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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4808-4818
Involvement of Caspases in Neutrophil Apoptosis: Regulation by
Reactive Oxygen Species
By
Bengt Fadeel,
Anders Åhlin,
Jan-Inge Henter,
Sten Orrenius, and
Mark B. Hampton
From the Institute of Environmental Medicine, Division of Toxicology,
Karolinska Institutet, Stockholm, Sweden; the Childhood Cancer Research
Unit, Karolinska Hospital, Stockholm, Sweden; the Department of
Pediatrics, Center for Inflammation Research, Karolinska Institutet at
Sach's Children's Hospital and Stockholm Söder Hospital,
Stockholm, Sweden.
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ABSTRACT |
Human neutrophils have a short half-life and are believed to die by
apoptosis or programmed cell death both in vivo and in vitro. We found
that caspases are activated in a time-dependent manner in neutrophils
undergoing spontaneous apoptosis, concomitant with other characteristic
features of apoptotic cell death such as morphologic changes,
phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment
of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs)
significantly accelerated this process. However, in cells treated with
the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA),
caspase activity was only evident after pharmacologic inhibition of the
nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase. Similarily, inhibition of the NADPH oxidase in constitutive
and Fas/APO-1-triggered apoptosis resulted in increased rather than
suppressed levels of caspase activity, suggesting that reactive oxygen
species may prevent caspases from functioning optimally in these cells.
Moreover, oxidants generated via the NADPH oxidase were essential for
PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an
important component of constitutive and Fas/APO-1-triggered neutrophil
apoptosis. However, these redox sensitive enzymes are suppressed in
activated neutrophils, and an alternate oxidant-dependent pathway is
used to mediate PS exposure and neutrophil clearance under these
conditions.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE PIVOTAL ROLE of apoptosis, or
programmed cell death, in the regulation of the immune system is
well-established.1,2 Apoptosis, which is distinct from
necrotic cell death occuring in response to various noxious stimuli,
takes place in a predictable and well-choreographed sequence of
morphological events, which ultimately results in the death of the cell
and removal by professional phagocytes.1 Several lines of
evidence indicate that the activation of aspartic acid-specific
cysteine proteases, known as caspases, plays a central role in the
execution of the apoptotic process.3,4 Overexpression of
various caspase family members induces apoptosis in cultured mammalian
cells.3 Furthermore, caspase inhibitors such as the cowpox
virus protein crmA and certain peptide methyl ketones and peptide
aldehydes have been shown to be potent inhibitors of apoptosis induced
by a variety of different stimuli.3 In addition, recent
studies have identified a number of proteins, including poly (adenosine
5 -diphosphate [ADP]-ribose) polymerase and nuclear lamin, that are
specifically cleaved by caspases in cells undergoing
apoptosis.3
Reactive oxygen species have been implicated as the final common
mediator of apoptosis in a variety of systems,5 yet the precise role of reactive oxygen species in the modulation of caspase activity remains unresolved. The evidence for a role of oxidative stress in the induction of apoptosis comes mainly from the numerous observations that antioxidants inhibit or delay the onset of apoptotic cell death in different systems.6,7 However, this does not imply that reactive oxygen species are absolutely required as mediators
of all types of apoptosis. Several groups have demonstrated that
apoptosis can occur independently of ambient oxygen
tension,7 and recent studies demonstrate that oxidants are
also able to inhibit apoptosis under certain
conditions,8-10 thereby adding to the complexity of the
current model of redox regulation in apoptosis. In this study, we have
focused on neutrophils, which are known to rapidly undergo apoptosis
and are also prolific generators of oxidants.
