Leukotriene B4 and platelet activating factor production in permeabilized human neutrophils: role of cytosolic PLA2 in LTB4 and PAF generation.

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 alpha-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 alpha-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 microM GTP gamma S, and stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 microM); 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 affect 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.

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils.

Neutrophils (PMN) contain two types of phospholipase A2 (PLA2), a 14 kDa 'secretory' Type II PLA2 (sPLA2) and an 85 kDa 'cytosolic' PLA2 (cPLA2), that differ in a number of key characteristics: (1) cPLA2 prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA2 is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA2 is active at nM calcium (Ca2+) concentrations; sPLA2 requires microM Ca2+ levels. (3) cPLA2 activity is regulated by phosphorylation; sPLA2 lacks phosphorylation sites. (4) cPLA2 is insensitive to reduction; sPLA2 is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to determine whether one or both forms of PLA2 were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [3H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to assess oleate and linoleate mass. A combination of 500 nM Ca2+, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [3H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA2 activation. Dithiothreitol treatment had little affect on [3H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA2 activity and the cytosolic buffer utilized did not support activity of recombinant sPLA2. These results strongly suggested that cPLA2 was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA2 in PMN.

Induction of cytosolic phospholipase A2 activity by phosphatidic acid and diglycerides in permeabilized human neutrophils: interrelationship between phospholipases D and A2.

Relationships between phospholipases are poorly understood, but phosphatidic acid (PA) and diglycerides (DGs), produced by phospholipase D (PLD) and phosphatidate phosphohydrolase actions, might function as second messengers coupling cell stimulation to cellular responses. This study investigates the role of PLD-mediated PA and DG formation in inducing phospholipase A2 (PLA2) activity in intact human neutrophils (PMNs) and in PMNs permeabilized with Staphylococcus aureus alpha-toxin. PMNs were labelled with [3H]arachidonic acid (AA) to assess AA release and metabolism and diacylglycerol formation, or with [3H]1-O-hexadecyl-2-lyso-glycerophosphatidylcholine for the determination of platelet-activating factor (PAF), PA and alkylacylglycerol production. In intact PMNs primed with tumour necrosis factor alpha before stimulation with N-formyl-Met-Leu-Phe, AA release and metabolism and PAF formation increased in parallel with enhanced PA and DG formation, and inhibition of PA and DG production led to a decrease in both AA release and PAF accumulation. In alpha-toxin-permeabilized PMNs, AA release and PAF production result from the specific activation of cytosolic PLA2 (cPLA2). In this system, PA and DG formation were always present when cPLA2 activation occurred; blocking PA and DG production inhibited AA release and PAF accumulation. Adding either PA or DG back to permeabilized cells (with endogenous PA and DG formation blocked) led to a partial restoration of AA release and PAF formation; a combination of PA and DGs reconstituted full cPLA2 activity. These results strongly suggest that products of PLD participate in activating cPLA2 in PMNs.