Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis

We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG), When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation, We found that piceatannol inhibited protein kinase C delta (PKC delta) and Raf-1 translocation from cytosol to plasma membrane by >90%, Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase, The phosphorylation of Syk preceded the activation of PI 3-kinase, Both wortmannin and piceatannol inhibited PI3-kinase, but only piceatannol inhibited Syk, In contrast to piceatannol, wortmannin did not inhibit PKC delta and Raf-1 translocation, To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKC delta and Raf-1(-) translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation, We conclude that Syk activation following Fc gamma receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.