SEARCH:   Advanced

Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-02-0522. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-02-0522v1 101/5/1987    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Maianski, N. A. Articles by Kuijpers, T. W. Search for Related Content PubMed PubMed Citation Articles by Maianski, N. A. Articles by Kuijpers, T. W. Related Collections Phagocytes Apoptosis Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 March 2003, Vol. 101, No. 5, pp. 1987-1995 PHAGOCYTES Tumor necrosis factor  induces a caspase-independent death pathway in human neutrophils Nikolai A. Maianski, Dirk Roos, and Taco W. Kuijpers From the Sanquin Research at Central Laboratory of the Netherlands Blood Transfusion Service, Landsteiner Laboratory, and Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and Medical Academy, Nizhniy Novgorod, Russia.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Tumor necrosis factor  (TNF-) is a cytokine with multiple roles in the immune system, including the induction and potentiation of cellular functions in neutrophils (PMNs). TNF- also induces apoptotic signals leading to the activation of several caspases, which are involved in different steps of the process of cell death. Inhibition of caspases usually increases cell survival. Here, we found that inhibition of caspases by the general caspase inhibitor zVAD-fmk did not prevent TNF--induced PMN death. After 6 hours of incubation, TNF- alone caused PMN death with characteristic apoptotic features (typical morphologic changes, DNA laddering, external phosphatidyl serine [PS] exposure in the plasma membrane, Bax clustering and translocation to the mitochondria, and degradation of mitochondria), which coincided with activation of caspase-8 and caspase-3. However, in the presence of TNF-, PMNs died even when caspases were completely inhibited. This type of cell death lacked nuclear features of apoptosis (ie, no DNA laddering but aberrant hyperlobulated nuclei without typical chromatin condensation) and demonstrated no Bax redistribution, but it did show mitochondria clustering and plasma membrane PS exposure. In contrast, Fas-triggered PMN apoptosis was completely blocked by zVAD-fmk. Experiments with scavengers of reactive oxygen species (ROS) and with inhibitors of mitochondrial respiration, with PMN-derived cytoplasts (which lack mitochondria) and with PMNs from patients with chronic granulomatous disease (which have impaired nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) indicated that TNF-/zVAD-fmk-induced cell death depends on mitochondria-derived ROS. Thus, TNF- can induce a "classical," caspase-dependent and a "nonclassical" caspase-independent cell death. (Blood. 2003;101:1987-1995) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Tumor necrosis factor  (TNF-) provides a wide variety of biologic signals, which are involved in the regulation of cell death and participate in the governing of immune homeostasis.1 TNF- has been shown to play a crucial role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis, adult respiratory distress syndrome, and sepsis.2-4 However, TNF- is also able to exert anti-inflammatory effects.5,6 The antiphlogistic potential of this cytokine can be ascribed, at least partly, to its ability to accelerate the apoptosis of neutrophils (PMNs), which are major effector cells of inflammation. Apoptotic cell death constitutes a powerful way of curtailing PMN-mediated reactions, providing a safe clearance of these potentially toxic cells.7 Moreover, the uptake of apoptotic cells by resident macrophages has an immunosuppressive effect, which gives an additional beneficial contribution to the control of inflammatory reactions.8 The proapoptotic effect of TNF- on PMNs has been well documented,9-14 although opposite results have also been published.15,16 Probably, this controversy can be explained by the findings that the effect of TNF- on PMN survival may depend on the concentration of the cytokine17 as well as on the duration of stimulation and the initial functional capacity of the PMNs before exposition to TNF-.11 The mechanism of apoptosis induction by TNF- is closely related to the cascade of apoptotic cysteine proteases known as caspases, which represent a group of enzymes responsible for initiation and execution of apoptosis.18,19 A death signal from the TNF- receptor is transduced to an adapter protein, TNF- receptor-associated death domain (TRADD), which uses the next adapter protein, Fas receptor-associated death domain (FADD), to organize the death-inducing signaling complex (DISC).20 DISC recruits and activates the upstream initiator caspase-8, providing therefore activation of downstream effector caspases and the final steps of the apoptotic program.20,21 Inhibition of caspases, for example, by certain peptide ketones, which mimic the active site of the enzyme,22 has been shown to dramatically increase cell survival in various cell types, including PMNs.13,14,23,24 In our study, investigating the effects of TNF- on PMN survival, we faced an unexpected phenomenon. We confirmed a central role for caspases in TNF--mediated apoptosis of PMNs, but at the same time we found that inhibition of caspases did not rescue PMNs from death in the presence of TNF- but instead enhanced an as yet unidentified form of PMN death. Our experiments indicate that in PMNs 2 death pathways are induced by TNF-: one is the predominant "classical" caspase-dependent apoptosis, whereas the other is a "nonclassical" death route, which becomes apparent when caspases are fully inhibited and involves mitochondria-derived reactive oxygen species (ROS).     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References PMN purification and culturing Heparinized venous blood was collected from healthy volunteers and 3 patients with chronic granulomatous disease (CGD) after obtaining informed consent, and PMNs were isolated as described.25 Briefly, 20 mL blood was diluted with 20 mL 10% trisodium citrate/phosphate-buffered saline (PBS). Mononuclear cells and platelets were removed by density gradient centrifugation over isotonic Percoll (Pharmacia, Uppsala, Sweden) with a specific gravity of 1.076 g/mL. Erythrocytes were lysed by short treatment of the pellet fraction with an ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA [ethylenediaminetetraacetic acid], pH 7.4). The remaining PMNs were washed once in PBS and used for further manipulations. In all cases cell purity was more than 97%. PMNs were resuspended at a final concentration of 2 × 106/mL in Iscoves modified Dulbecco medium (BioWhittaker, Brussels, Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco BRL, Paisley, United Kingdom), penicillin 100 IU/mL (Yamanouchi, Tokyo, Japan), streptomycin 100 µg/mL (Gibco BRL), and glutamine 300 µg/mL. One milliliter of cell suspension was put in each well of 24-well plates (NUNC Brand Products, Roskilde, Denmark) and was incubated for 6 hours in a 5% CO2 incubator at 37°C. PMNs were cultured with 20 ng/mL TNF- (Calbiochem, Bad Soden, Germany), with 150 µM z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk;, Alexis Biochemicals, San Diego, CA), with 5 mM N-acetyl-L-cysteine (NAC; Sigma, St Louis, MO) or with indicated combinations. Normal PMNs were also incubated with 500 ng/mL mouse anti-Fas (CD95) monoclonal antibodies (Abs; clone CH11; Immunotech, Marseille, France), with 10 mM 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron; Sigma), with 100 µM rotenone (Sigma), with 2 mM sodium azide (Calbiochem), with 300 µM thenoyltrifluoroacetone (TTFA; Sigma) alone or in a combination with zVAD-fmk or TNF-/zVAD-fmk, where indicated. When the combinations of reagents were used, they were added in culture medium simultaneously. The incubation time and the concentrations were found to be optimal in preliminary experiments (data not shown). Control PMNs were cultured without additions (no stimulus). Because