Effect of the inhibition of PLA2 and PAF on the neutrophilic respiratory burst and apoptosis

Abstract

Background Since the exact pathogenesis of sepsis-induced ARDS has not been elucidated, the mechanisms of enhanced neutrophilic respiratory burst were probed in endotoxin primed neutrophils associated with the roles of phospholipase A2 (PLA2), platelet activating factor (PAF) and apoptosis. Methods In isolated fresh human neutrophils, effects of the inhibition of PLA2 and PAF on the apoptosis were examined by the method of Annexin-FITC/dual PI flow cytometry. The roles of PLA2 and PAF on the neutrophilic respiratory burst were also examined by measuring oxidant generation in cytochrome-c reduction assay. Activities of the PLA2 and lysoPAF acetyltransferase (lysoPAF AT) of the neutrophils were determined to understand the effect of endotoxin on these enzymatic activities which may be related to the neutrophilic respiratory burst and apoptosis. In addition, the role roles of PLA2 and PAF in neutrophilic adhesion to bovine endothelial cells were examined in vitro by neutrophil adhesion assay. To investigate the effect of oxidants on pulmonary surfactant, cytochemical ultrastructural microscopy was performed. To inhibit PLA2 and PAF, non-specific PLA2 inhibitor mepacrine (100 nM) and WEB 2086 (100 nM) or ketotifen fumarate (10 kg/ml) were used respectively in all in vitro experimental sets. WEB 2086 is PAF receptor antagonist, and ketotifen fumarate is a lyso PAF AT inhibitor. Results The mapacrine treatment, provided after the endotoxin (ETX) treatment, resulted in increased apoptosis of neutrophils (p<0.001) while treatments of WEB 2086 and ketotifen did not. The inhibition of PLA2 and PAF decreased (p<0.001) production of oxidants from PMA-stimulated neutrophils. While endotoxin increased the PLA2 activity of neutrophils (p<0.01), mepacrine supressed (p<0.001) the activity, provided after treatment of ETX. The lyso PAF actyltransferase activity (lyso PAF AT) increased(p<0.01) after treatment of ETX. In contrast, mepacrine, WEB 2086 and ketotifen showed a tendency of decreasing the activity after treatment of ETX. The Treatment of ETX increased (p<0.001) neutrophil adhesion to endothelial cells, which was reversed by inhibition of PLA2 and PAF (p<0.001). The binding of oxidants to pulmonary surfactant was identified histologically. Conclusions The enhanced neutrophilic respiratory burst by ETX plays a pivotal role in the pathogenesis of ARDS in term of oxidayive oxidative stress. Increased production of oxidants form neutrophils is mediated by the activations of PLA2 and lyso PAF AT.