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 Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2001-12-0266.

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Blood, 1 January 2003, Vol. 101, No. 1, pp. 295-304

PHAGOCYTES

Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils

Chien-Ying Liu, Akihiro Takemasa, W. Conrad Liles, Richard B. Goodman, Mechthild Jonas, Henry Rosen, Emil Chi, Robert K. Winn, John M. Harlan, and Peter I. Chuang

From the Department of Medicine, Pathology, and Surgery, University of Washington, Seattle.


    Abstract
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

It is increasingly clear that there are caspase-dependent and -independent mechanisms for the execution of cell death and that the utilization of these mechanisms is stimulus- and cell type-dependent. Intriguingly, broad-spectrum caspase inhibition enhances death receptor agonist-induced cell death in a few transformed cell lines. Endogenously produced oxidants are causally linked to necroticlike cell death in these instances. We report here that broad-spectrum caspase inhibitors effectively attenuated apoptosis induced in human neutrophils by incubation with agonistic anti-Fas antibody or by coincubation with tumor necrosis factor-alpha (TNF-alpha ) and cycloheximide ex vivo. In contrast, the same caspase inhibitors could augment cell death upon stimulation by TNF-alpha alone during the 6-hour time course examined. Caspase inhibitor-sensitized, TNF-alpha -stimulated, dying neutrophils exhibit apoptoticlike and necroticlike features. This occurred without apparent alteration in nuclear factor-kappa B (NF-kappa B) activation. Nevertheless, intracellular oxidant production was enhanced and sustained in caspase inhibitor-sensitized, TNF-alpha -stimulated neutrophils obtained from healthy subjects. However, despite reduced or absent intracellular oxidant production following TNF-alpha stimulation, cell death was also augmented in neutrophils isolated from patients with chronic granulomatous disease incubated with a caspase inhibitor and TNF-alpha . These results demonstrate that, in human neutrophils, TNF-alpha induces a caspase-independent but protein synthesis-dependent cell death signal. Furthermore, they suggest that TNF-alpha activates a caspase-dependent pathway that negatively regulates reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. (Blood. 2003;101:295-304)

© 2003 by The American Society of Hematology.

    Introduction
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Nearly 1 billion neutrophils per kilogram of body mass are turned over daily in an average human adult.1 With a relatively short life span in vivo, mature human neutrophils appear to be endowed with robust machineries that drive cell death. Spontaneous neutrophil apoptosis is temporally associated with an increase in caspase-3 activity2 and is attenuated by caspase inhibitors.2,3 Agonistic activation of Fas, one of the death receptor family members constitutively expressed in circulating mature human neutrophils,4,5 also activates caspase-3 activity2 and provokes neutrophil apoptosis that is attenuated by caspase inhibitors.2,3 Thus, caspase activation mediates spontaneous and Fas-induced neutrophil apoptosis.

There is increasing evidence for caspase-independent mechanisms of apoptotic cell death.6 To distinguish these novel caspase-independent forms of cell death from the classically described apoptotic and necrotic cell death processes, terms such as paraptosis7 and aponecrosis8 have been proposed. The distinctions between apoptotic versus necrotic and programmed versus nonprogrammed cell death are increasingly blurred also,9,10 and neutrophils can undergo cell death in the absence of classical apoptotic or necrotic characteristics.2,11,12 Although it has been suggested that human neutrophils are relatively deficient in the numbers of mitochondria13 and caspases 2, 6, and 7,14,15 the execution of a death receptor agonist-induced caspase-independent pathway has not previously been described.

In addition to the Fas ligand-Fas system, the tumor necrosis factor-alpha (TNF-alpha )-TNF receptor system is an important participant in an inflammatory response. In contrast to a variety of other cell types, isolated mature, circulating human neutrophils are sensitive to Fas and TNF receptor agonist-induced cell death without requiring simultaneous inhibition of RNA or protein synthesis.16,17 Interestingly, TNF-alpha has opposing effects on neutrophil life span. While TNF-alpha activates caspase-3-like activities and induces neutrophil apoptosis at early time points (8 hours or fewer) in vitro,16,18 it can also extend neutrophil survival at later time points (12 hours or more).16,19 The characteristics of TNF-alpha -induced neutrophil apoptosis include morphologic features of apoptosis,16 internucleosomal DNA fragmentation,16 and caspase activation.18 During our investigation of the relationship between TNF-alpha -induced neutrophil activation and cell death, we attempted to block apoptosis with caspase inhibitors. Unexpectedly, we found that the cell-permeable, broad-spectrum caspase inhibitors, zVAD (benzoyloxycarbonyl-Val-Ala-Asp(OMe)-CH2F) and Boc-D (t-butyloxycarbonyl-Asp(OMe)-CH2F), not only failed to attenuate cell death but, instead, accentuated it. Because these results were unexpected,17 further detailed analyses were performed.

