrates. The mean percentage of SCMassociated 35S04-proteoglycanrelease that exceeded control medium values in 6-day cartilage cultures varied from 19.1 ? 1.8% (range 10.5-34.9) in OA specimens to 21.7 k 2.3% (range 6.6-36.9) in RA tissue cultures. The range variance observed in the 16 RA patients may reflect, in part, the medication still being administered to them at the time of surgery (5-10 mg prednisone, 7 patients; hydroxychloroquine I pa- CARTILAGE DESTRUCTION BY NSAID 261 35 P 30. 25 - - ; 20 - j 15 - 10 - 20- 0 IS- f 10- U I 0 -- -., SCM Alone SCM Generated m Presence ot Drug Post-Culture Addmon 01 Drua 10 SCM SCM Alone 35 SCM Generaled I" presence of Drug Past-Culture Addition 01 Drug to SCM r SCM Alone SCM Qsneraled Post-Culture Addition 01 in Prewnce of Drug Drug l o SCM Figure 3. The effect of nonsteroidal antiinflammatory drugs (NSAIDs) on the synthesis (synoviumconditioned medium [SCM] generated in the presence of drug) and activity (postculture addition of drug to SCM) of the catabolism-inducing factor produced in 6-day explant cultures of rheumatoid synovial tissue. Each joined series of data points represents the mean percentage of 35S04release (assays performed in replicates of 5 ) greater than corresponding control values (media with or without NSAID) derived from independent tissue specimens. Closed circles represent results that are significantly different from the effect of SCM alone. Table 1. In vitro effect of piroxicam on synovial membrane protein synthesis* Synovial tissue ~ Osteoarthritis Experiment 1 Experiment 2 Rheumatoid arthritis Experiment 1 Experiment 2 ~ ~ % difference Pi' Control Piroxicam 118.2 ? 1.7 83.7 2 3.5 118.5 -+ 2.8 90.4 2 1.6 0.25 2 2.4 8.01 f 1.9 0.925 0.107 341.4 f 10.7 199.3 2 1.7 316.5 f 6.6 214.5 2 5.2 -7.3 t 1.9 7.1 f 2.6 0.07 0.021 * Values are the mean f SEM cpm ( X 10-3/gm)of 3H-leucine-incorporatedcounts derived from the sum of nondialyzable cpm released into culture supernatants and the trichloroacetic acid-precipitable counts present in tissue homogenates. Three aliquots of synovial tissue of comparable wet weight were used for each phase per experiment. t Difference from control medium values as determined by 2-tailed t-test. 262 tient; penicilllamine 1 patient, methotrexate 1 patient; azathioprine 1 patient). These results are to be contrasted with a mean percentage release of 18.6 2 2.9% (range 13.3-19.2) produced by SCM derived from 3 normal synovial tissue specimens cultured under identical conditions (data not shown). We have previously shown that the labeled proteoglycans released in the presence of such SCM are heterogeneous in size and that monomers have a decreased capacity to aggregate with hyaluronic acid (31). Thus, significant enzymatic cleavage has occurred. Piroxicam used in vitro in a concentration comparable with a therapeutic serum level obtainable in humans has been shown to significantly suppress the synthesis of catabolism-inducing factor(s) by OA and, possibly, by RA synovial tissue. Because piroxicam significantly blocked factor activity in the majority of RA specimens, it remains uncertain whether inhibitory responses observed with conditioned media generated in the presence of piroxicam reflected a carryover effect of drug on factor activity. A suppression of synthesis in the absence of blocking of activity occurred in only 2 assays. Experiments assessing the effect of piroxicam on synovial tissue incorporation of 3H-leucine into newly synthesized protein indicate that this effect was not due to drug toxicity. Piroxicam did not inhibit total protein synthesis, which suggests a selectivity of the drug-induced suppressive response. Piroxicam alone had no significant effect on the release of proteoglycans from normal cartilage, further indicating a drugmediated effect at a synovial tissue level. Indomethacin and sodium salicylate, in the single therapeutic concentrations used, did not affect the synthesis of catabolism-inducing factor by OA synovium. Occasional suppression occurred in RA tissue cultures. N o consistent blocking effect on OA catabolic factor activity occurred using any of the NSAIDs studied. However, in contrast with indomethacin and sodium salicylate, piroxicam more consistently blocked activity produced by RA synovium. This suggests that there are potential differences between the catabolism-inducing factor generated from OA tissue and that generated from RA tissue, and that piroxicam may be an effective blocker for that produced by RA synovial tissue. Monocyte IL-1 heterogeneity is recognized (32-34), but there has been little effort to contrast the quality of that produced by synovial tissue from patients with different diseases. HERMAN ET AL Wood et a1 ( 3 9 , using