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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3106-3115
Bcl-Xl- and Bax- -Mediated Regulation of Apoptosis of
Human Neutrophils Via Caspase-3
By
Pamela Weinmann,
Peter Gaehtgens, and
Barbara Walzog
From the Department of Physiology, Freie Universität, Berlin,
Germany.
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ABSTRACT |
In this study, a mechanism is reported which determines the lifetime
of polymorphonuclear neutrophils (PMN). In human PMN freshly isolated
from the circulation, expression of bcl-Xl, bax- , and
bak, members of the bcl-2 family of apoptosis-associated genes, was
found using the reverse transcription-polymerase chain reaction technique. In contrast, no expression of bcl-2 was seen in PMN, whereas
the myeloid cell line HL-60 was positive for bcl-2 mRNA. Two gene
products, Bcl-Xl and Bax-, which are known to function as the regulatory machinery of programmed cell death (apoptosis), were
detected at the protein level in PMN. Moreover, differential expression
of these proteins was found upon induction or prevention of apoptosis
by cytokines: Whereas induction of apoptosis by tumor necrosis
factor- was associated with a reduction of expression of the
anti-apoptotic Bcl-Xl protein, prevention of apoptosis by
granulocyte-macrophage colony-stimulating factor led to a
downregulation of expression of the death-promoting Bax- protein.
This shift of balance of anti- and pro-apoptotic proteins was found to
control caspase-3 activity which, in turn, downregulated
Bcl-Xl expression in PMN undergoing apoptosis. Thus,
cytokines can affect the ratio of Bax-/Bcl-Xl expression
in human PMN and modulate the subsequent activity of caspase-3, which
functions as executer of the programmed cell death and may
promote apoptosis by a positive feed-forward mechanism that
downregulates Bcl-Xl.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
APOPTOSIS (programmed cell death) is
thought to contribute to the homeostasis of functional leukocyte
pools.1 It is an active and well-regulated process that is
characterized by specific phenomena such as cell shrinkage, chromatin
condensation, internucleosomal DNA fragmentation, membrane blebbing
and, finally, the decay into apoptotic bodies.2-4 Mature
human polymorphonuclear neutrophils (PMN) undergo apoptosis
spontaneously within hours to days, and this is thought to contribute
to the high turnover of these cells in the circulation.5
The life span of circulating PMN is relatively short when compared with
other leukocytes, but it can be further shortened or extended by
accelerating or delaying apoptosis.4 Upon induction of
apoptosis, the plasma membrane of the PMN remains intact, thus
preventing the release of proinflammatory and histotoxic contents of
these cells. Because apoptosis also leads to an impairment of PMN
responsiveness6 and allows specific recognition and
elimination of the cells by phagocytosis,7,8 the induction
or prevention of PMN apoptosis is currently discussed as a key event in
the control of inflammation.
Various inflammatory cytokines are able to affect PMN survival by
modulating apoptosis. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to inhibit PMN apoptosis both in vitro as well
as in the circulation.9,10 This effect occurs during systemic responses and is thought to strengthen the inflammatory reaction. Other cytokines such as tumor necrosis factor- (TNF-) serve as potent inductors of apoptosis.11 Adhesive
interactions via CD11/CD18 during PMN recruitment to sites of
inflammation also contribute to induction of apoptosis of emigrated
PMN, which may help to subside local inflammatory reactions by safe
elimination of emigrated PMN.12,13
Although PMN apoptosis seems to be critical for homeostasis as well as
for the control of inflammatory processes, the molecular mechanism that
underlies the control of PMN apoptosis is widely unknown. A previous
report presented evidence that activation of Lyn, a member of the Src
kinase family, may be involved in the GM-CSF-mediated delay of PMN
apoptosis.14 Because expression of bcl-2, a member of the
bcl-2 family of apoptosis-associated genes, was found to be absent in
PMN, the investigators stated that apoptosis-associated genes which are
critical for the control of apoptosis in many other cell systems are
probably not involved in the regulation of PMN apoptosis. However,
prolonged survival of PMN caused by inhibition of apoptosiswas
previously observed in bcl-2 transgenic mice,15 suggesting
that the targets of the Bcl-2 protein are present in PMN.
The mammalian Bcl-2 family of apoptosis-associated proteins consists of
members that inhibit apoptosis (Bcl-2, Bcl-Xl, Mcl-1, A1,
etc) and others that induce apoptosis (Bax, Bak, Bad,
Bcl-xs, Bik, etc), and the balance between pro-apoptotic
and anti-apoptotic members determines the fate of the cells in many
systems.16,17 Bcl-2 antagonizes cell death, like other
members of this protein family, by forming homodimers as well as
heterodimers with different homologues. The upregulation of
anti-apoptotic Bcl-2 or its close homologue Bcl-Xl is known
to inhibit apoptosis,18,19 whereas the downregulation of
Bcl-220 or its antagonization by dimerization with, eg,
Bax- promotes programmed cell death.20 The Bcl-2 family
regulates apoptosis, eg, by controlling the activity of caspases, the
executioners of apoptosis, via release of cytochrome C from
mitochondria.21 Although caspase-3 (CPP32) activity was identified in human PMN undergoing apoptosis,22 there is no information about the molecules that may control this protease in PMN.
The present study was undertaken to elucidate the putative role of the
Bcl-2 family of apoptosis-associated proteins in the control of PMN
apoptosis. Apoptosis of isolated human PMN was measured in GM-CSF- or
TNF--treated cells as well as in untreated control cells,
respectively, by flow cytometric analysis of DNA content, by DNA
fragmentation assay (DNA ladder), and by measuremnt of CD16 expression
on the cell surface. Expression of bcl-2, bcl-Xl, and
bax- was analyzed by reverse transcription-polymerase chain reaction
(RT-PCR) as well as by the Western blotting technique at the protein
level. Activation of caspase-3 was analyzed by detection of proteolytic
cleavage of the pro-caspase as well as by direct measurement of
caspase-3 acitivity.
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MATERIALS AND METHODS |
Isolation of human PMN and culture of HL-60 cells.
PMN were isolated from heparinized blood (10 I.E./mL) of healthy
donors. After erythrocyte sedimentation in the presence of 40%
(vol/vol) autologous plasma, the leukocyte-rich plasma was layered onto
a discontinuous Percoll gradient (Sigma Chemie,
Deisenhofen, Germany) as described23 and centrifuged at
600g for 20 minutes. The PMN-containing band was collected and
washed in Dulbecco's phosphate-buffered saline (PBS). Cells were
resuspended in RPMI-1640 medium supplemented with 10% fetal calf
serum. PMN viability was greater than 97% as assessed by the trypan
blue exclusion test; purity was greater than 98% as analyzed by
microscopy using Hemacolor staining (Merck, Darmstadt, Germany). The
human leukemia cell line HL-60 and the B-cell line BL-41 were grown in
RPMI-1640 medium supplemented with 10% fetal calf serum and
antibiotics (50 U/mL penicillin, 50 µg/mL streptomycin)
in 5% CO2 at 37°C.
Analysis of DNA content.
DNA content was analyzed by flow cytometry (FACScan; Becton Dickinson,
San Jose, CA) using propidium iodide. Briefly, PMN (5 × 105/100 µL) were washed with PBS supplemented
with 0.5 mmol/L EDTA and resuspended in 70% ethanol. PMN were
permeabilized overnight at  20°C, washed with PBS
supplemented with 0.5% EDTA and suspended in 250 µL PBS with 0.5 mmol/L EDTA. After addition of 20 µg/mL DNase-free RNase and 50 µg/mL propidium iodide (final concentrations), samples were incubated
for 15 minutes at room temperature and kept at 4°C until flow
cytometric analysis. In each sample, 104 cells were
counted, gated off-line for granulocytes, and analyzed using Cell Quest
software (Becton Dickinson).