In the present study, we show that TNF-alpha -stimulated neutrophils, when pretreated with broad-spectrum caspase inhibitors but not a cathepsin-selective inhibitor, can undergo apoptoticlike and necroticlike cell death during the 6-hour time course examined as demonstrated by flow cytometry as well as by biochemical, morphologic, and ultrastructural examinations. The paradoxical effect of zVAD occurred without apparent suppression in nuclear factor-kappa B (NF-kappa B) activation, a critical survival factor in neutrophils undergoing constitutive or TNF-alpha -induced cell death.17 These results share similarities with that reported in the murine fibroblast cell lines, L929,20 NIH3T3,21,22 and WEHI-S,23 and the human myelomonocytic leukemia cell line, U937.21 However, in contrast to L929 and NIH3T3 cells,22,24 agonistic anti-Fas antibody-induced cell death was effectively attenuated by zVAD in neutrophils. Protein synthesis inhibition, however, sensitized to TNF-alpha -induced killing and altered the cell death mechanism to a predominantly caspase-dependent one. Furthermore, we provide evidence that, in contrast to the cell lines mentioned above, reactive oxygen intermediates generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase are not required for zVAD-sensitized, TNF-alpha -induced neutrophil cell death to proceed.


    Materials and methods
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Reagents

Recombinant human TNF-alpha was purchased from R&D Systems (Minneapolis, MN). Mouse antihuman Fas monoclonal antibody (CH-11) was from Kamiya (Seattle, WA). The cell-permeable, irreversible, broad-spectrum caspase inhibitors Boc-D and zVAD; the cell-permeable cathepsin-L-selective inhibitor, zFF (benzyloxycarbony-Phe-Phe-CH2F); the fluorogenic caspase substrate, Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin); and cycloheximide (CHX) were purchased from Calbiochem (La Jolla, CA). The 2',7'-dichlorofluorescin-diacetate (DCFH-DA), and propidium iodide (PI) were obtained from Sigma (St Louis, MO). Fluorescein isothiocyanate (FITC)-conjugated annexin V (annexin V-FITC) and the mouse antihuman cytochrome c monoclonal antibody were purchased from BD PharMingen (San Diego, CA). Horseradish peroxidase (HRP)-conjugated rabbit antimouse IgG secondary antibody and the SuperSignal substrate were obtained from Pierce (Rockford, IL). All cell culture media and supplements were purchased from Life Technologies (Grand Island, NY).

Isolation of circulating mature human neutrophils

Peripheral blood was obtained from healthy adult donors and from 2 patients with documented X-linked chronic granulomatous disease (CGD, subtypes X91+ and X91-) under protocols approved by the Human Subjects Review Committee, University of Washington. Informed consent was provided according to the Declaration of Helsinki. Following erythrocyte sedimentation with hetastarch (Abbott Laboratories, Abbott Park, IL) or dextran T-500 (Amersham Pharmacia Biotech, Piscataway, NJ), neutrophils were separated from the leukocyte fraction over a discontinuous gradient (450g, 25°C, 30 minutes) utilizing dextran Ficoll-Paque Plus (Amersham Pharmacia Biotech) as previously described.25,26 After lysis of contaminating erythrocytes with buffered ammonium chloride,26 purified neutrophils were resuspended in a round-bottom polypropylene tube in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. The purity and viability of isolated neutrophils were consistently at least 98%.

Cell death assessment by light and electron microscopy

Unless indicated otherwise, 1 × 106 neutrophils at 5 × 106 cells per milliliter were used for each sample throughout our study. At predefined time points, total neutrophil viable cell counts that remained under various treatment conditions were determined by trypan blue dye exclusion with a hemacytometer. In parallel, cytospin slides were prepared, stained with Diff-Quick (Baxter, McGaw Park, IL), and examined by light microscopy for morphologic changes.

For transmission electron microscopy (TEM), 5 × 106 freshly isolated neutrophils in 1 mL culture medium were incubated under a specified condition and processed as previously described.27 Specifically, at predetermined time points, cells were fixed in 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde at 4°C, washed, and postfixed in distilled water containing 2% osmium tetroxide and a few drops of 2% aqueous potassium ferrocyanide. After block staining with 0.5% aqueous uranyl acetate for 15 minutes, the cells were embedded in 0.1 M sodium cacodylate buffer containing 2% agar. The cell pellet was then dehydrated in a graded series of ethanol and embedded in Eponate-12 resin (Ted Pella, Redding, CA). Finally, thin sections were cut on an LKB Nova ultramicrotome (LKB, Bromma, Sweden), stained with uranyl acetate and lead stain, and examined with a JEOL JEM 1200 EX (JEOL, Tokyo, Japan).

Cell death assessment by flow cytometry

Annexin V-FITC binds to exposed phosphatidylserine on early apoptotic (representing membrane-intact cells with externalized phosphatidylserine) and necrotic (representing "primary," nonapoptotic cells and "secondary,"28 late apoptotic cells with compromised membrane integrity) cells, and PI gains entry into necrotic cells.29 Utilizing these agents, early apoptotic and primary/secondary necrotic neutrophils were quantitatively determined by dual-parameter flow cytometry. Briefly, neutrophils were cultured under a specified condition in a humidified CO2 (5%) incubator at 37°C. At a predetermined time point, neutrophils were resuspended in 100 µL binding buffer (140 mM NaCl, 2.5 mM CaCl2, 1.5 mM MgCl2, and 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4) containing annexin V-FITC (20 µg/mL) and PI (2 µg/mL) for 15 minutes at room temperature. Samples were kept on ice and analyzed immediately with an EPICS XL-II flow cytometer (Beckman Coulter, Fullerton, CA).

