Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca²⁺-signalling induced by Fcγ receptor cross-linking in human neutrophils

The effect of wortmannin on IgG-receptor (FcγR)-mediated stimulation of human neutrophils was investigated. The Ca²⁺ influx induced by clustering of both Fcγ receptors was inhibited by wortmannin, as was the release of Ca²⁺ from intracellular stores. Wortmannin also inhibited, with the same efficacy, the accumulation of Ins(1,4,5)P₃ observed after FcγR stimulation, but did not affect the increase in Ins(1,4,5)P₃ induced by the chemotactic peptide, formyl-methionine-leucine-phenylalanine. Because wortmannin is, in the concentrations used here, an inhibitor of PtdIns 3-kinase, these results suggested a role for PtdIns 3-kinase upstream of Ca²⁺ signalling, induced by FcγR cross-linking. Support for this notion was obtained by investigating the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and by studying the kinetics of Ptdlns 3-kinase activation. We found translocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the mem-brane as soon as 5 s after FcγR cross-linking, even before the onset of the Ca²⁺ response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by co-cross-linking of either FcγRIIa and FcγRIIIb with the tyrosine phosphatase, CD45, indicating a requirement for protein tyrosine phosphorylation in the recruitment of Ptdγns 3-kinase to the plasma membrane. Taken together, our results suggest a role for PtdIns 3-kinase in early signal transduction events after FcγR cross-linking in human neutrophils.