Src Family Kinases and Syk Are Required for Neutrophil Extracellular Trap Formation in Response to β-Glucan Particles

We report that particles of β-glucan, one of the surface components of yeasts, are powerful inducers of neutrophil extracellular trap (NET) formation in human neutrophils. β-Glucan triggered a prolonged phosphorylation of Src family kinases and Syk that were suppressed by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d] pyrimidine (PP2) and a novel Syk inhibitor, PRT-060318, respectively. PP2 and PRT-060318 also inhibited β-glucan-induced NET formation and reactive oxygen species (ROS) generation, suggesting that both responses are triggered by a Src/Syk-regulated signaling pathway. Given that the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) markedly inhibited NET formation, our findings suggest that ROS are required for the full-blown formation of NETs in response to β-glucan particles. Contrary to β-glucan, ROS generation triggered by phorbol myristate acetate (PMA) was unaffected by PP2 and PRT-060318, but these compounds, as well as DPI, suppressed Src/Syk phosphorylation triggered by PMA. Whereas PP2 had no effect on PMA-induced NET formation, PRT-060318 had a significant, albeit partial, inhibitory effect, thus suggesting that ROS induce NET formation in part via activation of Syk. These findings were substantiated by the evidence that neutrophils from mice with the conditional deletion of Syk were defective in formation of NETs in response to β-glucan.