Cell surface expression of CD16.
Aliquots of cells (5 × 105/100 µL)
were incubated for 1 hour at 4°C in the dark with the fluorescein
isothiocyanate (FITC)-conjugated anti-CD16 monoclonal antibody (MoAb;
final dilution of 1:20), washed twice, and subjected to flow cytometry.
In each sample, 104 cells were counted, gated off-line for
granulocytes, and analyzed using Cell Quest software.
Internucleosomal DNA fragmentation assay.
PMN (107) were lysed for 10 minutes on ice in 600 µL
hypotonic lysis buffer (10 mmol/L EDTA, 0.2% Triton X-100 (Sigma
Chemie), 10 mmol/L Tris, pH 7.5). After centrifugation for
10 minutes at 4°C at 13,000g, DNA was isolated by
phenol/chloroform extraction and subsequent precipitation with 2.5 vol
ethanol containing 0.1 mol/L NaCl overnight at 20°C. After
centrifugation at 13,000g, the pellets were washed in 70%
ethanol, dried, and resuspended in 20 µL H2O. After
treatment with DNase-free RNase in a final concentration of 0.8 mg/mL
for 30 minutes at 37°C, samples were analyzed by gel
electrophoresis in 1.8% agarose and ethidium bromide staining.
RT-PCR.
Total RNA was isolated using the guanidine isothiocyanate
method24 using TRIZOL (Life Technologies, Eggenstein,
Germany). RNA (500 ng) was transcribed into cDNA using oligo(dT)
primers (Life Technologies) and 50 U reverse transcriptase Moloney
murine leukemia virus (MMLV; Promega, Madison, WI). PCR
amplification was performed using specific primer sets (TIP MOLBIOL,
Berlin, Germany) for bcl-2 (upstream primer:
5'-GGT-GCC-ACC-TGT-GGT-CCA-CCT, downstream primer:
5'-CTT-CAC-TTG-TGG-CCC-AGA-TAG-G, 458-bp product), bcl-X
(upstream primer: 5'-TTG-GAC-AAT-GGA-CTG-GTT-G, downstream primer: 5'-GTA-GAG-TGG-ATG-GTC-AGT-G, 765-bp product); bax-
(upstream primer: 5'-CTG-ACA-TGT-TTT-CTG-ACG-GC, downstream
primer: 5'-TCA-GCC-CAT-CTT-CTT-CCA-GA, 289-bp product); and bak
(upstream primer: 5'-TGA-AAA-ATG-GCT-TCG-GGG-CAA-GGC, downstream
5'-TCA-TGA-TTT-GAA-GAA-TCT-TCG-TAC-C, 642-bp product). For
control, a specific primer set for GADPH (upstream primer: 5'-GGT
CGG AGT CAA CGG ATT TGG T, downstream primer: 5'-TGT GGG CCA TGA
GGT CCA CCA C) was used, which yielded a 977-bp product. PCR (30 cycles: 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C) was performed using 1.25 U AmpliTaq DNA polymerase
(Perkin Elmer, Weiterstadt, Germany). PCR products were analyzed by
agarose gel electrophoresis and visualized with ethidium bromide under UV light.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and immunoblotting.
After stimulation as indicated, PMN were pelleted and lysed in 1X
Laemmli-buffer (2% [wt/vol] SDS, 6% (vol/vol)
2-mercaptoethanol, 10% (vol/vol) glycerol, and a trace amount of
bromphenol blue in 200 mmol/L Tris-HCl, pH 7.5). The samples were
immediately heated for 5 minutes at 100°C. Total cell
lysates (106 cells/sample) were subjected to SDS-PAGE on
gels containing 12% (wt/vol) acrylamide under reducing
conditions.25 Separated proteins were transferred to
nitrocellulose filters using semidry technique at 180 mA for 1 hour.
Filters were blocked by treatment with 3% BSA in TBS for 1 hour, and
subsequently incubated with the primary monoclonal or polyclonal
antibodies (Abs) in a final concentration of 1 µg/mL or at a final
dilution of 1:1,000, respectively, for 1 hour in TBS supplemented with
0.1% BSA. After three washes in TBS containing 0.1% Tween-20, filters
were incubated with peroxidase-conjugated secondary Abs (final
dilution, 1:1,000) in TBS supplemented with 0.1% BSA for 1 hour and
subsequently washed as described above. Detection was performed by
chemiluminescence using ECL-kit (Enhanced ChemiLuminescence; Amersham
Life Science, Braunschweig, Germany) and subsequent autoluminography by
exposure to x-ray films (XOMAT-AR, Kodak, Stuttgart,
Germany). Densitometry was performed using One-Dscan software (Scanalytics, Billerica, MA).
Measurement of caspase-3 activity.
PMN (5 × 106) were washed with PBS and lysed in 100 µL of 10 mmol/L potassium phosphate, 1 mmol/L EDTA, 0.5% Triton
X-100, 2 mmol/L phenlymethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 10 mmol/L dithiothreitol. After incubation for
10 minutes on ice, samples were centrifuged at 18,000g for 20 minutes at 4°C. The protein concentration of the supernatant was
determined by colorimetric measurement using the BCA protein assay
reagent (Pierce, Rockford, IL) according to supplier's instructions. An aliquot of each sample (40 µg) was diluted to a final volume of
200 µL in assay buffer consisting of 50 mmol/L HEPES, 10% sucrose, 0.1% CHAPS, and 10 mmol/L dithiothreitol supplemented
with 10 µmol/L of the fluorogenic caspase-3 substrate
T-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC).
Samples were incubated for 90 minutes at 30°C and measured at an
exitation wavelength of 400 nm and an emission wavelength of 505 nm
using a luminescence spectrometer (LS 50B; Perkin Elmer). Blanks were
measured in the absence of cell lysate to determine background fluorescence.
Antibodies.
The polyclonal rabbit anti-human Bcl-X (Ab-1) and the monoclonal mouse
anti-human Bak Ab (clone TC100) were obtained from Calbiochem (Bad
Soden, Germany). The polyclonal rabbit anti-human Bax (N-19) was
purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The
peroxidase-conjugated goat anti-mouse IgG and the peroxidase-conjugated
goat anti-rabbit IgG antibodies were obtained from Sigma (Deisenhofen,
Germany). The FITC-conjugated anti-human CD16 (Fc receptor type III)
MoAb (clone DJ130c) was purchased from Dako Diagnostica (Hamburg,
Germany). The polyclonal rabbit anti-human caspase-3 Ab was obtained
from PharMingen (San Diego, CA).
Reagents.
BSA, Chaps, dithiothreitol, leupeptin, ovalbumin, penicillin,
pepstatin, Percoll, phenlymethylsulfonyl fluoride, Ponceau S, propidium
iodide, streptomycin, sucrose, TNF-, Triton X-100, and Tween-20 were
obtained from Sigma. ECL Western blotting kit (RPN 2106) and
electrophoresis calibration standards for molecular mass determination
were purchased from Pharmacia (Freiburg, Germany). GM-CSF was obtained
from Boehringer (Mannheim, Germany). The fluorogenic caspase-3
substrate Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) and the caspase-3 inhibitor
Z-Asp-Glu-Val-Asp-7-fluoromethylketone (Z-DEVD-FMK) were purchased from
Calbiochem. Buffers, cell culture media, and fetal calf serum were
obtained from Biochrom (Berlin, Germany).
