Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils

Abstract

In this study, we determined the functional significance and mechanism of the exogenous hVPLA 2 -induced arachidonic acid (AA) release and leukotriene C 4 (LTC 4 ) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nM exogenous hVPLA 2 was able to elicit the significant release of AA and LTC 4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA 2 also augmented the release of AA and LTC 4 from eosinophils activated with formyl-Met-Leu-Phe ؉ cytochalasin B. A cellular fluorescent PLA 2 assay showed that hVPLA 2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA 2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA 2 induced neither the increase in intracellular calcium concentration nor cPLA 2 phosphorylation; consequently, cPLA 2 activity was not affected by hVPLA 2 . Pharmacological inhibition of cPLA 2 and the hVPLA 2 -induced activation of eosinophils derived from the cPLA 2 -deficient mouse corroborated that hVPLA 2 mediates the release of AA and leukotriene in a cPLA 2 -independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA 2 activation.

Transcellular Secretion of Group V Phospholipase A2 from Epithelium Induces β2-Integrin-Mediated Adhesion and Synthesis of Leukotriene C4 in Eosinophils

“…To date, there have been no prior reports on the identity of a specific physiological trigger originating from epithelial cells for direct eosinophil activation. Secretory gVPLA 2 has been associated with the secretion of LTB 4 in neutrophils (21,22) and synthesis of LTC 4 in eosinophils (20), but the role of gVPLA 2 in up-regulation of adhesion is not known. In these studies, we examined the effect of purified hVPLA 2 and endogenously secreted gVPLA 2 from activated epithelial cells on adhesion and secretion of LTC 4 in human eosinophils.…”

“…In another set of experiments, eosinophils also were pretreated with 1) 10 g/ml MCL-3G1, a specific mAb directed against gVPLA 2 (24,25); 2) 10 M LY311727, a global secretory PLA 2 inhibitor (15, 27); 3) 10 M TFMK, a selective gIVA-PLA 2 inhibitor (14,15,20,21,27); or 4) 10 g/ml anti-CD11b neutralizing mAb (15, 27) for 25 min before activation with 10 Ϫ7 M hVPLA 2 , and adhesion assay was performed as described.…”

“…The concentration of LTC 4 secreted during eosinophil activation was assayed by competitive enzyme immunoassay (Cayman Chemicals) using a method that we have reported previously (20,33,34). A total of 21 eosinophil isolations from different donors were used in this protocol.…”

“…In another set of experiments using an aliquot from the same eosinophil isolation, the specificity of focal clustering of CD11b in response to hVPLA 2 stimulation was assessed. Cells first were treated with MCL-3G1, a mAb directed against hVPLA 2 (20,24,25), for 30 min before hVPLA 2 stimulation and cytoslides were prepared as earlier described. The slides containing treated cells were stained with FITC-conjugated CD11b (clone Bear I) mAb, and fluorescence was analyzed by using an Axiovert confocal microscope (Zeiss) equipped with an external argon-krypton laser at 488 nm.…”

“…This response also corresponds to an increase in intracellular Ca 2ϩ concentration. In human eosinophils, hVPLA 2 also is internalized to cause lipid hydrolysis from perinuclear membranes (23); this results in LTC 4 synthesis and secretion (20). However, hVPLA 2 -mediated synthesis of LTC 4 in human eosinophils does not require either gIVA-PLA 2 activation or intracellular Ca 2ϩ concentration release (20).…”