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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 674-685
Caspases Mediate Tumor Necrosis Factor- -Induced Neutrophil
Apoptosis and Downregulation of Reactive Oxygen Production
By
Kouhei Yamashita,
Atsushi Takahashi,
Susumu Kobayashi,
Hirokazu Hirata,
Peter W. Mesner Jr,
Scott H. Kaufmann,
Shin Yonehara,
Kokichi Yamamoto,
Takashi Uchiyama, and
Masataka Sasada
From the Department of Hematology and Oncology, Clinical Sciences for
Pathological Organs, Graduate School of Medicine, the Department of
Viral Oncology, Institute for Virus Research, and the College of
Medical Technology, Kyoto University, Kyoto, Japan; and the Division of
Oncology Research, Mayo Clinic, Rochester, MN.
 |
ABSTRACT |
Tumor necrosis factor- (TNF-) exerts two separate effects on
neutrophils, stimulating effector functions while simultaneously inducing apoptosis. We examined here the involvement of caspases in
neutrophil apoptosis and the effect of TNF--induced apoptosis on
reactive oxygen production. Immunoblotting and affinity labeling showed
activation of caspase-8, caspase-3, and a caspase with a large subunit
of 18 kD (T18) in TNF--treated neutrophils. Active caspase-6 and -7 were not detectable in this cell type. Caspase-8 activated caspase-3
and T18 in neutrophil cytoplasmic extracts. zVAD-fmk blocked neutrophil
apoptosis, in parallel with the inhibition of caspase activation.
TNF--induced caspase activation was accompanied by a decrease in
the ability of neutrophils to release superoxide anion. Conversely,
TNF- treatment in the presence of zVAD-fmk caused a prolonged
augmentation of superoxide release. Granulocyte-macrophage colony-stimulating factor inhibited TNF--induced caspase activation and apoptosis, while reversing the diminution in superoxide release. These observations not only suggest that a caspase cascade mediates apoptotic events and downregulates oxygen radical production in TNF--treated neutrophils, but also raise the possibility that suppression of caspase activation with enhanced proinflammatory actions
of TNF- may underlie the pathogenesis of inflammatory diseases.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TUMOR NECROSIS factor- (TNF-) has
been implicated as a proinflammatory cytokine that plays critical roles
in the pathophysiology of inflammatory diseases, including bacterial sepsis, rheumatoid arthritis, Behçet's disease, and adult
respiratory distress syndrome (ARDS).1-4
TNF- can initiate cytokine cascades involving other downstream
proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, and
granulocyte-macrophage colony-stimulating factor (GM-CSF).1,2 However, recent studies suggest that TNF-
also plays a pivotal role in the resolution of inflammatory
responses.5 Mice deficient in TNF- suffer from
progressive inflammatory processes after bacterial
infections.6 The cellular and molecular basis by which
TNF- can mediate both proinflammatory and anti-inflammatory effects
remains unclear.
Anti-inflammatory actions of TNF- can be ascribed, at least in part,
to the ability of this cytokine to induce apoptotic cell death of
inflammatory effectors such as neutrophils.7 However,
information about the intracellular processes in apoptotic neutrophils
is limited to the descriptions of the characteristic morphological and
biochemical changes such as chromatin condensation,8 coalescence of nuclear lobes,9 condensation and shrinkage
of cytoplasm, internucleosomal DNA fragmentation,8 cell
surface exposure of phosphatidylserine (PS),10 and shedding
of CD16.11 The biochemical basis for these changes is
largely unknown.
It is known that neutrophils have two receptors for TNF-: 55-kD TNF
receptor 1 (TNF-R1) and 75-kD TNF receptor 2 (TNF-R2). TNF-R1 initiates
the TNF--induced death signal, and TNF-R2 facilitates the death
effect of TNF-R1 in neutrophils.12 TNF-R1 has a cytoplasmic death domain homologous to Fas, another apoptosis-inducing death receptor.13 In other cell types, binding of ligands to
TNF-R1 and Fas can induce the formation of signaling complexes,
TNF-R1-TRADD-FADD-pro-caspase-8 and Fas-FADD-pro-caspase-8,
respectively, with subsequent release of activated protease caspase-8
(MACH/FLICE/Mch5).14 The activation of caspase-8 is thought
to result in proteolytic activation of other caspase
proteases,15 which in turn mediate characteristic morphological and biochemical changes of death receptor-triggered apoptosis.16,17 The observation that gelsolin is cleaved in neutrophils treated with TNF- plus cycloheximide18
suggests that caspases have been activated in this cell type as well.
Nonetheless, it is unclear how many caspases are activated and whether
those active caspase(s) play a critical role in the execution of
TNF--induced apoptosis.
Although resolution of inflammation can be facilitated by recognition
and phagocytosis of apoptotic neutrophils by macrophages,8 it also remains to be determined whether neutrophil functions are
compromised by TNF--induced apoptotic processes before neutrophil phagocytosis by macrophages. TNF- can stimulate or enhance functions of neutrophils including phagocytosis, degranulation,19 and production of reactive oxygen species.20 A previous report
demonstrated the loss of various functional abilities of neutrophils
undergoing spontaneous apoptosis ex vivo.21 However,
culture of neutrophils for 24 hours in vitro might result in multiple
functional changes in addition to apoptotic processes. Thus, the
effects of TNF--associated apoptosis per se on neutrophil functions
remain incompletely understood.
