Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1beta and tumour necrosis factor-alpha.
OBJECTIVE
to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha), and to study the NADPH oxidase involvement in this production.


METHODS
Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1beta and TNF-alpha, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation.


RESULTS
Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase.


CONCLUSIONS
NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4).
Chenevier-Gobeaux
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Laboratoire de Biochimie A, Hôpital Cochin, Assistance Publique - Hôpitaux de Paris (AP-HP), 27 rue du Faubourg Saint-Jacques, 75679, Paris cedex 14, France. camille.gobeaux@cch.aphp.fr
Lemarechal
H
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Bonnefont-Rousselot
D
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Poiraudeau
S
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Ekindjian
O G
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Borderie
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D000368:Aged; D000369:Aged, 80 and over; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D053583:Interleukin-1beta / Q000502:physiology*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D053583:Interleukin-1beta; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C060505:neutrophil cytosolic factor 1