of longer transportation time, CGD neutrophils as well the healthy day-control cells were purified and cultured in a "delayed" manner (delayed cultures), ie, 4 to 5 hours after collection of the blood sample. Cytoplast preparation and culturing PMNs were isolated from the buffy coat of 500 mL fresh blood from volunteer donors, as described in "PMN purification and culturing." Cytoplasts were prepared from 108 PMNs as described previously.26 Briefly, PMNs were centrifuged through a discontinuous Ficoll-70 (Sigma) gradient (12.5%, 16%, 25%) prewarmed to 37°C, containing 5 µg/mL cytochalasin B (Sigma). Centrifugation was performed for 30 minutes at 34°C in a model L2-65B ultracentrifuge with an AH-629 rotor (Beckman Instruments, Fullerton, CA) at 81 000g. After centrifugation, the top band of cellular material was collected. This band was composed of more than 99% of cytoplasts, as assessed by light microscopy of cytospins stained with May-Grünwald-Giemsa solution. Cytoplasts were recognized by their absence of a nucleus. Following several washings in PBS, cytoplasts were resuspended at a final concentration of 8 × 106 per mL in the culture medium and were incubated overnight (16 hours) under conditions indicated in Figure 7. This duration of culture induced maximal differences in cytoplast apoptosis between tested conditions (data not shown). Measurement of cell death After 6 hours of incubation, PMNs were split into 2 portions, which were washed once in ice-cold PBS. One portion was stained with the annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay kit (Bender MedSystems, Vienna, Austria) and analyzed by fluorescence-activated cell sorter scan (FACScan; Becton Dickinson, San Jose, CA) as described previously.24 Dead cells were defined as positive for annexin-V-FITC or for annexin-V-FITC/PI staining. Cell death was expressed as a percentage of dead cells in relation to the total number of counted cells. The number of cells recovered after culture was similar under all conditions tested and was close to 90% of the initial input of cells. Another portion of PMNs (2-3 × 105 cells) was used for preparation of cytospins stained with May-Grünwald-Giemsa solution. The cytospins were estimated by light microscopy for morphologic changes in PMNs (described in "Results"). A minimum of 300 cells was scored for each sample, and the percentages of dead PMNs were determined. Cytoplast death was assessed by annexin-V-FITC binding as described earlier, without the PI step, with 4 × 105 cytoplasts for each preparation. Annexin-V+ cytoplasts were considered to be dead. Western blotting The cleavage of caspase-8 and caspase-3 was determined by Western blotting. Whole cell lysates were obtained by boiling 0.5 × 106 PMNs in sodium dodecyl sulfate (SDS) sample buffer with 2% mercaptoethanol for 5 minutes. Proteins were resolved on 15% SDS-polyacrylamide gel by electrophoresis (PAGE) and were electrotransferred to Immun-Blot polyvinylidene diflouride (PVDF) membrane (BioRad Laboratories, Hercules, CA). The blots were sequentially probed with monoclonal mouse antihuman-caspase-8 Abs (clone 1C12; Cell Signaling Technology, Beverly, MA), which recognize full-length caspase-8 as well as its fragments; with polyclonal rabbit antihuman-caspase-3 Abs (Pharmingen, San Diego, CA), which recognize both inactive procaspase-3 and its cleavage product; and with polyclonal rabbit antihuman-Bax Abs (Pharmingen). All indicated Abs were used at a final dilution of 1:1000. After exposure to each primary Ab, the blots were incubated with appropriate secondary Abs conjugated with horseradish peroxidase (Amersham, Arlington Height, IL) at a final dilution of 1:2500, followed by band visualization with an enhanced chemiluminescence kit as described by the manufacturer (Amersham). This reprobing was successful because of a different exposition time required for visualization of the proteins of interest. For caspase-8-related bands it was approximately 30 minutes, for caspase-3 and its cleavage product 5 minutes, and for Bax protein less than 1 minute. Agarose gel electrophoresis of DNA DNA was extracted from 5 × 106 freshly isolated PMNs or from PMNs treated under conditions indicated in Figure 3 by a PureGene DNA isolation kit (Gentra Systems, Minneapolis, MN) in accordance with the manufacturer's instructions. Isolated DNA was electrophoresed in a 1.2% agarose gel containing ethidium bromide, and the gels were photographed under ultraviolet light. Assessment of p38 MAP kinase phosphorylation in PMNs and cytoplasts After purification, PMNs and cytoplasts were resuspended in culture medium at final concentrations of 2 × 106/mL and 8 × 106/mL, respectively, and were incubated without or with 20 ng/mL TNF- for 10 minutes in a water-bath at 37°C. Thereafter, whole cell lysates were prepared, and Western blotting was performed as described earlier with 1 × 106 cytoplast or 0.5 × 106 PMN equivalents per lane. The blots were probed with phosphospecific polyclonal rabbit Abs against human p38 mitogen-activated protein (MAP) kinase (Cell Signaling Technology), which selectively recognize phosphorylated p38. To determine protein loading, reprobe was performed with polyclonal rabbit Abs against total p38 (Santa Cruz Biotechnology, Santa Cruz, CA), which bind to p38 irrespectively of its phosphorylation state. Confocal laser scanning microscopy (CLSM) For the mitochondrial staining, MitoTracker GreenFM (Molecular Probes, Eugene, OR) was used. To estimate mitochondrial morphology, unfixed PMNs were stained with 100 µM MitoTracker GreenFM and were analyzed by a confocal laser scanning microscope (LSM510; Carl Zeiss, Heidelberg, Germany) as described.24 To obtain simultaneous staining of mitochondria and Bax protein, PMNs were stained with 1 mM MitoTracker GreenFM. Thereafter, the cells were fixed with 2% paraformaldehyde, permeabilized in staining buffer containing 0.1% saponin (wt/vol; Calbiochem) and 1% (wt/vol) bovine serum albumin (Sigma), and were labeled by polyclonal rabbit antihuman-Bax Abs (final dilution 1:250; Pharmingen) followed by secondary staining with AlexaFluor-568-conjugated goat antirabbit immunoglobulin G (Molecular Probes) at a final concentration of 2.5 µg/mL as has been previously described.24 After staining, at least 300 PMNs were counted in each sample, and the percentages of cells with prevailing morphology (images shown in Figures 4-5) were determined, as indicated in the legends of Figures 4-5. Statistics Where applicable, values were compared by one-way analysis of variance (ANOVA) with Bonferroni posttest using GraphPad Prism version 3.0 software. Differences were accepted as significant at P < .05.     Results Top Abstract Introduction Materials and methods Results Discussion References TNF- alone induces classical apoptosis in PMNs Already after 6 hours of culture, untreated PMNs underwent spontaneous apoptosis, with 31.9% ± 2.5% of the cells being annexin-V+ (Figure 1A, No stimulus). Typical apoptotic morphology, including rounding of nuclei, pronounced chromatin condensation, and cell shrinkage, was displayed by 34.2% ± 2.0% of untreated PMNs (Figure 1B, top left; apoptotic cells shown by arrowheads). After 6 hours of treatment with TNF-, the fraction of annexin-V+ PMNs slightly but significantly increased to 40.0% ± 4.3% (Figure 1A, TNF-). When scored by morphologic changes, the proportion of PMNs with classical apoptotic features amounted to 74.0% ± 4.3% in the presence of TNF- (Figure 1B, top right). These data are consistent with previous observations that at early time points TNF- is indeed able to induce apoptosis in PMNs.11,13,14 View larger version (72K): [in this window] [in a new window]   Figure 1. Death of PMNs. (A-B) PMNs were cultured for 6 hours without additions, with 20 ng/mL TNF-, with 150 µM zVAD-fmk, or with the combination of these agents, and cell death was assessed (A) by FACScan analysis of annexin-V-FITC/PI