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m) and Cytochrome c Release Are Caspase Independent1
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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m) and the release of cytochrome c
into the cytoplasm appear to be early events in many systems, leading
to the activation of caspase-3 and, subsequently, nuclear apoptosis. We
show here that, in Jurkat targets treated in vitro with purified
granzyme B and perforin or granzyme B and adenovirus,
m collapse, reactive oxygen species production, and
cytochrome c release from mitochondria were observed.
Loss of m was also detected in an in vivo system
where green fluorescent protein-expressing targets were attacked by a
cytotoxic T cell line that kills predominantly through the granzyme
pathway. DNA fragmentation, phosphatidylserine externalization, and
reactive oxygen species production were inhibited in the presence of
the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl
ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl
ketone (zDEVD-fmk) in our in vitro system. Importantly, in either the
in vitro or in vivo systems, these inhibitors at concentrations up to
100 µM did not prevent m collapse. In addition,
cytochrome c release was observed in the in vitro system
in the absence or presence of zVAD-fmk. Thus the granzyme B-dependent
killing pathway in Jurkat targets involves mitochondrial alterations
that occur independently of caspases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The onset of apoptosis leads to the systematic disassembly of the target, resulting in cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. It is now widely accepted that the apoptotic events occurring within the target cell arise following the proteolytic activation by granzymes of endogenous cysteine proteases called caspases (for review see Refs. 8, 9). Indeed, granzyme B, the most potent of the cytolytic granzymes, has been shown in vitro to cleave and thereby activate caspase-3 (CPP32/Apopain/Yama) (10, 11, 12, 13). In addition, the closely related caspase-7 (Mch3/CMH-1/ICE-LAP3) is activated (14, 15) along with caspase 6 (Mch2) (16, 17), caspase-8 (FLICE/MACH/Mch5) (18), caspase-9 (ICE-LAP6/Mch6) (16, 19), and caspase-10 (Mch4) (20). Target cells treated with purified granzyme B and perforin contain processed caspase-1 (21), caspase-3, and caspase-7 (14). Moreover, cleaved, active caspase-3 and -8 are found in target cells following exposure to allogeneic CTL (11, 22). The activation of caspase-3 has been linked with the onset of hallmark apoptotic events such as DNA fragmentation and membrane alterations through the cleavage of cellular substrates such as the inhibitor of caspase-activated deoxyribonuclease (ICAD) (DNA fragmentation factor 45; Ref. 23) and gelsolin (24) as well as numerous others (8, 9, 25). Thus, recent models of granule-dependent CTL-mediated killing suggested that apoptosis arising within targets was the direct result of granzyme B-mediated caspase-3 activation. While granzyme B likely initiates apoptosis via caspase-3 in many instances, there have been reports suggesting that granule-dependent killing may also occur through caspase-independent means (26, 27)
Recently, mitochondria have been shown to undergo a number of profound
changes early within the apoptotic program
and appear to play a central role in apoptosis in a number of systems
(for review, see Refs. 28, 29, 30, 31). The mitochondrial changes
implicated include a disruption of electron transport and energy
metabolism, alterations in the cellular redox potential, the production
of reactive oxygen species (ROS),3 and a loss of the inner
membrane transmembrane potential (m), which
may be independent of or lead to the opening of permeability transition
(PT) pores (for a review of the mitochondrial permeability transition,
see Ref. 32). In addition, a number of mitochondrial
apoptogenic proteins including caspases-2, -3, and -9,
apoptosis-inducing factor (AIF), and cytochrome c are
released from the intermembrane space early during apoptosis in many
systems (33, 34, 35, 36, 37). AIF and cytochrome c have
been implicated in the activation of caspase-3, linking mitochondrial
events with caspase activation (33, 36, 38, 39). Current
models of granule-mediated target cell apoptosis do not implicate the
mitochondrial events outlined above. Indeed, the fact that granzyme B
cleaves caspase-3 directly in vitro (10, 12, 13) and that
proteolytically activated caspase-3 is found in target cells exposed
either to CTL or to purified granzyme B and perforin (10, 11), even in the presence of
benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk)
(22), suggests that mitochondria are not required during
caspase-dependent granule-mediated killing. However, it remained
possible that mitochondria played a role in the overall demise of the
cell, particularly in the caspase-independent pathway.