Cell death assessment by DNA fragmentation assays

Internucleosomal DNA fragmentation in neutrophils was assessed qualitatively by agarose gel electrophoresis as described.4 Briefly, neutrophils (10 × 106 cells in 2 mL supplemented RPMI medium) at a predetermined time point after treatment were washed twice with phosphate-buffered saline (PBS), pelleted, and lysed (0.2% Triton X-100, 10 mM EDTA [ethylenediaminetetraacetic acid], and 10 mM Tris [tris(hydroxymethyl)aminomethane] HCl, pH 7.5) for 10 minutes on ice. After centrifugation (12 000g, 10 minutes), DNA-containing supernatant was extracted with phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol), precipitated overnight with 70% ethanol at -20°C, pelleted, and dissolved in TE (10 mM Tris HCl, pH 7.5, and 1 mM EDTA). The samples were subsequently digested with 1 mg/mL DNase-free RNase at 37°C for 3 hours; 5 µg DNA from each sample was electrophoretically resolved in a 2% agarose gel containing 0.5 µg/mL ethidium bromide and visualized under ultraviolet light.

As a complementary approach, internucleosomal DNA fragmentation was quantitatively assayed by antibody-mediated capture and detection of cytoplasmic mononucleosome- and oligonucleosome-associated histone-DNA complexes (Cell Death Detection ELISA plus kit; Roche Molecular Biochemicals, Mannheim, Germany) that accumulated in dying neutrophils with intact cell membrane.30 Briefly, neutrophils (1 × 104 cells in 200 µL supplemented RPMI medium) at a predetermined time point after treatment were washed, resuspended in 200 µL of the lysis buffer supplied by the manufacturer, and incubated for 30 minutes at room temperature. After pelleting nuclei (200g, 10 minutes), 20 µL of the supernatant (cytoplasmic fraction) was used in the enzyme-linked immunosorbent assay (ELISA) following the manufacturer's standard protocol. Finally, absorbance at 405 nm and 490 nm (reference wavelength), upon incubating with a peroxidase substrate for 5 minutes, was determined with a microplate reader (Bio-Tec Instruments, Winooski, VT). Signals in the wells containing the substrate only were subtracted as background.

Caspase-3-like activity assay

Neutrophils (2 × 106 cells in 400 µL supplemented RPMI medium) at a predetermined time point after treatment were washed with PBS and lysed in 100 µL buffer (10 mM potassium phosphate, 1 mM EDTA, 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL leupeptin, 10 µg/mL pepstatin A, and 10 mM dithiothreitol [DTT]) for 10 minutes on ice. After centrifugation (15 000g, 20 minutes, 4°C), protein concentration of the supernatant was determined with the BCA Protein Assay Reagent (Pierce, Rockford, IL) following the manufacturer's instructions. Subsequently, 40 µg of the sample was diluted to a final volume of 200 µL with the assay buffer (50 mM HEPES, 10% sucrose, 0.1% CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid], and 10 mM DTT) containing the fluorogenic caspase-3-preferred substrate Ac-DEVD-AMC (100 µM) and incubated for 2 hours at 30°C in a 96-well plate. Fluorescence was determined (excitation, 360 nm; emission, 460 nm) with a CytoFluor series 4000 plate reader (Applied Biosystems, Foster City, CA). Background fluorescence was determined in wells containing the assay buffer only.

Subcellular fractionation and immunoblotting for cytochrome c release

The procedure for the preparation of mitochondria-poor cytosol fraction was modified from that previously reported.31 Specifically, neutrophils (10 × 106 cells in 2 mL supplemented RPMI medium) were harvested at a predetermined time point after treatment, washed with PBS, and resuspended in 500 µL buffer (20 mM HEPES-potassium hydroxide [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA [ethyleneglycoltetraacetic acid], 1 mM DTT, 250 mM sucrose, 1 mM PMSF, 1% aprotinin, 1 mM leupeptin, 1 µg/mL pepstatin A, and 1 µg/mL chymostatin). Following homogenization with a glass Pyrex homogenizer and a type B pestle (40 strokes), unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifugation (1000g, 20 minutes, 4°C). The supernatant was recentrifuged (100 000g, 1 hour, 4°C) to generate the mitochondria-poor cytosolic fraction. Fifty micrograms of the cytosolic protein extracts were then resolved by gradient (4% to 20%) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. The membrane was blocked overnight with 5% milk in TBST (50 mM Tris HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween 20) and then probed at room temperature for 1 hour with primary mouse monoclonal anticytochrome c antibody (1:1000 dilution) in TBS (50 mM Tris MCl [pH 7.4], 150 mM NaCl), supplemented with 0.05% Tween 20 and 1% bovine serum albumin. After 3 washes with TBST, the membrane was incubated with HRP-conjugated secondary antibody (1:5000 dilution) and the chemiluminescence of the expected protein bands (15 kDa) detected with the SuperSignal substrate, per the supplier's protocol.

NF-kappa B activation assays

For assay of NF-kappa B activation in neutrophils, a nonradioactive ELISA method (NF-kappa B Transcription Factor Assay Kit; Active Motif, Carlsbad, CA), which has been shown to be specific and quantitative as well as to correlate well with the traditional electrophoretic mobility shift assay (EMSA), was utilized.32 In brief, 5 × 106 neutrophils were cultured in 1 mL supplemented RPMI medium in the absence or presence of TNF-alpha (10 ng/mL) and without or with zVAD (100 µM) before incubation. At 30, 60, 90, and 120 minutes, neutrophils were washed twice with ice-cold PBS and lysed in 40 µL of the supplied lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Subsequently, 20 µg of protein lysate per sample was incubated for 1 hour in wells containing immobilized NF-kappa B consensus oligonucleotide in the absence or presence of competing, nonimmobilized, NF-kappa B consensus oligonucleotide. After extensive washing, total bound NF-kappa B p65/p50 heterodimer and p50/p50 homodimer were detected with the supplied anti-p50 antibody following the manufacturer's protocol. Wells incubated with lysis buffer alone were developed in parallel and the readout subtracted as background.

