Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA2

Abstract

We examined the regulatory role of cytosolic phospholipase A2 (cPLA2) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C4synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC4, which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-β2 and -γ2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca2+ concentration was inhibited by ET-18-OCH3, a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA2blocker, had no inhibitory effect. Both TFMK and ET-18-OCH3attenuated stimulated arachidonate release and LTC4secretion, suggesting that activation of both PLC and cPLA2is essential for LTC4 synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31–8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC4 secretion. 1,2-bis(2-Aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator, suppressed both EPO release and LTC4 secretion. We found that fMLP/B-induced LTC4 secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA2. However, cPLA2 does not regulate eosinophil degranulation.