To address directly the involvement of mitochondria in the
granzyme-mediated pathway, we examined the effect of granzyme B on
mitochondrial function in both in vitro and in vivo systems. Two
granzyme B-dependent in vitro assays were used. The first relied on
perforin, and the second utilized a replication-deficient adenovirus to
facilitate internalization of granzyme B (40). Identical
results were obtained with both in vitro systems. Jurkat cells induced
to undergo apoptosis in response to granzyme B in vitro exhibit signs
of m collapse, ROS production, and
cytochrome c release from mitochondria. Importantly, while
DNA fragmentation and phosphatidylserine externalization are inhibited
in the presence of the pan-caspase inhibitor zVAD-fmk, cytochrome
c release and m loss are
zVAD-fmk insensitive, indicating that mitochondrial disruption in these
cells is caspase independent. In addition, Jurkats exposed to
allogeneic CTL also exhibit m disruption,
which is zVAD-fmk insensitive. Thus, we demonstrate for the first time
that granzyme B-mediated death of Jurkat targets involves
caspase-independent mitochondrial disruption.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human T cell lymphoma line Jurkat (American Type Culture Collection (ATCC), Manassas, VA) was grown in RPMI 1640 (Life Technologies, Burlington, ON) supplemented with 10% (v/v) FBS (HyClone, Logan, UT) and 100 µM 2-ME. Jurkat stable transfectants containing the pEGFP-N1 plasmid (JpEGFP) were maintained in RPMI 1640 containing 10% (v/v) FBS, 100 µM 2-ME, and 0.8 mg/ml geneticin (Life Technologies). Human lymphocytes were isolated from peripheral blood by centrifugation through Ficoll (Pharmacia Biotech, Baie d’Urfé, Quebec, Canada). Lymphocytes were prepared according to Ref. 22 . Briefly, lymphocytes were incubated at 4 x 105 cells/ml with irradiated, EBV-transformed RPMI-8666 cells (5000 rad) at a 5:1 ratio (lymphocytes:RPMI-8666). Following 3 days in coculture, CTL were purified from RPMI-8666 cells by centrifugation through Ficoll and subsequently maintained in RPMI 1640 containing 10% (v/v) FBS and 90 U/ml of IL-2 (Chiron, Emeryville, CA). All cell lines were maintained at 2.5–5 x 105 cells/ml in a humidified atmosphere of 5% CO2. Human granzyme B was purified from human YT-indy cells according to (41). Chris Froelich, Northwestern University, supplied purified human perforin. Replication-deficient adenovirus (type 5) was a generous gift from Jack Gauldie, McMaster University. The anti-human Fas IgM Ab (clone CH11) was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-human cytochrome c (clone 7H8.2C12) was from PharMingen (Mississauga, Ontario, Canada). All other reagents were purchased from Sigma (Oakville, Ontario, Canada) unless otherwise stated.
Induction and measurement of apoptosis in vitro
Jurkat cells were washed three times in serum-free RPMI 1640 and
resuspended at 2 x 106 cells/ml in
serum-free RPMI 1640 containing 1% BSA (w/v). To 500 µL of cells at
2 x 106 cells/ml was added 250 µl of
high-Ca2+ HEPES (20 mM HEPES (pH 7.4), 150 mM
NaCl, 1% BSA (w/v), and 5 mM CaCl2). Samples
treated with granzyme B and/or adenovirus received a further 250 µl
of Ca2+-free HEPES (20 mM HEPES (pH 7.4), and 150
mM NaCl and 1% BSA (w/v)), bringing the total assay volume to 1 ml
with a final cell concentration of 1 x 106
cells/ml. Granzyme B and adenovirus were added directly to final
concentrations of 1 µg/ml and a multiplicity of infection (MOI) of 10
PFU/cell, respectively. Samples treated with perforin received 250 µl
of Ca2+-free HEPES containing sublytic doses of
perforin (100–200 hemolytic units (HU) Ref. 40 ; sublytic
refers to an amount of perforin giving less than 10% nonspecific
lysis). Cells were incubated at 37°C for 2 h. Following this
incubation, 200-µl aliquots containing 
2 x
105 cells each were removed to assess DNA
fragmentation, phosphatidylserine externalization, ROS production, and
mitochondrial m loss, as outlined
below.