Statistical analysis.
Data shown represent mean ± SD where applicable. Statistical
significance was determined using Student's t-test; P < .05 was considered statistically significant.
| |
RESULTS |
Regulation of apoptosis by cytokines.
To characterize apoptosis of mature human PMN, cells were freshly
isolated from the circulation and cultured in the presence of TNF-,
GM-CSF, or left untreated for control. The loss of DNA content, a
well-known marker of apoptosis,2 was measured by flow
cytometry using propidium iodide staining.
Figure 1 shows the original fluorescence
histograms obtained from these experiments. Unstimulated freshly
isolated PMN showed only fluorescence peak after up to 2 hours of
culture, which resulted from a uniform diploid DNA content of these
cells. Only a few more cells showed a loss of DNA content
when aged for 6 hours in culture, but a substantial amount of PMN
showed a diminished DNA content, ie, underwent apoptosis spontaneously
after 22 hours of culture. This effect was accelerated by stimulation
with TNF-, which induced a substantial loss of DNA content as early
as 2 hours and 6 hours after the onset of stimulation. In contrast,
GM-CSF inhibited the loss of DNA content associated with apoptosis when
compared with unstimulated control cells.
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| Fig 1.
Loss of DNA content in PMN undergoing apoptosis as
measured by flow cytometry of prodium iodide-stained PMN. Original
recordings of fluorescence histograms of PMN freshly isolated from the
circulation, aged for 2, 6, or 22 hours in culture without further
stimulation (control), or treated with 300 U/mL TNF- or 300 U/mL
GM-CSF, respectively. Numbers indicate apoptotic PMN in percent of
total cell number. Results are representative of 12 independent
experiments.
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Figure 2 shows the mean values of 12 independent experiments. The prolonged time-course revealed that more
than 80% of unstimulated PMN underwent apoptosis spontaneously within
34 hours of culture. TNF- was able to increase apoptosis
significantly within 2 hours and 6 hours of culture to 642% and 468%
of the values seen at the same time-points in the untreated controls
(100%). The relative ineffectiveness of TNF- to induce apoptosis at
later time-points (22 hours and 34 hours) was due to the fact that the
majority of unstimulated cells already underwent apoptosis
spontaneously within this time period. GM-CSF induced its maximal
effect at 6 hours after the onset of stimulation and reduced apoptosis
to 38% of control, but significant inhibition of apoptosis was also observed at earlier (2 hours) and later (22 hours and 34 hours) time-points. The effects of TNF- and GM-CSF were dose dependent (data not shown).
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| Fig 2.
Regulation of apoptosis by cytokines as measured by flow
cytometry of propidium iodide-stained PMN. PMN were aged for 2, 6, 22, or 34 hours in culture without further stimulation (control), or
treated with 300 U/mL TNF- or 300 U/mL GM-CSF, respectively. Data
represent apoptotic PMN in percent of total cell number (top panel) or
apoptotic PMN in percent of the values seen in the untreated samples at
the same time points (middle and bottom panels). Mean ± SD of 12 independent experiments. *P < .05 versus unstimulated
control. The effects of TNF- and GM-CSF were dose-dependent (data
not shown).
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Regulation of apoptosis was also apparent by loss of Fc receptor
expression and internucleosomal DNA degradation.
Figure 3 shows a flow cytometric analysis
of CD16 (Fc receptor type III) expression on the cell surface of PMN.
This surface molecule has previously been shown to be downregulated in
PMN undergoing apoptosis.26 PMN freshly isolated from the
circulation showed a high surface expression of CD16. Within 2 hours
and 6 hours of culture, the population with low CD16 expression
increased gradually when PMN were aged in culture, and reached 81% of
total cell number within 22 hours. This effect was accelerated by
TNF-, which decreased CD16 expression substantially as early as 2 hours after stimulation and led to about 74% of PMN with low CD16
expression within this time period. In contrast, GM-CSF inhibited the
loss of high CD16 surface expression during culture and diminished the
population with low CD16 expression markedly when compared with control
cells.
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| Fig 3.
Induction or prevention of apoptosis by cytokines as
measured by analysis of CD16 expression. Flow cytometric analysis of
DNA content using the FITC-labeled anti-CD16 MoAb. PMN freshly isolated
from the circulation (dotted line) were stimulated for 2, 6, or 22 hours with 300 U/mL TNF-, 300 U/mL GM-CSF, or left untreated
(control), respectively. Numbers indicate the cells with low CD16
expression in percent of total cell number. Results are representative
of three independent experiments.
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As shown in Fig 4, similar results were
obtained by analysis of internucleosomal DNA degradation, a ubiquitious
marker for apoptosis.3 PMN were stimulated for 6 hours by
TNF-, GM-CSF, or left untreated, respectively. Unstimulated PMN
showed only weak DNA degradation within this time period whereas
TNF--stimulated cells revealed strong DNA degradation. Almost no
DNA laddering was detectable in GM-CSF-treated cells. Thus, three
independent methods showed that the experimental conditions used were
suitable to detect spontaneous apoptosis as well as induction or
prevention of apoptosis by the cytokines TNF- and GM-CSF,
respectively.
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| Fig 4.
Apoptosis of PMN measured by internucleosomal DNA
degradation. Agarose gel of low-molecular-weight DNA of PMN stimulated
for 6 hours with 300 U/mL TNF-, 300 U/mL GM-CSF, or left untreated
(control), respectively. Results are representative of three
independent experiments.
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Gene expression of members of the bcl-2 family.
Expression of members of the bcl-2 family of apoptosis-associated genes
was studied by RT-PCR of RNA derived from PMN freshly isolated from the
circulation. Using specific primers, bcl-2 was found to be absent in
PMN (Fig 5A). This was also true for
unstimulated, GM-CSF- or TNF--stimulated PMN after 2, 6, 22, or 34 hours of culture (data not shown). In contrast, bcl-2 was detectable in the pro-myeloid cell line HL-60 as well as in the B-cell line BL-41,
which was used for positive control.27 However,
bcl-Xl, another anti-apoptotic member of this gene family,
was detectable in PMN as well as in HL-60 cells and BL-41 cells,
respectively. Analysis of PMN for expression of pro-apoptotic genes,
which are known to heterodimerize with the bcl-Xl gene
product, gave positive results (Fig 5B): RT-PCR products for both
bax- and bak were found in PMN. Both genes were also expressed in
HL-60 cells as well as in BL-41 cells, respectively. Because
bcl-Xl as well as its pro-apoptotic counterparts bax-
and bak have the capability to regulate apoptosis, we studied their
expression by Western blotting technique.
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| Fig 5.
Analysis of mRNA expression of members of the bcl-2
family. Total RNA of PMN freshly isolated from the circulation, HL-60,
or BL41 cells for positive control, respectively, was subjected to
RT-PCR using primers specific for bcl-2 or bcl-X (A), as well as for
bax- or bak (B). PCR products on a 1.5% agarose gel are shown.
Results are representative of three independent experiments.
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Effect of cytokines on differential protein expression of members of
the Bcl-2 family.
PMN were cultured for 0, 2, 6, or 22 hours in the presence of TNF-
or GM-CSF, or were left untreated. Whole-cell lysates were subjected to
SDS-PAGE and immunoblotted with an anti-Bcl-X antibody
(Fig 6A). Whereas unstimulated freshly
isolated PMN showed high expression of Bcl-Xl, the protein
level decreased with time of culture, suggesting that the loss of the
protective effect of Bcl-Xl may play a role in spontaneous
apoptosis. The reduction of Bcl-Xl expression in the
presence of TNF- was much stronger when compared with control cells.
