To be or not to be, the level of autophagy is the question: dual roles of autophagy in the survival response to starvation

Autophagy is an evolutionally conserved lysosomal pathway used to degrade and turn over long-lived proteins and cytoplasmic organelles. Since autophagy was discovered, it has been thought to act as a pro-survival response to several stresses, especially starvation, at the cell and organism levels by providing recycled metabolic substrates to maintain energy homeostasis. However, several recent studies suggest that autophagy also plays a pro-death role through an autophagic cell death pathway mostly at the cellular level. The mechanism by which autophagy could perform these seemingly opposite roles as a pro-survival and a pro-death mechanism remained elusive until recently. Using C. elegans as a model system, we found that physiological levels of autophagy promote optimal survival of C. elegans during starvation, but either insufficient or excessive levels of autophagy render C. elegans starvation-hypersensitive. Furthermore, we found that muscarinic acetylcholine receptor signaling is important in modulating the level of autophagy during starvation, perhaps through DAP kinase and RGS-2. Our recent study provides in vivo evidence that levels of autophagy are critical in deciding its promotion of either survival or death: Physiological levels of autophagy are pro-survival, whereas insufficient or excessive levels of autophagy are pro-death.     

Cytosolic accumulation of HSP60 during apoptosis with or without apparent mitochondrial release: evidence that its pro-apoptotic or pro-survival functions involve differential interactions with caspase-3

Most heat shock proteins (HSPs) have pro-survival functions. However, the role of HSP60, a mitochondrial matrix protein, is somewhat controversial with both pro-survival and pro-apoptotic functions reported. Here we show that in numerous apoptotic systems HSP60 protein accumulates in the cytosol. In BMD188-induced cell death, HSP60 accumulates in the cytosol with significant mitochondrial release. In contrast, in apoptosis induced by multiple other inducers, the cytosolic HSP60 accumulates without an apparent mitochondrial release. The short interfering RNA-mediated knockdown experiments revealed that in BMD188-induced apoptosis, HSP60 has a pro-death function and that the pro-death role of HSP60 seems to involve caspase-3 maturation and activation in the cytosol. In contrast, HSP60 appears to play a pro-survival role in other apoptotic systems where there is no apparent mitochondrial release as its knockdown promotes cell death. In these latter apoptotic systems HSP60 does not associate with active caspase-3. In both cases, HSP60 does not appreciably interact with Bax. Taken together, our results suggest the following: 1) cytosolic accumulation of HSP60 represents a common phenomenon during apoptosis induction; 2) cytosolic HSP60 accumulation during apoptosis occurs either with or without apparent mitochondrial release; and 3) the cytosolically accumulated HSP60 possesses either pro-survival or pro-death functions, which involves differential interactions with caspase-3.     

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine

There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 muM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.     

Molecular regulation of autophagy and apoptosis during ischemic and non-ischemic cardiomyopathy

A significant understanding of the genetic signaling pathways governing the extrinsic and intrinsic apoptotic pathways has been established. In recent years, the role of apoptosis in the heart during ischemic and non-ischemic cardiomyopathies has been under investigation and reported to contribute to ventricular remodeling and heart failure. Autophagy has been recently characterized as an essential cellular process in the heart, but whether autophagy functions as a pro-death or pro-survival program during disease conditions is still not completely understood. The mitochondrial death protein Bnip3 has been implicated in both apoptosis and autophagy, and its role in both processes is also discussed.     

Molecular regulation of autophagy and apoptosis during ischemic and non-ischemic cardiomyopathy

A significant understanding of the genetic signaling pathways governing the extrinsic and intrinsic apoptotic pathways has been established. In recent years, the role of apoptosis in the heart during ischemic and non-ischemic cardiomyopathies has been under investigation and reported to contribute to ventricular remodeling and heart failure. Autophagy has been recently characterized as an essential cellular process in the heart, but whether autophagy functions as a pro-death or pro-survival program during disease conditions is still not completely understood. The mitochondrial death protein Bnip3 has been implicated in both apoptosis and autophagy, and its role in both processes is also discussed.     

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.     

Role of cathepsin B in dengue virus-mediated apoptosis

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.     

