The death mediated by Siglec-9 upon GM-CSF priming is ROS-dependent. (A) The ROS scavenger NAC partially blocked Siglec-9-mediated death in normal GM-CSF-primed neutrophils. NAC and caspase inhibitors had an additive blocking effect, although no complete inhibition was achieved. Results of 20-hour cultures are shown (n = 4). ^*P < .05. (B) The inhibitor of ROS production DPI completely blocked Siglec-9-mediated death both in the absence (□) and presence (▪) of GM-CSF. Results of 20-hour cultures are shown (n = 4). (C) Comparison of normal (n = 6) and CGD (n = 2) neutrophils. Although spontaneous death of CGD neutrophils appeared to be delayed, they demonstrated normal responses to GM-CSF, anti-Fas mAb. However, the death effect of anti-Siglec-9 mAb appeared to be reduced, and the death priming effect of GM-CSF was not at all detectable in CGD neutrophils. Results of 24-hour cultures are shown. (D) Anti-Siglec-9 mAb stimulation resulted in increased production of ROS no matter whether GM-CSF was present or not. Neutrophils were stimulated for 4 hours (n = 3). Data in all panels are expressed as means ± SEM.