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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-02-0522.
 Previous Article | Table of Contents | Next Article 
Blood, 1 March 2003, Vol. 101, No. 5, pp. 1987-1995
PHAGOCYTES
Tumor necrosis factor  induces a caspase-independent death
pathway in human neutrophils
Nikolai A. Maianski,
Dirk Roos, and
Taco W. Kuijpers
From the Sanquin Research at Central Laboratory of the
Netherlands Blood Transfusion Service, Landsteiner Laboratory, and Emma
Children's Hospital, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands; and Medical Academy, Nizhniy
Novgorod, Russia.
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Abstract |
Tumor necrosis factor  (TNF-) is a cytokine with
multiple roles in the immune system, including the induction and
potentiation of cellular functions in neutrophils (PMNs). TNF- also
induces apoptotic signals leading to the activation of several
caspases, which are involved in different steps of the process of cell
death. Inhibition of caspases usually increases cell survival. Here, we
found that inhibition of caspases by the general caspase inhibitor zVAD-fmk did not prevent TNF--induced PMN death. After 6 hours of
incubation, TNF- alone caused PMN death with characteristic apoptotic features (typical morphologic changes, DNA laddering, external phosphatidyl serine [PS] exposure in the plasma membrane, Bax clustering and translocation to the mitochondria, and degradation of mitochondria), which coincided with activation of caspase-8 and
caspase-3. However, in the presence of TNF-, PMNs died even when
caspases were completely inhibited. This type of cell death lacked nuclear features of apoptosis (ie, no DNA laddering but aberrant
hyperlobulated nuclei without typical chromatin condensation) and
demonstrated no Bax redistribution, but it did show mitochondria clustering and plasma membrane PS exposure. In contrast, Fas-triggered PMN apoptosis was completely blocked by zVAD-fmk. Experiments with
scavengers of reactive oxygen species (ROS) and with inhibitors of
mitochondrial respiration, with PMN-derived cytoplasts (which lack
mitochondria) and with PMNs from patients with chronic granulomatous disease (which have impaired nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) indicated that
TNF-/zVAD-fmk-induced cell death depends on mitochondria-derived
ROS. Thus, TNF- can induce a "classical," caspase-dependent and
a "nonclassical" caspase-independent cell death.
(Blood. 2003;101:1987-1995)
© 2003 by The American Society of Hematology.
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Introduction |
Tumor necrosis factor (TNF-) provides a wide
variety of biologic signals, which are involved in the regulation of
cell death and participate in the governing of immune
homeostasis.1 TNF- has been shown to play a crucial
role in the pathogenesis of inflammatory diseases, such as rheumatoid
arthritis, adult respiratory distress syndrome, and
sepsis.2-4 However, TNF- is also able to exert
anti-inflammatory effects.5,6 The antiphlogistic potential
of this cytokine can be ascribed, at least partly, to its ability to
accelerate the apoptosis of neutrophils (PMNs), which are major
effector cells of inflammation. Apoptotic cell death constitutes a
powerful way of curtailing PMN-mediated reactions, providing a safe
clearance of these potentially toxic cells.7 Moreover, the
uptake of apoptotic cells by resident macrophages has an
immunosuppressive effect, which gives an additional beneficial contribution to the control of inflammatory reactions.8
The proapoptotic effect of TNF- on PMNs has been well
documented,9-14 although opposite results have also been
published.15,16 Probably, this controversy can be
explained by the findings that the effect of TNF- on PMN survival
may depend on the concentration of the cytokine17 as well
as on the duration of stimulation and the initial functional capacity
of the PMNs before exposition to TNF-.11
The mechanism of apoptosis induction by TNF- is closely related to
the cascade of apoptotic cysteine proteases known as caspases, which
represent a group of enzymes responsible for initiation and execution
of apoptosis.18,19 A death signal from the TNF- receptor is transduced to an adapter protein, TNF-
receptor-associated death domain (TRADD), which uses the next adapter
protein, Fas receptor-associated death domain (FADD), to organize the
death-inducing signaling complex (DISC).20 DISC recruits
and activates the upstream initiator caspase-8, providing therefore
activation of downstream effector caspases and the final steps of the
apoptotic program.20,21 Inhibition of caspases, for
example, by certain peptide ketones, which mimic the active site of the
enzyme,22 has been shown to dramatically increase cell
survival in various cell types, including PMNs.13,14,23,24
In our study, investigating the effects of TNF- on PMN survival, we
faced an unexpected phenomenon. We confirmed a central role for
caspases in TNF--mediated apoptosis of PMNs, but at the same time
we found that inhibition of caspases did not rescue PMNs from death in
the presence of TNF- but instead enhanced an as yet unidentified
form of PMN death. Our experiments indicate that in PMNs 2 death
pathways are induced by TNF-: one is the predominant "classical"
caspase-dependent apoptosis, whereas the other is a "nonclassical"
death route, which becomes apparent when caspases are fully inhibited
and involves mitochondria-derived reactive oxygen species (ROS).