Human neutrophils constitute an important line of host defense against
invading microorganisms, and the production of reactive oxygen species
in these cells is an essential step in the killing of ingested
bacteria.11 Neutrophils are known to have the shortest life
span among leukocytes and they spend only 6 to 10 hours in circulation
before marginating and entering tissues where they survive for 1 to 2 days.12 They rapidly undergo spontaneous apoptosis upon in
vitro culture, and the exposure of surface factors such as
phosphatidylserine (PS) labels the cells for engulfment by
macrophages.13 The apoptotic death of activated neutrophils is also proposed to be a critical component in the resolution of the
inflammatory process.14 Activated neutrophils are known to
generate extremely high amounts of reactive oxygen species, but these
are normally targeted at pathogens inside intracellular phagosomes,
thereby reducing damage to surrounding tissue. The major source of
reactive oxygen species in neutrophils is the nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase, a multicomponent enzyme that
catalyzes the transfer of electrons from NADPH to molecular
oxygen.15 Chronic granulomatous disease (CGD) is a rare
hereditary disease characterized by severe, protracted, and potentially
fatal bacterial and fungal infections.16 The basic defect
is an inability of phagocytic cells, including neutrophils, to produce
superoxide and hydrogen peroxide, as a result of a genetic deficiency
in any one of the components of the NADPH oxidase.17 A
recent study suggests that neutrophil apoptosis may be severely curtailed in these individuals.18
In recent years, Fas/APO-1 and the corresponding Fas/APO-1 ligand have
emerged as key regulators of the apoptotic response, particularily
within the immune system.19 Fas/APO-1 is a widely expresssed type I membrane protein belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor family19 and
mediates apoptosis upon cross-linking by either specific monoclonal
antibodies (MoAbs) or the endogenous ligand itself. Other investigators
have recently shown that neutrophils are susceptible to
Fas/APO-1-induced cell death,20,21 and interactions
between Fas/APO-1 and its ligand have been suggested to represent a key
element in controlling constitutive apoptosis in these
cells.20 However, the downstream events involved in this
signalling cascade have not been extensively studied in neutrophils.
Furthermore, the role of reactive oxygen species in the
Fas/APO-1-triggered apoptotic pathway remains
controversial.22-26
We sought to determine whether caspases are involved in neutrophil
apoptosis and to examine the possible role of reactive oxygen species
in the regulation of caspase activity. For this purpose, we studied two
different models of neutrophil cell death, the spontaneous apoptosis
model of resting neutrophils, which is accelerated upon ligation of the
Fas/APO-1 molecule by specific antibodies, and the activation-induced
model in which treatment of the cells with phorbol 12-myristate
13-acetate (PMA) results in the rapid generation of reactive oxygen
species and subsequent cell death. Our data clearly demonstrates the
activation of caspases in neutrophils undergoing spontaneous and
Fas/APO-1-triggered apoptosis, and we found that reactive oxygen
species derived from the NADPH oxidase were not involved in this
process. On the other hand, PS exposure after PMA treatment was blocked
both in neutrophils treated with diphenylene iodonium (DPI) and in
neutrophils from CGD patients, which lack the NADPH oxidase. PS
exposure in this model was caspase-independent, and it was not until
the NADPH oxidase was inhibited that caspase activity could be
measured. These findings indicate that the high level of reactive
oxygen species generated in activated neutrophils actually prevents
caspases from functioning, and an alternate death pathway therefore
appears to operate in these cells.
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MATERIALS AND METHODS |
Reagents.
The agonistic anti-Fas IgM MoAb (clone CH-11) and the antagonistic
anti-Fas IgG1 MoAb (clone ZB4) were purchased from Medical & Biological
Laboratories, Ltd (Nagoya, Japan). Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) and z-Val-Ala-Asp-chloromethylketone (zVAD-cmk) were purchased from Enzyme Systems Products (Dublin, CA), and
Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) was obtained
from Peptide Institute, Inc (Osaka, Japan). The Apoptest-fluorescein
isothiocyanate (FITC) kit containing annexin V-FITC was
from Nexins Research B.V. (Hoeven, The Netherlands). Ultrapure agarose
was purchased from Life Technologies (Paisley, Scotland). Propidium
iodide (PI), DPI, and PMA were from Sigma (St Louis, MO).
Isolation and culture of human neutrophils.
Peripheral neutrophils were isolated from heparinized blood from
healthy adult blood donors and also from three adult CGD patients27 by a method of dextran sedimentation and density gradient centrifugation as previously described.28 Residual erythrocytes were removed by hypotonic lysis. The CGD diagnosis was
established by the lack of chemiluminescence (CL) reaction and a
negative nitroblue tetrazolium reduction test.27 Isolated neutrophils (1 × 106/mL) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin in
24-well flat-bottomed plates (Costar, Cambridge, MA) at 37°C in a
humidified atmosphere containing 5% CO2. For PMA-treated
cultures, a 1% trypsin solution was used to detach adherent cells.
Preliminary experiments showed that trypsinization of neutrophils
undergoing spontaneous apoptosis did not significantly affect the
subsequent measurement of DEVD-AMC cleavage (data not shown).
Assessment of apoptotic morphology.
Apoptosis was assayed by estimating hematoxylin/eosin-stained
cytocentrifuge preparations of neutrophils for morphologic changes characteristic of apoptosis as previously described.14 A
minimum of 400 cells were scored for each sample and the percentages of apoptotic neutrophils were determined.
