Early signalling events implicated in leukotriene B₄-induced activation of the NADPH oxidase in eosinophils: Role of Ca²⁺, protein kinase C and phospholipases C and 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 B₄ (LTB₄). LTB₄ 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 LTB₄-induced H₂O₂ generation under conditions where the same response evoked by 4β-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90%. Paradoxically, Ro 31-8220 doubled the amount of H₂O₂ produced by LTB₄ which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB₄-induced Ins(1,4,5)P₃ accumulation and the duration of the Ca²⁺ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB₄-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 LTB₄ failed to stimulate the formation of Ptd[³H]BuOH in [³H]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 LTB₄ elevated the Ins(1,4,5)P₃ content and intracellular free Ca²⁺ concentration in intact cells, and co-chelation of extracellular and intracellular Ca²⁺ significantly attenuated LTB₄-induced H₂O₂ generation. Pretreatment of eosinophils with wortmannin did not affect LTB₄-induced H₂O₂ production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB₄ 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, evoked a significant respiratory burst in Ca²⁺-depleted cells.