Activation of phospholipase A2 in human neutrophils by polyunsaturated fatty acids and its role in stimulation of superoxide production

Although polyunsaturated fatty acids (PUFA) have been shown to stimulate neutrophil responses such as the oxygen-dependent respiratory burst (superoxide production), the mechanisms involved still remain undefined. Here we investigate the effect of PUFA on the phospholipase A₂ (PLA₂)-signal transduction process in human neutrophils. Exogenous eicosatetraenoic acid [arachidonic acid; C_20:4(n-6)] or docosahexaenoic acid [C_22:6(n-3)] promoted the release of [³H]C_20:4(n-6) from prelabelled neutrophils in a time- and dose-dependent manner, which is indicative of PLA₂ activation. The release of [³H]C_20:4(n-6) from the cells by C_20:4(n-6) and C_22:6(n-3) was suppressed by PLA₂ inhibitors. Other PUFA {eicosapentaenoic [C_20:5(n-3)], octadecatrienoic [γ-linolenic; C_18:3(n-6)] and octadecadienoic [linoleic; C_18:2(n-6)] acids} also had the ability to release [³H]C_20:4(n-6); however, certain C_20:4(n-6) derivatives [15-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid and C_20:4(n-6) methyl ester] and saturated fatty acids [octadecanoic (stearic; C_18:0) and eicosanoic (arachidic; C_20:0) acids] had no significant effect. Treatment of the neutrophils with exogenous C_22:6(n-3) caused the mass of endogenous unesterified C_20:4(n-6) to increase. Incubation of the leucocytes with C_20:4(n-6) or C_22:6(n-3) evoked activation of the 85 kDa cytosolic PLA₂ (cPLA₂) and the 14 kDa secretory PLA₂ (sPLA₂), but not the cytosolic Ca²⁺-independent PLA₂. In contrast, C_20:0 did not activate any of the PLA₂ isoforms. Activation of cPLA₂ by PUFA was found to precede that of sPLA₂. C_22:6(n-3), C_20:4(n-6) and other PUFA induced punctate localization of cPLA₂ in the cells, which was not observed with saturated fatty acids. Pretreatment of the leucocytes with PLA₂ inhibitors markedly decreased superoxide production induced by C_20:4(n-6). These results show that PUFA activate PLA₂ in neutrophils, which might have a mandatory role in biological responses.