Phospholipase A₂s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA₂α (cPLA₂α) is thought to be the dominant PLA₂ in eosinophils; however, eosinophils also have secreted PLA₂ (sPLA₂) activity that has not been fully defined.

To examine the expression of sPLA₂ group X (sPLA₂-X) in eosinophils, the participation of sPLA₂-X in the formation of CysLTs, and the mechanism by which sPLA₂-X initiates the synthesis of CysLTs in eosinophils.

Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti–sPLA₂-X antibody identified sPLA₂-X by Western blot. We used confocal microscopy to colocalize the sPLA₂-X to intracellular structures. An inhibitor of sPLA₂-X (ROC-0929) that does not inhibit other mammalian sPLA₂s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA₂α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT.

Eosinophils express the mammalian sPLA₂-X gene (PLA2G10). The sPLA₂-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 cPLA₂α also attenuated the fMLP-mediated formation of CysLT. The sPLA₂-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA₂α during cellular activation, indicating that sPLA₂-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA₂α. We further demonstrate that sPLA₂-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils.

These results demonstrate that sPLA₂-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 cPLA₂α. The sPLA₂-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.