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Materials and methods |
PMN purification and culturing
Heparinized venous blood was collected from healthy volunteers
and 3 patients with chronic granulomatous disease (CGD) after obtaining
informed consent, and PMNs were isolated as described.25 Briefly, 20 mL blood was diluted with 20 mL 10% trisodium
citrate/phosphate-buffered saline (PBS). Mononuclear cells and
platelets were removed by density gradient centrifugation over isotonic
Percoll (Pharmacia, Uppsala, Sweden) with a specific gravity of 1.076 g/mL. Erythrocytes were lysed by short treatment of the pellet fraction
with an ice-cold isotonic NH4Cl solution (155 mM
NH4Cl, 10 mM KHCO3, 0.1 mM
EDTA [ethylenediaminetetraacetic acid], pH 7.4). The
remaining PMNs were washed once in PBS and used for further
manipulations. In all cases cell purity was more than 97%. PMNs were
resuspended at a final concentration of 2 × 106/mL in
Iscoves modified Dulbecco medium (BioWhittaker, Brussels, Belgium)
supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco
BRL, Paisley, United Kingdom), penicillin 100 IU/mL (Yamanouchi, Tokyo,
Japan), streptomycin 100 µg/mL (Gibco BRL), and glutamine 300 µg/mL. One milliliter of cell suspension was put in each well of
24-well plates (NUNC Brand Products, Roskilde, Denmark) and was
incubated for 6 hours in a 5% CO2 incubator at 37°C.
PMNs were cultured with 20 ng/mL TNF- (Calbiochem, Bad Soden,
Germany), with 150 µM z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk;,
Alexis Biochemicals, San Diego, CA), with 5 mM N-acetyl-L-cysteine (NAC; Sigma, St Louis, MO) or with indicated combinations. Normal PMNs
were also incubated with 500 ng/mL mouse anti-Fas (CD95) monoclonal
antibodies (Abs; clone CH11; Immunotech, Marseille, France), with 10 mM
4,5-dihydroxy-1,3-benzene disulfonic acid (tiron; Sigma), with 100 µM
rotenone (Sigma), with 2 mM sodium azide (Calbiochem), with 300 µM
thenoyltrifluoroacetone (TTFA; Sigma) alone or in a combination with
zVAD-fmk or TNF-/zVAD-fmk, where indicated. When the combinations of
reagents were used, they were added in culture medium simultaneously.
The incubation time and the concentrations were found to be optimal in
preliminary experiments (data not shown). Control PMNs were cultured
without additions (no stimulus). Because of longer transportation time, CGD neutrophils as well the healthy day-control cells were purified and
cultured in a "delayed" manner (delayed cultures), ie, 4 to 5 hours
after collection of the blood sample.
Cytoplast preparation and culturing
PMNs were isolated from the buffy coat of 500 mL fresh blood
from volunteer donors, as described in "PMN purification and culturing." Cytoplasts were prepared from 108
PMNs as described previously.26 Briefly, PMNs were
centrifuged through a discontinuous Ficoll-70 (Sigma) gradient (12.5%,
16%, 25%) prewarmed to 37°C, containing 5 µg/mL cytochalasin B
(Sigma). Centrifugation was performed for 30 minutes at 34°C in a
model L2-65B ultracentrifuge with an AH-629 rotor (Beckman Instruments, Fullerton, CA) at 81 000g. After centrifugation, the top
band of cellular material was collected. This band was composed of more
than 99% of cytoplasts, as assessed by light microscopy of cytospins
stained with May-Grünwald-Giemsa solution. Cytoplasts were
recognized by their absence of a nucleus. Following several washings in
PBS, cytoplasts were resuspended at a final concentration of
8 × 106 per mL in the culture medium and were incubated
overnight (16 hours) under conditions indicated in Figure 7. This
duration of culture induced maximal differences in cytoplast apoptosis
between tested conditions (data not shown).
Measurement of cell death
After 6 hours of incubation, PMNs were split into 2 portions,
which were washed once in ice-cold PBS. One portion was stained with
the annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)
apoptosis assay kit (Bender MedSystems, Vienna, Austria) and analyzed
by fluorescence-activated cell sorter scan (FACScan; Becton Dickinson,
San Jose, CA) as described previously.24 Dead cells were
defined as positive for annexin-V-FITC or for annexin-V-FITC/PI staining. Cell death was expressed as a percentage of dead cells in
relation to the total number of counted cells. The number of cells
recovered after culture was similar under all conditions tested and was
close to 90% of the initial input of cells. Another portion of PMNs
(2-3 × 105 cells) was used for preparation of cytospins
stained with May-Grünwald-Giemsa solution. The cytospins were
estimated by light microscopy for morphologic changes in PMNs
(described in "Results"). A minimum of 300 cells was scored for
each sample, and the percentages of dead PMNs were determined.
Cytoplast death was assessed by annexin-V-FITC binding as described
earlier, without the PI step, with 4 × 105 cytoplasts
for each preparation. Annexin-V+ cytoplasts were considered
to be dead.