For EMSA, nuclear lysates were prepared as previously described33 from neutrophils (5 × 106 cells in 1 mL supplemented RPMI medium) incubated for 1 hour without or with TNF-alpha and in the absence or presence of zVAD. EMSA was performed with 5 µg nuclear protein extract per sample, utilizing a commercial Gel Shift Assay System (Promega, Madison, WI) and following the manufacturer's protocol. The resulting NF-kappa B consensus oligonucleotide-binding activities in the nuclear extracts in the absence or presence of 100-fold molar excess of unlabeled consensus oligonucleotide were resolved on a 6% nondenaturing polyacrylamide gel and exposed to film.

Measurement of intracellular oxidative activity

Neutrophil intracellular oxidative metabolism was assessed using the cell-permeable, fluorogenic DCFH-DA as described.34 Briefly, the neutrophil sample (1 × 106 cells in 200 µL supplemented RPMI medium) was supplemented with DCFH-DA (5 µM) 30 minutes before the completion of a predetermined duration of incubation under a specified condition. Upon intracellular hydrolysis and subsequent oxidization, fluorescent DCF was generated. Cells were washed with ice-cold PBS and resuspended in 0.5 mL PBS supplemented with 1% FBS. Accumulation of intracellular fluorescence in live cells was determined by flow cytometry.

Statistical analysis

Data are expressed as means ± SEM. For normally distributed data, a t test or paired t test was used to evaluate the differences between sets. For nonnormally distributed data, the Mann-Whitney U test was used. GraphPad Prism (version 2.01; GraphPad Software, San Diego, CA) was used for all statistical analyses. Statistical significance was defined as P < .05.


    Results
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Broad-spectrum caspase inhibitors augment TNF-alpha - but not Fas-induced neutrophil cell death

In contrast to several other primary human cells, agonistic stimulation of the neutrophil Fas or TNF receptors can induce caspase activation and cell death without the concurrent requirement of protein or RNA synthesis inhibition.2,4,5,16,18 The broad-spectrum, cell-permeable, irreversible caspase inhibitor, zVAD, was previously shown to attenuate caspase-3 activity in TNF-alpha -stimulated neutrophils in a dose-dependent manner, with maximal inhibition at 100 µM.14 At this concentration, zVAD also was reported to inhibit agonistic anti-Fas antibody-induced neutrophil cell death2,3 and to attenuate TNF-alpha -induced neutrophil cell death in the absence (assessed by morphology)17 or presence (assessed by DNA hypodiploidy and phosphatidylserine accessibility)14 of cycloheximide. In time-course experiments, we confirmed that zVAD effectively attenuated spontaneous and anti-Fas antibody-induced neutrophil cell death, measured both by total viable cell counts (Figure 1) and by dual-parameter flow cytometric analysis of cellular annexin V-FITC binding and PI accessibility (data not shown). Unexpectedly, zVAD failed to inhibit but instead augmented TNF-alpha -induced neutrophil cell death in the same experiments (Figure 1). To characterize further this surprising observation, a series of experiments were performed. In these analyses, pretreatment with zVAD sensitized neutrophils to TNF-alpha -induced cell death over 6 hours in a dose-dependent manner (Figure 2A), an effect that was minimal but appeared to be present at 5 µM and clearly discernible at zVAD concentrations at and above 25 µM. This sensitizing effect also occurred when zVAD was added up to 2 hours after TNF-alpha stimulation (data not shown). Similarly, with zVAD pretreatment, TNF-alpha at and above 1 ng/mL still induced neutrophil cell death in a dose-dependent manner (Figure 2B). Corroborating these results, TNF-alpha -induced cytosolic cytochrome c accumulation was enhanced with zVAD pretreatment (Figure 2C). In confirmatory experiments, the cleavage of a fluorogenic substrate for caspase-3-like proteases, Ac-DEVD-AMC, was abrogated in cell lysates prepared from neutrophils incubated with TNF-alpha and zVAD (Figure 3A). Importantly, zVAD in and of itself was not cytotoxic, because it clearly prolonged cell survival in spontaneously aging and anti-Fas antibody-stimulated neutrophils (Figure 1). Furthermore, the sensitization of neutrophils to TNF-alpha -induced cell death was not limited to zVAD, because another broad-spectrum, cell-permeable caspase inhibitor, Boc-D, produced a similar effect (Figure 3B). Some specificity for broad-spectrum caspase inhibition was suggested by the fact that zFF, a cell-permeable cathepsin L-selective inhibitor, did not significantly alter TNF-alpha -induced neutrophil cell death (Figure 3B). Taken together, although it is possible that zVAD exerts its effect nonspecifically in TNF-alpha -stimulated neutrophils, these experiments indicate that broad-spectrum caspase inhibitors are not cytotoxic per se, and their paradoxic effects in sensitizing to TNF-alpha -induced cell death is in contrast to Fas agonist stimulation.