staining and (B) by morphologic examination of cytospins stained with May-Grünwald-Giemsa stain (for quantitative data see C). Arrowheads indicate PMNs that have undergone spontaneous or TNF--induced apoptosis with typical apoptotic morphology; closed and open arrows depict PMNs with aberrant morphology appeared after TNF-/zVAD-fmk treatment (see "Results" for details). (C) Quantitative data obtained by FACScan analysis and cytospin evaluation of PMNs treated for 6 hours under the conditions as indicated. Dosage of additions: TNF- and zVAD-fmk, as indicated for A and B; anti-Fas monoclonal Abs, 500 ng/mL. *P < .05. Data represent means ± SEM of 4 to 8 separate experiments performed in duplicate. Inhibition of caspases in the presence of TNF- leads PMNs to an aberrant death different from apoptosis We subsequently studied the activation of initiator caspase-8 and executioner caspase-3 in PMNs by Western blotting. In freshly isolated PMNs caspase-8 was present as a 57- to 55-kd precursor protein (Figure 2A, lane 1), and caspase-3 appeared as a 32-kd proenzyme (Figure 2B, lane 1). These bands represent the full-length procaspases.13,27 PMNs that had undergone spontaneous apoptosis on culturing without stimuli displayed the initial activation of caspase-8 with the appearance of the large cleavage fragment of 43 to 41 kd (Figure 2A, lane 2). Caspase-3 was partially cleaved into the 17-kd fragment (Figure 2B, lane 2). Stimulation with TNF- caused a more pronounced activation of caspase-8 and caspase-3. The 57- to 55-kd procaspase-8 was completely degraded into smaller fragments, including the active 18-kd fragment28 (Figure 2A, lane 4), and the 32-kd procaspase-3 was entirely processed to the active 17-kd product (Figure 2B, lane 4). View larger version (79K): [in this window] [in a new window]   Figure 2. Cleavage of caspase-8 and caspase-3 in PMNs. Whole-cell lysates from freshly isolated PMNs (lane 1), from PMNs cultured for 6 hours without additions (lane 2), with 150 µM zVAD-fmk (lane 3), with 20 ng/mL TNF- (lane 4), or with the TNF-/zVAD-fmk combination (lane 5) were subjected to SDS-PAGE. Western blot was performed with anti-caspase-8 monoclonal Abs (A). Then the blot was reprobed with anti-caspase-3 polyclonal Abs (B). The expression of Bax protein determined by anti-Bax polyclonal Abs was used as a measurement for equal protein loading (C). Results are representative of 3 independent experiments. Next, we checked whether inhibition of caspases could abrogate the proapoptotic effects of TNF-. For this purpose the broad-spectrum caspase inhibitor zVAD-fmk was used. When used alone, this agent significantly reduced apoptotic membrane changes (18.6% ± 2.0% annexin-V+ PMNs; Figure 1A, zVAD-fmk) as well as morphologic features of apoptosis (9.4% ± 2.5% cells with apoptotic morphology; Figure 1B, bottom left), when compared with untreated PMNs (Figure 1A-B, No stimulus). Moreover, zVAD-fmk completely inhibited activation of caspase-8 (Figure 2A, lane 3) and caspase-3 (Figure 2B, lane 3), because no cleavage products were detectable, and the enzymes were only present as full-length precursors in the lysates from zVAD-fmk-treated PMNs. Unexpectedly, addition of zVAD-fmk to TNF- did not rescue PMNs from death. Instead, after such treatment, 52.4% ± 4.7% of the PMNs became annexin-V+, as shown in Figure 1A (TNF- + zVAD-fmk plot). These PMNs had an aberrant appearance (Figure 1B, bottom right): some cells (open arrow) were enlarged, with expanded disintegrated chromatin and visible vacuolization, whereas others (closed arrow) had hyperlobulated nuclei with moderately condensed chromatin (this picture is different from classical apoptotic features shown in Figure 1B, top left and top right). The proportion of such unusual cells in the TNF-/zVAD-fmk preparation was 40.0% ± 6.2%, whereas typical apoptotic morphology was noted in 9.4% ± 4.3% of the PMNs treated with this combination. The latter value is similar to the level of morphologic apoptosis found when PMNs were treated with zVAD-fmk alone, as summarized in Figure 1C. Notably, the fraction of aberrant cells was negligible among untreated or TNF--treated PMNs and very low in the presence of zVAD-fmk alone (< 3%). Of importance, the aberrant death of PMNs induced by TNF-/zVAD-fmk proceeded despite the absence of any detectable activation of caspase-8 (Figure 2A, lane 5) or caspase-3 (Figure 2B, lane 5). The equivalence of protein loading was established by Bax protein expression (Figure 2C), which has been previously shown to be stable in PMNs by us24 and various other groups.29-31 To investigate the role of protein synthesis in the TNF-/zVAD-fmk death pathway, PMNs were also tested in the presence of cycloheximide. However, transcription blockade showed no effect on TNF-/zVAD-fmk-induced cytotoxicity in PMNs (data not shown). Taken together, these results indicate that TNF- can induce 2 different death signals in PMNs, one caspase dependent and another caspase independent. No DNA laddering in TNF-/zVAD-fmk-treated PMNs To further characterize the caspase-independent cell death in PMNs, we investigated DNA laddering. Internucleosomal DNA degradation is a hallmark of apoptosis.32 Indeed, DNA from PMNs that had been incubated with TNF- for 6 hours demonstrated a typical laddering pattern, indicating internucleosomal cleavage characteristic for apoptosis (Figure 3). In contrast, the electrophoretic pattern of DNA extracted from TNF-/zVAD-fmk-treated PMNs was similar to that of fresh, untreated, or zVAD-fmk-treated cells, cultured for 6 hours (Figure 3). Thus, also in this respect, TNF-/zVAD-fmk-induced PMN death appeared to be different from typical apoptosis. View larger version (91K): [in this window] [in a new window]   Figure 3. DNA laddering in PMNs. DNA extracted from freshly isolated PMNs as well as from PMNs cultured for 6 hours without additions, with 20 ng/mL TNF-, with 150 µM zVAD-fmk or with the combination of these agents, was separated by agarose gel electrophoresis, and internucleosomal fragmentation (DNA laddering) was assessed. Results are representative of 3 separate experiments. zVAD-fmk prevents Fas-receptor-induced apoptosis in PMNs The Fas/Apo-1/CD95 system shares common death signaling pathways with the TNF--receptor. Both receptors belong to the TNF/nerve growth factor receptor family33 and can recruit the same adapter protein, FADD, forming the DISC to mediate death signals to the caspase cascade.20,21 As shown in Figure 1C, ligation of the Fas-receptor with agonistic anti-Fas monoclonal Abs CH-1123,34 led PMNs after a 6-hour culture to pronounced apoptosis, with 59.9% ± 4.7% of annexin-V+ cells and 83.3% ± 5.2% of cells with a typical apoptotic morphology (compare with Figure 1C, No stimulus). However, induction of apoptosis by anti-Fas monoclonal Abs was almost completely prevented by zVAD-fmk (Figure 1C; 21.9% ± 3.8% annexin-V+ and 17.5% ± 2.7% morphologically apoptotic PMNs; compare with Figure 1C, zVAD-fmk alone). Thus, despite the fact that Fas and TNF--receptors engage common upstream death pathways, Fas receptor-mediated death signals are strictly caspase dependent and can be blocked by a caspase inhibitor, whereas TNF- has the potential to bypass the caspase cascade, causing atypical death in PMNs in the presence of zVAD-fmk. TNF-/zVAD-fmk treatment causes in PMNs degradation of the mitochondria without Bax redistribution Our recent study has shown that PMNs contain mitochondria, which play an important role in the apoptotic program of these cells.24 To check whether the mitochondria are involved in caspase-independent death pathway, we undertook the next set of experiments. Specific mitochondrial fluorescent staining revealed that most of the untreated and zVAD-fmk-treated PMNs (Figure 4, top left and bottom left, respectively) after 6 hours of incubation preserved a tubular structure of the mitochondria, as was observed in fresh cells.24 When