Neutrophil oxidative burst activates the DNA damage response and regulates cytokine production. (A-D) NADPH oxidase-derived ROS activates the DNA damage response in stimulated neutrophils. (A) Phospho-ATM (red) and DNA (blue) staining of healthy neutrophils activated with PMA in the presence of 30 μM ROS scavenger pyrocatechol. neutrophils were stimulated for 90 minutes with PMA to induce ROS production, fixed, permeabilized, stained with anti-pATM (pS1981) antibody, and visualized by confocal microscopy. (B-D) Western blot analysis of DNA damage-response activation in lysates from neutrophils stimulated with PMA, opsonized zymosan, or 10 Gy irradiation in the absence or presence of pyrocatechol (B), or isolated from control and CGD patient (C). (D-E) IL-8 production from neutrophils deficient in ATM activity and ROS production. (D) IL-8 production from neutrophils isolated concurrently from a CGD patient, an AT patient, and a healthy control after 18 hour of LPS stimulation (single experiment). Data are plotted as the mean ± SEM of triplicate stimulations. (E) IL-8 production from healthy control neutrophils incubated with ROS scavenger pyrocatechol or ATM inhibitor KU-55933, and stimulated for 18 hours with LPS (n = 3). IL-8 concentration in supernatants was measured by ELISA. (F) IL-8 production from neutrophils incubated with pyrocatechol, pretreated for 1 hour with indicated concentrations of etoposide, and stimulated for 18 hours with LPS (n = 3). (E-F) Data are presented as the mean ± SEM of compiled experiments. Asterisks indicate significance: *P < .05, **P < .01, ***P < .001 by paired Student t test.