TLR3-dependent induction of nitric oxide synthase in RAW 264.7 macrophage-like cells via a cytosolic phospholipase A2/cyclooxygenase-2 pathway

dsRNA is a by-product of viral replication capable of inducing an inflammatory response when recognized by phagocyte cells. In this study, we identify group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) as an effector of the antiviral response. Treatment of RAW 264.7 murine macrophage-like cells with the dsRNA analog polyinosinic:polycytidylic acid (poly-IC) promotes the release of free arachidonic acid that is subsequently converted into PGE(2) by the de novo-synthesized cyclooxygenase-2 (COX-2) enzyme. These processes are blocked by the selective cPLA(2)alpha inhibitor pyrrophenone, pointing out to cPLA(2)alpha as the effector involved. In keeping with this observation, the cPLA(2)alpha phosphorylation state increases after cellular treatment with poly-IC. Inhibition of cPLA(2)alpha expression and activity by either small interfering RNA (siRNA) or pyrrophenone leads to inhibition of the expression of the inducible NO synthase (iNOS) gene. Moreover, COX-2-derived PGE(2) production appears to participate in iNOS expression, because siRNA inhibition of COX-2 also leads to inhibition of iNOS, the latter of which is restored by exogenous addition of PGE2. Finally, cellular depletion of TLR3 by siRNA inhibits COX-2 expression, PGE2 generation, and iNOS induction by poly-IC. Collectively, these findings suggest a model for macrophage activation in response to dsRNA, whereby engagement of TLR3 leads to cPLA(2)alpha-mediated arachidonic acid mobilization and COX-2-mediated PGE(2) production, which cooperate to induce the expression of iNOS.