Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils

Abstract

The exact mechanism of apoptosis in neutrophils (PMNs) and the explanation for the antiapoptotic effect of granulocyte colony-stimulating factor (G-CSF) in PMNs are unclear. Using specific fluorescent mitochondrial staining, immunofluorescent confocal microscopy, Western blotting, and flow cytometry, this study found that PMNs possess an unexpectedly large number of mitochondria, which are involved in apoptosis. Spontaneous PMN apoptosis was associated with translocation of the Bcl-2-like protein Bax to the mitochondria and subsequent caspase-3 activation, but not with changes in the expression of Bax. G-CSF delayed PMN apoptosis and prevented both associated events. These G-CSF effects were inhibited by cycloheximide. The general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented caspase-3 activation and apoptosis in PMNs, but not Bax redistribution. PMNderived cytoplasts, which lack a nucleus, granules, and mitochondria, spontaneously underwent caspase-3 activation and apoptosis (phosphatidylserine exposure), without Bax redistribution. zVAD-fmk inhibited both caspase-3 activation and phosphatidylserine exposure in cultured cytoplasts. Yet, G-CSF prevented neither caspase-3 activation nor apoptosis in cytoplasts, confirming the need for protein synthesis in the G-CSF effects. These data demonstrate that (at least) 2 routes regulate PMN apoptosis: one via Bax-tomitochondria translocation and a second mitochondria-independent pathway, both linked to caspase-3 activation. Moreover, G-CSF exerts its antiapoptotic effect in the first, that is, mitochondria-dependent, route and has no impact on the second.

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

“…3,5,6 However, in neutrophils, these organelles hardly play a role in energy metabolism as they do in other cells. This conclusion was derived from the experiment shown in Figure 1, where ATP concentrations in neutrophils were measured under various conditions.…”

“…from four independent experiments During the neutrophilic lineage maturation, HL-60 cells gain tubular mitochondria and lose cytochrome c expression Despite a limited role of neutrophil mitochondria in cellular metabolism, our recent studies have suggested that these organelles might be involved in neutrophil cell death. 5,6 In an attempt to clarify the role of mitochondria in apoptosis, we studied the expression of a number of proapoptotic mitochondrial proteins. 1,19 Unexpectedly, none of the tested proteins, including cytochrome c, AIF, Omi and Smac gave a positive signal in Western blot ( Figure 5, lane 1), whereas a mitochondrial isoform of superoxide dismutase (MnSOD) or the cytoplasmic protein Bax were readily detectable ( Figure 5, lane 1).…”

“…4 However, mitochondria have been recently visualized in neutrophils as a tubular network, by means of specific fluorescent dyes. 3,5 Furthermore, in neutrophils undergoing spontaneous or induced apoptosis, the mitochondria form clusters to which the proapoptotic Bax protein also is localized. [5][6][7] Such a subcellular redistribution of Bax to mitochondria upon apoptosis is known to result in the permeabilization of the outer mitochondrial membrane, with the subsequent release of additional proapoptotic proteins.…”

Dietary low or excess levels of lipids reduced growth performance, and impaired immune function and structure of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella) under the infection of Aeromonas hydrophila

“…In this study, compared with optimal dietary lipids levels, low or excess levels of lipids upregulate the caspase 3, caspase 7, caspase 8 and caspase 9 mRNA levels in the head kidney and spleen of young grass carp, suggesting that low or excess levels of lipids could increase apoptosis, whereas supplementation with optimal lipids could reverse this increase in the caspase mRNA levels in the head kidney and spleen of young grass carp. The B-cell lymphoma-2 (Bcl-2) family is an important class of regulatory proteins, which regulate apoptosis at the mitochondrial level [103]. In human cells, they can be divided into anti-apoptotic (such as Bcl-2) and pro-apoptotic (such as Bax) proteins according to their function [104].…”

Early mitochondrial alterations in ATRA-induced cell death

“…8 Furthermore, it reduces spontaneous apoptosis of neutrophils in vitro. 16,17 In vivo, the clinical effects of G-CSF are also related to its antiapoptotic properties. It is routinely used to overcome neutropenia after chemotherapy, and to treat severe congenital neutropenia in Kostmann syndrome.…”