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Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2001-12-0266.
Previous Article | Table of Contents | Next Article 
Blood, 1 January 2003, Vol. 101, No. 1, pp. 295-304
PHAGOCYTES
Broad-spectrum caspase inhibition paradoxically augments cell
death in TNF- -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.
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Abstract |
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- (TNF- )
and cycloheximide ex vivo. In contrast, the same caspase inhibitors
could augment cell death upon stimulation by TNF- alone during the
6-hour time course examined. Caspase inhibitor-sensitized, TNF- -stimulated, dying neutrophils exhibit apoptoticlike and necroticlike features. This occurred without apparent alteration in
nuclear factor- B (NF- B) activation. Nevertheless, intracellular oxidant production was enhanced and sustained in caspase
inhibitor-sensitized, TNF- -stimulated neutrophils obtained from
healthy subjects. However, despite reduced or absent intracellular
oxidant production following TNF- stimulation, cell death was also
augmented in neutrophils isolated from patients with chronic
granulomatous disease incubated with a caspase inhibitor and TNF- .
These results demonstrate that, in human neutrophils, TNF- induces a
caspase-independent but protein synthesis-dependent cell death signal.
Furthermore, they suggest that TNF- 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.
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Introduction |
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- (TNF- )-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- has opposing effects on neutrophil life span. While TNF- 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- -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- -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- -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- B
(NF- B) activation, a critical survival factor in neutrophils
undergoing constitutive or TNF- -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- -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- -induced neutrophil cell death to proceed.
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Materials and methods |
Reagents
Recombinant human TNF- 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- B activation assays
For assay of NF- B activation in neutrophils, a nonradioactive
ELISA method (NF- 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- (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- B consensus oligonucleotide in the absence or presence of competing, nonimmobilized, NF- B consensus
oligonucleotide. After extensive washing, total bound NF- 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- 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- 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.
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Results |
Broad-spectrum caspase inhibitors augment TNF- - 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- -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- -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- -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- -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- stimulation
(data not shown). Similarly, with zVAD pretreatment, TNF- at and
above 1 ng/mL still induced neutrophil cell death in a dose-dependent manner (Figure 2B). Corroborating these results, TNF- -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- 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- -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- -induced neutrophil cell death (Figure 3B). Taken together,
although it is possible that zVAD exerts its effect nonspecifically in
TNF- -stimulated neutrophils, these experiments indicate that
broad-spectrum caspase inhibitors are not cytotoxic per se, and their
paradoxic effects in sensitizing to TNF- -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- -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- (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- -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- 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- (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- -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- (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- (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-
alone.
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zVAD-sensitized, TNF- -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- -stimulated neutrophils in vitro. Interestingly, under light
microscopy, whereas neutrophils incubated with zVAD alone appeared
normal morphologically and TNF- -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- -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- -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- -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- -induced neutrophil cell
death occurred through processes that exhibited both apoptoticlike and
necroticlike features.

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| Figure 4.
zVAD-sensitized, TNF- -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-
(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- 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- -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- or buffered saline were examined by light
microscopy (magnification, × 1000). Note that TNF- alone induced
classic apoptotic nuclear changes. Similar to some of the features
observed by TEM (shown in panel C), zVAD-sensitized,
TNF- -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- 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-
(vii) alone. While some zVAD-preincubated neutrophils appeared to have
been activated with significant cytoplasmic vacuolizations as early as
1 hour after TNF- 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- , 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- alone also showed typical
apoptotic nuclear changes (vii). Interestingly, similar nuclear changes
could be seen at this time point in TNF- -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- -stimulated neutrophil cell death is
apoptosislike.
(A) Neutrophils were preincubated with or without zVAD (100 µM) for 1 hour before the addition of TNF- (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- only at the corresponding times.
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Protein synthesis inhibition alters cell death response in
TNF- -stimulated neutrophils
In addition to the induction of cell death early after stimulation
(8 hours or fewer), TNF- 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-
suggest certain functional heterogeneity in circulating neutrophils.
Alternatively, TNF- 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- -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- -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- -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- 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- -stimulated neutrophils.
(A) Compilation from a series of time-course studies of cell death in
TNF- -stimulated neutrophils with and without zVAD preincubation,
assayed by dual-parameter flow cytometry. Open circles represent the
TNF- -attributable effect on neutrophil survival (ie,
Survival = TNFLive, the percentage of
TNF- -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- -stimulated neutrophils relative to TNF- alone
(ie, 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- 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- 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- at
time 0. Cell survival was determined at 4 hours by dual-parameter flow
cytometry. *P < .05 compared with TNF + CHX;
n = 3.
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Similar to other TNF- -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- -induced cell death.14,17
Interestingly, under this condition, zVAD significantly attenuated
TNF- -induced neutrophil cell death (Figure 6B). In this regard, our
results are consistent with those reported
previously.14,17
NF- B activation is not suppressed in zVAD-sensitized,
TNF- -stimulated neutrophils
Ward et al reported that NF- B activation plays a critical
cytoprotective role during constitutive and TNF- -stimulated
neutrophil cell death.17 To determine if zVAD exerted its
cell death-enhancing effect in TNF- -stimulated neutrophils by
preventing NF- B activation, time-course studies of NF- 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- B
activation by TNF- .

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| Figure 7.
NF- B activation is not altered in zVAD-sensitized,
TNF- -stimulated neutrophils.
Neutrophils were preincubated in dimethyl sulfoxide (DMSO) or
zVAD for 30 minutes before stimulation, at time 0, with or without
TNF- . At indicated time points, cells were lysed and NF- 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-
alone; Tz, zVAD-preincubated, TNF- -stimulated; T* and
Tz*, assays performed in the presence of excess,
unlabeled/competitive consensus oligonucleotide.
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NADPH oxidase is not required for zVAD enhancement of cell death in
TNF- -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- -induced neutrophil cell death.45,46 Yamashita et al recently showed that, in the
presence of CHX, TNF- -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- -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- stimulation alone.
In the presence of zVAD, production of intracellular oxidants was
increased and sustained even at 4 hours after TNF- 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- -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- -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- -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- -stimulated neutrophils.

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| Figure 8.
The NADPH oxidase mediates intracellular oxidant
production but not cell death in zVAD-sensitized, TNF- -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- 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.
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Discussion |
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 |