Morphologic characterization of the Siglec-9-mediated caspase-independent neutrophil death. (A) Light microscopy: anti-Siglec-9 cross-linking alone induced neutrophil apoptosis (reduced cell volume and nuclear condensation). GM-CSF followed by Siglec-9 engagement resulted in both apoptotic and aberrant neutrophil death. The aberrant death was characterized by cytoplasmic vacuolization (arrows). Cells were stained with Giemsa-May-Grünwald (Diff-Quik). Original magnification, × 1000. Representative examples of 4 independent experiments are shown. (B) Transmission electron microscopy: panel i demonstrates a normal viable neutrophil; panel ii shows an apoptotic neutrophil with condensation of the nucleus and cell shrinkage. Panels iii and iv demonstrate the aberrant morphology of GM-CSF-primed and Siglec-9-stimulated neutrophils, which contained many vacuole-like double-membrane structures. Approximately 25% of these cells showed condensation of the nucleus and cell shrinkage. The mitochondria were partially swollen. The plasma membrane appeared to be intact. The cells in panels A and B were from 15-hour cultures.