Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-κB activation

The effect of secretory group II phospiholipase A(2) (sPLA(2)) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA(2) by itself barely stimulated nitrite production and MOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA(2) enzymes from sPLA(2)-transfected stable cells or for purified sPLA(2) from human synovial fluid. The effect of PLA(2) on INOS induction appears to be specific for the secretory type of PLA(2), LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA(2) such as 12-epi-scalaradial and rho-bromophenacyl bromide. In contrast, the cytosolic PLA(2)-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltriffuoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA(2), we observed that the sPLA(2)-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA(2)-transfected cells. The sPLA(2)-potentiated iNOS expression was associated with the activation of NF-KB, We found that the NF-KB inhibitor pyrrolidinedithiocarbamate prevented nitrite production, INOS induction, and mRNA accumulation by sPLA(2) plus LPS in Raw264.7 cells, Furthermore, EMSA analysis of the activation of the NF-KB involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA(2) plus LPS, Our findings indicated that sPLA(2), in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.