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  		Figure 1. zVAD attenuates anti-Fas antibody-induced neutrophil cell death but paradoxically augments TNF-alpha -induced neutrophil cell death. One million freshly isolated mature human neutrophils (5 × 106 cells per milliliter) were preincubated with or without the cell-permeable, broad-spectrum caspase inhibitor zVAD (100 µM) for 1 hour. Subsequently, at time 0, neutrophils were stimulated with TNF-alpha (10 ng/mL), anti-Fas antibody (CH-11; 100 ng/mL), or an equivalent volume of buffered saline. The total number of viable neutrophils that remained, as defined by trypan blue dye exclusion, was determined with a hemacytometer at indicated times. Data shown are means ± SEM and are representative of 3 experiments performed in triplicates. *P < .05 compared with no zVAD at the corresponding time point.



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  		Figure 2. zVAD sensitizes neutrophils to TNF-alpha -induced cell death in a dose-dependent manner. (A-B) Neutrophils were preincubated with or without zVAD at the indicated concentrations for 1 hour. At time 0, TNF-alpha was supplemented to the final concentrations shown. Cell death was analyzed at 6 hours by dual-parameter flow cytometry utilizing FITC-conjugated annexin V (annexin V-FITC), which binds specifically to phosphatidylserine on the cell membrane and marks early apoptotic, primary necrotic, and late apoptotic/secondary necrotic cells, and propidium iodide (PI), which enters dead cells with breached membrane integrity and marks primary and late apoptotic/secondary necrotic cells. Each panel represents data collected from 10 000 total events. In each panel, the left lower quadrant represents remaining live cells that do not bind annexin V-FITC and exclude PI. The right lower quadrant represents the accumulation of early apoptotic cells that have externalized membrane phosphatidylserine but still retain membrane integrity. The right upper quadrant represents the accumulation of both late apoptotic cells that have lost membrane integrity (secondary necrosis) and primary necrotic cells. The percentages of cells in each of these quadrants are indicated. (C) Neutrophils preincubated with or without zVAD (100 µM) for 1 hour were incubated in the absence or presence of TNF-alpha (10 ng/mL) for 3 additional hours and subsequently lysed and subcellularly fractionated (see "Materials and methods"). Accumulation of cytosolic cytochrome c (15 kDa) in the mitochondria-poor fraction was assessed by immunoblotting. Results shown above are representative of 3 independent experiments.



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  		Figure 3. Broad-spectrum caspase inhibitors but not a cathepsin L-selective inhibitor sensitize neutrophils to TNF-alpha -induced cell death. (A) Cleavage of the caspase-3-preferred fluorogenic substrate, Ac-DEVD-AMC, was measured (see "Materials and methods") in whole-cell lysates prepared from neutrophils preincubated with or without zVAD (100 µM) for 1 hour and then stimulated with or without TNF-alpha (10 ng/mL) for 2 additional hours. Results shown represent means ± SEM of 3 experiments. *P < .05 compared with unstimulated neutrophils at 2 hours. (B) Neutrophils were preincubated with zVAD (100 µM) or Boc-D (100 µM) or the cathepsin L-selective inhibitor zFF (100 µM) for 1 hour before the addition of TNF-alpha (10 ng/mL) or an equivalent volume of buffered saline at time 0. Cellular binding of annexin V-FITC (AV+) was subsequently assayed at 6 hours with flow cytometry. Results are means ± SEM of 3 independent experiments. *P < .05 compared with neutrophils stimulated with TNF-alpha alone.

zVAD-sensitized, TNF-alpha -stimulated neutrophils undergo cell death with apoptoticlike and necroticlike features

During the course of our studies, we frequently noted that the appearance of PI-impermeable and annexin V-positive (early apoptotic) cells heralded that of PI-permeable and annexin V-positive (primary necrotic or late apoptotic/secondary necrotic) cells (Figure 4A) in zVAD-sensitized, TNF-alpha -stimulated neutrophils in vitro. Interestingly, under light microscopy, whereas neutrophils incubated with zVAD alone appeared normal morphologically and TNF-alpha -stimulated neutrophils exhibited classic features of apoptosis, significant numbers of neutrophils concurrently incubated with both of these reagents showed apoptoticlike and necroticlike changes6 (Figure 4B). Examination by TEM confirmed that zVAD-sensitized, TNF-alpha -stimulated neutrophils developed atypical but apoptoticlike ultrastructural alterations (Figure 4C). These included the loss of normal membrane ruffles and "smoothing out" of the cell surface, extensive cytoplasmic fragmentations, and formation of many membrane-bound bodies. Additionally, features resembling cellular degranulation were occasionally seen. Apoptoticlike nuclear changes were also noted and included rounding up of the nuclear contour, dense condensation of the nuclear chromatin, as well as fragmentation of the nuclear lobes. Consistent with the results obtained by flow cytometry, most cells appeared to maintain membrane integrity at early time points in spite of the ultrastructural changes. While significant numbers of necroticlike cells were also observed, they were less frequent and were noted only later. Intriguingly, as demonstrated by DNA ladder formation (Figure 5A) and cytoplasmic oligonucleosome-associated histone assay (Figure 5B), we observed that zVAD not only failed to attenuate DNA fragmentation but appeared to augment it in several experiments. Similar enhancement of TNF-alpha -induced DNA ladder formation by zVAD was previously reported in the mouse fibroblast cell line NIH3T3.22 Furthermore, Li et al reported that endonuclease G was released from mitochondria in murine embryonic fibroblasts and directly resulted in nuclear DNA ladder formation during TNF-alpha -induced cell death in the presence of zVAD.35 Thus, these studies indicate that apoptoticlike cell death processes can proceed despite blockade of caspases by broad-spectrum inhibitors. Taken together, it appears that, during broad-spectrum caspase inhibition, TNF-alpha -induced neutrophil cell death occurred through processes that exhibited both apoptoticlike and necroticlike features.


