HETE-PEs regulate NET formation and superoxide generation by neutrophils. (A-D,G-L) Adherent neutrophils (24-well plate; 106 cells/well) were activated with PMA for 30 minutes, after a 10-minute preincubation with inhibitor or lipid. The supernatant was removed and centrifuged to ensure no cells were present, and NET formation was quantified by fluorescent assay for DNA. (A) Zileuton (10μM) increased NET formation in response to PMA. (B) MK886 (1μM) increased NET formation in response to PMA. (C) Exogenous 5-HETE-PE, but not SAPE (both 10μM), reduced NET formation in response to PMA (D) Exogenous 15-HETE-PE (10μM) reduced NET formation in response to PMA. (E) Superoxide generation was measured by reduction of cytochrome c at 550 nm with activation by 1μM fMLP, after10 minutes of preincubation with 5-HETE-PE or 5-HETE (10μM). (F) Superoxide generation was determined as in panel E with 10 minutes of preincubation with SAPE (1μM). (G-I) Representative fluorescent images of immunostaining of NETs by PMA-activated neutrophils. (J-L) Representative images of neutrophils pretreated with exogenous 15-HETE-PE before activation with PMA. Images show staining for histone H1 (green; G,J), myeloperoxidase (red; H,K), or overlay images (I,L). n = 4; mean ± SEM. Data presented from 1 experiment and representative of ≥ 3; * P < .05, ** P < .01, and *** P < .001 vs activated.