In the present study, we have examined the involvement of caspases in
TNF--induced neutrophil apoptosis. Because TNF- activates not
only death signals, but also survival signals that are mediated by the
activation of NF- B transcription factors,22,23 we used cycloheximide to block survival signals mediated through protein synthesis. We show that treatment of neutrophils with TNF- plus cycloheximide results in activation of caspases and concomitant loss of
the ability to produce superoxide anion. Inhibition of caspase
activation not only rescued the functional capacity of neutrophils, but
also resulted in prolonged enhancement of superoxide release.
Pretreatment of neutrophils with GM-CSF inhibited both TNF--induced
activation of caspases and the downregulation of reactive oxygen
production. These results are discussed with regard to the possibility
that TNF--induced apoptotic signaling leading to caspase activation
may work as a switch between proinflammatory and anti-inflammatory
actions of TNF-.
| |
MATERIALS AND METHODS |
Reagents.
N-(N -benzyloxycarbonylglutamyl-N -biotinyllysyl)-aspartic
acid [(2,6-dimethylbenzoyl)oxy] methyl ketone (zEK(bio)D-aomk)
(Peptide Institute, Osaka, Japan) and Acetyl-Asp-Gln-Thr-Asp-aldehyde
(DQTD-CHO) (Peptide Institute) were dissolved in dimethylsulfoxide
(DMSO) at 10 mmol/L and stored at  80°C. Stock solutions of
Acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-MCA),
Acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin (YVAD-MCA), Acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO; Peptide Institute), Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk; Enzyme
Systems, Dublin, CA), propidium iodide (Calbiochem, La Jolla, CA), and
3, 3 -dihexyloxacarbocyanine iodide [DiOC6(3); Molecular Probes, Eugene, OR] were prepared and stored as previously described.17,24 Cycloheximide (Research Organics,
Cleveland, OH) was dissolved at 1 mg/mL in water and stored at 4°C.
Recombinant human TNF- and GM-CSF were kindly provided by Dainippon
Pharmaceutical (Osaka, Japan) and Schering-Plough Research Institute
(Kenilworth, NJ), respectively.
Analysis of neutrophil apoptosis.
Human neutrophils were isolated from peripheral blood of healthy adult
volunteers by sedimentation through two-step Percoll (Pharmacia,
Uppsala, Sweden) gradients, as previously described.25 Freshly purified cells were resuspended in RPMI-1640 supplemented with
10% heat-inactivated fetal bovine serum, preincubated with or without
inhibitors such as zVAD-fmk or GM-CSF for 1 hour at 37°C, and
exposed to TNF- (10 U/mL) plus cycloheximide (10 µg/mL) for the
indicated time period at 37°C in a humidified atmosphere containing
5% CO2. Flow cytometric analyses of neutrophil apoptosis were performed using propidium iodide (PI)-staining of
ethanol-permeabilized cells for DNA fragmentation, staining with
phycoerythrin-conjugated annexin V (R&D Systems, Minneapolis, MN) for
PS externalization, and staining with DiOC6(3) for
mitochondrial permeability transition, as previously
described.17
Fluorometric analysis of caspase activities.
For preparation of cytoplasmic extracts, neutrophils (1 × 107) were lysed in 20 µL lysis buffer (50 mmol/L KCl, 50 mmol/L PIPES, pH 7.0, 10 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L dithiothreitol, 20 µmol/L cytochalasin B, 100 µmol/L
phenylmethyl sulfonyl fluoride [PMSF], and 1 mg/mL each of
chymostatin, leupeptin, antipain, and pepstatin) as previously
described.24 For real time recording of caspase activity,
lysates were mixed with 100 µL of reaction buffer24 and
the activity was measured by the release of 7-amino-4-methyl-coumarin (AMC) from 100 µmol/L DEVD-MCA using a fluorometric microplate reader
(Fluoroskan Ascent; Labsystems, Helsinki, Finland) with excitation and emission wavelengths of 355 nm and 460 nm, respectively.
Affinity labeling of active caspases.
Extracts (140 µg total protein) were incubated with 1 µmol/L
zEK(bio)D-aomk for 5 minutes at 37°C, resolved in a 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and
transferred to a nitrocellulose membrane (Hybond-ECL; Amersham,
Arlington Heights, IL). Blots were stained with streptavidin-conjugated horseradish peroxidase (HRP; Amersham; 1:300 dilution) for 3.5 hours at
room temperature. Signals were detected by enhanced chemiluminescence (ECL) kit (Amersham) as recommended by the manufacturer.
Cell-free activation of caspases.
Cytosolic extracts (140 µg total protein) were incubated either with
1 µg recombinant active caspase-8 purified as described24 or with 1 µmol/L horse heart cytochrome c (Sigma, St Louis, MO) plus
1 mmol/L dATP (Sigma) for 30 minutes at 37°C in a reaction volume
of 10 µL. Samples were assayed for caspase activation by affinity
labeling with zEK(bio)D-aomk or by fluorometric analysis using 100 µmol/L DEVD-MCA.
Western blotting.
