Figure 1

Extracellular xanthine oxidase-derived superoxide induces NETs. (A) Neutrophils were incubated with media alone (negative control), PMA (100 nM), or xanthine oxidase (10 mU/mL) and hypoxanthine (500 nM) to yield superoxide, for 1–8 h at 37°C. Cells were then fixed, immunolabeled with antibody to citrullinated Histone H3, detected with a conjugated fluorescent secondary antibody (green), then stained for DNA with Hoechst (blue). (B) Western blot of citrullinated Histone H3 in neutrophils following treatment as above, or (C) with or without the competitive xanthine oxidase inhibitor allopurinol. β-actin is shown as a loading control. Densitometric quantification charts show mean ± SEM cit-H3 to β-actin, expressed as fold over control. (D) Assay of superoxide generated, and (E) NET MPO-DNA complexes formed in media by xanthine oxidase (0, 5, 10 or 20 mU/mL) and hypoxanthine (500 nM) after 8 h at 37°C. Shown as mean fold over control. (F) NET formation kinetics in response to superoxide or PMA as measured by a fluorescent plate reader assay for extruded DNA fibers (n = 8). (G) Allopurinol inhibits superoxide-induced NET formation. NET release is expressed as percentage of total DNA (n = 8). *p < .05 vs control; ^#p < .05 vs superoxide treatment; **p < .001 vs control. All figures represent at least 3 independent experiments for each stimulus.