β1 integrin cross-linking inhibits CD16-induced phospholipase D and secretory phospholipase A2 activity and granule exocytosis in human NK cells: Role of phospholipase D in CD16-triggered degranulation

Recent data indicate that integrin-generated signals can modulate different receptor-stimulated cell functions in both a positive (costimulation) and a negative (inhibition) fashion. Here we investigated the ability of β₁ integrins, namely α₄β₁ and α₅β₁ fibronectin receptors, to modulate CD16-triggered phospholipase activation in human NK cells, β₁ integrin simultaneous crosslinking selectively inhibited CD16- induced phospholipase D (PLD) activation, without affecting either phosphatidylinositol-phospholipase C or cytosolic phospholipase A₂ (PLA₂) enzymatic activity. CD16-induced secretory PLA₂ (sPLA₂) protein release as well as its enzymatic activity in both cell-associated and soluble forms were also found to be inhibited upon β₁ integrin coengagement. The similar effects exerted by specific PLD pharmacological inhibitors (2,3- diphosphoglycerate, ethanol) suggest that in our experimental system, sPLA₂ secretion and activation are under the control of a PLD-dependent pathway. By using pharmacological inhibitors (2,3-diphosphoglycerate, wortmannin, ethanol) we also demonstrated that PLD activation is an important step in the CD16-triggered signaling cascade that leads to NK cytotoxic granule exocytosis. Consistent with these findings, fibronectin receptor engagement, by either mAbs or natural ligands, resulted in a selective inhibition of CD16-triggered, but not of PMA/ionomycin-induced, degranulation that was reversed by the exogenous addition of purified PLD from Streptomyces chromofuscus.