Whole cell extracts were obtained by boiling neutrophil pellets in SDS
sample buffer17 for 3 minutes. Proteins were resolved on
SDS-PAGE gels, transferred to nitrocellulose membranes, and visualized
by ECL. Antibodies used were 1:1,000 dilution of monoclonal antibody
against gelsolin (Sigma), 1:20 dilution of affinity-purified anti-caspase-3 antibodies,24,26 1:2,000 dilution of a
rabbit polyclonal antiserum against caspase-7 (kindly provided by Dr Gerald Cohen, University of Leicester, Leicester, UK),
1:1,000 dilution of monoclonal antibody against caspase-8 (MBL, Nagoya, Japan), and 1:1,000 dilution of monoclonal antibody against caspase-3 (Transduction Lab, Lexington, KY).
Release of superoxide anion from neutrophils.
Superoxide production was assessed by the superoxide
dismutase-inhibitable reduction of cytochrome c27 as
previously described.28
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RESULTS |
Apoptotic cell death of neutrophils induced by TNF- plus
cycloheximide.
The addition of cycloheximide markedly potentiated TNF--induced
apoptosis as previously described.7 TNF- alone induced DNA fragmentation characteristic of apoptosis in approximately 30% of
neutrophils within 3 hours (Fig 1A). After
3 hours of treatment with TNF- and cycloheximide, greater than 90%
of neutrophils showed hypodiploid DNA content (Fig 1A). Without
TNF-, cycloheximide alone induced minimal DNA fragmentation within 3 hours (data not shown).
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| Fig 1.
Time course of DNA fragmentation (A) and cell surface
exposure of phosphatidylserine (B) in neutrophils exposed to TNF-
plus cycloheximide. PI-stained, ethanol-permeabilized neutrophils (A)
or neutrophils stained with phycoerythrin-conjugated annexin V (B) were
subjected to flow cytometric analyses.
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Loss of plasma membrane asymmetry, resulting in cell surface exposure
of PS, is another biochemical hallmark of neutrophil apoptosis.10 Flow cytometric analysis using
phycoerythrin-labeled annexin V, which specifically binds to PS on the
cell surface, showed that PS is exposed on the surface of neutrophils
treated for 2 to 3 hours with TNF- plus cycloheximide (Fig 1B).
Despite these changes, approximately 100% of neutrophils maintained
plasma membrane integrity at 3 hours in TNF- plus cycloheximide as
determined by dye exclusion test (data not shown), ruling out the
possibility of necrotic cell death. These observations confirmed that
neutrophils treated with TNF- plus cycloheximide rapidly undergo
apoptotic cell death.
Activation of DEVD-cleaving caspase(s).
To determine whether caspases are activated in neutrophils during
TNF--induced cell death, we examined the fate of gelsolin, an
actin-binding protein essential for efficient neutrophil
locomotion.29 As previously described,18
blotting with a monoclonal anti-gelsolin antibody showed that this
caspase substrate was cleaved to a 41-kD fragment during
TNF-/cycloheximide-induced apoptosis
(Fig 2). Concomitant with this cleavage, a
protease activity capable of digesting DEVD-MCA, a fluorogenic caspase
substrate that is preferentially cleaved by caspase-3
(CPP32/Yama/apopain) and caspase-7
(Mch3/ICE-LAP3/ CMH-1),30 was detected in neutrophil
cytoplasmic extracts. Lysates of untreated neutrophils displayed low
level DEVD-MCA cleavage (Fig 3A), possibly reflecting
spontaneous apoptotic cell death.8 The activity increased
markedly 2 hours after addition of TNF- and reached a plateau at 3 hours (Fig 3A), in parallel with DNA fragmentation (Fig 1A) and PS
externalization (Fig 1B). Although the mature form of caspase-1
(IL-1 -converting enzyme) has been demonstrated in neutrophils by
immunoblotting,31 protease activity cleaving YVAD-MCA, a
substrate relatively specific for caspase-1,30 was not
detected in TNF--treated or untreated neutrophils (data not shown).
Preincubation of neutrophils with a caspase inhibitor, zVAD-fmk,32 inhibited the appearance of DEVD-MCA cleaving
activity in a dose-dependent manner (Fig 3B).
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| Fig 2.
Cleavage of gelsolin in apoptotic neutrophils. Whole cell
extracts obtained from 5 × 105 neutrophils treated with
(lanes 2 through 5) or without (lane 1) TNF- plus cycloheximide
(TNF/CHX) for the time indicated were subjected to SDS-PAGE (10% gel)
and immunoblotting using a monoclonal anti-gelsolin antibody.
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| Fig 3.
Fluorometric analyses of caspase activation. (A) Time
course of DEVD-cleaving activity. Cytoplasmic extracts (70 µg) from 1 × 107 neutrophils stimulated with TNF- plus
cycloheximide (TNF/CHX) for the indicated time periods were incubated
with 100 µmol/L DEVD-MCA for 30 minutes at 37°C. AMC release was
measured by a microplate reader as previously described.24
(B) Blockade of caspase activation by zVAD-fmk. Neutrophils were
preincubated with indicated concentrations of zVAD-fmk before
stimulation with TNF- plus cycloheximide for 3 hours. (C and D)
Cell-free activation of DEVD-cleaving caspase(s) by recombinant active
caspase-8 (C) or cytochrome c (D). Cytoplasmic extracts (70 µg) from
nonapoptotic neutrophils were incubated with 0.5 µg purified
recombinant caspase-8 (C) or with 1 µmol/L cytochrome c plus 1 mmol/L
dATP (D) for 30 minutes at 37°C. (B, C, and D) DEVD-cleaving
protease activities in the extracts were analyzed by real-time
recordings of AMC release using a fluorometric microplate reader
(Fluoroskan Ascent).