Western blotting
The cleavage of caspase-8 and caspase-3 was determined by
Western blotting. Whole cell lysates were obtained by boiling
0.5 × 106 PMNs in sodium dodecyl sulfate (SDS) sample
buffer with 2% mercaptoethanol for 5 minutes. Proteins were resolved
on 15% SDS-polyacrylamide gel by electrophoresis (PAGE) and were
electrotransferred to Immun-Blot polyvinylidene diflouride
(PVDF) membrane (BioRad Laboratories, Hercules, CA). The blots were
sequentially probed with monoclonal mouse antihuman-caspase-8 Abs
(clone 1C12; Cell Signaling Technology, Beverly, MA), which recognize
full-length caspase-8 as well as its fragments; with polyclonal rabbit
antihuman-caspase-3 Abs (Pharmingen, San Diego, CA), which recognize
both inactive procaspase-3 and its cleavage product; and with
polyclonal rabbit antihuman-Bax Abs (Pharmingen). All indicated Abs
were used at a final dilution of 1:1000. After exposure to each primary
Ab, the blots were incubated with appropriate secondary Abs conjugated
with horseradish peroxidase (Amersham, Arlington Height, IL) at a final
dilution of 1:2500, followed by band visualization with an enhanced
chemiluminescence kit as described by the manufacturer (Amersham). This
reprobing was successful because of a different exposition time
required for visualization of the proteins of interest. For
caspase-8-related bands it was approximately 30 minutes, for caspase-3
and its cleavage product 5 minutes, and for Bax protein less than
1 minute.
Agarose gel electrophoresis of DNA
DNA was extracted from 5 × 106 freshly isolated
PMNs or from PMNs treated under conditions indicated in Figure 3 by a
PureGene DNA isolation kit (Gentra Systems, Minneapolis, MN) in
accordance with the manufacturer's instructions. Isolated DNA was
electrophoresed in a 1.2% agarose gel containing ethidium bromide, and
the gels were photographed under ultraviolet light.
Assessment of p38 MAP kinase phosphorylation in PMNs and
cytoplasts
After purification, PMNs and cytoplasts were resuspended in
culture medium at final concentrations of 2 × 106/mL and
8 × 106/mL, respectively, and were incubated without or
with 20 ng/mL TNF- for 10 minutes in a water-bath at 37°C.
Thereafter, whole cell lysates were prepared, and Western blotting was
performed as described earlier with 1 × 106 cytoplast or
0.5 × 106 PMN equivalents per lane. The blots were
probed with phosphospecific polyclonal rabbit Abs against human p38
mitogen-activated protein (MAP) kinase (Cell Signaling
Technology), which selectively recognize phosphorylated p38. To
determine protein loading, reprobe was performed with polyclonal rabbit
Abs against total p38 (Santa Cruz Biotechnology, Santa Cruz, CA), which
bind to p38 irrespectively of its phosphorylation state.
Confocal laser scanning microscopy (CLSM)
For the mitochondrial staining, MitoTracker GreenFM (Molecular
Probes, Eugene, OR) was used. To estimate mitochondrial morphology, unfixed PMNs were stained with 100 µM MitoTracker GreenFM and were analyzed by a confocal laser scanning microscope (LSM510; Carl
Zeiss, Heidelberg, Germany) as described.24 To obtain
simultaneous staining of mitochondria and Bax protein, PMNs were
stained with 1 mM MitoTracker GreenFM. Thereafter, the cells were fixed
with 2% paraformaldehyde, permeabilized in staining buffer containing 0.1% saponin (wt/vol; Calbiochem) and 1% (wt/vol) bovine serum albumin (Sigma), and were labeled by polyclonal rabbit antihuman-Bax Abs (final dilution 1:250; Pharmingen) followed by secondary staining with AlexaFluor-568-conjugated goat antirabbit immunoglobulin G
(Molecular Probes) at a final concentration of 2.5 µg/mL as has been
previously described.24 After staining, at least 300 PMNs
were counted in each sample, and the percentages of cells with
prevailing morphology (images shown in Figures 4-5) were determined, as
indicated in the legends of Figures 4-5.
Statistics
Where applicable, values were compared by one-way analysis of
variance (ANOVA) with Bonferroni posttest using GraphPad Prism version
3.0 software. Differences were accepted as significant at
P < .05.
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Results |
TNF- alone induces classical apoptosis in PMNs
Already after 6 hours of culture, untreated PMNs underwent
spontaneous apoptosis, with 31.9% ± 2.5% of the cells being
annexin-V+ (Figure 1A, No
stimulus). Typical apoptotic morphology, including rounding of nuclei,
pronounced chromatin condensation, and cell shrinkage, was displayed by
34.2% ± 2.0% of untreated PMNs (Figure 1B, top left; apoptotic
cells shown by arrowheads). After 6 hours of treatment with TNF-,
the fraction of annexin-V+ PMNs slightly but significantly
increased to 40.0% ± 4.3% (Figure 1A, TNF-). When scored by
morphologic changes, the proportion of PMNs with classical apoptotic
features amounted to 74.0% ± 4.3% in the presence of TNF-
(Figure 1B, top right). These data are consistent with previous
observations that at early time points TNF- is indeed able to induce
apoptosis in PMNs.11,13,14
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| Figure 1.
Death of PMNs.
(A-B) PMNs were cultured for 6 hours without additions, with 20 ng/mL
TNF-, with 150 µM zVAD-fmk, or with the combination of these
agents, and cell death was assessed (A) by FACScan analysis of
annexin-V-FITC/PI staining and (B) by morphologic examination of
cytospins stained with May-Grünwald-Giemsa stain (for
quantitative data see C). Arrowheads indicate PMNs that have undergone
spontaneous or TNF--induced apoptosis with typical apoptotic
morphology; closed and open arrows depict PMNs with aberrant morphology
appeared after TNF-/zVAD-fmk treatment (see "Results" for
details). (C) Quantitative data obtained by FACScan analysis
and cytospin evaluation of PMNs treated for 6 hours under the
conditions as indicated. Dosage of additions: TNF- and zVAD-fmk, as
indicated for A and B; anti-Fas monoclonal Abs, 500 ng/mL.
