Activation of the DNA damage response downregulates neutrophil cytokine production. (A) Neutrophil IL-8 production in response to LPS stimulation in the presence of inhibitors of DNA damage-response proteins. Neutrophils were incubated with the indicated inhibitors (inhibitor targets in parentheses) and stimulated 18 hours with LPS in triplicate. IL-8 concentrations in supernatants were measured by ELISA. Data are plotted as the mean ± SEM of compiled experiments (n = 5). The black line represents the amount of IL-8 produced by LPS-stimulated control cells. (B) Neutrophil viability time course in the presence of p53 inhibitor pifithrin-µ (1 µM) measured by PE-annexin V/PI staining, as before. (C) Western blot analysis of phosphorylation of ATM and histone H2A.X in neutrophil lysates made after 1 hour of treatment with cisplatin, etoposide, and γ-irradiation. (D) Neutrophil IL-8 production in response to 18-hour LPS stimulation after 1 hour exposure to the indicated DNA damage–inducing agents. Data are plotted as the mean ± standard deviation. Results are representative of 3 experiments. (E) Neutrophil viability after 1 hour of incubation with indicated DNA damage–inducing treatment followed by 8 hours of stimulation with LPS or left unstimulated. Viability was measured as described by PE-annexin V/PI staining. Asterisks indicate significance: *P < .05, **P < .01, ***P< . 001 by paired Student t test.