Agarose gel electrophoresis of fragmented DNA.
DNA fragmentation was assessed by one-stage agarose gel electrophoresis
as described by Sorenson et al.29 Briefly, 2 × 106 cells were washed and resuspended in sample buffer
containing RNase (10 mg/mL) for 20 minutes. Samples were then loaded
onto a 1.8% agarose gel with a digestion gel (0.8% Ultrapure agarose, 2% sodium dodecyl sulfate (SDS), and 0.6 mg/mL proteinase K) at the
upper end. The gel was run at 20 V overnight and electrophoresis was
then continued for 3 hours at 90 V to separate the DNA fragments. The
gel was subsequently stained with ethidium bromide, visualized under
305 nm ultraviolet (UV) illumination, and photographed using Polaroid
665 positive/negative film (Polaroid Corp, Cambridge, MA).
Measurement of PS exposure.
PS exposure was measured by the binding of annexin V-FITC using the
protocol outlined in the Apoptest-FITC kit. Cells were also stained
with PI (100 µg/mL) before analysis with a FACScan flow cytometer
(Becton Dickinson, San Jose, CA) equipped with a 488 nm argon laser.
Ten thousand events were collected and analyzed using the CellQuest
software (Becton Dickinson). Low-fluorescence detritus was gated out
before analysis. Data are presented as dot plots showing the change in
mean fluorescence intensity of annexin V-FITC. For clarity, the
fluorescence profiles of 25% of the analyzed events are shown.
Determination of DEVD-AMC cleavage.
The measurement of DEVD-AMC cleavage was performed in a fluorometric
assay modified from Nicholson et al.30 Cell lysates and
substrate were combined in a standard reaction buffer (100 mmol/L
HEPES, 10% sucrose, 5 mmol/L dithiothreitol, 10 4%
octylphenoxy polyethoxy ethanol [NP-40], and 0.1%
3-[(3-cholomidopropyl) dimethylammonio] propane-1-sulphonic acid
[CHAPS], pH 7.25) and added to a microtiter plate. The
cleavage of the fluorogenic peptide substrate DEVD-AMC (50 µmol/L)
was monitored by AMC liberation in a Fluoroscan II plate reader
(Labsystems, Stockholm, Sweden) using 355 nm excitation and 460 nm
emission wavelengths. Fluorescence was measured every 70 seconds during
a 30-minute period, and fluorescence units were converted to pmol of
AMC using a standard curve generated with free AMC. Data from duplicate
samples were then analyzed by linear regression.
Statistics.
Means and standard deviations (SD) were calculated from at least three
independent experiments.
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RESULTS |
PS exposure, DNA laddering, and characteristic morphologic changes in
constitutive and Fas/APO-1-triggered apoptosis.
We determined apoptosis in freshly isolated neutrophils from healthy
donors by morphologic evaluation. Typical apoptotic cells, displaying
diminution in cell volume, increased cytoplasmic staining, and nuclear
pyknosis, were readily observed in our cultures, and the appearance of
such cells also correlated with a time-dependent increase in DNA
fragmentation (Fig 1A and B
and Table 1). One important mechanism for
the recognition of apoptotic cells is the exposure of PS on the outer
leaflet of the plasma membrane.31 This PS exposure was
evident in neutrophils by 3 hours, and the level increased to 48.5% ± 4.3% at 24 hours, in keeping with the assessment of morphologic
and biochemical end points of apoptosis that we measured (Fig 1C and
Table 1). There was a concomitant increase in the apoptotic (lower
right quadrant) and necrotic (upper right quadrant) populations at 24 hours, indicating that at later time points many of the apoptotic cells
have proceeded into secondary necrosis (Fig 1C).
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| Fig 1.
Spontaneous and Fas/APO-1-mediated apoptosis of freshly
isolated human neutrophils. (A) Cytospin preparations of neutrophils
were stained with hematoxylin/eosin and apoptotic cells were quantified
morphologically: (1) medium alone 0 hour, (2) medium alone 6 hours, (3)
anti-Fas MoAb 250 ng/mL 6 hours, (4) medium alone 24 hours. Bold arrows
indicate typical cells in the intermediate stages of apoptosis.
Percentages of apoptotic cells are reported in Table 1. (B) Agarose gel
electrophoresis of DNA showing a time-dependent increase in the
formation of oligonucleosomal DNA fragments in spontaneous (lane 1, 0 hour; lane 2, 3 hours; lane 3, 6 hours; lane 4, 12 hours; lane 5, 24 hours) and Fas/APO-1-triggered (lane 6, 3 hours; lane 7, 6 hours; lane
8, 12 hours) apoptosis. Lane M represents molecular weight marker (kB).
