Ca²⁺ signalling mechanisms of the β₂ integrin on neutrophils: involvement of phospholipase Cγ2 and Ins(1,4,5)P₃

Engagement of β₂ integrins triggers a tyrosine kinase-dependent intracellular mobilization and influx of Ca²⁺ in human neutrophils. However, the transduction pathway involved in generating this Ca²⁺ signal is obscure. In the present study we identified phospholipase Cγ2 (PLCγ2) as one of the major proteins that was phosphorylated on tyrosine in response to β₂ integrin activation. This β₂ integrin-induced phosphorylation of PLCγ2 occurred in parallel with an increased accumulation of Ins(1,4,5)P₃. The relevance of these observations for the β₂ integrin-induced Ca²⁺ signal was investigated using an inhibitor of PLC signalling pathways, 1-(6-{[17β-3-methoxyoestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U73122). U73122 dose-dependently (IC₅₀, approx. 0.15 μM) inhibited both the β₂ integrin-induced release of Ca²⁺ from intracellular stores and the subsequent influx of Ca²⁺ across the plasma membrane. These effects were not observed with the inactive analogue 1-(6-{[17β-3-methoxyoestra-1,3,5(10)-trien-17-yl]amino}hexyl)-pyrrolidine-2,5-dione (U73343). To gain further support for an involvement of PLC-induced Ins(1,4,5)P₃ formation in the β₂ integrin-induced Ca²⁺ signal, we searched for the molecular event(s) underlying the effects of U73122. Our experiments revealed that U73122 had no effect on either β₂ integrin-induced tyrosine phosphorylation of PLCγ2 (or any of the other proteins) or on the formation of Ins(1,4,5)P₃, but it reduced the Ins(1,4,5)P₃-induced release of ⁴⁵Ca²⁺ from intracellular stores of electropermeabilized cells. Taken together, the present data suggest that the β₂ integrin-induced Ca²⁺ signal in human neutrophils is generated through activation of a PLCγ2-dependent pathway.