EARLY SIGNALING EVENTS IMPLICATED IN LEUKOTRIENE-B-4-INDUCED ACTIVATION OF THE NADPH OXIDASE IN EOSINOPHILS - ROLE OF CA2+, PROTEIN-KINASE-C AND PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D

The early signalling events that may ultimately contribute to the assembly and subsequent activation of the NADPH oxidase in guinea-pig peritoneal eosinophils were investigated in response to leukotriene B-4 (LTB(4)). LTB(4) promoted a rapid, transient and receptor-mediated increase in the rate of H2O2 generation that was potentiated by R 59 022, a diradylglycerol (DRG) kinase inhibitor, implicating protein kinase C (PKC) in the genesis of this response. This conclusion was supported by the finding that the PKC inhibitor, Ro 31-8220, attenuated (by about 30 %) the peak rate of LTB(4)-induced H2O2 generation under conditions where the same response evoked by 4 beta-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90 %. Paradoxically, Ro 31-8220 doubled the amount of H2O2 produced by LTB(4) which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB(4)-induced Ins(1,4,5)P-3 accumulation and the duration of the Ca2+ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB(4)-induced oxidase activation indicated that phospholipase D (PLD) did not play a major role. Thus, although H2O2 generation was abolished by butan-1-ol, this was apparently unrelated to the inhibition of PLD, as LTB(4) failed to stimulate the formation of Ptd[H-3]BuOH in [H-3]butan-1-ol-treated eosinophils. Rather, the inhibition was probably due to the ability of butan-1-ol to increase the eosinophil cyclic AMP content. In contrast, Ca2+- and PLC-driven mechanisms were implicated in H2O2 generation, as LTB(4) elevated the Ins(1,4,5)P-3 content and intracellular free Ca2+ concentration in intact cells, and co-chelation of extracellular and intracellular Ca2+ significantly attenuated LTB(4)-induced H2O2 generation. Pretreatment of eosinophils with wortmannin did not affect LTB(4)-induced H2O2 production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB(4) activates the NADPH oxidase in eosinophils by PLD- and PtdIns 3-kinase-independent mechanisms that involve Ca2+, PLC and PKC. Furthermore, the activation of additional pathways that do not require Ca2+ is also suggested by the finding that LTB(4) evoked a significant respiratory burst in Ca2+-depleted cells.