DNA fragmentation was determined using TUNEL (42). TUNEL materials were supplied by Boehringer Mannheim (Laval, Quebec, Canada) and used as per the manufacturer’s instructions. Phosphatidylserine externalization (43, 44) was measured using either the ApoAlert Annexin V Apoptosis Kit (Clontech, Palo Alto, CA) or FITC-annexin V (PharMingen) as per the manufacturers’ instructions. Both TUNEL and annexin V binding were quantified using flow cytometric analysis by examining 10,000 events on a Becton Dickinson (San Jose, CA) FACScan with an excitation wavelength of 488 nm. The emission wavelengths for both the dUTP-FITC and FITC-annexin V were detected through the FL1 channel equipped with a 530-nm (20-nm band pass) filter. Data were acquired and analyzed with CELLQuest software (Becton Dickinson, Mississauga, Ontario, Canada). Cells deemed TUNEL or FITC-annexin V positive were those displaying fluorescence greater than the fluorescence of controls in the absence of apoptotic induction (control).
m was monitored using 40 nM
3,3'-dihexyloxacarbocyanine iodide (DiOC6(3))
(Molecular Probes, Eugene, OR), and ROS production was determined using
2 µM hydroethidine (HE) (Molecular Probes). DiOC6
(3), HE, and the positive control for m
loss, carbonyl cyanide m-chlorophenyl hydrazone (mClCCP,
5 µM), were added 15 min before the 2-h end-point of the apoptosis
assay, and cells were then maintained for a further 15 min at 37°C,
followed by flow cytometric analysis.
DiOC6(3) was detected through the FL1
channel, and HE was detected through the FL2 channel, equipped with a
580-nm (20-nm band pass) filter. DiOC6
(3)low cells were those displaying
DiOC6(3) fluorescence less than the fluorescence
of control cells in the absence of the apoptotic stimulus.
HE+ cells were those displaying HE fluorescence
greater than control cells in the absence of apoptotic induction.
Inhibitor studies
Jurkat targets (1 x 106 cells/ml) were pretreated for 30 min at 37°C with either zVAD-fmk or benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk; both inhibitors were from Kamiya Biomedical, Seattle, WA) at the concentrations indicated. Following this incubation, targets were treated with granzyme B and adenovirus or granzyme B and perforin as outlined above. Alternatively, cells were treated with anti-human Fas IgM Ab (500 ng/ml) for 6 h at 37°C. Apoptosis was determined as outlined above, but with the following alterations. Rather than measuring both DiOC6(3) and HE on the same dot plot, the FL1 and FL2 channels were separated, and DiOC6(3) and HE were quantified on separate histograms, which facilitated quantification of the individual signals. For the cyclosporine A (CsA) experiments, Jurkats were treated with 30 µM protoporphyrin IX (PPIX) or granzyme B (1 µg/ml) and adenovirus (MOI = 10 PFU/cell) in the presence or absence of 100 µM CsA for 2 h, and the effects on DiOC6(3) loss were determined by flow cytometry.
Preparation of GFP-containing Jurkat target cells (JpEGFP)
Jurkat cells were electroporated (250 V, 250 µF, time constant
10) with 10 µg of Qiagen-purified pEGFP-N1 plasmid (Clontech)
containing a neomycin-resistance gene. Following selection in geneticin
(1 mg/ml), individual clones were analyzed for green fluorescent
protein (GFP) expression using flow cytometry. A single clone (clone
10) displaying intermediate levels of fluorescence was chosen for the
in vivo experiments. This clone was designated JpEGFP.
In vivo killing assays
Chromium (51Cr) and
[3H]thymidine release assays have been
described previously (11). Jurkat targets were incubated
with human CTL (hCTL) in the presence or absence of 5 mM EGTA.
