Inhibition of calcium-independent phospholipase A2 prevents inflammatory mediator production in pulmonary microvascular endothelium

Inhalation of allergens can result in mast cell degranulation and release of granule contents, including tryptase, in the lung. Injury to human pulmonary microvascular endothelial cells (HMVEC-L) can also result in activation of the coagulation cascade and thrombin generation. We hypothesize that these proteases activate calcium-independent phospholipase A₂ (iPLA₂), in HMVEC-L, leading to the production of membrane phospholipids-derived inflammatory mediators. Both thrombin and tryptase stimulation of HMVEC-L increased iPLA₂ activity that was inhibited by pretreatment with the iPLA₂ selective inhibitor bromoenol lactone (BEL). Arachidonic acid and prostaglandin I₂ (PGI₂) release were also increased in tryptase and thrombin stimulated cells and inhibited by BEL pretreatment. Pretreating the endothelial cells with AACOCF₃ a cytosolic PLA₂ inhibitor did not inhibit tryptase or thrombin induced arachidonic acid and PGI₂ release. In addition thrombin and tryptase also increased HMVEC-L platelet activating factor (PAF) production that significantly contributes to the recruitment and initial adherence of polymorphonuclear neutrophils (PMN) to the endothelium. Tryptase or thrombin stimulated increase in PMN adherence to the endothelium was inhibited by pretreatment of HMVEC-L with BEL or pretreatment of PMN with CV3988, a PAF receptor specific antagonist. Collectively, these data support our hypothesis that iPLA₂ activity is responsible for membrane phospholipid hydrolysis in response to tryptase or thrombin stimulation in HMVEC-L. Therefore selective inhibition of iPLA₂ may be a pharmacological target to inhibit the early inflammation in pulmonary vasculature that occurs as a consequence of mast cell degranulation or acute lung injury.