Syk-deficient neutrophils display reduced integrin-mediated superoxide production in response to bacteria. (A) Flow cytometric detection of superoxide by fluorescence conversion of APF in WT or syk^−/− neutrophils after stimulation with APC-labeled S aureus (MOI = 5). (B) WT, syk^−/−, CD11b^−/−, or CD18^−/− neutrophils were plated in microtiter wells containing S aureus (MOI = 5) or (C) E coli (MOI = 10), in cytochrome c media. Production of superoxides (SOx) by respiratory burst was measured as reduction of cytochrome c. Data are representative of at least 3 independent experiments and are mean ± SD. (D) Western blot analysis of p40^phox phosphorylation (Thr 154) after priming of neutrophils for 30 minutes with 50 ng/mL TNF-α and, where indicated, 30 minutes with S aureus (Sa) MOI = 20, E coli (Ec) MOI = 40, or 10nM PMA. (E) Quantitation of phospho-p40^phox normalized to actin. (F-G) Intracellular activation of elastase was detected by flow cytometry after cleavage of ElastoLux. Neutrophils of the indicated genotype were stimulated with (F) S aureus or (G) E coli (MOI = 5) for the indicated time points. Data are representative of 3 independent experiments and are mean ± SD. *P < .05, **P < .01, ^#P < .001 compared with WT by ANOVA.