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

Abstract

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 4 beta-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, Ca(2+)- 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 cochelation 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 LTB4 evoked a significant respiratory burst in Ca(2+)-depleted cells.

Transepithelial Migration of Neutrophils in Response to Leukotriene B4 Is Mediated by a Reactive Oxygen Species-Extracellular Signal-Regulated Kinase-Linked Cascade

“…Given the inhibitory effect of DPI on LTB 4 -induced cell migration and ERK activation, we suggest that NADPH oxidase is the primary source of the ROS generated in response to LTB 4 . Consistent with that idea, several studies have shown that LTB 4 promotes robust, receptor-mediated activation of NADPH oxidase (35)(36)(37), which in eosinophils mediates activation of PKC, phospholipases C and D, and cPLA 2 (35,38).…”

Cytosolic phospholipase A2, lipoxygenase metabolites, and reactive oxygen species

“…These observations suggest that LTB4-induced ROS generation occurring via NOX is crucial to cell chemotaxis (34,35). In addition, Giembycz has reported that LTB4 promotes the robust, receptor-mediated activation of NADPH oxidase in guinea pig eosinophils (21,36). LTB4 has also been previously shown to promote the phosphorylation and translocation of p47phox, thereby stimulating NADPH oxidase (22).…”

Pharmacological comparison of LTB4‐induced NADPH oxidase activation in adherent and non‐adherent guinea‐pig eosinophils

“…Previous results suggest that LTB 4 can evoke a rapid, transient and concentration-dependent generation of H 2 O 2 from eosinophils in suspension (Perkins et al, 1995;Lindsay et al, 1998a,b;Lynch et al, 2000). Using guinea-pig eosinophils incubated upon 96-well plates coated with BSA, near identical rapid superoxide release, as measured by lucigenin-enhanced chemiluminescence was observed.…”

“…GF 109203X was particularly e ective thus implicating a role for both conventional and novel isoforms of PKC. Previous studies have demonstrated similar e ects of PKC on other functions such as Ca 2+ transients, homotypic aggregation, oxidative burst and eicosanoid production in guinea-pig and human-eosinophils (Perkins et al, 1995;Teixeira et al, 1997;Dent et al, 1998).…”

“…In previous studies of the intracellular mechanisms that mediate LTB 4 -induced NADPH oxidase activation, we have shown that this is partially mediated by lyn kinase, PKC and PLA 2 but occurs essentially independently of changes in the intracellular [Ca 2+ ], phospholipase D, PtdIns 3-kinase and ERK1/2 (Perkins et al, 1995;Lindsay et al, 1998a,b;Lynch et al, 2000). However, these studies were performed upon non-adherent guinea-pig eosinophils in which LTB 4 produces a rapid and transient (53 min) oxidant generation which is probably unrepresentative of the in vivo environment where eosinophils would be adherent to other cells and the extracellular matrix.…”