Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils

Background: Phospholipase A(2)s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA(2)alpha (cPLA(2)alpha) is thought to be the dominant PLA(2) in eosinophils; however, eosinophils also have secreted PLA2 (sPLA(2)) activity that has not been fully defined. Objectives: To examine the expression of sPLA(2) group X (sPLA(2)-X) in eosinophils, the participation of sPLA(2)-X in the formation of CysLTs, and the mechanism by which sPLA(2)-X initiates the synthesis of CysLTs in eosinophils. Methods: Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA(2)-X antibody identified sPLA(2)-X by Western blot. We used confocal microscopy to colocalize the sPLA(2)-X to intracellular structures. Aninhibitor of sPLA(2)-X (ROC-0929) that does not inhibit other mammalian sPLA(2)s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA(2)alpha, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Results: Eosinophils express the mammalian sPLA(2)-X gene (PLA2G10). The sPLA(2)-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA(2)alpha also attenuated the fMLP-mediated formation of CysLT. The sPLA(2)-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA(2)a during cellular activation, indicating that sPLA(2)-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA(2)alpha. We further demonstrate that sPLA(2)-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1 beta increased the expression of PLA2G10 by eosinophils. Conclusions: These results demonstrate that sPLA(2)-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA(2)alpha. The sPLA(2)-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.