Percentage lysis was calculated as follows: % lysis = 100 x
(sample-spontaneous lysis/total-spontaneous lysis). Measurement of
m was performed as follows. JpEGFP cells
were preloaded with the cationic lipophilic dye
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide
(DASPEI; Molecular Probes) at 50 nm for 15 min at 37°C. Dye-loaded
cells were treated for 30 min at 37°C in the absence or presence of
50 or 100 µM zVAD-fmk. Following this incubation, hCTL were added at
E:T ratios of 1:1, 0.5:1, and 0.25:1. The cell mixture was spun at
400 x g for 5 min to ensure contact between targets
and effectors and incubated at 37°C for 3 h. Loss of
m was monitored using flow cytometry by
gating on the green fluorescent JpEGFP population (530-nm filter) and
monitoring the loss of DASPEI fluorescence (580-nm filter) relative to
DASPEI fluorescence of cells in the absence of hCTL. Loss of
m was similarly determined for JpEGFP
treated with granzyme B (1 µg/ml) and adenovirus (MOI = 10
PFU/cell) for 3 h at 37°C in the presence or absence of
zVAD-fmk.
Analysis of cytochrome c release
Jurkat cells (1 x 106 cells/ml in serum-free RPMI 1640; 4 ml/sample) were treated with granzyme B (1 µg/ml) and adenovirus (MOI = 10 PFU/cell) for the times indicated. Anti-Fas-treated cells (also 4 ml of cells at 1 x 106 cells/ml in serum-free RPMI 1640) received 500 ng/ml of anti-human Fas IgM for the indicated times. Following treatment, cells were washed twice with PBS and resuspended in 200 µl digitonin lysis buffer (75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, 190 µg/ml digitonin). Digitonin is a weak nonionic detergent that, at low concentrations, selectively renders the plasma membrane permeable, releasing cytosolic components from cells but leaving other organelles intact (45). After 5 min on ice, cells were spun for 5 min at 14,000 rpm at 4°C in an Eppendorf microcentrifuge (Fisher Scientific, Nepean, Ontario, Canada). Supernatants were transferred to fresh tubes, and the pellets were resuspended in Triton lysis buffer (25 mM Tris-HCl (pH 8.0), 0.1% (v/v) Triton X-100). Aliquots (70 µl) of both pellet and supernatant for each sample were added to 30 µl of SDS-loading buffer (0.5 M Tris-HCl (pH 6.8), 1 M 2-ME, 10% (w/v) SDS, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue) and boiled for 10 min. Boiled samples (25 µl for pellets and 50 µl for supernatants) were loaded onto 15% polyacrylamide gels followed by electrophoresis and transfer to nitrocellulose membranes (Micron Separations, Westborough, MA). Membranes were blocked overnight at 4°C in PBS containing 0.1% (v/v) Tween 20 (Fisher Scientific, Nepean, Ontario, Canada) and 5% (w/v) milk proteins (Carnation, Don Mills, Ontario, Canada). Blocked membranes were incubated with a monoclonal anti-human cytochrome c Ab (1:2000 dilution in PBS containing 0.1% (v/v) Tween 20 and 5% milk proteins) for 1 h at room temperature. Membranes were washed three times (5 min each) with PBS containing 0.1% Tween 20, followed by incubation with an HRP-conjugated anti-mouse IgG secondary Ab (Bio-Rad, Mississauga, Ontario, Canada; 1:3000 dilution in PBS containing 0.1% Tween 20). Detection of cytochrome c on blots was performed using enzyme-linked chemiluminescence (ECL, Amersham, Oakville, Ontario, Canada).
HeLa cytochrome c release was determined identically except that, since HeLa are physically larger than Jurkat and since digitonin is a nonionic detergent requiring strict membrane to detergent ratios, only 2 x 106 HeLa cells were used per sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following the CTL-mediated induction of apoptosis in a target cell, several morphological and biochemical features become apparent. These features include DNA fragmentation (46) and membrane alterations such as phosphatidylserine externalization from the inner to the outer leaflet of the plasma membrane (43, 47). Cultured cells treated with granzyme B and perforin also exhibit these characteristic features of apoptosis (14). In addition, replication-deficient adenovirus has been shown to be a reliable substitute for perforin, in that the virus facilitates internalization of granzyme B into target cells (40, 48).