*P < .05. Data represent means ± SEM of 4 to 8 separate experiments performed in duplicate.
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Inhibition of caspases in the presence of TNF- leads PMNs to
an aberrant death different from apoptosis
We subsequently studied the activation of initiator caspase-8 and
executioner caspase-3 in PMNs by Western blotting. In freshly isolated
PMNs caspase-8 was present as a 57- to 55-kd precursor protein (Figure
2A, lane 1), and caspase-3 appeared as a
32-kd proenzyme (Figure 2B, lane 1). These bands represent the
full-length procaspases.13,27 PMNs that had undergone
spontaneous apoptosis on culturing without stimuli displayed the
initial activation of caspase-8 with the appearance of the large
cleavage fragment of 43 to 41 kd (Figure 2A, lane 2). Caspase-3 was
partially cleaved into the 17-kd fragment (Figure 2B, lane 2).
Stimulation with TNF- caused a more pronounced activation of
caspase-8 and caspase-3. The 57- to 55-kd procaspase-8 was completely
degraded into smaller fragments, including the active 18-kd
fragment28 (Figure 2A, lane 4), and the 32-kd procaspase-3
was entirely processed to the active 17-kd product (Figure 2B,
lane 4).
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| Figure 2.
Cleavage of caspase-8 and caspase-3 in PMNs.
Whole-cell lysates from freshly isolated PMNs (lane 1), from PMNs
cultured for 6 hours without additions (lane 2), with 150 µM zVAD-fmk
(lane 3), with 20 ng/mL TNF- (lane 4), or with the TNF-/zVAD-fmk
combination (lane 5) were subjected to SDS-PAGE. Western blot was
performed with anti-caspase-8 monoclonal Abs (A). Then the blot was
reprobed with anti-caspase-3 polyclonal Abs (B). The expression of Bax
protein determined by anti-Bax polyclonal Abs was used as a measurement
for equal protein loading (C). Results are representative of 3 independent experiments.
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Next, we checked whether inhibition of caspases could abrogate
the proapoptotic effects of TNF-. For this purpose the
broad-spectrum caspase inhibitor zVAD-fmk was used. When used alone,
this agent significantly reduced apoptotic membrane changes
(18.6% ± 2.0% annexin-V+ PMNs; Figure 1A, zVAD-fmk) as
well as morphologic features of apoptosis (9.4% ± 2.5% cells with
apoptotic morphology; Figure 1B, bottom left), when compared with
untreated PMNs (Figure 1A-B, No stimulus). Moreover, zVAD-fmk
completely inhibited activation of caspase-8 (Figure 2A, lane 3) and
caspase-3 (Figure 2B, lane 3), because no cleavage products were
detectable, and the enzymes were only present as full-length precursors
in the lysates from zVAD-fmk-treated PMNs.
Unexpectedly, addition of zVAD-fmk to TNF- did not rescue PMNs from
death. Instead, after such treatment, 52.4% ± 4.7% of the PMNs
became annexin-V+, as shown in Figure 1A (TNF- + zVAD-fmk plot). These PMNs had an aberrant appearance (Figure 1B,
bottom right): some cells (open arrow) were enlarged, with expanded
disintegrated chromatin and visible vacuolization, whereas others
(closed arrow) had hyperlobulated nuclei with moderately condensed
chromatin (this picture is different from classical apoptotic features
shown in Figure 1B, top left and top right). The proportion of such
unusual cells in the TNF-/zVAD-fmk preparation was
40.0% ± 6.2%, whereas typical apoptotic morphology was noted in
9.4% ± 4.3% of the PMNs treated with this combination. The latter
value is similar to the level of morphologic apoptosis found when PMNs
were treated with zVAD-fmk alone, as summarized in Figure 1C. Notably,
the fraction of aberrant cells was negligible among untreated or
TNF--treated PMNs and very low in the presence of zVAD-fmk alone
(< 3%). Of importance, the aberrant death of PMNs induced by
TNF-/zVAD-fmk proceeded despite the absence of any detectable
activation of caspase-8 (Figure 2A, lane 5) or caspase-3 (Figure 2B,
lane 5). The equivalence of protein loading was established by Bax
protein expression (Figure 2C), which has been previously shown to be
stable in PMNs by us24 and various other
groups.29-31
To investigate the role of protein synthesis in the
TNF-/zVAD-fmk death pathway, PMNs were also tested in the presence
of cycloheximide. However, transcription blockade showed no effect on
TNF-/zVAD-fmk-induced cytotoxicity in PMNs (data not shown).
Taken together, these results indicate that TNF- can induce 2 different death signals in PMNs, one caspase dependent and another
caspase independent.
No DNA laddering in TNF-/zVAD-fmk-treated
PMNs
To further characterize the caspase-independent cell death in
PMNs, we investigated DNA laddering. Internucleosomal DNA degradation is a hallmark of apoptosis.32 Indeed, DNA from PMNs that
had been incubated with TNF- for 6 hours demonstrated a typical
laddering pattern, indicating internucleosomal cleavage characteristic
for apoptosis (Figure 3). In contrast,
the electrophoretic pattern of DNA extracted from
TNF-/zVAD-fmk-treated PMNs was similar to that of fresh, untreated,
or zVAD-fmk-treated cells, cultured for 6 hours (Figure 3). Thus, also
in this respect, TNF-/zVAD-fmk-induced PMN death appeared to be
different from typical apoptosis.