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  		Figure 4. zVAD-sensitized, TNF-alpha -stimulated neutrophils undergo atypical cell death characterized by the emergence of early apoptoticlike cells that heralds the appearance of necroticlike cells. (A) Neutrophils were preincubated with or without zVAD (100 µM) for 1 hour. Subsequently, at time 0, neutrophils were stimulated with TNF-alpha (10 ng/mL) or equivalent volume of buffered saline. Cell death was analyzed by dual-parameter flow cytometry (see "Materials and methods") at indicated times. In each panel, the percentages of cells in the left lower (live), the right lower (early apoptotic), and the right upper (primary necrotic and late apoptotic/secondary necrotic) quadrants are indicated. Results shown are representative of 3 experiments. Note that, with typical neutrophil apoptosis such as constitutive or induced by TNF-alpha alone, there was progression from early apoptosis (annexin V-positive, PI-negative) to secondary necrosis (annexin V-positive, PI-positive) during the 6-hour time course examined. Note also that, with zVAD-preincubation, TNF-alpha -stimulated, dying (annexin V-FITC-positive) neutrophils were predominantly early apoptotic (PI-negative) at 2 and 3 hours but by 6 hours had progressed, leading to the accumulation of PI-positive cells (similar to that noted in Figure 2A-B). (B) Diff-Quick-stained cytocentrifuge preparations of neutrophils preincubated with or without zVAD and harvested at 4 hours after the addition of TNF-alpha or buffered saline were examined by light microscopy (magnification, × 1000). Note that TNF-alpha alone induced classic apoptotic nuclear changes. Similar to some of the features observed by TEM (shown in panel C), zVAD-sensitized, TNF-alpha -stimulated neutrophils frequently displayed apoptoticlike cell shrinkage and nuclear fragmentation and condensation (black arrow). In addition, necroticlike changes including cell and nuclear swelling that, at times, occurred in cells with fragmented nuclei (white arrow) were observed. (C) Neutrophils preincubated with zVAD were fixed at 2 (i,ii), 3 (iii,iv), and 6 (viii) hours after TNF-alpha stimulation and examined by TEM (magnification, × 10 000). Some of the notable cellular changes are shown. Also shown are neutrophils harvested after a 6-hour incubation with zVAD (v), cell culture medium (vi), or TNF-alpha (vii) alone. While some zVAD-preincubated neutrophils appeared to have been activated with significant cytoplasmic vacuolizations as early as 1 hour after TNF-alpha stimulation (not shown), many assumed atypical cell shapes and appeared to be undergoing extensive cytoplasmic fragmentation (i) and degranulation (ii) at 2 hours. Subtle but definite smoothing out of the outer nuclear envelopes was also noted (i,ii). Additionally, loss of the cell surface microvilli was frequently observed (i,iii). At 3 hours, nuclear lobes in many cells appeared to be fragmented, and nuclear chromatin condensed (iii). Furthermore, confirming the gross visual and light microscopic observations that significant cellular debris accumulated in neutrophil samples incubated with zVAD and TNF-alpha , many cellular fragments that appeared to remain membrane-bound were noted by TEM (iv). In addition, some necrotic cells were noted (not shown). In contrast, neutrophils incubated with zVAD alone appeared relatively normal, with preserved cell surface microvilli and nuclear morphology (v). Classically apoptotic neutrophils were observed when aged in culture medium alone (vi). Neutrophils stimulated with TNF-alpha alone also showed typical apoptotic nuclear changes (vii). Interestingly, similar nuclear changes could be seen at this time point in TNF-alpha -stimulated neutrophils in the presence of zVAD (viii).



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  		Figure 5. A component of the nuclear changes that occur during zVAD-sensitized, TNF-alpha -stimulated neutrophil cell death is apoptosislike. (A) Neutrophils were preincubated with or without zVAD (100 µM) for 1 hour before the addition of TNF-alpha (10 ng/mL) at time 0. Genomic DNA harvested from neutrophils incubated with the indicated reagents for 6 hours was then resolved by agarose gel electrophoresis to qualitatively assess internucleosomal DNA fragmentation (see "Materials and methods"). MW indicates 100-base pair (bp) ladder DNA molecular weight standard. (B) In separate experiments, internucleosomal DNA fragmentation was quantitatively determined by assaying for cytoplasmic mononucleosome- and oligonucleosome-associated histone accumulated in membrane-intact cells at the indicated time points (see "Materials and methods"). Results shown represent means ± SEM, n = 3. *P < .05 compared with unstimulated neutrophils at the corresponding times; #P < .05 compared with neutrophils stimulated with TNF-alpha only at the corresponding times.

