fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils
fMLP increases IL-8 release, ROS production, intracellular pH, and ERK1/2, p38 MAPK, and Akt phosphorylation. Human neutrophils were incubated with 100 nM fMLP or PAF for 4 h, and the IL-8 concentration in the supernatants was measured by ELISA (a). Neutrophils were incubated for 5 min at 37°C before fMLP or PAF was added. ROS production was monitored for 1200 s using a luminescence assay. RLU: relative luminescence unit (b), and superoxide production was measured following 30 min of incubation by a cytochrome c reduction assay (c). Curve dose response of ROS production in neutrophils stimulated with fMLP or PAF. AUC: area under curve for 700 s (AUC700) (d). BCECF-AM-loaded neutrophils were incubated for 5 min at 37°C, fMLP or PAF was added, and the signal was measured for 600 s in a spectrofluorometer (e). Neutrophils were incubated with fMLP or PAF for 2 min, and total protein was analysed by immunoblot with specific antibodies against the phosphorylated form of Akt, p38 MAPK, and ERK1/2. In this case the same membrane was used after stripping procedure for reprobed and total ERK1/2 antibody was used as a charge control (f). Mean ± SE, .
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