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| Figure 3.
DNA laddering in PMNs.
DNA extracted from freshly isolated PMNs as well as from PMNs cultured
for 6 hours without additions, with 20 ng/mL TNF-, with 150 µM
zVAD-fmk or with the combination of these agents, was separated by
agarose gel electrophoresis, and internucleosomal fragmentation (DNA
laddering) was assessed. Results are representative of 3 separate
experiments.
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zVAD-fmk prevents Fas-receptor-induced apoptosis
in PMNs
The Fas/Apo-1/CD95 system shares common death signaling pathways
with the TNF--receptor. Both receptors belong to the TNF/nerve growth factor receptor family33 and can recruit the
same adapter protein, FADD, forming the DISC to mediate death
signals to the caspase cascade.20,21 As shown in Figure
1C, ligation of the Fas-receptor with agonistic anti-Fas monoclonal Abs
CH-1123,34 led PMNs after a 6-hour culture to pronounced
apoptosis, with 59.9% ± 4.7% of annexin-V+ cells and
83.3% ± 5.2% of cells with a typical apoptotic morphology (compare
with Figure 1C, No stimulus). However, induction of apoptosis by
anti-Fas monoclonal Abs was almost completely prevented by zVAD-fmk
(Figure 1C; 21.9% ± 3.8% annexin-V+ and
17.5% ± 2.7% morphologically apoptotic PMNs; compare with Figure
1C, zVAD-fmk alone). Thus, despite the fact that Fas and TNF--receptors engage common upstream death pathways, Fas
receptor-mediated death signals are strictly caspase dependent and can
be blocked by a caspase inhibitor, whereas TNF- has the potential to
bypass the caspase cascade, causing atypical death in PMNs in the
presence of zVAD-fmk.
TNF-/zVAD-fmk treatment causes in PMNs degradation of the
mitochondria without Bax redistribution
Our recent study has shown that PMNs contain mitochondria, which
play an important role in the apoptotic program of these cells.24 To check whether the mitochondria are involved in
caspase-independent death pathway, we undertook the next set of
experiments. Specific mitochondrial fluorescent staining revealed that
most of the untreated and zVAD-fmk-treated PMNs (Figure
4, top left and bottom left, respectively) after 6 hours of incubation preserved a tubular structure
of the mitochondria, as was observed in fresh cells.24 When TNF- alone was present in the culture medium, the mitochondria changed into large unstructured aggregates (Figure 4, top right) typical for apoptosis.24 Interestingly, the proportion of
cells with clustered mitochondria closely correlated with the
proportion of cells with an apoptotic morphology (data not presented),
indicating that changes in the mitochondrial structure form an early
and reliable marker of apoptosis. The TNF-/zVAD-fmk combination also altered the appearance of the mitochondria (Figure 4, bottom right), leading to clustering and degradation of these organelles, although these mitochondrial aggregates were smaller in comparison to the aggregates in PMNs treated with TNF- alone.
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| Figure 4.
Staining patterns of mitochondria in PMNs.
PMNs were incubated for 6 hours without additions (No stimulus), with
20 ng/mL TNF-, with 150 µM zVAD-fmk, or with the TNF-/zVAD-fmk
combination. Then the cells were stained with MitoTracker GreenFM and
analyzed with CLSM. Each image represents the following proportion of
the total cell population (mean ± SEM): 73.8% ± 8.9% in No
stimulus; 66.7% ± 5.4% in TNF-; 89.0% ± 4.3% in zVAD-fmk;
77.0% ± 3.6% in TNF-/zVAD-fmk. Bar is 5 µm. Results are
representative of at least 4 independent experiments.
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When PMNs were costained for mitochondria and Bax protein, most of the
untreated cells showed after the 6-hour incubation a punctate
localization of Bax, remaining separate from mitochondria (Figure
5, top panel), as was also observed in
fresh PMNs.24 In PMNs after 6 hours of culture in the
presence of zVAD-fmk, Bax protein maintained a staining pattern similar
to fresh and to untreated cultured cells, visible as a punctate
distribution separate from mitochondria (data not shown). In contrast,
treatment with TNF- caused redistribution of Bax into large
clusters, which colocalized with mitochondria (Figure 5, middle panel;
a shift in fluorescence to yellow depicts colocalization). In
TNF-/zVAD-fmk-treated PMNs, Bax remained punctate and hardly
colocalized with the mitochondria (Figure 5, bottom panels).
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| Figure 5.
Subcellular redistribution of Bax protein in PMNs.
PMNs were cultured for 6 hours without additions (No stimulus), with 20 ng/mL TNF-, or with a combination of 20 ng/mL TNF- and 150 µM
zVAD-fmk. Then the cells were stained with MitoTracker GreenFM, fixed,
permeabilized, stained with polyclonal Abs specific for Bax, and
analyzed with CLSM. (Because of the fixation and permeabilization
procedures, the mitochondrial staining [green] showed a more diffuse
cytoplasmic pattern than the tubular structures shown in Figure 4, left
panels). Each image represents the following proportion of the total
cell population (mean ± SEM): 77.7% ± 5.4% in No stimulus;
63.3% ± 8.8% in TNF-; 88.3% ± 5.7% in TNF-/zVAD-fmk.