(C) Flow cytometric analysis of annexin V binding in neutrophils
undergoing spontaneous (panel 1, 6 hours; panel 2, 24 hours) and
Fas/APO-1-mediated (panel 3, 6 hours; panel 4, 24 hours) apoptosis.
The percentage of PS positive cells in each sample is indicated.
Similar results were obtained in four separate donors (see Table 1).
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Table 1.
Percentages of Cells Displaying Morphologic
Characteristics of Apoptosis and PS Exposure in Untreated and
Anti-Fas MoAb-Treated Neutrophils
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Neutrophils have been demonstrated to express the apoptosis-mediating
surface molecule Fas/APO-1, and incubation of neutrophils with the
agonistic anti-Fas MoAb significantly accelerates the apoptotic process
in these cells.20,21 We treated neutrophils with 250 ng/mL
of anti-Fas MoAb (clone CH-11) and observed a marked increase in
apoptosis, thus corroborating previous observations (Fig 1A through C
and Table 1). Neutrophils were also preincubated with the antagonistic
anti-Fas MoAb ZB4 (1 µg/mL) for 1 hour and then treated with the
agonistic antibody as before (Table 2). These results
clearly show that the ZB4 MoAb effectively inhibits anti-Fas
MoAb-induced PS exposure in cultured neutrophils, yet fails to block
constitutive apoptosis.
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Table 2.
Effect of Various Inhibitors on PS Externalization and
Caspase-3-Like In Vitro Activity in Constitutive and
Fas/APO-1-Triggered Apoptosis
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Caspases are activated in constitutive and Fas/APO-1-mediated
apoptosis.
Having established a system of constitutive and Fas/APO-1-triggered
apoptosis in neutrophils, we next sought to investigate whether the
caspases are activated in these cells. Caspase activity is measured in
vitro by the cleavage of the fluorogenic substrate DEVD-AMC in a
continous fluorometric assay. DEVD-AMC is a specific substrate for
caspase-3-like proteases and represents the common cleavage site for
this class of enzymes.4 Freshly isolated neutrophils were
incubated in the absence or presence of anti-Fas MoAb CH-11 (250 ng/mL)
and the maximum linear rate of AMC release was measured in
samples collected at various time points
(Fig 2A). A clear induction of caspase
activity, with peak values at 24 hours (38.8 ± 12.6; n = 3) was seen during constitutive apoptosis. Treatment of the cells with
the agonistic anti-Fas MoAb markedly augmented and accelerated this
process, with peak values exceeding those seen in the spontaneous model
and reached at 12 hours after stimulation (54.9 ± 9.2; n = 3).
DEVD-CHO (50 nmol/L), a specific inhibitor of caspase-3-like enzymes,
completely blocked DEVD-AMC cleavage thereby confirming the specificity
of the neutrophil lysates for DEVD-AMC (Fig 2B). Preincubation with the
ZB4 MoAb (1 µg/mL) effectively blocked the increase in DEVD-AMC
cleavage mediated by the Fas/APO-1-specific antibody (Table 2). On the other hand, DEVD-AMC cleavage in the constitutive model was unaffected by the addition of the antagonistic anti-Fas antibody. These findings are therefore indicative of the occurrence of both Fas/APO-1-dependent and -independent mechanisms of caspase activation in neutrophil apoptosis. Furthermore, Fas/APO-1-induced PS exposure in freshly isolated neutrophils was effectively inhibited by the general caspase
inhibitor zVAD-cmk (Table 2). Similar results were obtained in the
constitutive apoptosis model, thereby demonstrating that PS exposure is
caspase-dependent in these two models of neutrophil apoptosis (Table
2). As expected, the caspase inhibitor also blocked DEVD-AMC cleavage
in cells undergoing spontaneous and Fas/APO-1-triggered apoptosis,
although less efficiently at 12 hours (Table 2).
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| Fig 2.