The human T cell lymphoma line Jurkat was treated with granzyme B and
perforin or granzyme B and adenovirus for 2 h at 37°C. The
induction of apoptosis was then assessed by measuring DNA fragmentation
using TUNEL and phosphatidylserine externalization using FITC-annexin V
binding. The percentage of apoptotic cells was then quantified using
flow cytometric analysis by measuring the increases in TUNEL or
FITC-annexin V fluorescence in apoptotic cells relative to controls.
The data presented in Fig. 1
are a
graphical representation of the flow cytometry data for both TUNEL and
FITC-annexin V binding. Significant levels of both DNA fragmentation
(43%, 28%) and annexin V binding (41%, 27%) were seen when the
targets were treated with granzyme B and adenovirus or perforin,
respectively. Cells in the absence of treatment (control) were 5%
TUNEL or FITC-annexin V positive. Likewise, the TUNEL or FITC-annexin V
signals in Jurkats treated with granzyme B, or perforin, or adenovirus
alone were less than 10%. Both DNA fragmentation and
phosphatidylserine externalization were inhibited when granzyme B
enzymatic activity was blocked with 3,4-dichloroisocoumarin (DCI; data
not shown). Thus, targets treated with both enzymatically active
granzyme B and perforin or active granzyme B and adenovirus displayed a
5- to 10-fold increase in both DNA fragmentation and phosphatidylserine
externalization over untreated cells or cells treated with each
component alone.
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m loss and generate
ROS
The production of ROS and the loss of the
m have been identified as common early
events occurring in apoptosis induced by a variety of stimuli
(28, 29, 30). We monitored the integrity of the
m during apoptosis induced by granzyme B
and perforin, or granzyme B and adenovirus using the
potential-sensitive dye DiOC6 (3), which at low
concentration targets to the negatively charged environment of the
mitochondrial matrix in intact cells (28). During
apoptosis, dissipation of the m leads to
leakage of DiOC6 (3) from the matrix, which can
be measured by flow cytometry as a decrease in the fluorescence
intensity of DiOC6(3) (28, 32). This
is visualized as a shift in the dye-loaded population from the lower
right (DiOC6(3)+) to the
lower left (DiOC6(3)-)
quadrant of a dot plot. ROS production within the cell can be measured
simultaneously with m using HE, which is
oxidized to ethidium in the presence of ROS and exhibits red
fluorescence following intercalation into cellular DNA. ROS production
is visualized using flow cytometry as a shift from the lower left
(HElow) to the upper left
(HEhigh) quadrants of a dot plot.
In the absence of treatment (cells alone),
DiOC6(3) fluorescence was apparent
(DiOC6(3)+), indicating
retention of the dye in mitochondria and an intact
m (Fig. 2).
Jurkats treated with the protonophore mClCCP, a mitochondrial uncoupler
(49), exhibited a reduction in the retention of
DiOC6(3) seen as a shift in the population from
DiOC6 (3)+ to
DiOC6(3)-. This shift
indicated a compromise in m integrity. In
addition, there was an enhanced conversion of HE to ethidium
(HEhigh cells) indicative of ROS production in
these cells. When Jurkats were incubated with granzyme B, perforin or
adenovirus alone, neither m disruption nor
ROS production was observed. However, when cells were treated with
granzyme B and perforin or granzyme B and adenovirus together for
2 h, m collapse and ROS production
were apparent. As was seen for DNA fragmentation and phosphatidylserine
externalization, no m loss or ROS
production was observed when granzyme B enzymatic activity was
inhibited with DCI (data not shown). Thus, the combinations induce
mitochondrial disruption and ROS production in Jurkat targets in vitro.
Similar results have been seen for L1210 cells treated with granzyme B
and perforin and EL4 cells treated with granzyme B and adenovirus (data
not shown).
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m collapse is caspase-independent
As a result of the dependence of the Fas system on caspases
(50), Fas-mediated apoptosis is exquisitely sensitive to
peptide caspase inhibitors (51). When Jurkats were treated
with anti-Fas Abs for 6 h, all measures of apoptosis examined,
including DNA fragmentation, phosphatidylserine externalization,
m collapse, and ROS production were
abrogated in the presence of the pan-specific caspase inhibitor
zVAD-fmk at concentrations as low as 5 µM (Fig. 3A). Here,
m collapse and ROS production (HE
conversion to ethidium) were quantified on separate dot histograms
rather than on the same two-color dot plots. zDEVD-fmk, an inhibitor
primarily of caspase-3-like caspases, also completely abrogated all
measured signs of apoptosis albeit at higher inhibitor concentrations
(between 20–100 µM). Cells showed no signs of
m loss or ROS production in the presence of
either zVAD-fmk or zDEVD-fmk alone (data not shown).
