Modulation of human chondrocyte metabolism by recombinant human interferon.

Interferon gamma (IFN gamma) is found to be elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis, suggesting its implication in joint disease pathogenesis. In this study, we investigated the effects of IFN gamma on the production of cytokines (IL-6, IL-8, IL-10), prostaglandin E(2)(PGE(2)), proteoglycans (PG), nitric oxide (NO), interleukin-1 receptor antagonist (IL-1ra) and stromelysin by non-stimulated and IL-1 beta-treated human chondrocytes. The role played by NO in the responses of chondrocytes to IFN gamma was also examined by incubation of chondrocytes with N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Enzymatically isolated human chondrocytes were cultured for 48 h in the absence or presence of IL-1 beta, IFN gamma or N(G)-monomethyl-L-arginine (L-NMMA) added solely or in combination. The productions of IL-6, IL-8, IL-10, IL-1ra and stromelysin were measured by enzyme amplified sensitivity immunoassays (EASIA). PG and PGE(2)were quantified by specific radioimmunoassays (RIA). Nitrite concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. As expected, IL-1 beta highly stimulated NO, IL-6, IL-8, IL-10, IL-1ra, PGE(2)and stromelysin synthesis, but dramatically decreased PG production. NO, IL-6, IL-1ra and PGE(2)production by non-stimulated chondrocytes was dose-dependently increased by IFN gamma while PG production was inhibited. In the absence of IL-1 beta, IL-10 was undetectable in the culture supernatants. At the doses of 10 and 100 U/ml, IFN gamma markedly inhibited the constitutive and IL-1 beta-stimulated IL-8, IL-10 and stromelysin productions. Interestingly, IFN gamma synergized with IL-1 beta to increase NO, IL-6, IL-1ra and to depress PG production. As previously reported, the inhibition of NO synthesis by the competitive inhibitor L-NMMA led to enhancement of IL-6, IL-8 and PGE(2)production by IL-1 beta treated chondrocytes, but did not significantly modify IL-10, PG and MMP-3 productions. Inhibition of NO synthase significantly inhibited the stimulating effect of IFN gamma on IL-6 and IL-1ra but did not affect the inhibitory effect of IFN gamma on IL-8, PG or stromelysin production. These findings suggest that IFN gamma and IL-1 synergistically stimulate the production of IL-6, IL-1ra, NO and PGE(2)and inhibit PG synthesis. By contrast, IL-1 beta and IFN gamma have opposite effects on IL-8, IL-10 and stromelysin productions. These effects are not reversed by L-NMMA, suggesting that NO is not the principal mediator involved in responses of chondrocytes to IFN gamma.

Henrotin YE ，Zheng SX ，Labasse AH ，Deby GP ，Crielaard JM ，Reginster JY ... -  《OSTEOARTHRITIS AND CARTILAGE》

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

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. 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. 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. 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.

Mathy-Hartert M ，Deby-Dupont GP ，Reginster JY ，Ayache N ，Pujol JP ，Henrotin YE ... -  《OSTEOARTHRITIS AND CARTILAGE》

Effects of nimesulide and sodium diclofenac on interleukin-6, interleukin-8, proteoglycans and prostaglandin E2 production by human articular chondrocytes in vitro.

OBJECTIVES:The aim of this study was to investigate the effects of two nonsteroidal anti-inflammatory drugs (NSAIDs), nimesulide and sodium diclofenac, on the production of proteoglycans (PG), prostaglandin E2 (PGE2) and cytokines (IL-6 and IL-8) by human articular chondrocytes in vitro. METHODS:Enzymatically isolated chondrocytes were cultured under constant agitation in a well defined culture medium. Specific radioimmunoassays were used to quantify PG and PGE2 production. Cytokine production (IL-6 and IL-8) was assayed by enzyme amplified sensitivity immunoassays (EASIAs). RESULTS:At a concentration of 3 micrograms/ml, nimesulide did not affect the PG production by chondrocytes. This concentration was superior to the highest level of nimesulide found in the synovial fluid of patients with rheumatoid arthritis 3 hours after the last oral administration of nimesulide (100 mg twice daily for 7 days). At 6 micrograms/ml a significant reduction in the PG content was obtained in the cellular phase in 5 out of the 8 cultures investigated. No similar effect was observed in the culture supernatants. Above this concentration nimesulide inhibited PG production in a dose-dependent manner. At concentrations ranging from 0.005 to 1 microgram/ml diclofenac did not significantly alter PG production. At therapeutic concentrations PGE2 production was totally inhibited by nimesulide, thus suggesting that PG inhibition is not linked to PGE2 production. Nimesulide inhibited PGE2 production by unstimulated (IC50 = 6 ng/ml) and IL-1 beta-stimulated (IC50 = 6.9 ng/ml) chondrocytes. At these concentrations, PGE2 production was fully inhibited by diclofenac. Furthermore, both nimesulide and diclofenac at therapeutic concentrations significantly decreased spontaneous and IL-1 beta-stimulated IL-6 production by human chondrocytes, but did not modify IL-8 production. CONCLUSION:From the results of this study we conclude that nimesulide and diclofenac at therapeutic concentrations are potent inhibitors of PGE2 and IL-6 production while they do not modify proteoglycan or IL-8 production.

Henrotin YE ，Labasse AH ，Simonis PE ，Zheng SX ，Deby GP ，Famaey JP ，Crielaard JM ，Reginster JY ... -  《CLINICAL AND EXPERIMENTAL RHEUMATOLOGY》

Avocado/soybean unsaponifiables increase aggrecan synthesis and reduce catabolic and proinflammatory mediator production by human osteoarthritic chondrocytes.

Henrotin YE ，Sanchez C ，Deberg MA ，Piccardi N ，Guillou GB ，Msika P ，Reginster JY ... -  《JOURNAL OF RHEUMATOLOGY》

Aceclofenac increases the synthesis of interleukin 1 receptor antagonist and decreases the production of nitric oxide in human articular chondrocytes.

Maneiro E ，López-Armada MJ ，Fernández-Sueiro JL ，Lema B ，Galdo F ，Blanco FJ ... -  《JOURNAL OF RHEUMATOLOGY》