Protein synthesis inhibition alters cell death response in TNF-alpha -stimulated neutrophils

In addition to the induction of cell death early after stimulation (8 hours or fewer), TNF-alpha can also extend neutrophil survival when utilized at low concentrations (0.1 to 1 ng/mL)19 and/or when incubated with neutrophils for long periods (12 hours or more).16 These apparently diverse responses to TNF-alpha suggest certain functional heterogeneity in circulating neutrophils. Alternatively, TNF-alpha induces intracellular competing kinetics of neutrophil death and survival signals that, on temporal balance, determine cell fate.16,17,19 In an attempt to reconcile some of the differences between the findings presented above and those reported by others,17 time-course studies were compiled over a 4-month period. They revealed that the net magnitude of TNF-alpha -attributable decrease in cell survival relative to control, measured by dual-parameter flow cytometry, occurred at 2 hours after stimulation and remained statistically significant at 4 and 6 hours but waned and became more variable (Figure 6A). In contrast, zVAD not only failed to significantly protect neutrophils from TNF-alpha -induced cell death at 2 hours after stimulation but highly significantly augmented it in a sustained manner at 4 and 6 hours (Figure 6A). Of note, in some experiments, zVAD appeared to confer cytoprotection in TNF-alpha -stimulated neutrophils. Nonetheless, paradoxic enhancement of cell death still occurred at other time points in the same experiments. Moreover, parallel assays invariably showed significant reduction in total viable cell counts (data not shown), suggesting that a significant proportion of cells underwent necroticlike cell death in these experiments. However, because TNF-alpha induces both cell death and survival signals that are potentially complex, the effect of zVAD could not be extrapolated outside of the time points and the in vitro system examined. Thus, depending upon the time points assessed and the particular cell death assays performed, divergent interpretations could be reached.


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  		Figure 6. zVAD and cycloheximide alter the kinetics of cell death response in TNF-alpha -stimulated neutrophils. (A) Compilation from a series of time-course studies of cell death in TNF-alpha -stimulated neutrophils with and without zVAD preincubation, assayed by dual-parameter flow cytometry. Open circles represent the TNF-alpha -attributable effect on neutrophil survival (ie, Delta Survival = TNFLive, the percentage of TNF-alpha -treated neutrophils that do not bind annexin V-FITC and exclude PI, minus CLive; the percentage of live cells cultured in medium alone) at a given time point in a particular experiment. Closed circles represent zVAD-attributable effect on cell survival in TNF-alpha -stimulated neutrophils relative to TNF-alpha alone (ie, Delta Survival = (TNF + zVAD)Live minus TNFLive) at a given time point within the same experiment. Indicated also are median values of the net effects of TNF-alpha and zVAD on cell survival at each time point. The intra-assay variability for the assay was less than 2.2% (95% confidence interval) in absolute number. Additionally, a paired t test was used for comparing TNFLive versus CLive and (TNF + zVAD)Live versus TNFLive at each time point. Statistical significance (defined as P < .01) was reached in all comparisons except (TNF + zVAD)Live versus TNFLive at 2 hours (means ± SEM of 73.1% ± 2.7% versus 77.6% ± 2.3%, respectively, n = 19, P = 0.11). Thus, whereas the net cell killing effect exerted by TNF-alpha occurred early and decreased with time, it was not significantly protected by zVAD at 2 hours and, paradoxically, was significantly augmented and sustained by zVAD at 4 ((TNF + zVAD)Live versus TNFLive, 53.3% ± 3.5% versus 71.6% ± 3.2%, respectively) and 6 hours (52.3% ± 3.2% versus 66.8% ± 2.5%). (B) Neutrophils were preincubated with cycloheximide (CHX, 1 µg/mL) in the absence or presence of zVAD or zDEVD (100 µM) for 30 minutes before the addition of TNF-alpha at time 0. Cell survival was determined at 4 hours by dual-parameter flow cytometry. *P < .05 compared with TNF + CHX; n = 3.

Similar to other TNF-alpha -resistant cell types, inhibition of protein synthesis by CHX at a concentration that by itself imposes little effects on neutrophil survival17,19 greatly sensitizes the neutrophil to TNF-alpha -induced cell death.14,17 Interestingly, under this condition, zVAD significantly attenuated TNF-alpha -induced neutrophil cell death (Figure 6B). In this regard, our results are consistent with those reported previously.14,17

NF-kappa B activation is not suppressed in zVAD-sensitized, TNF-alpha -stimulated neutrophils

Ward et al reported that NF-kappa B activation plays a critical cytoprotective role during constitutive and TNF-alpha -stimulated neutrophil cell death.17 To determine if zVAD exerted its cell death-enhancing effect in TNF-alpha -stimulated neutrophils by preventing NF-kappa B activation, time-course studies of NF-kappa B activation were performed (Figure 7). As observed in L929 and NIH3T3 cells in which broad-spectrum caspase inhibitors also sensitized to TNF-induced cell death,20,22,36 zVAD did not appreciably suppress NF-kappa B activation by TNF-alpha .


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  		Figure 7. NF-kappa B activation is not altered in zVAD-sensitized, TNF-alpha -stimulated neutrophils. Neutrophils were preincubated in dimethyl sulfoxide (DMSO) or zVAD for 30 minutes before stimulation, at time 0, with or without TNF-alpha . At indicated time points, cells were lysed and NF-kappa B activation was quantitated utilizing a commercial kit (see "Materials and methods"). Results represent means ± SEM, n = 4. *P < .05 compared with DMSO or zVAD alone. The inset shows an EMSA at 1 hour following stimulation (see "Materials and methods"). C indicates DMSO alone; Z, zVAD alone; T, TNF-alpha alone; Tz, zVAD-preincubated, TNF-alpha -stimulated; T* and Tz*, assays performed in the presence of excess, unlabeled/competitive consensus oligonucleotide.

