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Under inflammatory conditions, neutrophil apoptosis is delayed due to survival-factor exposure, a mechanism that prevents the resolution of inflammation. One important proinflammatory cytokine involved in the regulation of neutrophil survival/activation is granulocyte-macrophage colony-stimulating factor (GM-CSF). Although GM-CSF mediates antiapoptotic effects in neutrophils, it does not prevent apoptosis, and the survival effect is both time dependent and limited. Here, we identified the proapoptotic Bcl-2 family member Bim as an important lifespan limiting molecule in neutrophils, particularly under conditions of survival factor exposure. Strikingly, GM-CSF induced Bim expression in both human and mouse neutrophils that was blocked by pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). Increased Bim expression was also seen in human immature bone marrow neutrophils as well as in blood neutrophils from septic shock patients; both cell populations are known to be exposed to GM-CSF under in vivo conditions. The functional role of Bim was investigated using Bim-deficient mouse neutrophils in the presence and absence of the survival cytokines interleukin (IL)-3 and GM-CSF. Lack of Bim expression resulted in a much higher efficacy of the survival cytokines to block neutrophil apoptosis. Taken together, these data demonstrate a functional role for Bim in the regulation of neutrophil apoptosis and suggest that GM-CSF and other neutrophil hematopoietins initiate a proapoptotic counterregulation that involves upregulation of Bim.

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.

Apoptosis involves mitochondrial steps such as the release of the apoptogenic factor cytochrome c which are effectively blocked by Bcl-2. Although Bcl-2 may have a direct action on the mitochondrial membrane, it also resides and functions on the endoplasmic reticulum (ER), and there is increasing evidence for a role of the ER in apoptosis regulation as well. Here we uncover a hitherto unrecognized, apoptotic crosstalk between the ER and mitochondria that is controlled by Bcl-2. After triggering massive ER dilation due to an inhibition of secretion, the drug brefeldin A (BFA) induces the release of cytochrome c from mitochondria in a caspase-8- and Bid-independent manner. This is followed by caspase-3 activation and DNA/nuclear fragmentation. Surprisingly, cytochrome c release by BFA is not only blocked by wild-type Bcl-2 but also by a Bcl-2 variant that is exclusively targeted to the ER (Bcl-2/cb5). Similar findings were obtained with tunicamycin, an agent interfering with N-linked glycosylations in the secretory system. Thus, apoptotic agents perturbing ER functions induce a novel crosstalk between the ER and mitochondria that can be interrupted by ER-based Bcl-2.

Following exposure of cells to stimuli that trigger programmed cell death (apoptosis), cytochrome c is rapidly released from mitochondria into the cytoplasm where it activates proteolytic molecules known as caspases that specifically cleave the amino-acid sequence DEVD and are crucial for the execution of apoptosis. The protein Bcl-2 interferes with this activation of caspases by preventing the release of cytochrome c. Here we study these molecular interactions during apoptosis induced by the protein Bax, a pro-apoptotic homologue of Bcl-2. We show that in cells transiently transfected with bax, Bax localizes to mitochondria and induces the release of cytochrome c, activation of caspase-3, membrane blebbing, nuclear fragmentation, and cell death. Caspase inhibitors do not affect Bax-induced cytochrome c release but block caspase-3 activation and nuclear fragmentation. Unexpectedly, Bcl-2 also fails to prevent Bax-induced cytochrome c release, although it co-localizes with Bax to mitochondria. Cells overexpressing both Bcl-2 and Bax show no signs of caspase activation and survive with significant amounts of cytochrome c in the cytoplasm. These findings indicate that Bcl-2 can interfere with Bax killing downstream of and independently of cytochrome c release.