Crosstalk between apoptosis,necrosis and autophagy

Apoptosis and necrosis are the two major modes of cell death, the molecular mechanisms of which have been extensively studied. Although initially thought to constitute mutually exclusive cellular states, recent findings reveal cellular contexts that require a balanced interplay between these two modes of cellular demise. Several death initiator and effector molecules, signaling pathways and subcellular sites have been identified as key mediators in both processes, either by constituting common modules or alternatively by functioning as a switch allowing cells to decide which route to take, depending on the specific situation. Importantly, autophagy, which is a predominantly cytoprotective process, has been linked to both types of cell death, serving either a pro-survival or pro-death function. Here we review the recent literature that highlights the intricate interplay between apoptosis, necrosis and autophagy, focusing on the relevance and impact of this crosstalk in normal development and in pathology. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic.     

Targeted regulation of autophagy using nanoparticles: New insight into cancer therapy

Normal cells depend on autophagy to maintain cellular homeostasis by recycling damaged organelles and misfolded proteins and degrading toxic agents. Similar to apoptosis, targeting autophagy has been under attention in cancer therapy. However, autophagy has both pro-survival and pro-death functions in tumors, and its targeting requires further elucidation. The current review focuses on using nanoparticles for targeting autophagy in cancer treatment. Nanocarriers can deliver autophagy regulators along with chemotherapeutic agents leading to intracellular accumulation in cancer cells and synergistic cancer therapy. Furthermore, genetic tools such as siRNA and shRNA can be used for targeting molecular components that regulate autophagy, such as the ATG12-ATG5-ATG16L1 complex. A number of nanostructures, such as gold and zinc oxide nanoparticles, can be used to enhance oxidative stress-mediated apoptosis and autophagy, reducing cancer progression. Further, using nanoparticles to modulate autophagy potentiates the anti-tumor effects of cisplatin and gefitinib during chemotherapy. Polymeric nanoparticles, lipid-based nanostructures and carbon-based nanomaterials are among other nanoparticles capable of regulating autophagy in cancer cells. Of note, various regulatory components of autophagy such as ATGs, Beclin-1 and LC3-II can be affected by nanomaterials. Based on the role of nanomaterial-induced autophagy as pro-survival or pro-death, further targeting can potentiate the fight against cancer cells.     

Endosomal-lysosomal proteolysis mediates death signalling by TNFalpha,not by etoposide,in L929 fibrosarcoma cells: evidence for an active role of cathepsin D

In several 'in vitro' models of apoptosis, lysosomal proteolysis has been shown to play an active role in mediating the death signal by cytokines or antiblastic drugs. Depending on the experimental cell model and the cytotoxic stimulus applied, an increased expression and the cytosolic translocation of either cathepsin D or B have been reported in apoptotic cells. We have analysed the involvement of these lysosomal proteases in a canonical apoptotic cell model, namely L929 fibroblasts, in which apoptosis was induced by cytotoxic agents acting through different mechanisms: (i) the cytokine TNFalpha, which triggers the cell suicide via interaction with its membrane receptor, and (ii) the topoisomerase II-inhibitor etoposide (VP16), which directly causes DNA damage. In both cases the activity of cathepsins B and D increased in apoptosing cultures. CA074-Me, a specific inhibitor of cathepsin B, and Leupeptin, a broad inhibitor of serine and cysteine proteases (among which is cathepsin B), did not exert any protection from TNFalpha. In contrast, pre-loading the cells with pepstatin A, a specific inhibitor of cathepsin D, protected L929 cells from TNFalpha cytotoxicity by more than 50%. However, no protection was observed if pepstatin A was added concomitantly with the cytokine. Inhibition of either cathepsin B or D did not impede apoptosis induced by etoposide. Lysosomal integrity was preserved and cathepsin D remained still confined in vesicular structures in apoptotic cells treated with either TNFalpha or etoposide. It follows that proteolysis by cathepsin D is likely to represent an early event in the death pathway triggered by TNFalpha and occurs within the endosomal-lysosomal compartment.     

Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.     

Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose

Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.     

Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program.     