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Affinity labeling of active caspases in apoptotic neutrophils.
To examine which caspase(s) are activated in TNF--induced
apoptosis, we used an affinity labeling technique.33
Caspase activation involves the proteolytic processing of inactive
precursors (pro-caspases) to active proteases that contain two large
subunits (molecular weight [Mr] ~20 kD) and two small subunits (Mr
~10 kD) in a tetramer.34 zEK(bio)D-amok, an affinity
labeling reagent that mimicks the peptide sequences preferred by
caspases30 and irreversibly binds to the active site
cysteines within the large subunits of most active
caspases,35 can distinguish active caspases from one
another based on the differences in their apparent molecular weights.24,26 As shown in Fig 4A, 1 µmol/L
zEK(bio)D-amok labeled three major polypeptides, designated T20, T18,
and T17 according to their apparent molecular mass in kilodaltons, in
cytoplasmic extracts from neutrophils treated for 1 to 3 hours with
TNF- plus cycloheximide (lanes 2 through 4). T20 and T17 comigrated with F20 and F17 in Fas-stimulated Jurkat cells (Fig 4A, lane 5),
labeled polypeptides corresponding to caspase-3-p20 and caspase-3-p17, respectively.24 Reprobing the same blot with
anti-caspase-3 antibodies confirmed that T20 and T17 correspond to
caspase-3-p20 and -p17 (data not shown), although we could not rule out
the possibility that other active caspases comigrating with caspase-3 might also be present. The identity of T18 remained unclear, although its size is in accord with that of caspase-10 (Mch4/FLICE2; 18.4 kD)
predicted from cDNA. The apparent molecular mass of T18 is not
consistent with predicted sizes of caspase-1 (21.4 kD), -2 (19.7 kD),
-4 (19.9 kD), -5 (22.8 kD), -8 (19.0 kD), or -9 (22.2 or 37.8 kD).36 zEK(bio)D-aomk did not show a band
comigrating with F22/caspase-7 (Fig 4A), and immunoblotting using a
rabbit antiserum against caspase-7 could not detect pro-caspase-7 in neutrophils (Fig 5A). A
polypeptide comigrating with F19/caspase-6 (Mch2) was also absent in
apoptotic neutrophils (Fig 4A).
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| Fig 4.
Affinity labeling of active caspases in neutrophils. (A)
Time course of caspase activation. Cytoplasmic extracts were obtained
from 1 × 107 neutrophils treated with TNF- plus
cycloheximide (TNF/CHX) for the indicated time periods (lanes 1 through
4 and 6) or from 5 × 106 Jurkat cells stimulated with
anti-Fas antibody (CH-11, 100 ng/mL) for 3 hours (lane 5). Extracts
were incubated with (lanes 1 through 5) or without (lane 6) 1 µmol/L zEK(bio)D-aomk for 5 minutes at 37°C. (B) Preferential
competition of zEK(bio)D-aomk binding to caspase-3 by DQTD-CHO.
Cytoplasmic extracts from 1 × 107 neutrophils treated
with TNF- plus cycloheximide for 3 hours were preincubated with
DQTD-CHO for 15 minutes at 37°C before labeling with 1 µmol/L
zEK(bio)D-aomk. (C) zVAD-fmk inhibition of caspase activation.
Neutrophils preincubated for 1 hour with indicated concentrations of
zVAD-fmk were stimulated with TNF- plus cycloheximide (lanes 2 through 5) or left untreated (lane 1) for 3 hours. (Upper lanes)
zEK(bio)D-aomk-labeled caspases were detected with HRP-conjugated
streptavidin. (Lower lanes) The same blot was reprobed with a rabbit
anti-caspase-3 antibody. (D) Sensitivities of active caspases to
zVAD-fmk. zVAD-fmk at indicated concentrations was added to neutrophils
stimulated with TNF- plus cycloheximide for 3 hours. After
incubation for 1 hour at 37°C, cytoplasmic extracts were prepared
for affinity labeling analysis. (E) Cell-free activation of endogenous
caspases in neutrophil extracts by recombinant caspase-8 and by
cytochrome c. Cytoplasmic extracts from neutrophils were treated with
cytochrome c plus dATP (lanes 1 through 3) or recombinant caspase-8
(lanes 4 through 6) for the indicated time period. Lane 7, cytoplasmic
extracts from Fas-stimulated Jurkat cells. All samples were affinity
labeled with 1 µmol/L zEK(bio)D-aomk.
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| Fig 5.
Immunoblotting for procaspase processing. Whole cell
extracts were prepared from 1 × 106 neutrophils (PMNs)
stimulated with TNF- plus cycloheximide (TNF/CHX) for the indicated
time periods. (A) Pro-caspase-7 is not detectable in neutrophils.
Whole cell extracts from 5 × 105 Jurkat cells treated
with (lane 5) or without (lane 6) anti-Fas antibody were also analyzed.
(B) Processing of pro-caspase-8 (upper panel) and pro-caspase-3
(lower panel) in TNF--treated neutrophils. Lane 6, neutrophils were
preincubated with zVAD-fmk before exposure to TNF- plus
cycloheximide. The same blot was sequentially probed with
anti-caspase-8 (upper panel) and anti-caspase-3 (lower panel)
monoclonal antibodies.
