Leukotriene B4 mediates p47phox phosphorylation and membrane translocation in polyunsaturated fatty acid-stimulated neutrophils

Abstract

Polyunsaturated fatty acids (PUFAs) and leukotriene B₄ (LTB₄) are involved in many inflammatory and physiological conditions. The role of arachidonic acid (AA) and linoleic acid (LA) in promoting the assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits is well known, but the involvement of LTB₄ and other 5-lipoxygenase (5-LO) pathway metabolites of AA in hydrogen peroxide (H₂O₂) production by PUFA-stimulated polymorphonuclear leukocytes (PMNs) has not been investigated. We examined this question by determining H₂O₂ production as well as phosphorylation and membrane translocation of the p47phox subunit of NADPH oxidase. Elicited peritoneal PMNs from rats and from 5-LO-deficient or wild-type mice were pretreated with or without inhibitors of LT biosynthesis and antagonists of the receptors for LTB₄ and cysteinyl LTs for 20 min before stimulation with AA (at 5 and 20 μM) or LA (at 20 μM). PUFAs elicited H₂O₂ production in a dose-dependent manner, and pharmacologic or genetic inhibition of LT synthesis decreased H₂O₂ production by ∼40% when compared with untreated controls. LTB₄ was the moiety responsible for H₂O₂ production, as revealed by studies using receptor antagonists and its exogenous addition. LTB₄ itself also promoted p47phox phosphorylation and translocation. These results identify a heretofore unrecognized role for activation of 5-LO and subsequent production of LTB₄ in stimulation of PMN NADPH oxidase activation by PUFAs.