TNF- alone was present in the culture medium, the mitochondria changed into large unstructured aggregates (Figure 4, top right) typical for apoptosis.24 Interestingly, the proportion of cells with clustered mitochondria closely correlated with the proportion of cells with an apoptotic morphology (data not presented), indicating that changes in the mitochondrial structure form an early and reliable marker of apoptosis. The TNF-/zVAD-fmk combination also altered the appearance of the mitochondria (Figure 4, bottom right), leading to clustering and degradation of these organelles, although these mitochondrial aggregates were smaller in comparison to the aggregates in PMNs treated with TNF- alone. View larger version (30K): [in this window] [in a new window]   Figure 4. Staining patterns of mitochondria in PMNs. PMNs were incubated for 6 hours without additions (No stimulus), with 20 ng/mL TNF-, with 150 µM zVAD-fmk, or with the TNF-/zVAD-fmk combination. Then the cells were stained with MitoTracker GreenFM and analyzed with CLSM. Each image represents the following proportion of the total cell population (mean ± SEM): 73.8% ± 8.9% in No stimulus; 66.7% ± 5.4% in TNF-; 89.0% ± 4.3% in zVAD-fmk; 77.0% ± 3.6% in TNF-/zVAD-fmk. Bar is 5 µm. Results are representative of at least 4 independent experiments. When PMNs were costained for mitochondria and Bax protein, most of the untreated cells showed after the 6-hour incubation a punctate localization of Bax, remaining separate from mitochondria (Figure 5, top panel), as was also observed in fresh PMNs.24 In PMNs after 6 hours of culture in the presence of zVAD-fmk, Bax protein maintained a staining pattern similar to fresh and to untreated cultured cells, visible as a punctate distribution separate from mitochondria (data not shown). In contrast, treatment with TNF- caused redistribution of Bax into large clusters, which colocalized with mitochondria (Figure 5, middle panel; a shift in fluorescence to yellow depicts colocalization). In TNF-/zVAD-fmk-treated PMNs, Bax remained punctate and hardly colocalized with the mitochondria (Figure 5, bottom panels). View larger version (35K): [in this window] [in a new window]   Figure 5. Subcellular redistribution of Bax protein in PMNs. PMNs were cultured for 6 hours without additions (No stimulus), with 20 ng/mL TNF-, or with a combination of 20 ng/mL TNF- and 150 µM zVAD-fmk. Then the cells were stained with MitoTracker GreenFM, fixed, permeabilized, stained with polyclonal Abs specific for Bax, and analyzed with CLSM. (Because of the fixation and permeabilization procedures, the mitochondrial staining [green] showed a more diffuse cytoplasmic pattern than the tubular structures shown in Figure 4, left panels). Each image represents the following proportion of the total cell population (mean ± SEM): 77.7% ± 5.4% in No stimulus; 63.3% ± 8.8% in TNF-; 88.3% ± 5.7% in TNF-/zVAD-fmk. Bar is 5 µm. This figure is representative of at least 4 independent experiments. Taken together, these results demonstrate that TNF- can act via a classical apoptotic route, inducing subcellular Bax redistribution and its aggregation with mitochondria, which have been shown to be significant events during the execution of apoptosis in various cell types,35-38 including PMNs.24 At the same time, TNF- stimulation under conditions that preclude caspase activation does not lead to Bax changes, but it does cause degradation of mitochondria. Again, this finding may indicate the presence of a caspase-independent, mitochondria-dependent route of cell death in PMNs that is different from apoptosis. PMN-derived cytoplasts lacking mitochondria do not display TNF-/zVAD-fmk-induced death phenomenon To further elucidate the role of mitochondria in TNF-/zVAD-fmk-induced death, we used PMN-derived cytoplasts, which are cellular vesicles from which mitochondria have been eliminated.24,39 First, we determined whether the TNF- receptors were functional on the cytoplast surface. As a read-out, the TNF- receptor-mediated phosphorylation of p38 MAP kinase was used.40 Figure 6 (top panel) demonstrates that TNF- induced phosphorylation of p38 MAP kinase both in cytoplasts and PMNs. Next, cytoplasts were cultured under various conditions. Untreated and TNF--treated cytoplasts after culture exposed phosphatidyl serine on the outer layer of the plasma membrane, which was evident by annexin-V positivity of 54.2% ± 4.9% and 50.3% ± 5.7% cytoplasts, respectively (Figure 7, top left and top right plots, respectively). zVAD-fmk reduced the number of annexin-V+ cytoplasts to 7.2% ± 1.9% (Figure 7, bottom left plot). Similar values were found in the experiments with PMNs (Figure 1C). However, in contrast to PMNs, cytoplasts treated with a combination TNF-/VAD-fmk remained "alive," with only 11.0% ± 1.3% annexin-V+ cells (Figure 7, bottom right plot). Thus, addition of TNF- to zVAD-fmk had no effect on cytoplast survival. This finding indicates that TNF- was not able to induce a caspase-independent death in cytoplasts in the absence of the mitochondria, despite the intact receptor signaling, and the caspase inhibitor completely preserved its prosurvival effect. Also, this finding demonstrates that the TNF-/zVAD-fmk combination itself is not nonspecifically toxic for the cells. View larger version (37K): [in this window] [in a new window]   Figure 6. p38 MAP kinase phosphorylation in cytoplasts. Freshly isolated cytoplasts or fresh PMNs were treated for 10 minutes with control medium or with 20 ng/mL TNF-. Thereafter, whole cell lysates were prepared and subjected to SDS-PAGE. Western blot was performed with monoclonal Abs that specifically recognized the phosphorylated form of p38 (top panel). Reprobing with anti-total p38 monoclonal Abs (bottom panel), which recognizes p38 regardless of its phosphorylation state, gives an estimation of the equal protein loading. Results are representative of 4 independent experiments. View larger version (52K): [in this window] [in a new window]   Figure 7. Death of cytoplasts. Cytoplasts cultured overnight without additions or with 20 ng/mL TNF-, with 150 µM zVAD-fmk, or with the combination of these agents were stained with annexin-V-FITC and were analyzed by FACScan. Cytoplasts with annexin-V staining were counted as dead cytoplasts (bottom right quadrant of each plot). *P < .05 versus No stimulus and TNF-. Values represent means ± SEM of 5 separate experiments performed in duplicate. NADPH oxidase system-independent ROS are involved in the TNF-/zVAD-fmk-induced cytotoxic effects The data shown earlier indicated that the mitochondria may participate in the process of unusual TNF-/zVAD-fmk-induced PMN death. How do these organelles contribute to this death pathway? One possibility is ROS production by the mitochondria in response to TNF- stimulation, which mediates, at least in part, cytotoxic effects of this cytokine.41-43 NAC, a well-characterized ROS scavenger,34,44,45 had no effect on spontaneous apoptosis of PMNs, reduced TNF--induced apoptosis, and almost completely abrogated the TNF-/zVAD-fmk death effects (Figure 8 and data not shown). NAC significantly (P < .05) reduced the number of annexin-V+ PMNs in TNF-/zVAD-fmk-treated PMNs and completely prevented the appearance of morphologically aberrant cells (not shown). The ROS scavenger tiron,43 which is unrelated to NAC, also prevented TNF-/zVAD-fmk-induced cell death (data not shown). The mitochondrial origin of ROS was further supported by experiments, in which we used inhibitors of the mitochondrial electron transport (respiratory) chain, ie, inhibitors of the mitochondrial ROS production.41 Rotenone stopped the death-inducing effects of the TNF-/zVAD-fmk combination, preventing plasma membrane flip-flop (Figure 8; P < .05) and aberrant morphologic changes in the TNF-/zVAD-fmk-treated PMNs (data not shown). Two other mitochondrial inhibitors, sodium azide and TTFA, demonstrated a similar effect of