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To assess which labeled caspase(s) might be responsible for the
cleavage of gelsolin (Fig 2), we performed a competition
experiment.33 DQTD-CHO was synthesized based on the
cleavage site sequence within gelsolin.18 As shown in Fig
4B, DQTD-CHO at 10 to 100 nmol/L inhibited the binding of
zEK(bio)D-aomk to T20/caspase-3-p20 and T17/caspase-3-p17. In contrast,
the labeling of T18 persisted up to 1 µmol/L DQTD-CHO. This indicated
that caspase-3 has selective affinity for the gelsolin cleavage site
sequence and suggested that caspase-3 is responsible for gelsolin
cleavage in TNF--treated neutrophils. These results are consistent
with the previous claim that gelsolin is a caspase-3
substrate.18 We cannot, of course, rule out the possibility
that some other caspase (eg, caspase-8) also contributes to this
cleavage, although we note that the DQTD tetrapeptide would be expected
to have very low affinity for this caspase.30
Additional affinity labeling experiments were performed to further
evaluate the observation that zVAD-fmk suppressed the appearance of
DEVD-MCA cleaving activity in TNF-/cycloheximide-treated neutrophils (Fig 3B). When added before TNF- and cycloheximide, zVAD-fmk at 10 µmol/L suppressed the appearance of T20/caspase-3-p20, T18, and
T17/caspase-3-p17 (Fig 4C, upper panel). Reprobing the same blot with
anti-caspase-3 showed that the processing of pro-caspase-3 to mature
caspase-3-p20 and caspase-3-p17 was blocked by 10 µmol/L zVAD-fmk
(Fig 4C, lower panel). Thus, the TNF--induced processing of
pro-caspase-3 rather than activity of the processed enzyme was blocked
by treating neutrophils with 10 µmol/L zVAD-fmk, as previously
reported for Fas-stimulated Jurkat T cells.37 The sensitivities of the labeled caspases to zVAD-fmk were assessed by
exposing neutrophils to zVAD-fmk after caspases had been activated by 3 hours of TNF- plus cycloheximide stimulation. T20/caspase-3-p20 and
T17/caspase-3-p17 were suppressed at 5 and 10 µmol/L, respectively, whereas overexposure of the film showed that complete inhibition of T18
requires 20 µmol/L zVAD-fmk (Fig 4D). Thus, 10 µmol/L zVAD-fmk blocked the activation of T18 rather than the protease activity of T18.
These observations suggested that a caspase(s) upstream of caspase-3
and T18 is the major target for zVAD-fmk.
Signaling upstream of caspase-3 and T18.
Ligation of TNF-R1 by TNF- may induce the formation of
TNF-R1-TRADD-FADD-pro-caspase-8 complex, resulting in the activation of caspase-8.13 Active caspase-8 tends to escape detection
in cell extracts by affinity labeling methods,24 most
likely because active caspase-8 at low concentrations is sufficient for
apoptotic execution, owing to the amplifying nature of downstream
caspase cascades.38 Indeed, immunoblotting demonstrated
that pro-caspase-8 is present in neutrophils and is processed during
TNF--induced apoptosis (Fig 5B). Moreover, addition of active
recombinant caspase-8 to neutrophil cytosol resulted in activation of
caspase-3-p20 within 30 minutes (Fig 4E, lanes 5 and 6), concomitant
with the appearance of DEVD-MCA cleaving activity (Fig 3C).
Overexposure of the film showed two additional bands corresponding to
caspase-3-p17 and T18 at 1 hour (data not shown). Evidence of
caspase-8-induced processing of pro-caspase-3 to caspase-3-p20 before
the appearance of T18 argued against the possibility that caspase-3 is
indirectly activated by caspase-8 via activation of T18. Instead, these
observations suggested that active caspase-8 released from the
TNF-R1-TRADD-FADD-pro-caspase-8 complex is responsible for subsequent
activation of caspase-3 and T18 in neutrophils. Consistent with this
conclusion, we observed that processing of pro-caspase-8 in
TNF--treated neutrophils was blocked by zVAD-fmk (Fig 5B, lane 6),
suggesting that the blockade of pro-caspase-8 autoprocessing by
zVAD-fmk39 is responsible for the lack of the downstream
caspases, caspase-3 and T18, in human neutrophils.
Caspase-8 is able to stimulate mitochondria, inducing cytochrome c
release40 or triggering permeability transition with subsequent release of AIF (apoptosis-inducing factor).41
Both cytochrome c and AIF can activate caspase-3.41,42
Because mitochondrial permeability transition was minimal in
neutrophils treated for 3 hours with TNF- plus cycloheximide (see
below), the present study focused on cytochrome c. The addition of
cytochrome c plus dATP to cytoplasm from untreated neutrophils resulted
in the activation of caspase-3-p20 within 30 minutes (Fig 4E, lanes 2 and 3), concomitant with the appearance of DEVD-MCA cleaving activity
(Fig 3D). This observation indicated that neutrophil cytoplasm contains
the machinery43 for the activation of caspase-3 in response
to the apoptosis-associated release of cytochrome c from
mitochondria.44,45 Thus, it is possible that caspase-8
activates caspase-3 indirectly by inducing mitochondrial release of
cytochrome c.
Role for caspase activation in neutrophil apoptosis.