NADPH oxidase is not required for zVAD enhancement of cell death in TNF-alpha -stimulated neutrophils

The role of oxidants as effectors during execution of cell death is controversial. In cell lines in which broad-spectrum caspase inhibition exerts a sensitizing effect to death receptor agonist-induced cell death, oxidants appear to play an important mechanistic role.20,21,37,38 While most of these reports propose that mitochondria are the major source of oxygen radicals mediating cell death, Khwaja et al suggested that the NADPH oxidase system is critically involved in NIH3T3 cells.21 Moreover, one recent study suggests that oxidants play only a minor role during paradoxic cell death in a murine macrophagelike cell line when incubated with lipopolysaccharide (LPS) and zVAD.39 Thus, generalizations cannot be made with respect to either the source or the requirement of intracellular oxidants during zVAD-sensitized, death receptor agonist-induced cell death.

In activated human neutrophils, the NADPH oxidase system assumes a critical role as the generator of superoxide and hydrogen peroxide during the respiratory burst.40 Patients with defects in the components of this system manifest clinically with impairment in host defense and develop chronic granulomatous disease.41 Importantly, several studies suggest that oxidants produced by this system participate in constitutive and Fas and TNF receptor agonist-induced neutrophil cell death.3,19,42-44 However, one study proposes that the role of intracellular oxidants as effectors during neutrophil cell death is context-dependent and that NADPH oxidase is not required for spontaneous and Fas activation-induced cell death.2 Two additional studies suggest that oxidants do not play significant roles during TNF-alpha -induced neutrophil cell death.45,46 Yamashita et al recently showed that, in the presence of CHX, TNF-alpha -primed neutrophil oxidant production stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP), opsonized zymosan, or phorbol myristate acetate (PMA) was further augmented by zVAD.14 To gain insights into the role of intracellular oxidants and the requirement of the NADPH oxidase system during zVAD-sensitized, TNF-alpha -induced neutrophil cell death in the absence of CHX or additional stimulants of the respiratory burst, we compared oxidant production in neutrophils isolated from healthy subjects and patients with X-linked CGD (Figure 8A). Cell death assays were performed in parallel. We observed that intracellular oxidant production in normal neutrophils peaked about 60 to 90 minutes and subsided to the background level by 150 to 180 minutes after TNF-alpha stimulation alone. In the presence of zVAD, production of intracellular oxidants was increased and sustained even at 4 hours after TNF-alpha stimulation, whereas zVAD alone showed little effect. In contrast, intracellular oxidant remained constant at resting levels throughout in patient 1 with X-linked CGD and complete absence of NADPH oxidase activity (X91+). Because patient 2 had a variant form of X-linked CGD with a partial defect in NADPH oxidase activity (X91-), TNF-alpha -stimulated oxidant production, alone or in the presence of zVAD, was intermediate. (The means of the mean florescence intensities, representing intracellular oxidant production, from 2 to 4 experiments in TNF-alpha -stimulated neutrophils in the absence or presence of zVAD, expressed as fold of that measured in unstimulated neutrophils at 4 hours, were 1.02 and 2.21 [healthy donors], 1.01 and 0.98 [patient 1], and 1.14 and 1.77 [patient 2], respectively.) Interestingly, zVAD still augmented cell death in TNF-alpha -stimulated neutrophils from both of these patients (Figure 8B,C), with apoptoticlike and necroticlike morphologic changes identical to those described above in normal neutrophils. Although there might have been minute quantities of intracellular oxidants produced47 that were not detected by the methodology used, the NADPH oxidase system was clearly the dominant generator of intracellular oxidants produced but was not required for cell death in zVAD-sensitized, TNF-alpha -stimulated neutrophils.


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  		Figure 8. The NADPH oxidase mediates intracellular oxidant production but not cell death in zVAD-sensitized, TNF-alpha -stimulated neutrophils. Neutrophils isolated from healthy donors or 2 patients with X-linked chronic granulomatous disease (X-CGD) were preincubated with or without zVAD for 30 minutes. Subsequently, TNF-alpha was added at time 0. At 1 and 4 hours, intracellular production of reactive oxygen species was quantified by the accumulation of DCF fluorescence with flow cytometry (see "Materials and methods"). (A) Results from a healthy donor and patient 1 with documented X91+ subtype of X-CGD and complete absence of NADPH oxidase activity. Dotted contours indicate resting neutrophils; bolded contours, neutrophils stimulated with TNF alone; filled-in contours, neutrophils stimulated with TNF in the presence of zVAD. (B) Results of neutrophil survival in both patients by dual-parameter flow cytometry performed at 4 hours. Cytocentrifuge preparations of the neutrophils from patient 1 were stained with Diff-Quick at 6 hours (C; magnification, × 400). Note that the apoptoticlike (closed arrow) and the necroticlike (open arrow) cell death that occurred in patient 1 was similar to that described above.


    Discussion
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

An increasing number of studies have described caspase-independent cell death upon agonistic death receptor stimulation. However, few have documented death receptor-mediated, caspase-independent cell death in nontransformed primary mammalian cells. Thus far, human peripheral blood T48,49 and B50 lymphocytes are the only primary human cell types in which caspase-independent cell death has been reported. Several observations suggest that the mechanisms for executing caspase-independent cell death must also exist in circulating mature human peripheral blood neutrophils. The most apparent is that, although there is modest prolongation of survival afforded by broad-spectrum, cell-permeable, irreversible caspase inhibitors, neutrophils eventually undergo constitutive or Fas agonist-induced cell death.3 Also, PMA induces rapid neutrophil cell death that is characterized by minimal induction of caspase-3-like activities and insensitivity to broad-spectrum caspase inhibition.2 Furthermore, PMA-induced neutrophil cell death is characterized b