On the biosynthesis of specialized pro-resolving mediators in human neutrophils and the influence of cell integrity

Abstract

Neutrophils are key players in inflammation initiation and resolution. Little attention has been paid to the detailed biosynthesis of specialized pro-resolving mediators (SPM) in these cells. We investigated SPM formation in human polymorphonuclear leukocytes (PMNL), in broken PMNL preparations and recombinant human 5-lipoxygenase (5-LO) supplemented with the SPM precursor lipids 15-Hydroxyeicosatetraenoic acid (15-HETE), 18-Hydroxyeicosapentaenoic acid (18-HEPE) or 17-Hydroxydocosahexaenoic acid (17-HDHA). In addition, the influence of 5-LO activating protein (FLAP) inhibition on SPM formation in PMNL was assessed. Intact human PMNL preferred ARA over DHA for lipid mediator formation. In contrast, in incubations supplemented with the SPM precursor lipids DHA-derived 17-HDHA was preferred over 15-HETE and 18-HEPE. SPM formation in the cells was dominated by 5(S),15(S)-diHETE (800 pmol/20 mio cells) and Resolvin D5 (2300 pmol/20 mio cells). Formation of lipoxins (<10 pmol/20 mio cells), E-series (<70 pmol/20 mio cells) and other D-series resolvins (<20 pmol/20 mio cells) was low and only detected after addition of the precursor lipids. Upon destruction of cell integrity, formation of lipoxins and 5(S),15(S)-diHETE increased while formation of 17-HDHA- and 18-HEPE-derived SPMs was attenuated. Recombinant 5-LO did not accept the precursors for SPM formation and FLAP inhibition prevented the formation of the 5-LO-dependent SPMs. Together with the data on FLAP inhibition our results point to unknown factors that control SPM formation in human neutrophils and also render lipoxin and 5(S),15(S)-diHETE formation independent of membrane association and FLAP when cellular integrity is destroyed.

Keywords: Leukotriene; Lipoxin; Lipoxygenase; Neutrophil; Resolvin; Specialized pro-resolving mediator.