Bar is 5 µm. This figure is representative of at least 4 independent
experiments.
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Taken together, these results demonstrate that TNF- can act via a
classical apoptotic route, inducing subcellular Bax redistribution and
its aggregation with mitochondria, which have been shown to be
significant events during the execution of apoptosis in various cell
types,35-38 including PMNs.24 At the same
time, TNF- stimulation under conditions that preclude caspase
activation does not lead to Bax changes, but it does cause degradation
of mitochondria. Again, this finding may indicate the presence of a
caspase-independent, mitochondria-dependent route of cell death in PMNs
that is different from apoptosis.
PMN-derived cytoplasts lacking mitochondria do not display
TNF-/zVAD-fmk-induced death phenomenon
To further elucidate the role of mitochondria in
TNF-/zVAD-fmk-induced death, we used PMN-derived cytoplasts, which
are cellular vesicles from which mitochondria have been
eliminated.24,39 First, we determined whether the TNF-
receptors were functional on the cytoplast surface. As a read-out, the
TNF- receptor-mediated phosphorylation of p38 MAP kinase was
used.40 Figure 6 (top panel)
demonstrates that TNF- induced phosphorylation of p38 MAP kinase
both in cytoplasts and PMNs. Next, cytoplasts were cultured under
various conditions. Untreated and TNF--treated cytoplasts after
culture exposed phosphatidyl serine on the outer layer of the plasma
membrane, which was evident by annexin-V positivity of
54.2% ± 4.9% and 50.3% ± 5.7% cytoplasts, respectively
(Figure 7, top left and top right plots,
respectively). zVAD-fmk reduced the number of annexin-V+
cytoplasts to 7.2% ± 1.9% (Figure 7, bottom left plot). Similar values were found in the experiments with PMNs (Figure 1C). However, in
contrast to PMNs, cytoplasts treated with a combination TNF-/VAD-fmk remained "alive," with only 11.0% ± 1.3%
annexin-V+ cells (Figure 7, bottom right plot). Thus,
addition of TNF- to zVAD-fmk had no effect on cytoplast survival.
This finding indicates that TNF- was not able to induce a
caspase-independent death in cytoplasts in the absence of the
mitochondria, despite the intact receptor signaling, and the caspase
inhibitor completely preserved its prosurvival effect. Also, this
finding demonstrates that the TNF-/zVAD-fmk combination itself is
not nonspecifically toxic for the cells.
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| Figure 6.
p38 MAP kinase phosphorylation in cytoplasts.
Freshly isolated cytoplasts or fresh PMNs were treated for 10 minutes
with control medium or with 20 ng/mL TNF-. Thereafter, whole cell
lysates were prepared and subjected to SDS-PAGE. Western blot was
performed with monoclonal Abs that specifically recognized the
phosphorylated form of p38 (top panel). Reprobing with anti-total p38
monoclonal Abs (bottom panel), which recognizes p38 regardless of its
phosphorylation state, gives an estimation of the equal protein
loading. Results are representative of 4 independent experiments.
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| Figure 7.
Death of cytoplasts.
Cytoplasts cultured overnight without additions or with 20 ng/mL
TNF-, with 150 µM zVAD-fmk, or with the combination of these
agents were stained with annexin-V-FITC and were analyzed by FACScan.
Cytoplasts with annexin-V staining were counted as dead cytoplasts
(bottom right quadrant of each plot). *P < .05 versus No
stimulus and TNF-. Values represent means ± SEM of 5 separate
experiments performed in duplicate.
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NADPH oxidase system-independent ROS are involved in the
TNF-/zVAD-fmk-induced cytotoxic effects
The data shown earlier indicated that the mitochondria may
participate in the process of unusual TNF-/zVAD-fmk-induced PMN death. How do these organelles contribute to this death pathway? One
possibility is ROS production by the mitochondria in response to
TNF- stimulation, which mediates, at least in part, cytotoxic effects of this cytokine.41-43 NAC, a well-characterized
ROS scavenger,34,44,45 had no effect on spontaneous
apoptosis of PMNs, reduced TNF--induced apoptosis, and almost
completely abrogated the TNF-/zVAD-fmk death effects (Figure
8 and data not shown). NAC significantly (P < .05) reduced the number of annexin-V+
PMNs in TNF-/zVAD-fmk-treated PMNs and completely prevented the
appearance of morphologically aberrant cells (not shown). The ROS
scavenger tiron,43 which is unrelated to NAC, also
prevented TNF-/zVAD-fmk-induced cell death (data not shown). The
mitochondrial origin of ROS was further supported by experiments, in
which we used inhibitors of the mitochondrial electron transport
(respiratory) chain, ie, inhibitors of the mitochondrial ROS
production.41 Rotenone stopped the death-inducing effects
of the TNF-/zVAD-fmk combination, preventing plasma membrane
flip-flop (Figure 8; P < .05) and aberrant morphologic
changes in the TNF-/zVAD-fmk-treated PMNs (data not shown). Two
other mitochondrial inhibitors, sodium azide and TTFA, demonstrated a
similar effect of rescuing PMNs from TNF-/zVAD-fmk-mediated cell
death (data not shown). Importantly, these mitochondrial inhibitors,
when added alone, influenced neither the basal level of PMN apoptosis
nor PMN adenosine triphosphate (ATP) levels, measured by a
luciferase-based assay46 (not shown). The latter result
can be explained by the fact that PMNs mainly use glycolysis rather
than mitochondrial oxidative phosphorylation for their energy
supply.47
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| Figure 8.