rescuing PMNs from TNF-/zVAD-fmk-mediated cell death (data not shown). Importantly, these mitochondrial inhibitors, when added alone, influenced neither the basal level of PMN apoptosis nor PMN adenosine triphosphate (ATP) levels, measured by a luciferase-based assay46 (not shown). The latter result can be explained by the fact that PMNs mainly use glycolysis rather than mitochondrial oxidative phosphorylation for their energy supply.47 View larger version (33K): [in this window] [in a new window]   Figure 8. Effect of NAC and rotenone on TNF-/zVAD-fmk-induced PMN cell death. PMNs were cultured for 6 hours with a combination of 20 ng/mL TNF- and 150 µM zVAD-fmk or with TNF-/zVAD-fmk in combination with 5 mM NAC or 100 µM rotenone. Afterwards, PMNs were stained with annexin-V/PI and analyzed by FACScan. Values represent the percentage of cells (mean ± SEM) for each respective quadrant. Data obtained in 5 independent experiments. The most powerful source of ROS in PMNs is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, which provides a rapid and a dramatic increase in ROS generation known as the respiratory burst. To check whether ROS produced by NADPH oxidase participates in the TNF-/zVAD-fmk-mediated cell death, we investigated PMNs from 3 patients with CGD. Because of a genetic defect in the NADPH oxidase in PMNs from these patients, their cells cannot generate ROS.48 In our experiments, PMNs from patients with CGD displayed a behavior in terms of the death rate similar to the normal cells under the conditions tested as illustrated by annexin-V/PI staining in Table 1. CGD PMNs had levels of spontaneous apoptosis comparable to the healthy day-control cells incubated in the delayed cultures (see "Materials and methods"). zVAD-fmk protected CGD cells from apoptosis as it did in healthy PMNs, and the TNF-/zVAD-fmk combination induced a similar amount of phosphatidyl serine exposure and aberrant morphology in CGD PMNs, as was observed in normal PMNs (Table 1 and data not shown). Hence, the NADPH oxidase system plays no role in the TNF-/zVAD-fmk-induced death of PMNs. Interestingly, the ROS scavenger NAC also rescued CGD PMNs from the TNF-/zVAD-fmk-induced death by preventing the membrane changes (Table 1) and the appearance of unusual morphology (not shown), indicating that CGD PMNs have the ability to produce some ROS from an alternative source.                                View this table: [in this window] [in a new window]   Table 1. TNF-/zVAD-fmk-induced cell death of CGD PMNs     Discussion Top Abstract Introduction Materials and methods Results Discussion References Most forms of programmed cell death proceed through the activation of caspases, which can be blocked by the general caspase inhibitor zVAD-fmk. In this study we describe an as yet unidentified form of PMN death induced by TNF- in the presence of caspase inhibition. TNF- alone induced activation of the classical apoptotic route in PMNs, which is accompanied by activation of initiator and executioner caspases, Bax translocation to mitochondria, mitochondrial clustering, internucleosomal cleavage of DNA, and typical apoptotic changes in morphology and plasma membranes. When caspase activity was blocked, TNF--treated PMNs displayed no Bax redistribution and no DNA fragmentation, and increased cell death as indicated by the plasma membrane exposure of phosphatidyl serine in the outer layer (flip-flop). Moreover, these PMNs showed an aberrant morphology, with hyperlobulated nuclei and expanded, disintegrated chromatin. Apparently, under conditions when caspases do not function, an alternative, TNF--induced nonclassical, caspase-independent pathway of cell death is revealed in PMNs. Further experiments demonstrated the involvement of ROS in the TNF-/zVAD-fmk-induced cytotoxicity (Figure 8), independent of protein synthesis. During the last decade, the physiologic role of ROS has been gradually reevaluated. These agents moved from a category of merely unwanted side products of oxidative metabolism to a cohort of important messenger molecules.49-51 Intracellular sources of ROS are mainly represented by electron-transfer processes in mitochondria49,52 and enzymatic oxidation provided by various oxidases.50 Among oxidases, the NADPH oxidase system is one of the most powerful generators of ROS, being used by PMNs for the killing of ingested microorganisms.48 We found that PMNs from patients with CGD, who have an impaired NADPH oxidase system, died in the same caspase-independent way after TNF-/zVAD-fmk treatment as did healthy PMNs. This observation ruled out the involvement of NADPH oxidase-derived ROS in this nonclassical form of PMN death. Experiments with PMN-derived cytoplasts underscored that these ROS originated from mitochondria, because cytoplasts, having no mitochondria, did not show any enhanced exposure of phosphatidyl serine after TNF-/zVAD-fmk stimulation, in contrast to intact PMNs. A number of inhibitors of the mitochondrial electron transport chain, including rotenone, sodium azide, and TTFA, were also able to prevent the TNF-/zVAD-fmk-induced features of cell death, again pointing to the mitochondria as the main origin of ROS production. These findings are in line with previous reports on the cytotoxic effects of TNF- in cell lines,41-43 caused by ROS from mitochondria. Excessive amounts of ROS may cause direct oxidative damage of nucleic acids, proteins, and lipids53 or may make proteins more susceptible to proteolysis.54,55 Probably, such events take place during TNF-/zVAD-fmk-induced PMN death, resulting in the observed cellular changes that could be prevented by ROS inhibitors. Importantly, mitochondria are not only a source of ROS but also a target for ROS.52 ROS produced in mitochondria may lead to self-damage of these organelles, causing apoptosis or necrosis.53 This could be an explanation for our data, which showed Bax-independent mitochondrial changes in PMNs after TNF-/zVAD-fmk treatment. Undoubtedly, ROS production requires a tight control, and our results suggest that caspases might be involved in this regulation.44,56 Possibly, mitochondrial proteins involved in electron transport within these organelles and providing the excess production of oxygen radicals could be a direct substrate for caspases, because mitochondrial caspases with as yet unidentified intramitochondrial functions have been described.57 Deactivation of this system by caspases could normally prevent the accumulation of ROS. Alternatively, caspases may play a role in the elimination of damaged lipids and proteins, which accumulate after TNF- stimulation and may normally act as a natural sink for ROS.56 Several studies have shown that blockade of caspases in some cell lines sensitize them to TNF--mediated cytotoxicity.44,56,58 The researchers refer to this type of cell death as necrosis,56 "a nonapoptotic" cell death,44 or "a transitional stage between apoptosis and necrosis."58 Such descriptions underline the complicated nature of the phenomenon but, at the same time, dictate the necessity to use an adequate set of tools for the registration of cell death. For example, staining with propidium iodide alone56 does not seem to be sufficient to discriminate between necrotic and apoptotic cell death, because these basically different types of death both lead to the final disruption of the plasma membrane.59 The death rate of PMNs treated with a combination of TNF- and caspase inhibitors has obviously been underestimated,13,14 because only a DNA fragmentation assay has been used as a read-out for cell death, whereas our present findings clearly show the absence of this hallmark of apoptosis under these conditions. We conclude from our data that TNF- is able to trigger 2 pathways of cell death in PMNs, and the availability of downstream caspases appears to determine the mode of cell death. In the presence of an intact caspase cascade TNF- mainly induces the classical form