MODULATION OF IL-1-INDUCED CARTILAGE INJURY BY NO SYNTHASE INHIBITORS- A COMPARATIVE-STUDY WITH RAT CHONDROCYTES AND CARTILAGE ENTITIES

1 Nitric oxide (NO) is produced in diseased joints and may be a key me diator of IL-1 effects on cartilage. Therefore, we compared the potenc y of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethyl isothiourea (AETU)] and classical [N-omega-monomethyl-L-arginine (L-NM MA), N-omega-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin- 1 beta (rhIL-1 beta) on rat cartilage anabolism. Three different cultu re systems were used: (1) isolated chondrocytes encapsulated in algina te beads; (2) patellae and (3) femoral head caps. 2 Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presenc e of rhIL-1 beta with a daily change of incubation medium to obtain op timal responses on proteoglycan synthesis and NO production. Proteogly can synthesis was assessed by incorporation of radiolabelled sodium su lphate [(Na2SO4)-S-35] and NO production by cumulated nitrite release during the period of study. 3 Chondrocytes and patellae, as well as fe moral head caps, responded concentration-dependently to IL-1 beta chal lenge (0 to 250 U ml(-1) and 0 to 15 U ml(-1) respectively) by a large increase in nitrite level and a marked suppression of proteoglycan sy nthesis. Above these concentrations of IL-1 beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitr ite release still increased thus suggesting different concentration-re sponse curves. 4 When studying the effect of NOS inhibitors (1 to 1000 mu M) on NO production by cartilage cells stimulated with IL-1 beta ( 25 U ml(-1) or 5 U ml(-1)), we observed that: (i) their ability to red uce nitrite level decreased from chondrocytes to cartilage samples, ex cept for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA greater than or equal to SMT>AG greater than or equal to L-NAME and (iii) AETU was cytotoxic w hen used in the millimolar range. 5 When studying the effect of NOS in hibitors on proteoglycan synthesis by cartilage cells treated with IL- 1 beta, we observed that: (i) they had more marked effects on proteogl ycan synthesis in chondrocytes than in cartilage samples; (ii) they co uld be roughly classified in the following rank order of potency: L-NA ME greater than or equal to L-NMMA>>AG>SMT>>AETU and (iii) potentiatio n of the IL-1 effect by AETU was consistent with cytotoxicity in the m illimolar range. 6 D-isomers of L-arginine analog inhibitors (1000 mu M) were unable to correct nitrite levels or proteoglycan synthesis in IL-1 beta treated cells. L-arginine (5000 mu M) tended to reverse the correcting effect of L-NMMA (1000 mu M) on proteoglycan synthesis, thu s suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. 7 Dexamethasone (0.1 to 100 mu M) (i) failed to inhibit NO production in femoral head caps and cho ndrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1 beta on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat c hondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. 8 In conclusion, our results sug gest that (i) NO may account only partially for the suppressive effect s of IL-1 beta on proteoglycan synthesis, particularly in cartilage sa mples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cel ls and (iii) L-arginine analog inhibitors are more promising than S-su bstituted isothioureas for putative therapeutical uses.