This suggests that the mechanism which allows TNF- to enhance
apoptosis may be associated with an accelerated downregulation of
Bcl-Xl. In contrast, the level of Bcl-Xl in
GM-CSF-stimulated PMN was not altered when compared with unstimulated
cells, indicating that the inhibition of apoptosis by this cytokine is
not mediated by (up-)regulation of Bcl-Xl expression.
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| Fig 6.
Differential expression of Bcl-Xl and Bax-
upon induction or prevention of apoptosis by cytokines. PMN were
stimulated for indicated times with 300 U/mL TNF-, 300 U/mL GM-CSF,
or left untreated (control), respectively. Whole-cell lysates were
subjected to SDS-PAGE and Western blot was performed using the
anti-Bcl-X (A) or anti-Bax- Abs and a peroxidase-conjugated
secondary antibody (B). Numbers indicate mean OD of each lane obtained
from three representative and independent experiments. Bak was present
in BL41 cells but not detectable in PMN at any time point (data not
shown).
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Next, protein expression of Bax- was studied (Fig 6B). Whereas
unstimulated PMN showed a gradual decrease of Bax- expression with
time of culture, protein expression remained almost constant upon
stimulation with TNF-. In contrast, GM-CSF induced a downregulation of Bax- when compared with control cells, suggesting that the downregulation of this death-promoting protein is involved in PMN
survival mediated by GM-CSF. Although present in BL41 cells, Bak was
not detectable in PMN, which may reflect a low expression of this
protein (data not shown). Thus, rather differential expression of
Bcl-Xl and Bax-, which occurred during induction, or
prevention of apoptosis by cytokines may be critical for control of
apoptosis: Whereas TNF- seems to alter the
Bax-/Bcl-Xl ratio by downregulating Bcl-Xl
and stabilizing Bax- expression, and thereby may promote apoptosis,
GM-CSF seems to promote survival by modulating the Bax-/Bcl-Xl ratio via downregulation of Bax-. Data
were confirmed by analysis of protein expression using densitometry.
To further characterize the balance of Bax- and Bcl-Xl
in PMN undergoing apoptosis, the Bax-/Bcl-Xl ratio was
calculated using the mean optical density (OD) of Bax-
and Bcl-Xl obtained from three independent experiments
(Fig 7). During spontaneous apoptosis a
transient increase of the Bax-/Bcl-Xl ratio occurred within 2 hours of culture, which may account for induction of spontaneous apoptosis. When compared with control cells, TNF- led to
a sustained increase of the Bax-/Bcl-Xl ratio within 2 hours when compared with control cells. In contrast, a decrease of the
Bax-/Bcl-Xl ratio was observed in the presence of GM-CSF within 2 hours and 6 hours of culture when compared with the ratio obtained from unstimulated control cells at the same time-points. Thus,
the Bax-/Bcl-Xl ratio may be responsible for the
induction or prevention of apoptosis. To test the hypothesis that the
altered Bax-/Bcl-Xl ratio may represent the functional
relevant check-point for the regulation apoptosis, the activation of
caspase-3 was studied, which finally executes the apoptotic program in
PMN.28
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| Fig 7.
Alteration of the Bax-/Bcl-Xl ratio upon
induction or prevention of apoptosis. The Bax-/Bcl-Xl
ratio was calculated from mean OD of Bax- and Bcl-Xl
obtained in the three independent cell experiments described in Fig
6.
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Activation of caspase-3 in PMN undergoing apoptosis.
PMN were cultured for 2, 6, or 22 hours in the presence of TNF- or
GM-CSF, or were left untreated. Whole-cell lysates were subjected to
SDS-PAGE and immunoblotted with a caspase-3 antibody. Results were
confirmed by densitometry (Fig 8A). The
expression of the proteolytically inactive pro-caspase of 32 kD was substantially decreased in the presence of TNF-
when compared with unstimulated control cells at the same time points
(2, 6, and 22 hours), suggesting an enhanced cleavage into the active
form. In contrast, an increased pro-caspase level was observed in the
presence of GM-CSF when compared to control cells, indicating that
GM-CSF may exert its protective effect by inhibiting proteolytic
cleavage of the pro-caspase-3 into the active form. To confirm this
result, the enzymatic activity of caspase-3 was measured in the cell
lysates obtained from unstimulated control cells as well as from
TNF-- and GM-CSF-stimulated cells, respectively (Fig 8B). Using a
fluorogenic substrate bearing the caspase-3-specific cleavage site
DEVD, caspase-3 activity was detected as early as 2 hours after the
onset of culture in unstimulated control cells. Activity of caspase-3
was markedly enhanced upon stimulation by TNF-. As expected, GM-CSF
induced a downregulation of caspase-3 activity when compared with
unstimulated control cells. In all samples, caspase-3 activity was
almost completely inhibited after pretreatment of the cells with 200 µmol/L of the specific caspase-3 inhibitor Z-DEVD-FMK, demonstrating
the specificity of the employed assay. This shows that the modulatory
role of the cytokines on the processing of the pro-caspase corresponded to the enzymatic activity of caspase-3 in the cell lysates. Thus, two
independent methods showed that the cytokine-mediated alteration of the
Bax-/Bcl-Xl ratio resulted in the modulation of
caspase-3 activity.
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| Fig 8.
Effect of cytokines on caspase-3 activation. PMN were
stimulated for indicated times with 300 U/mL TNF-, 300 U/mL GM-CSF,
or left untreated (control), respectively. (A) After stimulation for
indicated times, whole-cell lysates were subjected to SDS-PAGE and
Western blot was performed using the anti-caspase-3 Ab and a
peroxidase-conjugated secondary antibody. Numbers indicate mean OD of
each lane obtained from three representative and independent
experiments. (B) Caspase-3 activity was measured in whole-cell lysates
obtained from PMN, which were treated for 1 hour at 37°C with 200 µmol/L of the caspase-3 inhibitor Z-DEVD-FMK or left untreated
(vehicle) before stimulation for 2 hours as indicated. Caspase-3
activity is shown as mean fluorescence intensity ± SD obtained from
three independent experiments. *P < .05; #P < .05 versus unstimulated control; n.s., not significant.
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Next, additional experiments were performed to elucidate the molecular
mechanism that underlies the alteration of the
Bax-/Bcl-Xl ratio, ie, the downregulation of
Bcl-Xl upon TNF- stimulation. Because caspase-3 has
recently been shown to convert the anti-apoptotic Bcl-2 protein, a
homologue of Bcl-Xl, into a death-promoting
factor,29 the effect of caspase-3 inhibition was studied on
the expression of Bcl-Xl (Fig
9). PMN were treated with 200 µmol/L of the cell-permeable caspase-3
inhibitor Z-DEVD-FMK for 1 hour at 37°C and were stimulated with
TNF- or GM-CSF, or left untreated. Subsequent Western blot analysis
of Bcl-Xl expression showed that spontaneous as well as
TNF--induced downregulation of Bcl-Xl was almost
completely abolished in the presence of the caspase-3 inhibitor
(compare with Fig 6A). This suggests that the observed downregualtion
of Bcl-Xl is due to an enhanced protein turnover, probably
via direct proteolytic cleavage of Bcl-Xl by caspase-3.
Thus, caspase-3 seems to play a pivotal role in mediating apoptosis of
human PMN.
View larger version (21K):
[in this window]
[in a new window]
| Fig 9.