Caspase-3-like activity in neutrophils undergoing
spontaneous and Fas/APO-1-mediated apoptosis, as measured by cleavage
of the specific fluorogenic substrate DEVD-AMC (50 µmol/L). (A) Time
course of caspase-3-like activation in spontaneous ( ) and anti-Fas
MoAb-triggered (250 ng/mL;  ) apoptosis of neutrophils. The cells
were lysed at various times (0 to 72 hours) after initiation of in
vitro culture and the release of AMC was monitored. The maximum rate of
AMC release (pmol/min) was estimated by linear regression
(r2 > 0.99). Data shown are the mean and range of
duplicate determinations from a representative experiment. (B) The
competitive inhibitor DEVD-CHO (50 nmol/L) was added to cell lysates in
vitro and shown to block DEVD-AMC cleavage in cells undergoing
spontaneous apoptosis (control,  ; plus inhibitor,  ). Cells (1 × 106) were incubated for 15 hours before the fluorometric
assay. Each data point represents the average value of determinations
performed in triplicate samples.
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Role of the NADPH oxidase in spontaneous and Fas/APO-1-mediated
apoptosis.
To examine whether reactive oxygen species are involved in caspase
activation in neutrophils, the flavoprotein inhibitor, DPI, an
inhibitor of the NADPH oxidase,32 was used. We found that treatment of freshly isolated neutrophils with 10 µmol/L DPI resulted in an increase in caspase activity, as determined by
DEVD-AMC cleavage, both in the constitutive and Fas/APO-1-triggered models (Fig 3). Peak values for spontaneous
DEVD-AMC cleavage were 36.1 ± 10.5 (without DPI) and 59.5 ± 8.3 (with DPI), and peak values for Fas/APO-1-triggered DEVD-AMC
cleavage were 50.9 ± 7.6 (without DPI) and 66.2 ± 8.7 (with
DPI) (mean ± SD; n = 3). In addition, DPI treatment enhanced PS
exposure in these two model systems, consistent with the view that this
event is caspase-mediated (Table 2). These results suggest that NADPH
oxidase-derived reactive oxygen species are not involved in activating
the caspases, and in contrast are partially suppressing caspase
activity in neutrophils triggered to undergo apoptosis.
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| Fig 3.
Treatment of neutrophils with the NADPH oxidase inhibitor
DPI leads to an increase in caspase-3-like activity. Neutrophils
undergoing spontaneous or Fas/APO-1-triggered apoptosis ( and  ,
respectively) were cultured in the absence or presence of DPI (10 µmol/L;  and  ) for various times (0 to 72 hours). Anti-Fas MoAb
was used at 250 ng/mL. Determination of AMC release was performed in a
fluorometric assay. The maximum rate of AMC release (pmol/min) was
estimated by linear regression (r2 > 0.99). The mean and
range of duplicate determinations from a representative experiment are
shown. Solvent alone (ie, dimethyl sulfoxide [DMSO]) did not affect
DEVD-AMC cleavage (data not shown).
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Reactive oxygen species promote PS exposure, but suppress caspase
activity, in activated neutrophils.
The protein kinase C activator, PMA, activates the NADPH oxidase in
neutrophils, leading to a burst of reactive oxygen species similar to
what is seen when neutrophils ingest invading
pathogens.33,34 We found that PMA (200 nmol/L) rapidly
induced morphologic changes, which were distinct from those observed in
constitutive and Fas/APO-1-triggered apoptosis, with an increase in
cell size and the appearance of numerous vacuoles throughout the
cytoplasm (Fig 4A). Moreover, in agreement
with a previous report by Takei et al,35 we were unable to
detect any DNA fragmentation in PMA-treated cells (data not shown).
However, PS exposure was evident at 3 hours and was markedly
accelerated when compared with untreated neutrophils (untreated cells:
6.4 ± 3.1; PMA-treated cells: 38.5 ± 6.4; mean ± SD; n = 3;
Fig 4B), demonstrating that changes similar to those observed in
apoptotic cells are taking place.
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| Fig 4.
PMA treatment of neutrophils results in rapid morphologic
changes accompanied by externalization of PS. (A) Cytospin preparation
of neutrophils cultured in medium alone (1) or treated for 3 hours with
200 nmol/L PMA (2). Note the presence of numerous vacuoles in the
cytoplasm and the coalescence of the nuclear lobes after PMA treatment.
Original magnification × 100. (B) PS exposure in unstimulated (panel
1) and PMA-treated (panel 2, 200 nmol/L) neutrophils after 3 hours of
culture. PS exposure was determined by flow cytometric analysis of
annexin V binding as described in Materials and Methods. The percentage
PS exposure is indicated. PMA treatment also caused a marked increase
in forward scatter of these cells, indicative of an increase in cell
size (data not shown). Similar results were obtained with three
independent donors.