Proliferative versus apoptotic functions of caspase-8 Hetero or homo: the caspase-8 dimer controls cell fate

Caspase-8, the initiator of extrinsically-triggered apoptosis, also has important functions in cellular activation and differentiation downstream of a variety of cell surface receptors. It has become increasingly clear that the heterodimer of caspase-8 with the long isoform of cellular FLIP (FLIP(L)) fulfills these pro-survival functions of caspase-8. FLIP(L), a catalytically defective caspase-8 paralog, can interact with caspase-8 to activate its catalytic function. The caspase-8/FLIP(L) heterodimer has a restricted substrate repertoire and does not induce apoptosis. In essence, caspase-8 heterodimerized with FLIP(L) prevents the receptor interacting kinases RIPK1 and -3 from executing the form of cell death known as necroptosis. This review discusses the latest insights in caspase-8 homo- versus heterodimerization and the implication this has for cellular death or survival. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.     

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage.

Among the cellular responses to photodamage initiated by photodynamic therapy (PDT) are autophagy and apoptosis. While autophagy is a reversible process that can be both a survival and a death pathway, apoptosis is irreversible, leading only to cell death. In this study, we followed the fate of mouse leukemia L1210 cells after photodamage to the endoplasmic reticulum (ER) using a porphycene photosensitizer, where Bcl-2 was among the PDT targets. In wild-type cells, we observed a rapid wave of autophagy, presumed to represent the recycling of some damaged organelles, followed by apoptosis. Using shRNA technology, we created a Bax knockdown line (L1210/Bax(-)). In the latter cell line, we found a marked decrease in apoptosis after photodamage or pharmacologic inactivation of Bcl-2 function, but this did not affect PDT efficacy. Loss of viability was associated with a highly-vacuolated morphology consistent with autophagic cell death. Previous studies indicated pro-survival attributes of autophagy after low-dose PDT, suggesting that autophagy may be responsible for the 'shoulder' on the dose-response curve. It appears that attempts at extensive recycling of damaged organelles are associated with cell death, and that this phenomenon is amplified when apoptosis is suppressed.     

Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.     

Autophagic and apoptotic response to stress signals in mammalian cells

Autophagy is a highly conserved catabolic programme for degrading proteins and organelles. This process has been shown to act as a pro-survival or pro-death mechanism in different physiological and pathological conditions. Several stress stimuli can induce autophagy, such as nutrient deprivation or critical steps in development of lower and higher eukaryotes. Apoptosis is an orchestrated form of cell death in which cells are actively involved in their own demise. Again, stress is a positive regulator of apoptosis and, in particular, of its apoptosome-mediated mitochondrial pathway. Besides discussing the individual roles played by the key molecules involved in autophagy in mammals in response to stress signals, we discuss here the interrelations between autophagy and apoptosis under these conditions.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis

Autophagy and apoptosis cooperate to modulate cell survival. Neutrophils are short-lived cells and apoptosis is considered to be the major mechanism of their death. In the present study, we addressed whether autophagy regulates neutrophil apoptosis and investigated the effects of autophagy inhibition on apoptosis of human neutrophils. We first showed that the established autophagy inhibitors 3-methyladenine (MA) and chloroquine (CQ) markedly accelerated spontaneous neutrophil apoptosis as was evidenced by phosphatidylserine exposure, DNA fragmentation and caspase-3 activation. Apoptosis induced by the autophagy inhibitors was completely abrogated by a pan-caspase inhibitor Q-VD-OPh. Unexpectedly, both MA and CQ significantly delayed neutrophil apoptosis induced by TNF-α, although the inhibitors did attenuate late pro-survival effect of the cytokine. The effect was specific for TNF-α because it was not observed in the presence of other inflammation-associated cytokines (IL-1β or IL-8). The autophagy inhibitors did not modulate surface expression of TNF-α receptors in the absence or presence of TNF-α. Both MA and CQ induced a marked down-regulation of a key anti-apoptotic protein Mcl-1 but did not affect significantly the levels of another anti-apoptotic protein Bcl-X(L). Finally, to confirm the effects of the pharmacological inhibition of autophagy by a genetic approach, we evaluated the consequences of siRNA-mediated autophagy suppression in neutrophil-like differentiated HL60 cells. Knockdown of ATG5 in the cells resulted in accelerated spontaneous apoptosis but attenuated TNF-α-induced apoptosis. Together, these data suggest that autophagy regulates neutrophil apoptosis in an inflammatory context-dependent manner and mediates the early pro-apoptotic effect of TNF-α in neutrophils.