The role of a cytosolic phospholipase A 2 -α (cPLA 2 -α) in neutrophil arachidonic acid release, plateletactivating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA 2 -α activity was blocked with the specific cPLA 2 -α inhibitor, Pyrrolidine-1 (human cells), or by cPLA 2 -α gene disruption (mice). cPLA 2 -α inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcγII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA 2 -α inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B 4 . Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA 2 -α (−/−) mice compared with wild type littermates. These studies identify a novel * This work was supported by grants from The Physicians of Ontario through The P.S.I. Foundation (Grant 01-12 (to B. B. R.) and 98-049

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A 2 (PLA 2 ) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA 2 (GV and GX sPLA 2 ) mRNA and contain GV and GX sPLA 2 proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA 2 s are undetectable. GV sPLA 2 is a component of both azurophilic and specific granules, whereas GX sPLA 2 is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA 2 and most, if not all, of the PLA 2 activity in the extracellular fluid of fMLPstimulated neutrophils is due to GV sPLA 2 . GV sPLA 2 does not contribute to fMLP-stimulated leukotriene B 4 production but may support the anti-bacterial properties of the neutrophil, because 10 -100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA 2 -␣ (group IV PLA 2 ␣), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B 4 in fMLP-and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

Phospholipase A2 (PLA2) has been purified to homogeneity from human arthritic synovial fluid. The activity resolved into multiple peaks by preparative HPLC. The most abundant peak (A) was present in synovial fluid from patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. A second major peak (B) was variable and lower in relative abundance, but was distinguishable from peak A by its stimulated activity in the presence of either 0.5 M Tris or 0.1% sodium deoxycholate (DOC), in addition to its longer HPLC column retention time. Both peaks required Ca2+ and showed optimal activity in DOC/phosphatidylcholine (PC) mixed micelle assays between pH 8.0 and 9.0. Both peaks showed higher activity with PC as substrate than with PI, however peak A exhibited higher activity with PE than PC. Upon preparative SDS-polyacrylamide gel electrophoresis, both peaks of PLA2 activity were resolved as proteins of approximately 14,000 Da. The N-terminal sequence obtained from purified peak A material matched that of a recent similar isolate (Hara et al. (1988) J. Biochem. 104, 326-328).