of apoptosis. However, when caspase activity is blocked, eg, by zVAD-fmk, other signals result from TNF- stimulation. This nonclassical and caspase-independent pathway of PMN death, which lacks most of the characteristic apoptotic features, is mediated by mitochondria-derived ROS. In PMNs, this signaling pathway seems to be restricted to the TNF- receptor, because the Fas receptor-mediated as well as the spontaneous apoptosis in PMNs were both completely blocked by zVAD-fmk. Our present data raise another issue. Caspases are attractive targets for pharmacologic intervention in vivo in disease states that have been associated with enhanced apoptosis.60,61 Caspase inhibitors, predominantly zVAD-fmk-like active-site mimetic peptide ketones, have been extensively used in animal models of human diseases. These inhibitors have shown beneficial effects in various types of ischemia-reperfusion injury,62-64 but also in infectious conditions, including bacterial meningitis and sepsis.65,66 The promising approach of using caspase inhibitors as anti-inflammatory agents should, however, be considered with caution because of the possible adverse effects.61 For example, during ischemia-reperfusion injury and particularly during generalized infections, inflammation proceeds through a massive activation of PMNs and generation of inflammatory cytokines. Under many generalized inflammatory conditions, TNF--induced apoptosis of PMNs through the activation of caspases provides a "silent turnover" of these potentially hazardous cells and leads to a suppression of inflammation,8 limiting the extent of inflammatory reactions. Instead, inhibition of caspases may lead to a deterioration of the injury, caused by an as yet unforeseen atypical neutrophil death as shown in our study, with a potentially uncontrolled release of their contents, in contrast to the classical apoptotic cells. Moreover, under the circumstances of caspase inhibition, PMN cell death is exaggerated, and clearance mechanisms may be insufficiently able to minimize PMN-related damage. To date, the existence per se of caspase-independent cell death has been shown for several untransformed cell types, including T lymphocytes,67,68 neurons,69-71 erythropoietic cells,72 and fibroblasts.73 Many researchers refrain to designate this type of cell turnover as "apoptosis," because of a lack of some typical apoptotic features. Our data on PMNs support this view. The biologic significance and physiologic role as well as the precise mechanisms of this phenomenon warrant further study.     Acknowledgments We are grateful to Dr P. Hordijk for his comments while preparing the manuscript, to Dr R. S. Weening for his help in obtaining blood from CGD patients, and to Dr S. Albracht for his gift of mitochondrial inhibitors.     Footnotes Submitted February 15, 2002; accepted October 3, 2002. Prepublished online as Blood First Edition Paper, October 10, 2002; DOI 10.1182/blood-2002-02-0522. Supported by a grant from Nuffic (N.A.M.). T.W.K. is a research fellow of the Royal Dutch Academy of Sciences. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Taco Kuijpers, Central Laboratory of the Netherlands Blood Transfusion Service (CLB), Department of Experimental Immunohematology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: t_kuijpers{at}clb.nl.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Tracey KJ, Cerami A. Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol. 1993;9:317-343[CrossRef][Medline] [Order article via Infotrieve]. 2. Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell. 1996;85:307-310[CrossRef][Medline] [Order article via Infotrieve]. 3. Parsons PE, Moore FA, Moore EE, Ikle DN, Henson PM, Worthen GS. Studies on the role of tumor necrosis factor in adult respiratory distress syndrome. Am Rev Respir Dis. 1992;146:694-700[Medline] [Order article via Infotrieve]. 4. Windsor AC, Walsh CJ, Mullen PG, et al. Tumor necrosis factor-alpha blockade prevents neutrophil CD18 receptor upregulation and attenuates acute lung injury in porcine sepsis without inhibition of neutrophil oxygen radical generation. J Clin Invest. 1993;91:1459-1468[Medline] [Order article via Infotrieve]. 5. Marino MW, Dunn A, Grail D, et al. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci U S A. 1997;94:8093-8098[Abstract/Free Full Text]. 6. Liu J, Marino MW, Wong G, et al. TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination. Nat Med. 1998;4:78-83[CrossRef][Medline] [Order article via Infotrieve]. 7. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989;83:865-875[Medline] [Order article via Infotrieve]. 8. Savill J, Fadok V. Corpse clearance defines the meaning of cell death. Nature. 2000;407:784-788[CrossRef][Medline] [Order article via Infotrieve]. 9. Takeda Y, Watanabe H, Yonehara S, Yamashita T, Saito S, Sendo F. Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-alpha. Int Immunol. 1993;5:691-694[Abstract/Free Full Text]. 10. Watson RW, Redmond HP, Wang JH, Bouchier-Hayes D. Bacterial ingestion, tumor necrosis factor-alpha, and heat induce programmed cell death in activated neutrophils. Shock. 1996;5:47-51[Medline] [Order article via Infotrieve]. 11. Murray J, Barbara JA, Dunkley SA, et al. Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro. Blood. 1997;90:2772-2783[Abstract/Free Full Text]. 12. Kettritz R, Gaido ML, Haller H, Luft FC, Jennette CJ, Falk RJ. Interleukin-8 delays spontaneous and tumor necrosis factor-alpha-mediated apoptosis of human neutrophils. Kidney Int. 1998;53:84-91[CrossRef][Medline] [Order article via Infotrieve]. 13. Yamashita K, Takahashi A, Kobayashi S, et al. Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production. Blood. 1999;93:674-685[Abstract/Free Full Text]. 14. Weinmann P, Gaehtgens P, Walzog B. Bcl-XL- and Bax--mediated regulation of apoptosis of human neutrophils via caspase-3. Blood. 1999;99:3106-3115. 15. Colotta F, Re F, Polentarutti N, Sozzani S, Mantovani A. Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood. 1992;80:2012-2020[Abstract/Free Full Text]. 16. Keel M, Ungethum U, Steckholzer U, et al. Interleukin-10 counterregulates proinflammatory cytokine-induced inhibition of neutrophil apoptosis during severe sepsis. Blood. 1997;90:3356-3363[Abstract/Free Full Text]. 17. van den Berg JM, Weyer S, Weening JJ, Roos D, Kuijpers TW. Divergent effects of tumor necrosis factor-alpha on apoptosis of human neutrophils. J Leukoc Biol. 2001;69:467-473[Abstract/Free Full Text]. 18. Reed JC. Mechanisms of apoptosis. Am J Pathol. 2000;157:1415-1430[Abstract/Free Full Text]. 19. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407:770-776[CrossRef][Medline] [Order article via Infotrieve]. 20. Kischkel FC, Hellbardt S, Behrmann I, et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 1995;14:5579-5588[Medline] [Order article via Infotrieve]. 21. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science. 1998;281:1305-1308[Abstract/Free Full Text]. 22. Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem. 1998;273:32608-32613[Abstract/Free Full Text]. 23. Fadeel B, Ahlin A, Henter JI, Orrenius S, Hampton MB. Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species. Blood. 1998;92:4808-4818[Abstract/Free Full Text]. 24. Maianski NA, Mul FPJ, van Buul JD, Roos D, Kuijpers TW. Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils. Blood. 2002;99:672-679[Abstract/Free Full Text]. 25. Roos D, de Boer M. Purification and cryopreservation of phagocytes from human blood. Methods Enzymol. 1986;132:225-243[Medline] [Order article via Infotrieve]. 26. Roos D, Voetman AA. Preparation and cryopreservation of cytoplasts from human phagocytes. Methods Enzymol. 1986;132:250-257[Medline] [Order article via Infotrieve]. 27. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 1996;85:817-827[CrossRef][Medline] [Order article via Infotrieve]. 28. Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998;17:1675-1687[CrossRef][Medline] [Order article via Infotrieve]. 