Blockade of caspase activation with zVAD-fmk inhibited apoptotic DNA
fragmentation (Fig 6A) and the surface
exposure of PS (Fig 6B) in a dose-dependent manner. Despite significant
loss of T18 activity at 1 µmol/L zVAD-fmk (Fig 4C), apoptotic changes were minimally interrupted (Fig 6A and B). In contrast, DNA
fragmentation and PS externalization was potently suppressed by 10 µmol/L zVAD-fmk (Fig 6A and B), a concentration that significantly
blocked the activation of caspase-3 (Fig 4C). These observations
implied that caspases activated in response to TNF- play critical
roles in the execution of neutrophil apoptosis.
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| Fig 6.
Blockade of DNA fragmentation (A) and PS externalization
(B) by zVAD-fmk. Neutrophils were preincubated for 1 hour with the
indicated concentration of zVAD-fmk before stimulation with TNF-
plus cycloheximide for 3 hours. The plots represent three independent
experiments with essentially identical results.
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Negative regulation of superoxide release downstream of caspase
activation.
Consistent with previous reports on neutrophil priming by
TNF-,20 neutrophils show increased reactive oxygen
production (Fig 7A through C), especially
in response to formyl-Met-Leu-Phe (FMLP; Fig 7A), after 1 hour of
treatment with TNF- plus cycloheximide. However, the progression of
TNF--induced apoptotic processes after 2 hours was associated with
a diminution in neutrophil functional capacities. At 2 to 3 hours, the
release of superoxide anion from neutrophils stimulated with FMLP (Fig
7A), opsonized zymosan (OZ; Fig 7B), and phorbol myristate acetate
(PMA; Fig 7C) was suppressed in parallel with the appearance of
apoptotic changes (Fig 1).
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| Fig 7.
Downregulation of superoxide release in TNF--induced
neutrophils and reversal by zVAD-fmk. Neutrophils incubated with or
without TNF- plus cycloheximide (TNF/CHX) for the indicated time in
the absence or presence of indicated concentrations of zVAD-fmk were
stimulated with either FMLP (100 nmol/L), OZ (1 mg/mL), or PMA (20 ng/mL) for 15 minutes at 37°C. Superoxide generated during that 15 minute period was determined by cytochrome c reduction. Data shown
represent the average values from three separate experiments performed
in duplicate.
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This change might reflect the collapse of mitochondrial membrane
potential46 with decline in intracellular ATP
level,40 causing a general derangement in neutrophil
metabolism, including oxygen radical generation. To assess this
possibility, neutrophils were examined by flow cytometry after
incubation with DiOC6(3), a fluorescent probe whose
sequestration to mitochondria depends on an intact membrane
potential.47 Compared with DNA fragmentation and PS
externalization, which reached plateaus at 3 hours, loss of
mitochondrial membrane potential was a delayed event, as reported in
staurosporine-induced apoptosis of HL-6044 and CEM
cells40 as well as UVB-induced apoptosis of HeLa
cells.40 In our studies, neutrophils with a low
mitochondrial potential were first noted at 3 hours and increased over
time. Only approximately 40% of cells showed diminished binding of
DiOC6(3) after 12 hours in TNF- plus cycloheximide
(Fig 8). Combined with recent studies showing that ATP/ADP ratio is maintained in neutrophils undergoing apoptosis48 and that apoptotic execution requires
ATP,49-51 loss of ATP production seemed to be an unlikely
explanation for the depressed superoxide production.
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| Fig 8.
Delayed collapse of mitochondrial membrane potential in
TNF--treated neutrophils. Neutrophils treated with TNF- plus
cycloheximide for the indicated time periods were incubated with
DiOC6(3) and subjected to flow cytometry.
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The ability of neutrophils to release superoxide in response to PMA,
FMLP, and OZ was restored by the blockade of apoptotic processes with
zVAD-fmk (Fig 7A through C). Interestingly, in the presence of 10 to
100 µmol/L zVAD-fmk, enhanced production of superoxide was not
limited to the first hour. Even at 2 and 3 hours after addition of
TNF- plus cycloheximide, superoxide release was significantly higher
in neutrophils treated with zVAD-fmk plus TNF- than neutrophils left
untreated (Fig 7A through C). This observation indicated that TNF-
introduces a priming signal for enhanced oxygen radical production that
is usually masked by TNF--induced apoptotic processes.
Effect of GM-CSF on TNF--induced apoptosis, downregulation of
respiratory burst, and caspase activation.
TNF- can initiate cytokine cascades involving multiple
proinflammatory cytokines, including GM-CSF.2 Indeed,
lipopolysaccharide-induced production of GM-CSF is depressed in TNF-
knockout mice.6 We therefore examined the ability of
combined treatment with GM-CSF and TNF- to modulate neutrophil
functions. GM-CSF simultaneously inhibited TNF--induced apoptosis
(Fig 9A) and
enhanced PMA-triggered release of superoxide from neutrophils treated
with TNF- plus cycloheximide (Fig 9B). As shown in Fig 9C, the
activation of caspases, as assessed by DEVD-MCA cleavage, was
suppressed in a dose-dependent manner by preincubation of neutrophils
with GM-CSF. Affinity labeling with zEK(bio)D-aomk showed that GM-CSF
at 10 ng/mL blocked the appearance of active caspase-3-p20, -p17, and T18 (Fig 9D). Thus, GM-CSF inhibits TNF--induced apoptotic
processes upstream of caspase activation. Interestingly, when
TNF--induced caspase activation was completely blocked with 100 µmol/L zVAD-fmk, the addition of GM-CSF caused no further
augmentation of superoxide production (Fig 9E). This result, which
rules out a major contribution by other GM-CSF-triggered signal(s),
supports the view that GM-CSF intensifies reactive oxygen generation in
TNF--stimulated neutrophils mainly by inhibiting caspase
activation.
