Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes.
OBJECTIVE
To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1beta or lipopolysaccharide (LPS).


METHODS
Human chondrocytes in monolayer culture were incubated for 3 h with ROS generating molecules such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 microM), 3-morpholinosydnonimine (SIN-1, 100 microM), with chemically synthesised peroxynitrite (ONOO-, 10 microM) or hydrogen peroxide (H2O2, 100 microM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 microg/ml) or IL-1beta (1.10(-11) M). IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression was analysed by real time and quantitative RT PCR. IL-6, IL-8 and prostaglandin (PG) E2 productions were assayed by specific immunoassays. Nitrite was measured in the culture supernatants by the Griess procedure.


RESULTS
LPS and IL-1beta stimulated IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression. SNAP significantly downregulated LPS induced overall gene expressions, whereas SIN-1 had no effect. ONOO- inhibited iNOS and COX-2 gene expression but not that of the cytokine genes. When chondrocytes were incubated with IL-1beta, SIN-1 and ONOO dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1beta induced gene expressions.


CONCLUSIONS
These data provide an evidence that ROS may have anti-inflammatory properties by depressing inflammatory gene expression. Further, we demonstrate that ROS effects are dependent on the nature of radical species and the signalling pathway that is activated. These findings should be taken into consideration for the management of antioxidant therapy in treatment of inflammatory joint diseases.
Mathy-Hartert
M
M
University of Liege, Institute of Pathology, CHU Sart-Tilman, B-4000 Liège, Belgium.
Martin
G
G
Devel
P
P
Deby-Dupont
G
G
Pujol
J P
JP
Reginster
J Y
JY
Henrotin
Y
Y
D002358:Cartilage, Articular / Q000166:cytology; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D053938:DNA Fragmentation / Q000187:drug effects; D015232:Dinoprostone / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation / Q000235:genetics*; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D004247:DNA; D007770:L-Lactate Dehydrogenase; D015232:Dinoprostone