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 mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [N-omega-monomethyl-L-arginine (L-NMMA), 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 culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. 2 Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1 beta with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [(Na2SO4)-S-35] and NO production by cumulated nitrite release during the period of study. 3 Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1 beta challenge (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 synthesis. Above these concentrations of IL-1 beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response 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 reduce nitrite level decreased from chondrocytes to cartilage samples, except 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 when used in the millimolar range. 5 When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1 beta, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME greater than or equal to L-NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar 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, thus 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 chondrocytes 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 chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. 8 In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1 beta on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.