Mcl-1-Mediated Impairment of the Intrinsic Apoptosis Pathway in Circulating Neutrophils from Critically Ill Patients Can Be Overcome by Fas Stimulation

Abstract

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Inhibition of neutrophil apoptosis by PAI-1

“…Exposure of neutrophils to TNF-related apoptosis-inducing ligand (TRAIL) resulted in enhanced apoptosis (Fig. 6F), consistent with previous studies showing the importance of extrinsic induced apoptotic pathways in neutrophil death (27,31,41). Inclusion of PAI-1 in the neutrophil cultures diminished the proapoptotic effects of TRAIL (Fig.…”

Molecular Mechanisms Underlying Delayed Apoptosis in Neutrophils from Multiple Trauma Patients with and without Sepsis

“…This prolonged neutrophil survival is associated with the accumulation of activated neutrophils contributing to an ongoing inflammation, and consecutive host tissue damage, with subsequent organ failure in critical ill patients. Profound inhibition of apoptosis has been already reported in neutrophils from patients with systemic inflammatory response syndrome (SIRS) (2,10), in sepsis (11)(12)(13)(14), as well as after burn injuries (15) and acute respiratory distress syndrome (8,16,17). These observations suggest that the resolution of the inflammatory response is highly dependent from the induction of neutrophil programmed cell death.…”

“…To measure the mitochondrial membrane potential, neutrophils were stained with the lipophilic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1; Sigma Aldrich, Taufkirchen, Germany) as previously described (10). Then, cells immediately analyzed by flow cytometry.…”

“…Apoptosis was normalized to total double-stranded (ds) DNA, which has been quantified by PicoGreen (Invitrogen) staining. In addition, neutrophil apoptosis was measured by propidium iodide staining and flow cytometry as previously described (10). Nuclei to the left of the G1 peak (sub-G1) containing hypodiploid DNA, corresponding to fragmented DNA, were considered apoptotic.…”

Cytochrome c modulates the mitochondrial signaling pathway and polymorphonuclear neutrophil apoptosis in bile duct-ligated rats

“…Matsuda et al (11) showed that cytokine-modulated inhibition of PMN apoptosis at a local site augments PMN functions and induces excessive inflammatory response. The anti-apoptotic B cell lymphoma-2 (Bcl-2) family members Bcl-X-linked, A1 and myeloid cell leukemia-1, have been demonstrated to inhibit PMN apoptosis and promote survival by antagonizing the pro-apoptotic proteins (35). However, Fas ligation or ligation of the tumor necrosis-related apoptosis-inducing ligand receptor on PMN may also trigger apoptosis (36)(37)(38).…”