Effect of caspase-3 inhibition on expression of
Bcl-Xl. PMN were treated for 1 hour at 37°C with 200 µmol/L of the caspase-3 inhibitor Z-DEVD-FMK before stimulation for
indicated times with 300 U/mL TNF-, 300 U/mL GM-CSF, or left
untreated (control), respectively. Whole-cell lysates were subjected to
SDS-PAGE and Western blot was performed using the anti-Bcl-X Ab and a
peroxidase-conjugated secondary antibody. Results are representative of
three independent experiments.
|
|
To prove the biological significance of caspase-3 activation, apoptosis
of PMN was measured in the presence of the caspase-3 inhibitor
Z-DEVD-FMK (Fig 10). After treatment of
the PMN with 200 µmol/L the caspase-3 inhibitor for 1 hour at
37°C, apoptosis was almost reduced to the level observed in freshly
isolated PMN. This was true for both, spontaneous as well as
TNF--induced apoptosis of PMN, and shows that activation of
caspase-3 is critical for the induction of apoptosis. As expected, no
further reduction of apoptosis was observed when PMN were stimulated
with GM-CSF after inhibition of caspase-3, suggesting that inhibition
of apoptosis by this cytokine may be due to prevention of caspase-3
activation. Thus, alteration of the Bax-/Bcl-Xl ratio
seems to represent the key event in the regulation of apoptosis of
human PMN by controlling the execution of the apoptotic program via
caspase-3.
View larger version (28K):
[in this window]
[in a new window]
| Fig 10.
Prevention of apoptosis by inhibition of caspase-3. PMN
were treated for 1 hour at 37°C with 200 µmol/L of the caspase-3
inhibitor Z-DEVD-FMK or left untreated (vehicle) before stimulation for
2 hours with 300 U/mL TNF-, 300 U/mL GM-CSF, or left untreated
(control), respectively. Data represent apoptotic PMN in percent of
total cell number. Mean ± SD of three independent experiments.
*P < .05; n.s., not significant.
|
|
| |
DISCUSSION |
In the present study, a mechanism that can determine the lifetime of
PMN is reported. Gene expression of bcl-Xl, bax-, and bak, members of the bcl-2 family that is known to control the apoptotic
machinery, were identified in mature human PMN isolated from the
circulation. The anti-apoptotic bcl-Xl gene as well as the
death-promoting bax- gene were also found in significant amounts at
the protein level. Although present in control cells, expression of bak
was not detectable in PMN at the protein level, suggesting that its
expression may be rather low when compared with Bcl-Xl and
Bax-. The myeloid cell line HL-60 was not only positive for
expression of the three above-mentioned genes, but also for bcl-2 mRNA,
which was absent in mature PMN. This is in agreement with a previous
finding showing that bcl-2 is downregulated in HL-60 cells upon
granulocytic differentiation.30 The presence of bcl-2
expression in pro-myeloid cells and the absence in mature PMN is
consistent with the observation that bcl-2 is downregulated, eg, in
lymphocytes under negative selection but highly expressed in pro B
cells and mature B cells that are selected to survive.31 Thus, the absence of bcl-2 expression can be interpreted as
determination to undergo apoptosis. However, the expression of other
members of the bcl-2 family of apoptosis-associated genes implies that mature PMN do not simply die due to the absence of bcl-2 expression, but that the apoptotic programat least its time-courseis subject to
regulation. Bcl-Xl shows some functional redundancy with
Bcl-2 in suppressing cell death, but only Bcl-Xl, and not
Bcl-2, was observed to inhibit apoptosis in WEHI-231.7 cells in
response to cross-linking of IgM and other stimuli.32
Similarly, Bcl-Xl may fulfill a special anti-apoptotic
function in mature human PMN. However, both Bcl-2 and
Bcl-Xl are known to act as anti-apoptotic counterparts of
Bax-,33 and this may explain the observation that
expression of bcl-2 in PMN of transgenic mice prevents apoptosis, although it is absent in normal PMN.20
Both the anti-apoptotic Bcl-Xl, the death-antagonizing
splice variant of the bcl-X gene, and the pro-apoptic Bax-, the
death-promoting splice variant of the bax gene, are known to form
homodimers and heterodimers, respectively.33 The shift of
balance of the Bax-/Bcl-Xl ratiowhich can be achieved
by upregulation or downregulation of both interacting partnershas
previously been shown to determine survival and apoptosis in other cell
systems.34 In our system, the survival factor GM-CSF
induced a downregulation of pro-apoptotic Bax-, and TNF- led to a
decrease of survival-promoting Bcl-Xl. Thus, a
downregulation of the death or survival promoting proteins seems to
modulate the Bax-/Bcl-Xl ratio in PMN.
When compared with other leukocytes, the life span of PMN is extremely
short, and its regulation was found to occur within hours upon cytokine treatment. Downregulation may provide an appropriate mechanism that is
fast enough to meet this requirement.
The fact that downregulation of Bcl-Xl was almost
completely abolished upon inhibition of caspase-3 suggests that
caspase-3 is critically involved in the regulation of
Bcl-Xl expression. Thus, rather an enhanced turnover of
Bcl-Xl than its transcriptional regulation seems to control
the level of Bcl-Xl expression. This is
consistent with previous findings showing that the death-preventing protein Bcl-2a close homologue of Bcl-Xlserves as a
substrate for caspase-3 in cells undergoing apoptosis. Moreover, the
cleavage product of Bcl-2 was found to exert a death-promoting
function, which further strengthened the apoptotic
process.29 Thus, the enhanced downregulation of
Bcl-Xl expression, which promoted the shift of the
Bax-/Bclxl ratio and induced apoptosis upon TNF- stimulation, presumably underlies direct regulation by caspase-3. This
observation not only elucidates the molecular mechanism that may
underlie the regulation of Bcl-Xl downregulation, but also may explain the acceleration of apoptotic cell death upon TNF- stimulation by providing a powerful feed-forward mechanism.
Similarly, this may be relevant for spontaneous apoptosis, which was
also found to be associated with a downregulation Bcl-Xl. However, Bax- expression was also downregulated upon spontaneous apoptosis, suggesting the existence of a multi-step control mechanism. The fact that PMN underwent apoptosis spontaneously suggests an excess
of death-promoting factors when PMN age in culture. The observed
decrease of Bcl-Xl may enhance the overall susceptibility of PMN to undergo apoptosis, and the increase of the
Bax-/Bcl-Xl ratio may induce spontaneous apoptosis.
Also, a possible role of other yet unidentified members of the Bcl-2
family has to be considered to contribute to the regulation of
apoptosis of PMN besides Bax- and Bcl-Xl.
In general, apoptosis is divided into three different steps. The
initiation phase, which allows the intracellular signaling, precedes
the effector phase, which includes regulation of apoptosis by the Bcl-2
family. Subsequently, irreversible cell death occurs upon
activation of caspases, which function as the executers of apoptosis.35 The present study gives evidence that
caspase-3 may be involved in the final degradation process in apoptosis of PMN, which is in agreement with previous findings.22
Caspase-3 activation is generally thought to requirebesides two
further apoptosis protease-activating factors (Apafs-1 and -3)the
release of cytochrome c (Apaf-2) from mitochondria into the cytosol.