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PMA-treated neutrophils were also assessed for caspase activity in
vitro and were found to yield no detectable DEVD-AMC cleavage activity
at any time point tested. However, when cells were pretreated with DPI
(10 µmol/L), there was a time-dependent induction of caspase
activity, as evidenced in the in vitro enzyme assay
(Fig 5A). Interestingly, we found that DPI
(10 µmol/L), but not zVAD-cmk (10 µmol/L) was capable of completely
diminishing PS exposure in these cells (PMA alone: 38.5 ± 6.4; PMA
plus DPI: 2.4 ± .1.1; PMA plus zVAD-cmk: 39.8 ± .4.7; mean ± SD; n = 3; Fig 5B), suggesting that caspase activity is not
required for PS exposure under these conditions. Rather, the
PMA-induced increase in the level of reactive oxygen species appears to
be critical for externalization of PS. Taken together, these results
suggest that reactive oxygen species are simultaneously inhibiting
caspase activity and mediating PS exposure in PMA-activated
neutrophils.
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| Fig 5.
NADPH oxidase-derived oxygen metabolites inhibit
caspase-3-like activity and promote PS exposure in PMA-activated
neutrophils. (A) PMA-stimulated neutrophils were cultured for the
indicated times in the presence () or absence () of the NADPH
oxidase inhibitor DPI (10 µmol/L), and the rate of AMC liberation was
determined in a fluorometric assay. Treatment of the cells with DPI did
not affect the morphologic changes induced by PMA (data not shown).
Solvent alone (ie, DMSO) had no discernible effect on either morphology
or DEVD-AMC cleavage (data not shown). Data are presented as the mean
and SD of three separate experiments performed in duplicate. (B) PS
exposure was determined in neutrophils by flow cytometry after in vitro
culture for 3 hours. Panel 1 shows cells treated with PMA alone (200 nmol/L). As seen in panel 2, addition of the NADPH oxidase inhibitor
DPI (10 µmol/L) competely blocks PS exposure, whereas the general
caspase inhibitor zVAD-cmk (10 µmol/L) has no effect in this system
(panel 3). Reproducible results were obtained in three separate
donors.
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Absence of PS exposure in PMA-treated neutrophils from CGD patients.
To confirm the results obtained with DPI treatment of normal
neutrophils, we isolated cells from three adult CGD patients, whose
neutrophils do not generate any superoxide.27 These
patients suffered from the autosomal recessive form of the disease with genetic defects in the cytosolic components of the NADPH oxidase, ie,
p67phox (phox, phagocyte oxidase) and p47phox,
respectively. Upon isolation, cells were treated with PMA (200 nmol/L)
and evaluated morphologically and the levels of PS exposure and
DEVD-AMC cleavage were determined. PMA treatment was found to induce
similar morphological changes as those seen in neutrophils from healthy
individuals, ie, an increase in cell size and the formation of
intracellular vacuoles (data not shown). PS exposure, however, was
completely absent at 3 hours (Table 3).
Moreover, caspase-3-like activity was evident in PMA-treated CGD
neutrophils at later time points (Table 3). The PS exposure evidenced
in PMA-treated cells at later time points most likely results from the
delayed caspase activation. These data therefore corroborate our
findings obtained with DPI treatment of PMA-activated neutrophils from
healthy individuals.
Spontaneous apoptosis of neutrophils from CGD patients has previously
been reported to be impaired, and these cells were purportedly resistant to anti-Fas-mediated apoptosis.18 Contrary to
these findings, we detected PS exposure and morphological changes
indicative of apoptosis in CGD neutrophils undergoing spontaneous as
well as Fas/APO-1-triggered apoptosis (Table 3 and data not shown), albeit with slower kinetics when compared with healthy individuals. In
addition, caspase-3-like activity was detected under both constitutive and Fas/APO-1-triggered conditions (Table 3), indicating that resting
neutrophils from CGD patients are not resistant to undergoing apoptosis.
| |
DISCUSSION |
Caspases have been ascribed an essential role in the execution of the
apoptotic process. We were interested in evaluating the role of
reactive oxygen species in the regulation of these cysteine proteases
and chose to investigate human neutrophils, as these cells readily
undergo spontaneous apoptosis and also have the capacity to generate a
powerful oxidative burst upon activation. In this study, we show
caspase activation in neutrophils undergoing constitutive and
Fas/APO-1-mediated apoptosis, but no caspase activation in neutrophils
stimulated with PMA. Pretreatment with the NADPH oxidase inhibitor,
DPI, allowed the caspases to function in PMA-stimulated neutrophils,
indicating that caspase activity is suppressed by NADPH oxidase-derived
reactive oxygen species. These findings are therefore suggestive of a
specialized caspase-independent pathway of cell death in activated
neutrophils.
