Spontaneous in Contrast to CD95-Induced Neutrophil Apoptosis Is Independent of Caspase Activity

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Abstract
Background The influence of caspase inhibitors on spontaneous and on CD95-triggered apoptosis was investigated in neutrophils from healthy volunteers and compared with neutrophils from patients with severe sepsis. Methods To further elucidate the mechanisms of neutrophil apoptosis, isolated neutrophils from healthy volunteers (n = 9) were either stimulated with the agonistic anti-CD95 antibody (100 ng/mL) or left unstimulated in the presence or absence of the caspase inhibitors zIETD-fmk (10 μmol/L), zDEVD-fmk (10 μmol/L), or zVAD-fmk (20 μmol/L). Apoptosis was determined by measuring DNA fragmentation and Annexin-V binding in FACS, and caspase-3-like activity by DEVD-afc cleavage assay. Results were compared with those from patients with severe sepsis (n = 15). Results Reduced spontaneous neutrophil apoptosis in patients with sepsis (−48.7%) was completely restored by incubation with agonistic anti-CD95 antibody (p < 0.05). Inhibition of caspases did not influence spontaneous neutrophil apoptosis in both groups. However, zVAD-fmk reduced anti-CD95 antibody-induced apoptosis in neutrophils from controls by −22.6% (p < 0.05) and in patients with sepsis by −43.1% (p < 0.05). Conclusion These results indicate that spontaneous in contrast to CD95-induced neutrophil apoptosis is independent of caspase activity.