Regulation by reactive oxygen species of interleukin-1beta, nitric oxide and prostaglandin E(2) production by human chondrocytes.
OBJECTIVE
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.
Mathy-Hartert
M
M
Bone and Cartilage Metabolism Research Unit, Belgium.
Deby-Dupont
G P
GP
Reginster
J-Y L
JY
Ayache
N
N
Pujol
J-P
JP
Henrotin
Y E
YE
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008565:Membrane Proteins; D008875:Middle Aged; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D020370:Osteoarthritis, Knee / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone