Oxidase activity in fMLP-stimulated, TNF-α-primed mouse neutrophils. (A) Bone marrow neutrophils obtained from the indicated strains of mice were prepared in parallel, primed with 500 U/mL TNF-α, and preincubated with luminol and HRP, as described in “Materials and methods.” Cells (3.75 × 105 per well) were stimulated with 10 μM fMLP, and chemiluminescence was recorded using a Berthold MicroLumatPlus luminometer. Incubations were performed in duplicate, and data (mean ± range) from 1 of 2 representative experiments are shown. (B) Experiments were performed as described in panel A except that cells were incubated with the luminol and HRP in the presence of 0 to 30 μM TGX-221. Data represent the accumulated light emission over 3 minutes expressed as a percentage of the value with no inhibitor (mean ± SEM from 3 separate experiments). (C) Wild-type murine neutrophils (5 × 106/point, performed in triplicate) were primed with 500 U/mL TNF-α and preincubated for 5 minutes with 0 to 30 μM AS-252424 before stimulation with 10 μM fMLP. Oxidative burst activity was quantitated by a reduction of cytochrome c. Data represent mean ± SEM from 3 separate experiments.