Effect of NAC and rotenone on TNF-/zVAD-fmk-induced
PMN cell death.
PMNs were cultured for 6 hours with a combination of 20 ng/mL TNF-
and 150 µM zVAD-fmk or with TNF-/zVAD-fmk in combination with 5 mM
NAC or 100 µM rotenone. Afterwards, PMNs were stained with
annexin-V/PI and analyzed by FACScan. Values represent the percentage
of cells (mean ± SEM) for each respective quadrant. Data obtained
in 5 independent experiments.
|
|
The most powerful source of ROS in PMNs is the nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase system, which provides a
rapid and a dramatic increase in ROS generation known as the respiratory burst. To check whether ROS produced by NADPH oxidase participates in the TNF-/zVAD-fmk-mediated cell death, we
investigated PMNs from 3 patients with CGD. Because of a genetic defect
in the NADPH oxidase in PMNs from these patients, their cells cannot generate ROS.48 In our experiments, PMNs from patients
with CGD displayed a behavior in terms of the death rate similar to the
normal cells under the conditions tested as illustrated by annexin-V/PI
staining in Table 1. CGD PMNs had levels
of spontaneous apoptosis comparable to the healthy day-control cells
incubated in the delayed cultures (see "Materials and methods").
zVAD-fmk protected CGD cells from apoptosis as it did in healthy PMNs, and the TNF-/zVAD-fmk combination induced a similar amount of phosphatidyl serine exposure and aberrant morphology in CGD PMNs, as
was observed in normal PMNs (Table 1 and data not shown). Hence, the
NADPH oxidase system plays no role in the TNF-/zVAD-fmk-induced death of PMNs. Interestingly, the ROS scavenger NAC also rescued CGD
PMNs from the TNF-/zVAD-fmk-induced death by preventing the membrane changes (Table 1) and the appearance of unusual morphology (not shown), indicating that CGD PMNs have the ability to produce some
ROS from an alternative source.
| |
Discussion |
Most forms of programmed cell death proceed through the activation
of caspases, which can be blocked by the general caspase inhibitor
zVAD-fmk. In this study we describe an as yet unidentified form of PMN
death induced by TNF- in the presence of caspase inhibition. TNF-
alone induced activation of the classical apoptotic route in PMNs,
which is accompanied by activation of initiator and executioner
caspases, Bax translocation to mitochondria, mitochondrial clustering,
internucleosomal cleavage of DNA, and typical apoptotic changes in
morphology and plasma membranes. When caspase activity was blocked,
TNF--treated PMNs displayed no Bax redistribution and no DNA
fragmentation, and increased cell death as indicated by the plasma
membrane exposure of phosphatidyl serine in the outer layer
(flip-flop). Moreover, these PMNs showed an aberrant morphology, with
hyperlobulated nuclei and expanded, disintegrated chromatin.
Apparently, under conditions when caspases do not function, an
alternative, TNF--induced nonclassical, caspase-independent pathway
of cell death is revealed in PMNs.
Further experiments demonstrated the involvement of ROS in the
TNF-/zVAD-fmk-induced cytotoxicity (Figure 8), independent of
protein synthesis. During the last decade, the physiologic role of ROS
has been gradually reevaluated. These agents moved from a category of
merely unwanted side products of oxidative metabolism to a cohort of
important messenger molecules.49-51 Intracellular sources
of ROS are mainly represented by electron-transfer processes in
mitochondria49,52 and enzymatic oxidation provided by
various oxidases.50 Among oxidases, the NADPH oxidase
system is one of the most powerful generators of ROS, being used by
PMNs for the killing of ingested microorganisms.48 We
found that PMNs from patients with CGD, who have an impaired NADPH
oxidase system, died in the same caspase-independent way after
TNF-/zVAD-fmk treatment as did healthy PMNs. This observation ruled
out the involvement of NADPH oxidase-derived ROS in this nonclassical form of PMN death. Experiments with PMN-derived cytoplasts underscored that these ROS originated from mitochondria, because cytoplasts, having
no mitochondria, did not show any enhanced exposure of phosphatidyl
serine after TNF-/zVAD-fmk stimulation, in contrast to intact PMNs.
A number of inhibitors of the mitochondrial electron transport chain,
including rotenone, sodium azide, and TTFA, were also able to prevent
the TNF-/zVAD-fmk-induced features of cell death, again pointing to
the mitochondria as the main origin of ROS production. These findings
are in line with previous reports on the cytotoxic effects of TNF-
in cell lines,41-43 caused by ROS from mitochondria.
Excessive amounts of ROS may cause direct oxidative damage of nucleic
acids, proteins, and lipids53 or may make proteins more
susceptible to proteolysis.54,55 Probably, such events
take place during TNF-/zVAD-fmk-induced PMN death, resulting in the
observed cellular changes that could be prevented by ROS inhibitors.