This is thought to be regulated by the proteins of the Bcl-2
family associated with the mitochondrial membrane.36,37
But there exists some evidence that caspase-3 may be also activated via Bcl-Xl independently of cytochrome c.38
Activation of caspase-3 has recently been shown to be required for DNA
fragmentation in cells undergoing apoptosis in response to
TNF-,39 which is consistent with our finding that
TNF- promotes DNA laddering in human PMN. The observed
downregulation of the 32-kD pro-enzyme is well known to reflect the
activation of the enzyme, which is due to proteolytic cleavage in two
fragments with molecular masses of 20 kD and 11 kD,
respectively.40,41 The fact that processing of the
pro-caspase was promoted in the presence of TNF- and inhibited in
the presence of GM-CSF suggests that induction or prevention of
apoptosis by these cytokines depends on activation of caspase-3. This
was confirmed by direct measurement of caspase-3 activity, which showed
that TNF- and GM-CSF inversely regulate its proteolytic activity
toward a specific DEVD-containing substrate. Thus, this study not only
gives evidence that caspase-3 activation is critical for the final
degradation process of apoptosis as well as for the downregulation of
Bcl-Xl expression, but it also shows that the functional
endpoint of GM-CSF-mediated inhibition of apoptosis involves the
Bax-/Bcl-Xl-mediated prevention of caspase-3
activation. Thus, the critical checkpoint for both, induction as well
as the prevention of apoptosis, seems to represent the control of
caspase-3. The biological relevance of this pathway was confirmed by
the finding that inhibition of caspase-3 activity almost completely
prevented apoptosis of human PMN.
In summary, induction of apoptosis by TNF- was found to be
associated with a reduction of expression of the anti-apoptotic Bcl-Xl protein whereas prevention of apoptosis by GM-CSF
led to a downregulation of expression of the pro-apoptotic Bax-
protein. This shift of balance of the Bax-/Bcl-Xl ratio
led to an induction of caspase-3 activation by TNF- and a prevention
of caspase-3 activation in the presence of GM-CSF. Thus, cytokines
affected the ratio of Bax-/Bcl-Xl expression and
modulated the subsequent activity of caspase-3, which function as
executers of the programmed cell death. The regulation of the
Bcl-Xl/Bax- ratio critically involves activation of
caspase-3, which downregulated Bcl-Xl, probably by direct
proteolytic cleavage, and thereby provides a positive feedback
mechanism that may be responsible for the TNF--mediated
acceleration of apoptosis. The reported
Bax-/Bcl-Xl-mediated control of apoptosis via caspase-3
seems to represent an important checkpoint with strong biological
relevance, because apoptosis was almost completely absent upon
inhibition of this pathway.
| |
ACKNOWLEDGMENT |
The expert technical assistance of S. Sölter is acknowledged.
| |
FOOTNOTES |
Submitted August 24, 1998; accepted December 22, 1998.
Supported by Deutsche Forschungsgemeinschaft (SFB 366/C3).
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 Barbara Walzog, PhD, Freie
Universität Berlin, Department of Physiology, Arnimallee 22, D-14195 Berlin, Germany.
| |
REFERENCES |
1.
Gregory CD
(ed):
Apoptosis and the immune system. New York, NY, Wiley-Liss, 1995.
2.
Kerr JFR, Wyllie AH, Currie AR:
Apoptosis: A basic phenomenon with wide-ranging implications in tissue kinetics.
Br J Cancer
26:239, 1972[Medline]
[Order article via Infotrieve]
3.
Wyllie AH:
Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.
Nature
284:555, 1980[Medline]
[Order article via Infotrieve]
4.
Squier MKT, Sehnert AJ, Cohen JJ:
Apoptosis in leukocytes.
J Leukoc Biol
57:2, 1995[Abstract]
5.
Payne CM, Glasser L, Tischler ME, Wyckoff D, Cromey D, Fiederlein R, Bohnert O:
Programmed cell death of the normal human neutrophil: An in vitro model of senescence.
Microsc Res Tech
28:327, 1994[Medline]
[Order article via Infotrieve]
6.
Whyte MKB, Meagher LC, MacDermot J, Haslett C:
Impairment of function in aging neutrophils is associated with apoptosis.
J Immunol
150:5124, 1993[Abstract]
7.
Savill J, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C:
Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.
J Clin Invest
83:865, 1989
8.
Hall SE, Savill JS, Henson PM, Haslett C:
Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin.
J Immunol
153:3218, 1994[Abstract]
9.
Cox G, Gauldie J, Jordana M:
Bronchial epithelial cell-derived cytokines (G-CSF and GM-CSF) promote the survival of peripheral blood neutrophils in vitro.
Am J Respir Cell Mol Biol
7:507, 1992
10.
Chitinis D, Dickerson C, Munster AM, Winchurch RA:
Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients.
J Leukoc Biol
59:835, 1996[Abstract]
11.
Ohata H, Yatomi Y, Sweeney EA, Hakomori S, Igarashi Y:
A possible role of sphingosine in induction of apoptosis by tumor necrosis factor- in human neutrophils.
FEBS Lett
355:267, 1994[Medline]
[Order article via Infotrieve]
12.
Coxon A, Rieu P, Barkalow FJ, Askari S, Sharpe AH, von Andrian UH, Arnaout MA, Mayadas TN:
A novel role for the  2 integrin CD11b/CD18 in neutrophil apoptosis: A homeostatic mechanism in inflammation.
Immunity
5:653, 1996[Medline]
[Order article via Infotrieve]
13.
Walzog B, Jeblonski F, Zakrzewicz A, Gaehtgens P:
2 integrins (CD11/CD18) promote apoptosis of human neutrophils.
FASEB J
11:1177, 1997[Abstract]
14.
Wei S, Liu JH, Epling-Burnette PK, Gamero AM, Ussery D, Pearson EW, Elkabani ME, Diaz JI, Djieu JY:
Critical role for Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor.
J Immunol
157:5155, 1996[Abstract]
15.
Lagasse E, Weissman IL:
Bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages.
J Exp Med
179:1047, 1994[Abstract/Free Full Text]
16.
Kroemer G:
The proto-oncogene Bcl-2 and its role in regulating apoptosis.
Nat Med
3:614, 1997[Medline]
[Order article via Infotrieve]
17.
Oltvai ZN, Korsmeyer SJ:
Checkpoints of dueling dimers foil death wishes.
Cell
79:189, 1994[Medline]
[Order article via Infotrieve]
18.
Vaux DL, Cory S, Adams JM:
Bcl-2 gene promotes hematopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.
Nature
335:440, 1988[Medline]
[Order article via Infotrieve]
19.
Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindstan T, Turka LA, Mao X, Nunez G, Thompson CB:
bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.
Cell
74:597, 1993[Medline]
[Order article via Infotrieve]
20.
Oltvai ZN, Milliman CL, Korsmeyer SJ:
Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death.
Cell
74:609, 1993[Medline]
[Order article via Infotrieve]
21.
Kroemer G, Dallaporta B, Resche-Rigon M:
The mitochondrial death/life regulator in apoptosis and necrosis.
Annu Rev Physiol
60:619, 1998[Medline]
[Order article via Infotrieve]
22.
Sanghavi DM, Thelen M, Thornberry NA, Casciola-Rosen L, Rosen A:
Caspase-mediated proteolysis during apoptosis: Insight from apoptotic neutrophils.
FEBS Lett
422:179, 1998[Medline]
[Order article via Infotrieve]
23.
Hjorth R, Jonsson AK, Vretblad P:
A rapid method for purification of human granulocytes using Percoll. A comparison with dextran sulfate.
J Immunol Methods
43:95, 1981[Medline]
[Order article via Infotrieve]
24.
Chomczynski P, Sacchini N:
Single-step method of RNA isolation by acid guanidinium-phenol-chloroform extraction.
Anal Biochem
162:156, 1987[Medline]
[Order article via Infotrieve]
25.