View larger version (37K):
[in this window]
[in a new window]
| Fig 9.
GM-CSF inhibits apoptosis (A), reverses downregulation of
superoxide release (B), and suppresses caspase activation (C and D) in
TNF--treated neutrophils. Neutrophils were pretreated with 1, 10, and 100 ng/mL GM-CSF for 1 hour before the addition of TNF- plus
cycloheximide. DNA fragmentation (A), superoxide release in response to
PMA (B), and the presence of DEVD-cleaving (C) or
zEK(bio)D-aomk-binding (D) active caspases were analyzed after
incubation for the indicated time period (B) or for 2 hours (A, C, and
D). (E) Cells were pretreated with 100 µmol/L zVAD-fmk or 10 ng/mL
GM-CSF singly and in combination before addition of
TNF-/cycloheximide for 2 hours followed by measurement of
PMA-induced superoxide release. Values represent the means + SEM of
three independent experiments performed in duplicate.
|
|
| |
DISCUSSION |
The present study demonstrated that TNF--induced apoptotic cell
death of neutrophils is accompanied by activation of multiple caspases.
Concomitant with caspase activation, TNF--stimulated superoxide
generation decreased. Interestingly, inhibition of caspase activation
simultaneously suppressed apoptosis and prolonged the TNF--induced
augmentation of superoxide generation. These findings not only have
important implications for understanding the balance between
proinflammatory and anti-inflammatory effects of TNF-, but also
might need to be considered in assessing the therapeutic potential of
caspase inhibitors.
A combination of affinity labeling and immunoblotting showed the
presence of multiple caspases in neutrophils treated with TNF- and
cycloheximide. These included caspase-8, caspase-3, and another caspase
with a large subunit of 18 kD (T18), most likely corresponding to
caspase-10. In contrast to Fas-stimulated Jurkat T cells,17
active caspase-7 and caspase-6 were not detected. The differences in
the activated caspases may contribute to the heterogeneity in
biochemical and morphological changes, such as the absence of
fragmentation into apoptotic bodies in apoptotic neutrophils.10 Moreover, the absence of active caspase-7 in apoptotic neutrophils provides an explanation for the dependence of
neutrophil apoptosis on caspase-3. Recently published gene targeting
studies showed that neutrophil apoptosis is impaired in
caspase-3-deficient mice,52 but did not disclose an
explanation for this result. Caspase-7 is highly homologous to
caspase-3 and has similar substrate preferences.30 In many
cell types, caspase-7 appears to be able to take over when caspase-3 is
knocked out.53 Our observation that expression of
pro-caspase-7 is very low or absent in neutrophils can explain the
inability of this cell type to compensate for the loss of caspase-3.
Our experiments seemed to indicate that TNF-R1 ligation initiates a
caspase cascade by inducing autoprocessing of pro-caspase-8 in
neutrophils as it does in other cell types.39,54-56 In
subsequent experiments, we showed that addition of active caspase-8 to
cytosol from nonapoptotic neutrophils was capable of activating
caspase-3 and T18 directly. Caspase-8-induced release of cytochrome c
from mitochondria may also contribute to the activation of caspase-3. However, these cell-free conditions did not completely reproduce the
pattern of activated caspases generated in TNF--treated
neutrophils. In particular, the autodigestion of caspase-3-p20 to yield
caspase-3-p1757 was not efficient in neutrophil cytosol
treated with caspase-8, cytochrome c plus dATP, or a combination of
these agents. A recent report demonstrating mitochondrial distribution
of pro-caspase-358 raises the possibility that
mitochondria in intact neutrophils may facilitate the caspase-3
autocatalysis by providing a surface for effective enzyme-substrate
interactions.
Apoptosis-associated caspase activation is associated with changes in
neutrophil function. Neutrophil functions that are dependent on the
integrity of cytoskeleton, eg, shape changes, spreading, chemotaxis,
degranulation, and phagocytosis, have previously been shown to decline
in aging neutrophils undergoing spontaneous apoptosis.21 These changes might be related to the disruption of cytoskeletal networks due to caspase-catalyzed proteolysis of
gelsolin,18 catenin,59 and focal adhesion
kinase60 as well as the caspase-dependent dephosphorylation
of ezrin-radixin-moesin (ERM).61
Spontaneous apoptosis is also accompanied by suppression of superoxide
generation in response to the receptor-dependent stimuli FMLP and
OZ21 but not the receptor-independent stimulus PMA. In
contrast, we observed that TNF--induced apoptosis was associated with decreased PMA-triggered superoxide release from neutrophils as
well. Although further studies are required to determine the mechanism
by which PMA-induced superoxide release is suppressed, current
available observations are sufficient to rule out several potential
explanations. Neither cytoskeletal disruption nor loss of mitochondrial
membrane potential appears to account for this phenomenon. Further,
even though ceramide inhibits superoxide release from PMA-stimulated
neutrophils,62 a previous report has shown that TNF-
does not stimulate ceramide accumulation in neutrophils.63
Several potential explanations for the TNF--associated decrease in
PMA-induced superoxide release cannot currently be ruled out. One
possibility is that decreased amounts of ATP are available for reactive
oxygen production due to diversion of most neutrophil ATP stores to
apoptotic processes. Alternatively, cytochrome c released from
mitochondria might oxidize superoxide to oxygen.64 Finally,
proteolysis of D4-GDP-dissociation inhibitor (GDI) by caspase(s)65 may depress superoxide production. D4-GDI, a
polypeptide that is homologous to Rho-GDI, is preferentially expressed
at high levels in hematopoietic cells, including granulocytes. Because macrophages deficient in D4-GDI show decreased respiratory burst activity,66 it is possible that D4-GDI proteolysis
contributes to the suppression of PMA-induced superoxide production.