Importantly, mitochondria are not only a source of ROS but also a
target for ROS.52 ROS produced in mitochondria may lead to
self-damage of these organelles, causing apoptosis or
necrosis.53 This could be an explanation for our data,
which showed Bax-independent mitochondrial changes in PMNs after
TNF-/zVAD-fmk treatment. Undoubtedly, ROS production requires a
tight control, and our results suggest that caspases might be involved
in this regulation.44,56 Possibly, mitochondrial proteins
involved in electron transport within these organelles and providing
the excess production of oxygen radicals could be a direct substrate for caspases, because mitochondrial caspases with as yet unidentified intramitochondrial functions have been described.57
Deactivation of this system by caspases could normally prevent the
accumulation of ROS. Alternatively, caspases may play a role in the
elimination of damaged lipids and proteins, which accumulate after
TNF- stimulation and may normally act as a natural sink for
ROS.56
Several studies have shown that blockade of caspases in some cell lines
sensitize them to TNF--mediated
cytotoxicity.44,56,58 The researchers refer to this type
of cell death as necrosis,56 "a nonapoptotic" cell
death,44 or "a transitional stage between apoptosis and
necrosis."58 Such descriptions underline the complicated nature of the phenomenon but, at the same time, dictate the necessity to use an adequate set of tools for the registration of cell death. For
example, staining with propidium iodide alone56 does not seem to be sufficient to discriminate between necrotic and apoptotic cell death, because these basically different types of death both lead
to the final disruption of the plasma membrane.59 The
death rate of PMNs treated with a combination of TNF- and caspase
inhibitors has obviously been underestimated,13,14 because
only a DNA fragmentation assay has been used as a read-out for cell
death, whereas our present findings clearly show the absence of this hallmark of apoptosis under these conditions.
We conclude from our data that TNF- is able to trigger 2 pathways of
cell death in PMNs, and the availability of downstream caspases appears
to determine the mode of cell death. In the presence of an intact
caspase cascade TNF- mainly induces the classical form of apoptosis.
However, when caspase activity is blocked, eg, by zVAD-fmk, other
signals result from TNF- stimulation. This nonclassical and
caspase-independent pathway of PMN death, which lacks most of the
characteristic apoptotic features, is mediated by mitochondria-derived
ROS. In PMNs, this signaling pathway seems to be restricted to the
TNF- receptor, because the Fas receptor-mediated as well as the
spontaneous apoptosis in PMNs were both completely blocked by zVAD-fmk.
Our present data raise another issue. Caspases are attractive targets
for pharmacologic intervention in vivo in disease states that have been
associated with enhanced apoptosis.60,61 Caspase inhibitors, predominantly zVAD-fmk-like active-site mimetic peptide ketones, have been extensively used in animal models of human diseases.
These inhibitors have shown beneficial effects in various types of
ischemia-reperfusion injury,62-64 but also in infectious conditions, including bacterial meningitis and
sepsis.65,66 The promising approach of using caspase
inhibitors as anti-inflammatory agents should, however, be considered
with caution because of the possible adverse effects.61
For example, during ischemia-reperfusion injury and particularly during
generalized infections, inflammation proceeds through a massive
activation of PMNs and generation of inflammatory cytokines. Under many
generalized inflammatory conditions, TNF--induced apoptosis of PMNs
through the activation of caspases provides a "silent turnover" of
these potentially hazardous cells and leads to a suppression of
inflammation,8 limiting the extent of inflammatory
reactions. Instead, inhibition of caspases may lead to a deterioration
of the injury, caused by an as yet unforeseen atypical neutrophil death
as shown in our study, with a potentially uncontrolled release of their
contents, in contrast to the classical apoptotic cells. Moreover, under
the circumstances of caspase inhibition, PMN cell death is exaggerated,
and clearance mechanisms may be insufficiently able to minimize
PMN-related damage. To date, the existence per se of
caspase-independent cell death has been shown for several untransformed
cell types, including T lymphocytes,67,68 neurons,69-71 erythropoietic cells,72 and
fibroblasts.73 Many researchers refrain to designate this
type of cell turnover as "apoptosis," because of a lack of some
typical apoptotic features. Our data on PMNs support this view. The
biologic significance and physiologic role as well as the precise
mechanisms of this phenomenon warrant further study.
| |
Acknowledgments |
We are grateful to Dr P. Hordijk for his comments while preparing
the manuscript, to Dr R. S. Weening for his help in obtaining blood from CGD patients, and to Dr S. Albracht for his gift of mitochondrial inhibitors.
| |
Footnotes |
Submitted February 15, 2002; accepted October 3, 2002.
Prepublished
online as Blood First Edition Paper, October 10, 2002; DOI
10.1182/blood-2002-02-0522.
Supported by a grant from Nuffic (N.A.M.). T.W.K. is a research
fellow of the Royal Dutch Academy of Sciences.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Taco Kuijpers, Central Laboratory of
the Netherlands Blood Transfusion Service (CLB), Department of
Experimental Immunohematology, Plesmanlaan 125, 1066 CX Amsterdam, The
Netherlands; e-mail: t_kuijpers{at}clb.nl.
| |
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Tracey KJ, Cerami A.
Tumor necrosis factor, other cytokines and disease.
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1993;9:317-343[CrossRef][Medline]
[Order article via Infotrieve].
2.
Feldmann M, Brennan FM, Maini RN.
Rheumatoid arthritis.
Cell.
1996;85:307 |
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Abstract
Introduction