29. Moulding DA, Quayle JA, Hart CA, Edwards SW. Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood. 1998;92:2495-2502[Abstract/Free Full Text]. 30. Moulding DA, Akgul C, Derouet M, White MR, Edwards SW. BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis. J Leukoc Biol. 2001;70:783-792[Abstract/Free Full Text]. 31. Epling-Burnette PK, Zhong B, Bai F, et al. Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils. J Immunol. 2001;166:7486-7495[Abstract/Free Full Text]. 32. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980;284:555-556[CrossRef][Medline] [Order article via Infotrieve]. 33. Nagata S, Golstein P. The Fas death factor. Science. 1995;267:1449-1456[Abstract/Free Full Text]. 34. Kasahara Y, Iwai K, Yachie A, et al. Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils. Blood. 1997;89:1748-1753[Abstract/Free Full Text]. 35. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997;139:1281-1292[Abstract/Free Full Text]. 36. Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol. 1998;143:207-215[Abstract/Free Full Text]. 37. Khaled AR, Kim K, Hofmeister R, Muegge K, Durum SK. Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH. Proc Natl Acad Sci U S A. 1999;96:14476-14481[Abstract/Free Full Text]. 38. Murphy KM, Ranganathan V, Farnsworth ML, Kavallaris M, Lock RB. Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ. 2000;7:102-111[CrossRef][Medline] [Order article via Infotrieve]. 39. Roos D, Voetman AA, Meerhof LJ. Functional activity of enucleated human polymorphonuclear leukocytes. J Cell Biol. 1983;97:368-377[Abstract/Free Full Text]. 40. Suzuki K, Hino M, Hato F, Tatsumi N, Kitagawa S. Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. Blood. 1999;93:341-349[Abstract/Free Full Text]. 41. Schulze-Osthoff K, Bakker AC, Vanhaesebroeck B, Beyaert R, Jacob WA, Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992;267:5317-5323[Abstract/Free Full Text]. 42. Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A. 1995;92:8115-8119[Abstract/Free Full Text]. 43. Moreno-Manzano V, Ishikawa Y, Lucio-Cazana J, Kitamura M. Selective involvement of superoxide anion, but not downstream compounds hydrogen peroxide and peroxynitrite, in tumor necrosis factor-alpha-induced apoptosis of rat mesangial cells. J Biol Chem. 2000;275:12684-12691[Abstract/Free Full Text]. 44. Khwaja A, Tatton L. Resistance to the cytotoxic effects of tumor necrosis factor alpha can be overcome by inhibition of a FADD/caspase-dependent signaling pathway. J Biol Chem. 1999;274:36817-36823[Abstract/Free Full Text]. 45. Pani G, Colavitti R, Borrello S, Galeotti T. Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes. Biochem J. 2000;347:173-181[CrossRef][Medline] [Order article via Infotrieve]. 46. Peachman KK, Lyles DS, Bass DA. Mitochondria in eosinophils: functional role in apoptosis but not respiration. Proc Natl Acad Sci U S A. 2001;98:1717-1722[Abstract/Free Full Text]. 47. Borregaard N, Herlin T. Energy metabolism of human neutrophils during phagocytosis. J Clin Invest. 1982;70:550-557[Medline] [Order article via Infotrieve]. 48. Roos D, Curnutte JT. Chronic granulomatous disease. In: Ochs HD,Smith CIE,Puck JM, eds. Primary Immunodeficiency Diseases. A Molecular and Genetic Approach. Oxford University Press: New York; 1999:353-374. 49. Papa S, Skulachev VP. Reactive oxygen species, mitochondria, apoptosis and aging. Mol Cell Biochem. 1997;174:305-319[CrossRef][Medline] [Order article via Infotrieve]. 50. Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol. 1998;10:248-253[CrossRef][Medline] [Order article via Infotrieve]. 51. Lenaz G. Role of mitochondria in oxidative stress and ageing. Biochim Biophys Acta. 1998;1366:53-67[Medline] [Order article via Infotrieve]. 52. Lee HC, Wei YH. Mitochondrial role in life and death of the cell. J Biomed Sci. 2000;7:2-15[CrossRef][Medline] [Order article via Infotrieve]. 53. Richter C, Gogvadze V, Laffranchi R, et al. Oxidants in mitochondria: from physiology to diseases. Biochim Biophys Acta. 1995;1271:67-74[Medline] [Order article via Infotrieve]. 54. Davies KJ, Lin SW, Pacifici RE. Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein. J Biol Chem. 1987;262:9914-9920[Abstract/Free Full Text]. 55. Dean RT, Pollak JK. Endogenous free radical generation may influence proteolysis in mitochondria. Biochem Biophys Res Commun. 1985;126:1082-1089[CrossRef][Medline] [Order article via Infotrieve]. 56. Vercammen D, Beyaert R, Denecker G, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med. 1998;187:1477-1485[Abstract/Free Full Text]. 57. Costantini P, Bruey JM, Castedo M, et al. Pre-processed caspase-9 contained in mitochondria participates in apoptosis. Cell Death Differ. 2002;9:82-88[CrossRef][Medline] [Order article via Infotrieve]. 58. Luschen S, Ussat S, Scherer G, Kabelitz D, Adam-Klages S. Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression. J Biol Chem. 2000;275:24670-24678[Abstract/Free Full Text]. 59. Kroemer G, Petit P, Zamzami N, Vayssiere JL, Mignotte B. The biochemistry of programmed cell death. FASEB J. 1995;9:1277-1287[Abstract]. 60. Haunstetter A, Izumo S. Toward antiapoptosis as a new treatment modality. Circ Res. 2000;86:371-376[Free Full Text]. 61. Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature. 2000;407:810-816[CrossRef][Medline] [Order article via Infotrieve]. 62. Cursio R, Gugenheim J, Ricci JE, et al. A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis. FASEB J. 1999;13:253-261[Abstract/Free Full Text]. 63. Daemen MA, van `t Veer C, Denecker G, et al. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest. 1999;104:541-549[Medline] [Order article via Infotrieve]. 64. Mocanu MM, Baxter GF, Yellon DM. Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury. Br J Pharmacol. 2000;130:197-200[CrossRef][Medline] [Order article via Infotrieve]. 65. Braun JS, Novak R, Herzog KH, Bodner SM, Cleveland JL, Tuomanen EI. Neuroprotection by a caspase inhibitor in acute bacterial meningitis. Nat Med. 1999;5:298-302[CrossRef][Medline] [Order article via Infotrieve]. 66. Grobmyer SR, Armstrong RC, Nicholson SC, et al. Peptidomimetic fluoromethylketone rescues mice from lethal endotoxic shock. Mol Med. 1999;5:585-594[Medline] [Order article via Infotrieve]. 67. Uzzo RG, Dulin N, Bloom T, Bukowski R, Finke JH, Kolenko V. Inhibition of NFkappaB induces caspase-independent cell death in human T lymphocytes. Biochem Biophys Res Commun. 2001;287:895-899[CrossRef][Medline] [Order article via Infotrieve]. 68. Sharif-Askari E, Alam A, Rheaume E, et al. Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation. EMBO J. 2001;20:3101-3113[CrossRef][Medline] [Order article via Infotrieve]. 69. Herkert M, Shakhman O, Schweins E, Becker CM. Beta-bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization. Eur J Neurosci. 2001;14:821-828[CrossRef][Medline] [Order article via Infotrieve]. 70. Volbracht C, Leist M, Kolb SA, Nicotera P. Apoptosis in caspase-inhibited neurons. Mol Med. 2001;7:36-48[Medline] [Order article via Infotrieve]. 71. Chechlacz M, Vemuri MC, Naegele JR. Role of DNA-dependent protein kinase in neuronal survival. J Neurochem. 2001;78:141-154[CrossRef][Medline] [Order article via Infotrieve]. 72. Somervaille TC, Linch DC, Khwaja A. Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax. Blood. 2001;98:1374-1381[Abstract/Free Full Text]. 73. Miyazaki K, Yoshida H, Sasaki M, et al. Caspase-independent cell death and mitochondrial disruptions observed in the Apaf1-deficient cells. J Biochem (Tokyo). 