Abstract

The specific type of phospholipase A₂ (PLA₂) involved in formation of leukotriene B₄ (LTB₄) and platelet activating factor (PAF) in inflammatory cells has been controversial. In a recent report we characterized activation of the ‘cytosolic’ form of PLA₂ (cPLA₂) in human neutrophils (PMN) permeabilized with Staphylococcus aureus α-toxin under conditions where the secretory form of PLA₂ (sPLA₂) was inactive. In the current study, generation of both LTB₄ and PAF in porated PMN are demonstrated. PMN, prelabeled with [³H]arachidonic acid (³H-AA, to assess AA release and LTB₄ production) or with 1-O-[9′,10′-³H]hexadecyl-2-lyso-glycero-3-phosphocholine (³H-lyso-PAF, for determination of lyso-PAF and PAF formation), were permeabilized with α-toxin in a ‘cytoplasmic’ buffer supplemented with acetyl CoA. Maximum production of both PAF and LTB₄ required addition of 500 nM Ca²⁺, G-protein activation induced with 10 μM GTPγS, and stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 μM); LTB₄ production was confirmed by radioimmunoassay. Removal of acetyl CoA from the system had little effect on LTB₄ generation but blocked PAF production with a concomitant increase in lyso-PAF formation. LTB₄ and PAF production occurred in parallel over time and at differing ATP and Ca²⁺ concentrations. Further work demonstrated that: (i) maximum production of both inflammatory mediators required a hydrolyzable form of ATP; (ii) blocking phosphorylation with staurosporin inhibited production of both; (iii) the reducing agent, dithiotreitol, had little effect on LTB₄ formation but slightly enhanced PAF generation. This study clearly shows that cPLA₂ activation can provide precursors for both LTB₄ and PAF, that maximum PAF and LTB₄ formation occur under conditions that induced optimal cPLA₂ activation, that a close coupling between LTB₄ and PAF formation exists, and that, after substrate generation, no additional requirements are necessary for LTB₄ and PAF generation in the permeabilized PMN system.