Regulation by reactive oxygen species of interleukin-1beta, nitric oxide and prostaglandin E(2) production by human chondrocytes.

OBJECTIVES: To determine the effects of two drugs, N-monomethyl-L-arginine (L-NMMA) and N-acetylcysteine (NAC), on interleukin-1beta (IL-1beta), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production by human chondrocytes. The effect of aceclofenac (ACECLO), a non-steroidal antiinflammatory drug (NSAID), was also examined.

METHODS: Human chondrocytes were enzymatically isolated from osteoarthritic knee cartilage and then maintained in culture in suspension for 48h in the absence or in the presence of lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5mM), NAC (1mM) or ACECLO (6.10(-6)M). IL-1beta and PGE(2) productions were quantified by specific immunoassays. Nitrite was measured in the culture supernatants by a spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) and IL-1beta gene expressions were quantified by transcription of mRNA followed by real time and quantitative polymerase chain reaction. COX-2 protein expression was analysed by Western blot.

RESULTS: LPS markedly increased the expression of IL-1beta, iNOS and COX-2 genes. In parallel, NO(2) and PGE(2) amounts found in the culture supernatants were significantly enhanced whereas IL-1beta was immunologically undetectable. The addition of L-NMMA (0.5mM) fully blocked LPS-induced NO production but greatly increased PGE(2) production, suggesting a negative effect of NO on PGE(2) synthesis. Inversely, NO production was stimulated by NAC while PGE(2) production was not affected. Interestingly, NAC increased the IL-1beta and iNOS mRNA levels but did not significantly modify COX-2 mRNA expression. L-NMMA did not significantly affect the expression of IL-1beta, iNOS and COX-2. The amount of COX-2 protein did not change in the presence of the antioxidants. Finally, ACECLO fully blocked the production of PGE(2) by chondrocytes without affecting the levels of COX-2 mRNA.

CONCLUSIONS: The stimulation of IL-1beta, NO and PGE(2) production by LPS is differentially controlled by reactive oxygen species (ROS). In fact, L-NMMA and NAC have different mechanisms of action on the regulation of NO and PGE(2) productions. L-NMMA fully inhibits NO but increases PGE(2) production whereas NAC up-regulates NO but does not modify PGE(2) synthesis. The stimulating effect of L-NMMA on PGE(2) production is not controlled at the transcriptional level. These findings suggest that antioxidant therapy could have different effects according to the oxygen radical species targeted.