The Apoptosome Pathway to Caspase Activation in Primary Human Neutrophils Exhibits Dramatically Reduced Requirements for Cytochrome c

Abstract

Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the “apoptosome” as a result of the cell stress–associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell–free extracts readily supported Apaf-1–dependent caspase activation, suggesting that these cells may assemble cytochrome c–independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1–dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1–dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.

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

“…It is released from mitochondria upon an apoptotic insult and participates in the assembly of the apoptosome to promote caspase-9 activation. 31,32 However, the present data and the results from other studies 7,24,25 indicate that neutrophils express an extremely low amount of cytochrome c. In our hands, cytochrome c was only detectable by Western blot after concentration procedures such as subcellular fractionation or immunoprecipitation (see Figures 5,7 and 8b). Moreover, during the DMSO-induced neutrophilic differentiation of HL-60 cells, we observed a gradual depletion of The equivalent of B30 mg total protein was subjected to SDS-PAGE, and Western blot was performed with specific Abs for indicated proteins.…”

“…This underscores that in neutrophils, which possess a powerful proteolytic potential, the absence of protein detection does not necessarily indicate that a protein is not expressed. Indeed, the use of the DFP-treated neutrophil lysates enabled us to detect the mitochondrial proapoptotic effector Smac and caspase-9 ( Figures 7 and 9), whereas other investigators 7,24 have failed to detect these in neutrophils.…”

“…The latter is currently under investigation, although we expect that cytochrome c-independent mitochondrial complexes are likely to preserve some activity in neutrophils, because mitochondria-derived ROS have been shown to mediate the TNF-a-induced caspase-independent neutrophil cell death. 6 In this respect, also a high expression of the mitochondrial anti-oxidant enzyme MnSOD in neutrophils seems to be logical (see Figure 5, lane 4 and Murphy et al 24 ), because this enzyme may have the important physiological role of inactivating excessive ROS in the absence of the functional 'natural' sink of electrons -complex IV. Increased oxidant injury due to loss of the protective function of MnSOD has been proposed to play a role in glucocorticoid-induced apoptosis of eosinophils, 34 which are believed to have a defective mitochondrial respiration like neutrophils.…”

“…While this manuscript was in preparation, Murphy et al 24 have published a study in which they proposed that, in neutrophils, the cytochrome-c-dependent apoptotic pathway displays a dramatic reduction in the requirement for cytochrome c. It was suggested that neutrophils have a lowered threshold requirement for cytochrome c, and their low content of cytochrome c is partially compensated by the increased expression of Apaf-1. 24 This is in keeping with the present results, which show a relatively high content of Apaf-1 in neutrophils in comparison to HL-60 cells (Figure 8a), and with the hypothesis that upregulation of Apaf-1 contributes to the increased sensitivity of apoptosome activation to cytochrome c. 36 On the other hand, neutrophil-derived cytoplasts, which are devoid of any detectable cytochrome c (Figure 8), still displayed intact activation of caspase-9 ( Figure 9). Perhaps, the apoptosome pathway of caspase-9 activation in neutrophils demands even less cytochrome c than has been proposed.…”

Delayed apoptosis of tumor associated neutrophils in the absence of endogenous IFN‐β

“…23 Regarding neutrophil apoptosis, the intrinsic apoptosis pathway exhibits a low threshold requirement for Cytochrome c, which is compensated by a high cytosolic expression of Apaf-1. 46 We were not able to detect differences in Cytochrome c release, comparing neutrophils of tumor-bearing WT or Ifnb1 2/2 mice. However, Apaf-1 gene expression was significantly upregulated in the presence of IFN-b.…”