It is becoming increasingly apparent that caspases are redox-sensitive
enzymes. They possess an active site cysteine, which is predicted to be
susceptible to oxidation, and thiol reductants such as dithiothreitol
are routinely included in experimental assay buffers. Previous work
from our laboratory has demonstrated that dithiocarbamate disulfides
are potent inhibitors of caspase-1 and caspase-3, the inhibitory effect
being accomplished most likely through thiol-disulfide
exchange.36 Similarily, Takahashi et al37
recently reported the inhibition of caspases by phenylarsine oxide, a
compound that is known to covalently modify vicinal thiol groups. We
have also documented the ability of hydrogen peroxide to inhibit
caspase activity in Jurkat T cells,8 and several investigators have recently shown the inhibitory effect of nitric oxide
on caspase function.9,10 Our current results provide the
first evidence that endogenous oxidant production may prevent the
activation of caspases within the same cell.
While caspases may function optimally in a reducing environment, this
does not preclude a role for low level oxidative stress in the
initiation of an apoptotic response. Kasahara et al18 showed an impairment in spontaneous and Fas/APO-1-mediated apoptosis in CGD neutrophils and concluded that oxidants have a critical role in
the induction process. We could detect PS exposure and caspase-3-like
activity in the CGD cells we tested, although the response was slower
when compared with normal individuals; furthermore, there was a
moderate increase rather than inhibition of caspase-3-like activity
and PS exposure in normal cells in the presence of DPI. Therefore, we
conclude that NADPH oxidase-derived reactive oxygen species do not play
a significant role as mediators of spontaneous neutrophil apoptosis.
Narayanan et al38 measured an increase in intracellular
oxidants in unstimulated neutrophils and showed that while DPI had no
effect, cyanide lowered oxidant production, implicating
mitochondrial-derived reactive oxygen species in apoptosis. However,
the dichlorofluorescein assay used for measuring intracellular hydrogen
peroxide requires peroxidase activity for substrate oxidation, and
cyanide is likely to act by inhibiting the peroxidase rather than the
source of hydrogen peroxide. Thiol antioxidants39 and a
hypoxic environment40 were reported to inhibit
Fas/APO-1-triggered and spontaneous neutrophil apoptosis,
respectively, again suggesting that reactive oxygen species play an
important role in the process. However, hypoxia was proposed to act by
altering the expression of various cellular antioxidants,40
rather than lowering the production of oxygen metabolites per se. Thiol
antioxidants have been shown to inhibit apoptosis in T
lymphocytes,41 but rather than directly scavenging reactive
oxygen species, they could act by altering the redox status of thiol
enzymes, such as the caspases, in the pathway leading to apoptosis.
While evidence for reactive oxygen species involvement in spontaneous
neutrophil apoptosis remains inconclusive, it seems clear that cell
death after PMA treatment is dependent on oxidant production. PMA
failed to trigger PS exposure in neutrophils obtained from the CGD
patients, and PMA-induced PS exposure was completely prevented by DPI
treatment of normal cells, thus demonstrating that the burst of oxygen
metabolites after PMA treatment is coupled to the externalization of
PS. Coxon et al42 have recently reported that phagocytosis
of opsonized particles, mediated by the  2 integrin CD11b/CD18,
causes activation-induced apoptosis in neutrophils, which is dependent
on NADPH oxidase-derived reactive oxygen species. Similarily,
neutrophils have been reported to undergo apoptosis after ingestion of
Escherichia coli.43 While inhibition of these events by reduced glutathione and N-acetylcysteine supports the involvement of reactive oxygen species,43 the nonspecific
thiol effects described above mean that more conclusive studies with DPI or CGD neutrophils are required. Interestingly, neutrophils from
the CGD patients in the present study were susceptible to spontaneous
and Fas/APO-1-triggered apoptosis in vitro. Therefore, it appears
reasonable to speculate that while neutrophils from these patients are
not inherently resistant to the induction of apoptosis, their failure
to externalize PS in response to activation may result in the
inefficient clearance by macrophages from the site of inflammation,
thus accounting for the formation of characteristic granulomatous
lesions and subsequent tissue destruction. Further work is needed to
evaluate the contribution of these events to the emergence of clinical
symptoms in CGD patients.
