Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

Abstract

Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

“…The caspase-8 inhibitor z-IETD-fmk and the caspase-9 inhibitor z-LEHD-fmk demonstrated similar blocking efficacies, consistent with previously published work suggesting that these caspases also play a role in neutrophil apoptosis. 15,17 Taken together, NA accelerated spontaneous neutrophil apoptosis in a caspase-dependent manner in vitro.…”

“…5 In contrast, immature neutrophils exhibit a much longer life-span in vitro, although they also undergo cell death at later time points. 15,16 Addition of NA to purified cell populations resulted in a detectable cytotoxic effect in mature neutrophils, but not in eosinophils or immature neutrophils (including HL-60 cells), in a concentration-dependent manner in 24-h cultures (Figure 3a). Longer culture times (48-h) revealed same results, except that spontaneous death of mature granulocytes was more advanced (data not shown).…”

“…Mature blood neutrophils were isolated from peripheral blood of healthy donors by Ficoll-Hypaque centrifugation. 11,15,35,36 Briefly, peripheral blood mononuclear cells were separated by centrifugation on Ficoll-Hypaque (Seromed-Fakola AG, Basel, Switzerland). The lower phase, mainly granulocytes and erythrocytes, was treated with erythrocyte lysis solution (155 mmol/l NH 4 Cl, 10 mmol/l KHCO 3 , and 0.1 mmol/l EDTA, pH 7.3).…”

“…15,16 Briefly, following centrifugation on a two-step discontinuous percoll density gradient, cells were negatively isolated using anti-CD7 and anti-CD36 mAbs to eliminate contaminating lymphoid and erythroid precursors. The resulting cell population contained 497% cells of the neutrophil lineage as determined by myeloperoxidase staining, flow cytometric analysis of lineage-associated surface proteins, 15 as well as with DiffQuik and light microscopy. The proportion of mature neutrophils was less than 5%.…”

“…Cell death was assessed by uptake of 1 mM ethidium bromide and flow cytometric analysis (FACSCalibur). 11,15,35,36 To determine whether cell death was apoptosis, DNA fragmentation and redistribution of PS were measured. 11,15,35,36 Immunoprecipitation.…”