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 i nflammatory cells has been controversial. In a recent report we charac terized activation of the 'cytosolic' form of PLA(2) (cPLA(2)) in huma n neutrophils (PMN) permeabilized with Staphylococcus aureus alpha-tox in under conditions where the secretory form of PLA(2) (sPLA(2)) was i nactive. In the current study, generation of both LTB(4) and PAF in po rated PMN are demonstrated. PMN, prelabeled with [H-3]arachidonic acid (H-3-AA, to assess AA release and LTB(4) production) or with ,10'-H-3 ]hexadecyl-2-lyso-glycero-3-phosphocholine (H-3-lyso-PAF, for determin ation of lyso-PAF and PAF formation), were permeabilized with alpha-to xin in a 'cytoplasmic' buffer supplemented with acetyl CoA. Maximum pr oduction of both PAF and LTB(4) required addition of 500 nM Ca2+, G-pr otein activation induced with 10 mu M GTP gamma S, and stimulation wit h the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 mu M); LTB(4) production was confirmed by radioimmunoassay. Removal of acetyl CoA f rom the system had little effect on LTB(4) generation but blocked PAF production with a concomitant increase in lyso-PAF formation. LTB(4) a nd PAF production occurred in parallel over time and at differing ATP and Ca2+ concentrations. Further work demonstrated that: (i) maximum p roduction of both inflammatory mediators required a hydrolyzable form of ATP; (ii) blocking phosphorylation with staurosporin inhibited prod uction of both; (iii) the reducing agent, dithiotreitol, had little ef fect on LTB(4) formation but slightly enhanced PAF generation. This st udy clearly shows that cPLA(2) activation can provide precursors for b oth LTB(4) and PAF, that maximum PAF and LTB(4) formation occur under conditions that induced optimal cPLA(2) activation, that a close coupl ing between LTB(4) and PAF formation exists, and that, after substrate generation, no additional requirements are necessary for LTB(4) and P AF generation in the permeabilized PMN system.