ASSOCIATION BETWEEN LEUKOTRIENE B4-INDUCED PHOSPHOLIPASE-D ACTIVATIONAND DEGRANULATION OF HUMAN NEUTROPHILS

We have explored the role of phospholipase D (PLD) activation in leuko triene B4 (LTB4)-induced Ca2+ mobilization and degranulation of human neutrophils. Stimulation of H-3!alkyl-acyl-phosphatidylcholine-labele d neutrophils with LTB4 resulted in a rapid accumulation of H-3!alkyl -phosphatidic acid (PA) as well as a somewhat slower accumulation of  H-3!alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB4 increased H-3!alkyl-p hosphatidylethanol formation and simultaneously decreased LTB4-induced PA and DG accumulation. This pattern of lipid metabolism is consisten t with the conclusion that LTB4 stimulates PLD activity in human neutr ophils. Additional studies in which the extracellular and intracellula r concentrations of Ca2+ were varied indicated that maximal LTB4-induc ed PLD activation was dependent upon Ca2+ and potentiated by inhibitor s of protein kinase C. The time-course and concentration-response curv es 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 acti vation was similar to that for LTB4-induced degranulation. Preincubati on of the cells with ethanol inhibited LTB4-induced PA and DG accumula tion, as well as degranulation, suggesting that one or both of these m etabolites 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 respo nse to LTB4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectiv ely, these data indicate that LTB4-induced activation of PLD in human neutrophils is mediated by a Ca2+-dependent mechanism, but not by prot ein kinase C. In addition, PLD activation in these cells may induce de granulation, but not Ca2+ mobilization.