Association between leukotriene B4-induced phospholipase D activation and degranulation of human neutrophils.

We have explored the role of phospholipase D (PLD) activation in leukotriene B4 (LTB4)-induced Ca2+ mobilization and degranulation of human neutrophils. Stimulation of [3H]alkyl-acyl-phosphatidylcholine-labeled neutrophils with LTB4 resulted in a rapid accumulation of [3H]alkyl-phosphatidic acid (PA) as well as a somewhat slower accumulation of [3H]alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB4 increased [3H]alkyl-phosphatidylethanol formation and simultaneously decreased LTB4-induced PA and DG accumulation. This pattern of lipid metabolism is consistent with the conclusion that LTB4 stimulates PLD activity in human neutrophils. Additional studies in which the extracellular and intracellular concentrations of Ca2+ were varied indicated that maximal LTB4-induced PLD activation was dependent upon Ca2+ and potentiated by inhibitors of protein kinase C. The time-course and concentration-response curves for LTB4-induced PLD activation were different from those for LTB4-induced Ca2+ mobilization, as measured by fura-2 fluorescence. On the other hand, the concentration-response curve for LTB4-induced PLD activation was similar to that for LTB4-induced degranulation. Preincubation of the cells with ethanol inhibited LTB4-induced PA and DG accumulation, as well as degranulation, suggesting that one or both of these metabolites were important for this response. In contrast, ethanol had no effect on LTB4-induced Ca2+ mobilization. Propranolol, an inhibitor of phosphatidate phosphohydrolase, abolished DG accumulation in response to LTB4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectively, these data indicate that LTB4-induced activation of PLD in human neutrophils is mediated by a Ca(2+)-dependent mechanism, but not by protein kinase C. In addition, PLD activation in these cells may induce degranulation, but not Ca2+ mobilization.