Implication of cytosolic phospholipase A2 (cPLA2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A2 (cPLA2) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA2 inhibitors prior to IL-1β or TNF-α treatment. Total cellular AA concentrations and PLA2 activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA2 inhibitors inhibited both IL-1β and TNF-α-induced superoxide production in RA and OA cells. Basal PLA2 activity was significantly more important in RA cells than in OA cells; PLA2 activity was increased in IL-1β and TNF-α pre-treated RA cells, and cPLA2 inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1β and TNF-α, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H+ channel.