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B), as was seen for
anti-Fas-treated cells (Fig. 3A). While 20 µM zVAD-fmk
or zDEVD-fmk were sufficient to abrogate DNA fragmentation,
concentrations up to 100 µM were necessary to inhibit
phosphatidylserine externalization and ROS production. Importantly, in
these cells m loss was not prevented in the
presence of 100 µM of either inhibitor. Similar results were observed
when Jurkats were treated with granzyme B and perforin (data not
shown). Thus, while the onset of classic apoptotic morphology induced
by granzyme B and adenovirus was caspase dependent in a 2-h assay,
m collapse was caspase independent.
CsA does not block granzyme B-mediated m loss
Granzyme B-dependent killing involves a decrease in
m that occurs through a caspase-independent
process (Fig. 3B). The immunosuppressive agent CsA has been
shown to inhibit the m loss caused by PT
pore opening in many but not all systems (32). One system
in which CsA has been shown to prevent m
loss involves treatment with the PT pore-inducer PPIX
(52). As was shown previously (52), cells
treated with mClCCP or PPIX experience a loss of
m as measured by a decrease in
DiOC6 (3) fluorescence (Fig. 4). The PPIX-induced
m loss was prevented in the presence of 100
µM CsA. By contrast, CsA did not prevent the loss of
DiOC6 (3) from cells treated with granzyme B and
adenovirus, suggesting the granzyme B-dependent
m loss observed occurred through a
CsA-insensitive mechanism.
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m
collapse
It was necessary to confirm that the results observed in Jurkats
treated with granzyme B and perforin, or granzyme B and adenovirus in
vitro, were also occurring in vivo in response to allogeneic CTL. CTL
have been shown to kill targets predominantly through two mechanisms,
the granule-mediated and Fas pathways (1, 4). To determine
which pathway prevailed in our in vivo system, we relied on the
differential sensitivity of the two pathways to
Ca2+ chelation, the granule-mediated pathway
being Ca2+ dependent. Jurkat targets were exposed
to hCTL at a 2:1 E:T ratio in the absence or presence of 5 mM EGTA
(Fig. 5A). The amount of
[3H]thymidine release, a measure of DNA
fragmentation, decreased dramatically from 90% to less than 20% in
the presence of EGTA. Similar results were observed for
51Cr release, a measure of plasma membrane
damage. Furthermore, when Jurkats were pretreated with a blocking
anti-Fas IgG Ab to prevent Fas-mediated killing, no impairment of
either DNA fragmentation or 51Cr release was
observed relative to control targets in the absence of Ab (data not
shown). Thus, we conclude that the cytolysis of Jurkats mediated by
allogeneic CTL in our system involves predominantly the
granule-dependent pathway.
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m disruption seen in
cells treated in vitro with granzyme B and perforin, or granzyme B and
adenovirus, was occurring in vivo, studies were undertaken in which
Jurkats were preloaded with DiOC6(3) and then
incubated with hCTL at a fixed E:T ratio. Jurkats were loaded before,
rather than following, experimentation in an effort to differentiate
the targets from the effectors. However, using these means, it proved
difficult to distinguish targets from effectors by flow cytometry. To
alleviate this problem, Jurkat targets were generated that expressed a
green fluorescent protein (JpEGFP; see Materials and
Methods). The green fluorescence of these cells allowed them to be
distinguished from effectors by flow cytometry. By gating on
green-fluorescent cells and by utilizing a red-fluorescent
potential-sensitive dye, DASPEI, which has been used previously to
monitor mitochondrial membrane potential (53, 54), it was
possible to examine mitochondrial events in these cells. JpEGFP targets
were pretreated with DASPEI before addition of hCTL to further ensure
that the signals observed were coming only from the targets. Using in
vitro experiments, the disruption of m in
targets treated with granzyme B and adenovirus appears the same whether
examined with DiOC6(3) or DASPEI and
whether the dyes are added before or following the addition of the
apoptotic stimulus (data not shown). Thus, we are confident that
preloading JpEGFP targets with DASPEI is a viable technique for
examining m changes in vivo.