Laemmli UK:
Cleavage of structural proteins during assembly of the head of bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
26.
Dransfield I, Buckle A-M, Savill JS, Mcdowall A, Haslett C, Hogg N:
Neutrophil apoptosis is associated with a reduction in CD16 (FcgammaRIII) expression.
J Immunol
153:1254, 1994[Abstract]
27.
Bargou R, Bommert K, Weinmann P, Daniel P, Wagener PT, Mapara MY, Dörken B:
Induction of Bax- precedes apoptosis in a human B lymphoma cell line: Potential role of the bcl-2 gene family in surface IgM-mediated apoptosis.
Eur J Immunol
25:770, 1995[Medline]
[Order article via Infotrieve]
28.
Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwaiatkowski DJ, Williams LT:
Caspase-3-generated fragment of gelsolin: Effector of morphological change in apoptosis.
Science
278:294, 1997[Abstract/Free Full Text]
29.
Cheng EH-Y, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM:
Conversion of Bcl-2 to a Bax-like death effector by caspases.
Science
278:1966, 1998[Abstract/Free Full Text]
30.
Benito A, Grillot D, Nunez G, Fernandez-Luna JL:
Regulation and function of Bcl-2 during differentiation-induced cell death in HL-60 promyelocytic cells.
Am J Pathol
146:481, 1995[Abstract]
31.
Li YS, Hayakawa K, Hardy RR:
The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver.
J Exp Med
178:951, 1993[Abstract/Free Full Text]
32.
Gottschalk AR, Boise LH, Thompson CB, Quintans J:
Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not bcl-2.
Proc Natl Acad Sci USA
91:7350, 1994[Abstract/Free Full Text]
33.
Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, Korsmeyer SJ:
Multiple Bcl-2 family members demonstrate selective dimerization with Bax.
Proc Natl Acad Sci USA
92:7834, 1995[Abstract/Free Full Text]
34.
Yang E, Korsmeyer SJ:
Molecular thanatopsis: A discourse on the BCL2 family and cell death.
Blood
88:386, 1996[Free Full Text]
35.
Nicholson DW, Thornberry NA:
Caspases: killer proteases.
Trends Biochem Sci
22:299, 1997[Medline]
[Order article via Infotrieve]
36.
Reed JC:
Cytochrome c: Can't live with itCan't live without it.
Cell
91:559, 1997[Medline]
[Order article via Infotrieve]
37.
Zou H, Henzel WJ, Lui X, Lutschg A, Wang X:
Apaf-1, a human homologous to C. elegans CED4, participates in cytochrome C-dependent activation of caspase-3.
Cell
90:405, 1997[Medline]
[Order article via Infotrieve]
38.
Li F, Srinivasan A, Wang Y, Armstrong RC, Tomaselli KJ, Fritz LC:
Cell-specific induction of apoptosis by microinjection of cytochrome C. Bcl-xL has activity independent of cytochrome c.
J Biol Chem
272:30299, 1997[Abstract/Free Full Text]
39.
Janicke RU, Sprengart ML, Wati MR, Porter AG:
Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis.
J Biol Chem
273:9357, 1998[Abstract/Free Full Text]
40.
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, Miller DK:
Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.
Nature
376:37, 1995[Medline]
[Order article via Infotrieve]
41.
Fernadez-Alnemri T, Litwak G, Alnemri ES:
CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme.
J Biol Chem
269:30761, 1994[Abstract/Free Full Text]
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|
|
H. Koller, K. Hochegger, G. J. Zlabinger, K. Lhotta, G. Mayer, and A. R. Rosenkranz
Apoptosis of human polymorphonuclear neutrophils accelerated by dialysis membranes via the activation of the complement system
Nephrol. Dial. Transplant.,
December 1, 2004;
19(12):
3104 - 3111.
[Abstract]
[Full Text]
[PDF]
|
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P. Boutet, D. Boulanger, L. Gillet, A. Vanderplasschen, R. Closset, F. Bureau, and P. Lekeux
Delayed Neutrophil Apoptosis in Bovine Subclinical Mastitis
J Dairy Sci,
December 1, 2004;
87(12):
4104 - 4114.
[Abstract]
[Full Text]
[PDF]
|
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S. Doi, H. Soda, M. Oka, J. Tsurutani, T. Kitazaki, Y. Nakamura, M. Fukuda, Y. Yamada, S. Kamihira, and S. Kohno
The histone deacetylase inhibitor FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway in small cell lung cancer cells
Mol. Cancer Ther.,
November 1, 2004;
3(11):
1397 - 1402.
[Abstract]
[Full Text]
[PDF]
|
|
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|
|
|
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C. C. Yost, M. M. Denis, S. Lindemann, F. J. Rubner, G. K. Marathe, M. Buerke, T. M. McIntyre, A. S. Weyrich, and G. A. Zimmerman
Activated Polymorphonuclear Leukocytes Rapidly Synthesize Retinoic Acid Receptor-{alpha}: A Mechanism for Translational Control of Transcriptional Events
J. Exp. Med.,
September 7, 2004;
200(5):
671 - 680.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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V. Asensi, E. Valle, A. Meana, J. Fierer, A. Celada, V. Alvarez, J. Paz, E. Coto, J. A. Carton, J. A. Maradona, et al.
In Vivo Interleukin-6 Protects Neutrophils from Apoptosis in Osteomyelitis
Infect. Immun.,
July 1, 2004;
72(7):
3823 - 3828.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
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F. Altznauer, S. Martinelli, S. Yousefi, C. Thurig, I. Schmid, E. M. Conway, M. H. Schoni, P. Vogt, C. Mueller, M. F. Fey, et al.
Inflammation-associated Cell Cycle-independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils
J. Exp. Med.,
May 17, 2004;
199(10):
1343 - 1354.
[Abstract]
[Full Text]
[PDF]
|
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|
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S. J. Gardai, D. A. Hildeman, S. K. Frankel, B. B. Whitlock, S. C. Frasch, N. Borregaard, P. Marrack, D. L. Bratton, and P. M. Henson
xPhosphorylation of Bax Ser184 by Akt Regulates Its Activity and Apoptosis in Neutrophils
J. Biol. Chem.,
May 14, 2004;
279(20):
21085 - 21095.
[Abstract]
[Full Text]
[PDF]
|
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|
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C. D. Garlichs, S. Eskafi, I. Cicha, A. Schmeisser, B. Walzog, D. Raaz, C. Stumpf, A. Yilmaz, J. Bremer, J. Ludwig, et al.
Delay of neutrophil apoptosis in acute coronary syndromes
J. Leukoc. Biol.,
May 1, 2004;
75(5):
828 - 835.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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F. Goepel, P. Weinmann, J. Schymeinsky, and B. Walzog
Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis
J. Leukoc. Biol.,
May 1, 2004;
75(5):
836 - 843.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
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F. Altznauer, S. Conus, A. Cavalli, G. Folkers, and H.-U. Simon
Calpain-1 Regulates Bax and Subsequent Smac-dependent Caspase-3 Activation in Neutrophil Apoptosis
J. Biol. Chem.,
February 13, 2004;
279(7):
5947 - 5957.
[Abstract]
[Full Text]
[PDF]
|
|
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|
|
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S. A. Madsen, L.-C. Chang, M.-C. Hickey, G. J. M. Rosa, P. M. Coussens, and J. L. Burton
Microarray analysis of gene expression in blood neutrophils of parturient cows
Physiol Genomics,
January 15, 2004;
16(2):
212 - 221.