Takei et al35 recently reported that PMA induces rapid
killing of neutrophils with features distinct from typical apoptosis or
necrosis. Because extensive intracellular vacuolation made it difficult
to observe nuclear changes, our best indicator of PMA-induced cell
death was the rapid externalization of PS. Although difficult to
classify as conventional apoptosis solely on the basis of PS exposure,
the expression of surface markers is critical for neutrophil clearance
by macrophages,13 making this event an essential
physiologic process. Cell death induced by PMA may therefore be
considered a type of activation-induced death, akin to apoptosis,
albeit occuring more rapidly. We have shown that the flavoprotein
inhibitor, DPI, efficiently blocks PS exposure in PMA-treated
neutrophils. On the other hand, PS exposure readily occurs in CGD
neutrophils undergoing spontaneous and Fas/APO-1-mediated apoptosis,
suggesting that different mechanisms for PS exposure operate in resting
and activated neutrophils. Caspase activation has previously been shown
to be necessary for PS exposure in certain cell types44 and
Brown et al45 reported that PS exposure in spontaneous
neutrophil apoptosis is blocked by zVAD-fmk. However, caspase-independent PS exposure in apoptotic cell death is not unprecendented. Work from our laboratory has shown that PS exposure in
TNF-treated monocytic U937 cells is blocked by calpain inhibitors, but
not by the caspase inhibitor zVAD-cmk.46 Fabisiak et
al47 recently reported on the selective oxidation
and externalization of PS in paraquat-induced apoptosis in the murine
myeloid cell line 32D, thereby supporting the view that oxidative
stress may directly initiate PS exposure. The mechanisms involved
warrant further investigation.
Recent reports suggest that constitutive apoptosis of neutrophils is,
at least in part, due to an autocrine or paracrine Fas/APO-1-mediated mechanism.20 This conclusion was based on the fact that
neutrophils appeared to express low levels of the Fas/APO-1 ligand on
their surface and that the antagonistic anti-Fas MoAb, ZB4, partially inhibited the decrease in cell viability of cells undergoing
spontaneous apoptosis. However, we could not detect any effect of the
ZB4 MoAb on caspase activity or PS exposure in constitutive neutrophil apoptosis. On the other hand, apoptosis triggered by the agonistic anti-Fas MoAb was efficiently blocked in our system. In a previous study, we defined the epitope targeted by the ZB4 and CH-11
antibodies,48 and by molecular modeling, we could show that
this epitope most likely differed from the receptor-ligand
interface.49 These findings would tend to suggest that the
antagonistic MoAb may be less efficient in blocking the endogenous
ligand. However, steric interference of ligand binding may conceivably
occur despite the targeting of an epitope distinct from the
receptor-ligand interface. Indeed, the ZB4 MoAb was recently shown to
inhibit apoptosis induced by ligation of the T-cell receptor with
anti-CD3, a treatment known to initiate death by upregulating Fas/APO-1 ligand expression.50 Therefore, our current data favors the view that constitutive neutrophil apoptosis is likely to proceed by
Fas/APO-1-independent mechanisms.
In summary, our findings suggest that neutrophils possess two different
modes of cell death. The constitutive mode of cell death, which is
likely to be involved in the turnover of senescent neutrophils in vivo,
appears to use a caspase-dependent pathway characteristic of apoptotic
cell death in many other systems. While the majority of neutrophils die
in this way, a small number are called on to destroy invading
pathogens. It is critical that these cells, loaded with their
potentially deleterious contents, are safely cleared from an
inflammatory site with minimal damage to surrounding tissues. The
present data indicates that the powerful burst of reactive oxygen
species generated to kill the infectious agent will prevent the redox
sensitive caspases from functioning, thereby necessitating a novel
oxidant-dependent pathway of neutrophil death. Further work is needed
to elucidate the mechanisms of this pathway and to determine if
abnormalities in its functioning are involved in the pathology of
various inflammatory conditions.
| |
ACKNOWLEDGMENT |
We would like to thank Afshin Samali for constructive advice during the
course of this study and Boris Zhivotovsky, David Burgess, Isabella
Pörn-Ares, and Dean Jones for valuable discussions. We are also
grateful for the cooperation of the CGD patients (LNG, AA, and MA) in
the study.
| |
FOOTNOTES |
Submitted December 29, 1997;
accepted August 5, 1998.
Supported by the Swedish Medical Research Council, the Children's
Cancer Foundation of Sweden and the Institute of Environmental Medicine
at Karolinska Institutet. M.H. was supported by the New Zealand
Foundation for Research, Science and Technology.
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 Mark B. Hampton, PhD,
Pathology Research, Massachusetts General Hospital, 149 13th St,
Charlestown, MA 02129.
| |
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Top
Abstract
Introduction