TLR3-Dependent Induction of Nitric Oxide Synthase in RAW 264.7 Macrophage-Like Cells via a Cytosolic Phospholipase A sub(2)/Cyclooxygenase-2 Pathway

• Published in: Journal of Immunology 2007-10, Vol.179 (7), p.4821-4828
• Main Authors: Pindado, Jose, Balsinde, Jesus, Balboa, Maria A
• Format: Article
• Language: English
• ISSN: 0022-1767; 1365-2567

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 sub(2) (cPLA sub(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 sub(2) by the de novo-synthesized cyclooxygenase-2 (COX-2) enzyme. These processes are blocked by the selective cPLA sub(2) alpha inhibitor pyrrophenone, pointing out to cPLA sub(2) alpha as the effector involved. In keeping with this observation, the cPLA sub(2) alpha phosphorylation state increases after cellular treatment with poly-IC. Inhibition of cPLA sub(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 sub(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 PGE sub(2). Finally, cellular depletion of TLR3 by siRNA inhibits COX-2 expression, PGE sub(2) 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 sub(2) alpha -mediated arachidonic acid mobilization and COX-2-mediated PGE sub(2) production, which cooperate to induce the expression of iNOS.