[Abstract]
[Full Text]
[PDF]
|
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e. Moisan, e. Kouassi, and D. Girard
Mechanisms involved in methylmercuric chloride (MeHgCl)-induced suppression of human neutrophil apoptosis
Human and Experimental Toxicology,
December 1, 2003;
22(12):
629 - 637.
[Abstract]
[PDF]
|
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S. Faderl, D. Harris, Q. Van, H. M. Kantarjian, M. Talpaz, and Z. Estrov
Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces antiapoptotic and proapoptotic signals in acute myeloid leukemia
Blood,
July 15, 2003;
102(2):
630 - 637.
[Abstract]
[Full Text]
[PDF]
|
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|
|
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M. Miyamoto, M. Emoto, Y. Emoto, V. Brinkmann, I. Yoshizawa, P. Seiler, P. Aichele, E. Kita, and S. H. E. Kaufmann
Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17
J. Immunol.,
May 15, 2003;
170(10):
5228 - 5234.
[Abstract]
[Full Text]
[PDF]
|
|
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|
|
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T. Hasegawa, K. Suzuki, C. Sakamoto, K. Ohta, S. Nishiki, M. Hino, N. Tatsumi, and S. Kitagawa
Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia
Blood,
February 1, 2003;
101(3):
1164 - 1171.
[Abstract]
[Full Text]
[PDF]
|
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P. Weinmann, K. Scharffetter-Kochanek, S. B. Forlow, T. Peters, and B. Walzog
A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice
Blood,
January 15, 2003;
101(2):
739 - 746.
[Abstract]
[Full Text]
[PDF]
|
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C.-Y. Liu, A. Takemasa, W. C. Liles, R. B. Goodman, M. Jonas, H. Rosen, E. Chi, R. K. Winn, J. M. Harlan, and P. I. Chuang
Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils
Blood,
January 1, 2003;
101(1):
295 - 304.
[Abstract]
[Full Text]
[PDF]
|
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K. Yasui, Y. Sekiguchi, M. Ichikawa, H. Nagumo, T. Yamazaki, A. Komiyama, and H. Suzuki
Granulocyte macrophage-colony stimulating factor delays neutrophil apoptosis and primes its function through Ia-type phosphoinositide 3-kinase
J. Leukoc. Biol.,
November 1, 2002;
72(5):
1020 - 1026.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. S. Cowburn, K. A. Cadwallader, B. J. Reed, N. Farahi, and E. R. Chilvers
Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival
Blood,
September 18, 2002;
100(7):
2607 - 2616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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N. Perskvist, M. Long, O. Stendahl, and L. Zheng
Mycobacterium tuberculosis Promotes Apoptosis in Human Neutrophils by Activating Caspase-3 and Altering Expression of Bax/Bcl-xL Via an Oxygen-Dependent Pathway
J. Immunol.,
June 15, 2002;
168(12):
6358 - 6365.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Lavastre, M. Pelletier, R. Saller, K. Hostanska, and D. Girard
Mechanisms Involved in Spontaneous and Viscum album Agglutinin-I-Induced Human Neutrophil Apoptosis: Viscum album Agglutinin-I Accelerates the Loss of Antiapoptotic Mcl-1 Expression and the Degradation of Cytoskeletal Paxillin and Vimentin Proteins Via Caspases
J. Immunol.,
February 1, 2002;
168(3):
1419 - 1427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. A. Maianski, F. P. J. Mul, J. D. van Buul, D. Roos, and T. W. Kuijpers
Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils
Blood,
January 15, 2002;
99(2):
672 - 679.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Moulding, C. Akgul, M. Derouet, M. R. H. White, and S. W. Edwards
BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis
J. Leukoc. Biol.,
November 1, 2001;
70(5):
783 - 792.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Durand, Y. Renaudineau, J.-O. Pers, P. Youinou, and C. Jamin
Cross-Linking of Human Fc{gamma}RIIIb Induces the Production of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor by Polymorphonuclear Neutrophils
J. Immunol.,
October 1, 2001;
167(7):
3996 - 4007.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. K. Epling-Burnette, B. Zhong, F. Bai, K. Jiang, R. D. Bailey, R. Garcia, R. Jove, J. Y. Djeu, T. P. Loughran Jr., and S. Wei
Cooperative Regulation of Mcl-1 by Janus Kinase/STAT and Phosphatidylinositol 3-Kinase Contribute to Granulocyte-Macrophage Colony-Stimulating Factor-Delayed Apoptosis in Human Neutrophils
J. Immunol.,
June 15, 2001;
166(12):
7486 - 7495.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. V. B. K. Subrahmanyam, S. Yamaga, Y. Prashar, H. H. Lee, N. P. Hoe, Y. Kluger, M. Gerstein, J. D. Goguen, P. E. Newburger, and S. M. Weissman
RNA expression patterns change dramatically in human neutrophils exposed to bacteria
Blood,
April 15, 2001;
97(8):
2457 - 2468.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Suzuki, T. Hasegawa, C. Sakamoto, Y.-M. Zhou, F. Hato, M. Hino, N. Tatsumi, and S. Kitagawa
Cleavage of Mitogen-Activated Protein Kinases in Human Neutrophils Undergoing Apoptosis: Role in Decreased Responsiveness to Inflammatory Cytokines
J. Immunol.,
January 15, 2001;
166(2):
1185 - 1192.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Tudan, J. K. Jackson, L. Blanis, S. L. Pelech, and H. M. Burt
Inhibition of TNF-{alpha}-Induced Neutrophil Apoptosis by Crystals of Calcium Pyrophosphate Dihydrate Is Mediated by the Extracellular Signal-Regulated Kinase and Phosphatidylinositol 3-Kinase/Akt Pathways Up-Stream of Caspase 3
J. Immunol.,
November 15, 2000;
165(10):
5798 - 5806.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. B. Rucker III, P. Dierisseau, K.-U. Wagner, L. Garrett, A. Wynshaw-Boris, J. A. Flaws, and L. Hennighausen
Bcl-x and Bax Regulate Mouse Primordial Germ Cell Survival and Apoptosis during Embryogenesis
Mol. Endocrinol.,
July 1, 2000;
14(7):
1038 - 1052.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. B. Brown, M. C. H. Clarke, L. Magowan, H. Sanderson, and J. Savill
Constitutive Death of Platelets Leading to Scavenger Receptor-mediated Phagocytosis. A CASPASE-INDEPENDENT CELL CLEARANCE PROGRAM
J. Biol. Chem.,
February 25, 2000;
275(8):
5987 - 5996.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Dibbert, M. Weber, W. H. Nikolaizik, P. Vogt, M. H. Schoni, K. Blaser, and H.-U. Simon
Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: A general mechanism to accumulate effector cells in inflammation
PNAS,
November 9, 1999;
96(23):
13330 - 13335.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. WALZOG, P. WEINMANN, F. JEBLONSKI, K. SCHARFFETTER-KOCHANEK, K. BOMMERT, and P. GAEHTGENS
A role for {beta}2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response
FASEB J,
October 1, 1999;
13(13):
1855 - 1865.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. G. Pryde, A. Walker, A. G. Rossi, S. Hannah, and C. Haslett
Temperature-dependent Arrest of Neutrophil Apoptosis. FAILURE OF Bax INSERTION INTO MITOCHONDRIA AT 15 {degrees}C PREVENTS THE RELEASE OF CYTOCHROME c
J. Biol. Chem.,
October 20, 2000;
275(43):
33574 - 33584.
[Abstract]
[Full Text]
[PDF]
|
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TOP
ABSTRACT
INTRODUCTION