Leukotriene B-4 and platelet activating factor production in permeabilized human neutrophils: Role of cytosolic PLA(2) in LTB(4) and PAF generation

The specific type of phospholipase A(2) (PLA(2)) involved in formation of leukotriene B-4 (LTB(4)) and platelet activating factor (PAF) in inflammatory cells has been controversial. In a recent report we characterized activation of the 'cytosolic' form of PLA(2) (cPLA(2)) in human neutrophils (PMN) permeabilized with Staphylococcus aureus alpha-toxin under conditions where the secretory form of PLA(2) (sPLA(2)) was inactive. In the current study, generation of both LTB(4) and PAF in porated PMN are demonstrated. PMN, prelabeled with [H-3]arachidonic acid (H-3-AA, to assess AA release and LTB(4) production) or with 1-O-[9',10'-H-3]hexadecyl-2-lyso-glycero-3-phosphocholine (H-3-lyso-PAF, for determination of lyso-PAF and PAF formation), were permeabilized with alpha-toxin in a 'cytoplasmic' buffer supplemented with acetyl CoA. Maximum production of both PAF and LTB(4) required addition of 500 nM Ca2+, G-protein activation induced with 10 mu M GTP gamma S, and stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 mu M); LTB(4) production was confirmed by radioimmunoassay. Removal of acetyl CoA from the system had little effect on LTB(4) generation but blocked PAF production with a concomitant increase in lyso-PAF formation. LTB(4) 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 LTB(4) formation but slightly enhanced PAF generation. This study clearly shows that cPLA(2) activation can provide precursors for both LTB(4) and PAF, that maximum PAF and LTB(4) formation occur under conditions that induced optimal cPLA(2) activation, that a close coupling between LTB(4) and PAF formation exists, and that, after substrate generation, no additional requirements are necessary for LTB(4) and PAF generation in the permeabilized PMN system.