2001;129:963-969[Abstract/Free Full Text]. © 2003 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: S. Moriceau, C. Kantari, J. Mocek, N. Davezac, J. Gabillet, I. C. Guerrera, F. Brouillard, D. Tondelier, I. Sermet-Gaudelus, C. Danel, et al. Coronin-1 Is Associated with Neutrophil Survival and Is Cleaved during Apoptosis: Potential Implication in Neutrophils from Cystic Fibrosis Patients J. Immunol., June 1, 2009; 182(11): 7254 - 7263. [Abstract] [Full Text] [PDF] A. Nagarsekar, R. S. Greenberg, N. G. Shah, I. S. Singh, and J. D. Hasday Febrile-Range Hyperthermia Accelerates Caspase-Dependent Apoptosis in Human Neutrophils J. Immunol., August 15, 2008; 181(4): 2636 - 2643. [Abstract] [Full Text] [PDF] A. Cross, R. J. Moots, and S. W. Edwards The dual effects of TNF{alpha} on neutrophil apoptosis are mediated via differential effects on expression of Mcl-1 and Bfl-1 Blood, January 15, 2008; 111(2): 878 - 884. [Abstract] [Full Text] [PDF] C. Kantari, M. Pederzoli-Ribeil, O. Amir-Moazami, V. Gausson-Dorey, I. C. Moura, M.-C. Lecomte, M. Benhamou, and V. Witko-Sarsat Proteinase 3, the Wegener autoantigen, is externalized during neutrophil apoptosis: evidence for a functional association with phospholipid scramblase 1 and interference with macrophage phagocytosis Blood, December 1, 2007; 110(12): 4086 - 4095. [Abstract] [Full Text] [PDF] R. D. Dinnen, L. Drew, D. P. Petrylak, Y. Mao, N. Cassai, J. Szmulewicz, P. Brandt-Rauf, and R. L. Fine Activation of Targeted Necrosis by a p53 Peptide: A NOVEL DEATH PATHWAY THAT CIRCUMVENTS APOPTOTIC RESISTANCE J. Biol. Chem., September 14, 2007; 282(37): 26675 - 26686. [Abstract] [Full Text] [PDF] T. W. Kuijpers, R. v. Bruggen, N. Kamerbeek, A. T. J. Tool, G. Hicsonmez, A. Gurgey, A. Karow, A. J. Verhoeven, K. Seeger, O. Sanal, et al. Natural history and early diagnosis of LAD-1/variant syndrome Blood, April 15, 2007; 109(8): 3529 - 3537. [Abstract] [Full Text] [PDF] A. D. Gregory, L. A. Hogue, T. W. Ferkol, and D. C. Link Regulation of systemic and local neutrophil responses by G-CSF during pulmonary Pseudomonas aeruginosa infection Blood, April 15, 2007; 109(8): 3235 - 3243. [Abstract] [Full Text] [PDF] B. E. Steinberg and S. Grinstein Unconventional Roles of the NADPH Oxidase: Signaling, Ion Homeostasis, and Cell Death Sci. Signal., March 27, 2007; 2007(379): pe11 - pe11. [Abstract] [Full Text] [PDF] H.-R. Kim, H.-J. Chae, M. Thomas, T. Miyazaki, A. Monosov, E. Monosov, M. Krajewska, S. Krajewski, and J. C. Reed Mammalian dap3 is an essential gene required for mitochondrial homeostasis in vivo and contributing to the extrinsic pathway for apoptosis FASEB J, January 1, 2007; 21(1): 188 - 196. [Abstract] [Full Text] [PDF] A. De Geer, R. Kiessling, V. Levitsky, and J. Levitskaya Cytotoxic T Lymphocytes Induce Caspase-Dependent and -Independent Cell Death in Neuroblastomas in a MHC-Nonrestricted Fashion J. Immunol., December 1, 2006; 177(11): 7540 - 7550. [Abstract] [Full Text] [PDF] A. Daryadel, R. F. Grifone, H.-U. Simon, and S. Yousefi Apoptotic Neutrophils Release Macrophage Migration Inhibitory Factor upon Stimulation with Tumor Necrosis Factor-{alpha} J. Biol. Chem., September 15, 2006; 281(37): 27653 - 27661. [Abstract] [Full Text] [PDF] H.-M. Shen and S. Pervaiz TNF receptor superfamily-induced cell death: redox-dependent execution FASEB J, August 1, 2006; 20(10): 1589 - 1598. [Abstract] [Full Text] [PDF] Y. Xu, S. O. Kim, Y. Li, and J. Han Autophagy Contributes to Caspase-independent Macrophage Cell Death J. Biol. Chem., July 14, 2006; 281(28): 19179 - 19187. [Abstract] [Full Text] [PDF] L. Zheng, N. Bidere, D. Staudt, A. Cubre, J. Orenstein, F. K. Chan, and M. Lenardo Competitive Control of Independent Programs of Tumor Necrosis Factor Receptor-Induced Cell Death by TRADD and RIP1. Mol. Cell. Biol., May 1, 2006; 26(9): 3505 - 3513. [Abstract] [Full Text] [PDF] V. Temkin, Q. Huang, H. Liu, H. Osada, and R. M. Pope Inhibition of ADP/ATP Exchange in Receptor-Interacting Protein-Mediated Necrosis. Mol. Cell. Biol., March 1, 2006; 26(6): 2215 - 2225. [Abstract] [Full Text] [PDF] Y. Guo, C. Chen, Y. Zheng, J. Zhang, X. Tao, S. Liu, D. Zheng, and Y. Liu A Novel Anti-human DR5 Monoclonal Antibody with Tumoricidal Activity Induces Caspase-dependent and Caspase-independent Cell Death J. Biol. Chem., December 23, 2005; 280(51): 41940 - 41952. [Abstract] [Full Text] [PDF] S. von Gunten, S. Yousefi, M. Seitz, S. M. Jakob, T. Schaffner, R. Seger, J. Takala, P. M. Villiger, and H.-U. Simon Siglec-9 transduces apoptotic and nonapoptotic death signals into neutrophils depending on the proinflammatory cytokine environment Blood, August 15, 2005; 106(4): 1423 - 1431. [Abstract] [Full Text] [PDF] S. Karmakar, R. D. Cummings, and R. P. McEver Contributions of Ca2+ to Galectin-1-induced Exposure of Phosphatidylserine on Activated Neutrophils J. Biol. Chem., August 5, 2005; 280(31): 28623 - 28631. [Abstract] [Full Text] [PDF] T. W. Kuijpers, M. Alders, A. T. J. Tool, C. Mellink, D. Roos, and R. C. M. Hennekam Hematologic abnormalities in Shwachman Diamond syndrome: lack of genotype-phenotype relationship Blood, July 1, 2005; 106(1): 356 - 361. [Abstract] [Full Text] [PDF] S. Sim, T.-S. Yong, S.-J. Park, K.-i. Im, Y. Kong, J.-S. Ryu, D.-Y. Min, and M. H. Shin NADPH Oxidase-Derived Reactive Oxygen Species-Mediated Activation of ERK1/2 Is Required for Apoptosis of Human Neutrophils Induced by Entamoeba histolytica J. Immunol., April 1, 2005; 174(7): 4279 - 4288. [Abstract] [Full Text] [PDF] A. S. Cowburn, J. F. White, J. Deighton, S. R. Walmsley, and E. R. Chilvers z-VAD-fmk augmentation of TNF{alpha}-stimulated neutrophil apoptosis is compound specific and does not involve the generation of reactive oxygen species Blood, April 1, 2005; 105(7): 2970 - 2972. [Abstract] [Full Text] [PDF] M. Hu, X. Lin, Q. Du, E. J. Miller, P. Wang, and H. H. Simms Regulation of polymorphonuclear leukocyte apoptosis: role of lung endothelium-epithelium bilayer transmigration Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L266 - L274. [Abstract] [Full Text] [PDF] S. Mocellin, M. Provenzano, C. R. Rossi, P. Pilati, R. Scalerta, M. Lise, and D. Nitti Induction of Endothelial Nitric Oxide Synthase Expression by Melanoma Sensitizes Endothelial Cells to Tumor Necrosis Factor-Driven Cytotoxicity Clin. Cancer Res., October 15, 2004; 10(20): 6879 - 6886. [Abstract] [Full Text] [PDF] R. Blomgran, L. Zheng, and O. Stendahl Uropathogenic Escherichia coli Triggers Oxygen-Dependent Apoptosis in Human Neutrophils through the Cooperative Effect of Type 1 Fimbriae and Lipopolysaccharide Infect. Immun., August 1, 2004; 72(8): 4570 - 4578. [Abstract] [Full Text] [PDF] N. A. Maianski, D. Roos, and T. W. Kuijpers Bid Truncation, Bid/Bax Targeting to the Mitochondria, and Caspase Activation Associated with Neutrophil Apoptosis Are Inhibited by Granulocyte Colony-Stimulating Factor J. Immunol., June 1, 2004; 172(11): 7024 - 7030. [Abstract] [Full Text] [PDF] F. Goepel, P. Weinmann, J. Schymeinsky, and B. Walzog Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis J. Leukoc. Biol., May 1, 2004; 75(5): 836 - 843. [Abstract] [Full Text] [PDF] M. Okada, S. Adachi, T. Imai, K.-i. Watanabe, S.-y. Toyokuni, M. Ueno, A. S. Zervos, G. Kroemer, and T. Nakahata A novel mechanism for imatinib mesylate-induced cell death of BCR-ABL-positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity Blood, March 15, 2004; 103(6): 2299 - 2307. [Abstract] [Full Text] [PDF] H. Liu, Y. Ma, L. J. Pagliari, H. Perlman, C. Yu, A. Lin, and R. M. Pope TNF-{alpha}-Induced Apoptosis of Macrophages Following Inhibition of NF-{kappa}B: A Central Role for Disruption of Mitochondria J. Immunol., February 1, 2004; 172(3): 1907 - 1915. [Abstract] [Full Text] [PDF] K. C. Jung, W. S. Park, H. J. Kim, E. Y. Choi, M.-C. Kook, H.-W. Lee, and Y. Bae TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-Linking J. Immunol., January 15, 2004; 172(2): 795 - 802. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-02-0522v1 101/5/1987    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Maianski, N. A. Articles by Kuijpers, T. W. Search for Related Content PubMed PubMed Citation Articles by Maianski, N. A. Articles by Kuijpers, T. W. Related Collections Phagocytes Apoptosis Social Bookmarking                   What's this?

	Copyright © 2003 by American Society of Hematology         Online ISSN: 1528-0020