enzymatically disaggregated OA and RA synovium, have recently reported a difference in the isoelectric focusing profiles of IL-1 obtained from these 2 sources. Unfractionated SCM was used in the experiments reported here. No attempt was made to identify the responsible catabolism-inducing cytokine(s). We have, however, been able to show that comparably derived OA and RA SCM does contain IL-1 activity, as assessed using a C3HIHeJ mouse thymocyte proliferation assay. In addition, chromatographic analyses performed on OA SCM have shown that catabolisminducing activity primarily elutes in a molecular weight range of 12,000-20,000, consistent with IL-1 (36). The pathophysiologic significance of IL-1 produced by OA and RA synovial tissue currently remains speculative. IL-1 may have potential deleterious effects that contribute to cartilage and bone destruction beyond its property of inducing the synthesis and release of matrix-degrading proteinases from chondrocytes. It is able to stimulate chondrocyte release of prostaglandins and plasminogen activator (23,37), suppress chondrocyte glycosaminoglycan synthesis (38), induce the release of prostaglandins, plasminogen activator, and matrix-degrading proteinases from dendritic synoviocytes (39), induce bone resorption (40,41), and suppress bone collagen and osteocalcin synthesis (42). Therefore, pharmacologic intervention to modulate the synthesis and/or activity of IL-1 would appear to be highly desirable. Few studies have addressed the effect of NSAIDs on the synthesis or activity of catabolisminducing factors produced by monocyte/macrophages or synovial tissue. Ridge et a1 (43) reported that indomethacin had no effect on such factor synthesis by lipopolysaccharide-stimulated rabbit peritoneal macrophages. In contrast, Phadke et a1 (44) observed that the addition of aspirin and indomethacin (but not phenylbutazone or fenoprofen) to such macrophageconditioned media could suppress chondrocyte collagenase and other neutral proteinase release without. affecting cell viability. Why there is a discrepancy between the latter study and our observation that indomethacin was essentially ineffectual in blocking catabolism-inducing activity present in SCM is not clear. It could perhaps reflect a difference in the quality of factor(s) produced by the different cell/tissue sources studied. Rainsford has recently reported the effect of various NSAIDs on purified porcine leukocyte IL-1 activity (45). Therapeutic concentrations of diflunisal, CARTILAGE DESTRUCTION BY NSAID indomethacin, mefenamic acid, naproxen, piroxicam, salicylate, and sulindac were without effect. Pharmacologic concentrations of benoxaprofen, diclofenac, fenclofenac, indomethacin, and sulindac sulfide did effectively block activity. However, a compromise of chondrocyte viability was not excluded as a basis for reduction in IL- 1-induced proteoglycan release from cartilage. That piroxicam (0.1-500 pM) did not block the activity of leukocyte-derived IL- 1 is consistent with our results using OA SCM, but differs from that employing conditioned media derived from RA tissues. Although the cartilage substrate (bovine nasal) and duration of cartilage culture (4 days) differed from our experimental design, results again suggest a possible difference in catabolism-inducing factors produced by the 2 sources studied. In experiments employing RA synovium cocultured with bovine nasal cartilage (46) and SCM derived from a rabbit model of antigen-induced arthritis (47), indomethacin appeared to have no effect on either the synthesis or activity of catabolism-inducing factors. Jones et a1 (48), using normal bovine synovium, found that indomethacin had a variable effect on SCMmediated cartilage breakdown. Sheppeard et a1 (49), using conditioned medium derived from normal porcine synovium, noted that aspirin failed to affect catabolism-inducing activity. The majority of the above studies have failed to demonstrate the effectiveness of therapeutic concentrations of NSAIDs of diverse classes in modulating either the synthesis or activity of catabolism-inducing activity generated from leukocyte or synovial tissue sources. The current experimental data, however, indicate that a member of at least 1 class of these compounds, piroxicam, has the potential capacity to suppress such factor synthesis by OA and RA synovium, as well as block the activity of that produced by RA tissue. Sheppeard and Couchman (50) have recently reported findings that substantiate the uniqueness of this drug. They observed that the addition of a therapeutic concentration of piroxicam to normal porcine synovial tissue significantly suppressed SCM-induced cartilage breakdown. 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