Whatever the mechanism of decreased PMA-induced superoxide production,
it is clear that caspase activation plays a role. Treatment of
neutrophils with zVAD-fmk at 10 µmol/L prevented TNF--induced DNA
fragmentation, PS externalization, and suppression of superoxide production, in parallel with the blockade of pro-caspase-3 processing. Recent studies with other cell types support the idea that caspase-3 mediates DNA fragmentation and PS externalization in apoptotic neutrophils. Caspase-3 can activate caspase-activated DNase
(CAD)67 by cleaving the inhibitor of CAD
(ICAD).68 Caspase-3 is upstream of PS externalization in
Fas-stimulated Jurkat T cells.17 The present results
suggest that caspase-3-mediated proteolysis also plays a role in
apoptosis-associated suppression of superoxide generation. In addition,
the observation that zVAD-fmk enhances and prolongs TNF--primed
superoxide production raises the possibility that caspase inhibition, a
strategy currently being considered for the treatment of a variety of
pathological conditions, might be associated with enhanced production
of proinflammatory and potentially harmful neutrophil products such as
superoxide anions.
GM-CSF, like zVAD-fmk, appears to suppress TNF--induced apoptosis
upstream of active caspase-3 and T18. Several different GM-CSF-induced
biochemical changes might contribute to this suppression of apoptosis.
Although GM-CSF downregulates neutrophil TNF receptors, probably by
activating a sheddase,69 the downregulation is not complete, with greater than 50% of TNF binding retained after 2 hours
of exposure to GM-CSF.70 Lyn, a src family
tyrosine kinase, has been reported to play a critical role in the
GM-CSF blockade of spontaneous neutrophil apoptosis.71
Phosphatidylinositol 3-kinase (PI3K), which has been shown to inhibit
apoptosis in other cell types,72 is tyrosine phosphorylated
in its 85-kD regulatory subunit and activated in GM-CSF-stimulated
neutrophils.73 Bfl-1,74 a survival-promoting
Bcl-2 family protein expressed in myeloid cells,75 might be
activated downstream of PI3K. ERK2, which has been reported to block
apoptosis in other cell types,76 is tyrosine phosphorylated
in GM-CSF-stimulated neutrophils.77 Finally, GM-CSF
stimulates neutrophils to produce platelet-activating factor
(PAF),78 which is able to prevent TNF--induced
neutrophil apoptosis without downregulating TNF
receptors.12 Further study is required to determine the
relative contributions of each of these mechanisms to the
GM-CSF-induced suppression of neutrophil apoptosis.
TNF- signaling in vivo occurs in the context of complex cytokine
networks and important cell-to-cell interactions. The functional consequences of the GM-CSF-induced block in caspase activation are
potentially important in understanding the dual role of TNF- as a
proinflammatory and anti-inflammatory cytokine. Because the present
study employed a simplified model system using cycloheximide to focus
on the TNF- death pathway, the results must be interpreted cautiously. Nonetheless, our data imply that TNF- introduces dual
signals to neutrophils with respect to their effector functions: negative apoptotic signal(s) downstream of caspase activation and
positive priming signal(s) independent of caspases or protein synthesis. Our data also indicate that other cytokines, eg, GM-CSF can
affect the balance between these signals. Differential modulation of
diverse TNF--induced signals by interactions with other cytokines and inflammatory mediators79 may explain the ability of
TNF- to mediate a wide variety of inflammatory diseases. GM-CSF can suppress apoptotic signals by inhibiting caspase activation and potentiate priming signals for enhanced reactive oxygen production. Such functional interactions between TNF- and GM-CSF may contribute to neutrophil-mediated tissue damages in inflammatory diseases such as
ARDS.80 Caspase cascade(s) in neutrophils provide a point
of convergence for complex intracellular signals from multiple exogenous stimuli.81 As a critical regulatory point in
apoptotic and anti-inflammatory signals, caspase activation may work as a switch that determines the balance between proinflammatory and anti-inflammatory responses.
| |
ACKNOWLEDGMENT |
The authors thank Gerald M. Cohen for anti-caspase-7 antibodies;
Akinori Maeda for help in flow cytometric analysis; and Yuri A. Lazebnik, Kohsuke Asagoe, and Katsumi Takada for helpful discussion and
thoughtful suggestions.
| |
FOOTNOTES |
Submitted April 9, 1998;
accepted September 21, 1998.
A.T. is a Research Resident of the Japanese Foundation of Aging and
Health. S.H.K. is a Leukemia Society Scholar.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Atsushi Takahashi, MD, First Division,
Department of Internal Medicine, Faculty of Medicine, Kyoto University,
54 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606-01, Japan; e-mail:
atakahas{at}kuhp.kyoto-u.ac.jp.
| |
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Abstract
Introduction