Ca2+ signalling mechanisms of the beta 2 integrin on neutrophils: involvement of phospholipase C gamma 2 and Ins(1,4,5)P3.

Engagement of beta 2 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 C gamma 2 (PLC gamma 2) as one of the major proteins that was phosphorylated on tyrosine in response to beta 2 integrin activation. This beta 2 integrin-induced phosphorylation of PLC gamma 2 occurred in parallel with an increased accumulation of Ins(1,4,5)P3. The relevance of these observations for the beta 2 integrin-induced Ca2+ signal was investigated using an inhibitor of PLC signalling pathways, 1-(6-?[17 beta-3-methoxyoestra-1,3.5(10)-trien-17-yl] amino?hexyl)-1H-pyrrole-2,5-dione({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122). {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 dose-dependently (IC50, approx. 0.15 microM) inhibited both the beta 2 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-?[17 beta-3-methoxyoestra-1,3,5(10)-trien-17-yl] amino?hexyl)-pyrrolidine-2,5-dione ({"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343). To gain further support for an involvement of PLC-induced Ins(1,4,5)P3 formation in the beta 2 integrin-induced Ca2+ signal, we searched for the molecular event(s) underlying the effects of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Our experiments revealed that {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 had no effect on either beta 2 integrin-induced tyrosine phosphorylation of PLC gamma 2 (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 beta 2 integrin-induced Ca2+ signal in human neutrophils is generated through activation of a PLC gamma 2-dependent pathway.