Siglec-9 cross-linking accelerates spontaneous neutrophil death, which is enhanced following GM-CSF priming. Cell death was assessed by ethidium bromide uptake and flow cytometric analysis. (A) Time-dependent acceleration of neutrophil death by Siglec-9 and Fas receptor cross-linking, respectively. GM-CSF-enhanced Siglec-9-mediated, but not Fas-triggered, cell death (n = 4). ▪ indicates GM-CSF + anti-Siglec-9; ⋄, anti-Fas; ▵, GM-CSF + anti-Fas; •, anti-Siglec-9 alone; ○, medium; and □, GM-CSF alone. (B) Concentration-effect curve of anti-Siglec mAb in 20-hour neutrophil cultures. Maximal death effects in the absence of cytokine priming were seen at 35 μg/mL. GM-CSF preincubation for 20 minutes and its presence in subsequent culture resulted in higher death efficacy and potency of the mAb (n = 3). Symbols are as in panel A. (C, top) Priming by GM-CSF was time dependent. Addition of GM-CSF after Siglec-9 cross-linking had no effect. Cells were cultured for 20 hours. (Bottom) Stimulation with GM-CSF resulted in rapid tyrosine phosphorylation of Siglec-9 and SHP-1. In these experiments, neutrophil lysates were immunoprecipitated (IP) with anti-ptyr mAb followed by immunoblotting with anti-Siglec-9 and anti-SHP-1 mAbs. One representative out of 4 independent experiments is shown. (D) GM-CSF, IFN-α, and IFN-γ, but not G-CSF, MIF, and IL-1β were able to enhance the Siglec-9-mediated neutrophil death. Results of 24-hour cultures are shown (n = 4). Cytokines were preincubated for 20 minutes before cross-linking. □ indicates control IgG1; and ▪, anti-Siglec-9. ^**P < .01; ^***P < .001. In panels A, B, and D, data are expressed as means ± SEM.