Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis

We investigated the requirement for Syk activation to initiate downstream s ignaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab -coated erythrocytes (EIgG), When PMN were challenged with EIgG, Syk phosph orylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective in hibitor, blocked EIgG phagocytosis and Syk phosphorylation, We found that p iceatannol inhibited protein kinase C delta (PKC delta) and Raf-1 transloca tion from cytosol to plasma membrane by >90%, Extracellular signal-regulate d protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blo cked. We also investigated phosphatidylinositide 3-kinase (PI3-kinase) acti vity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, i nhibitors of PI 3-kinase, The phosphorylation of Syk preceded the activatio n 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 down stream of Syk activation, we assessed whether the cell-permeable diacylglyc erol analogue didecanoylglycerol could normalize PMN phagocytosis, PKC delt a and Raf-1(-) translocation, and ERK1 and ERK2 phosphorylation inhibited b y piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phag ocytosing PMN normalized all three without a concomitant effect on PI 3-kin ase activity and Syk phosphorylation, We conclude that Syk activation follo wing Fc gamma receptor engagement initiates downstream signaling events lea ding to mitogen-activated protein kinase activation independent of PI 3-kin ase activation.