To confirm that the JpEGFP targets responded to allogeneic CTL in the
same fashion as Jurkats, JpEGFP were exposed to hCTL at an E:T ratio of
2:1 and analyzed for [3H]thymidine release and
51Cr release (Fig. 5B). The data show
clearly that JpEGFP cells undergo apoptosis in response to hCTL,
displaying levels of DNA fragmentation and membrane disruption
comparable to parent Jurkat targets. The JpEGFP clones were also
sensitive to EGTA in a fashion similar to parent Jurkats (data not
shown).
Having determined that JpEGFP cells underwent apoptosis in response to
hCTL attack, we wanted to know whether they also experienced the loss
in m seen when Jurkats were treated with
granzyme B and perforin or granzyme B and adenovirus (Fig. 2). To this
end, JpEGFP cells were preloaded with DASPEI and then exposed to hCTL
for 3 h at the E:T ratios indicated (Fig. 6A).
m disruption was observed in the
green-fluorescent target population as a loss in DASPEI fluorescence
seen at E:T ratios as low as 0.25:1. As observed previously (Fig. 3),
loss of m was not inhibited by zVAD-fmk. At
a zVAD-fmk concentration of 100 µM, m
loss was largely resistant (70%) at all E:T ratios tested. It is
possible that the small amount of sensitivity (30%) observed was due
to the small amount of Ca2+-independent killing
seen in the presence of EGTA (Fig. 5A).
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m loss
was again largely resistant to zVAD-fmk (80%) in DASPEI-loaded
JpEGFP targets treated with granzyme B and adenovirus (Fig. 6B). Hence, in our in vivo system,
m loss mediated through the granzyme
pathway is predominantly caspase independent. Cytochrome c release from mitochondria during granzyme B-mediated apoptosis is caspase independent
The release of cytochrome c from mitochondria appears
to be an early event during apoptosis induced by a variety of stimuli
(34, 35). Cytosolic cytochrome c has been shown
to promote the formation of a complex with Apaf-1 and procaspase-9 in
an ATP-dependent fashion (33, 39). The formation of this
apoptosome facilitates cleavage and activation of caspase-9, which then
proteolytically activates caspase-3 (38). Cytochrome
c release has been shown to occur in Jurkats following
treatment with anti-Fas Abs (reviewed in Refs. 31, 50, 55). Since Jurkats are type II cells (56),
cytochrome c release and subsequent apoptosis mediated
through the Fas pathway is exquisitely sensitive to the caspase
inhibitor zVAD-fmk. To confirm the caspase dependence of Fas-mediated
cytochrome c release in our system, Jurkat targets were
treated with anti-Fas Abs for up to 6 h in the absence and
presence of 100 µM zVAD-fmk. Following treatment, cells were
collected by centrifugation, and the pellets were resuspended in
digitonin lysis buffer. At low concentrations, the nonionic detergent
digitonin selectively renders the plasma membrane permeable while
leaving other organelles intact (45). Following
permeabilization, cells were centrifuged to separate the membrane
fraction (including mitochondria) from the cytosol fraction. Proteins
of both supernatants (cytosol) and pellets (membranes) were resolved on
15% polyacrylamide gels followed by transfer to nitrocellulose
membranes. Blots were probed with a monoclonal anti-cytochrome
c Ab. As a control for the presence of cytochrome
c, purified rat mitochondria (Rat Mito.) were also loaded
onto these gels (Fig. 7A). The
data indicate that, in the absence of zVAD-fmk, cytochrome c
translocates from the membrane fraction to the cytosol (Fig. 7A, lanes 10-12). This translocation
is apparent within 4 h following the initiation of treatment (Fig. 7A, lane 11). However, in the presence of
zVAD-fmk, cytochrome c release is completely abrogated, even
at 6 h following treatment (Fig. 7A, lanes
13–15). Thus, as has been previously reported (50),
Fas-induced cytochrome c release is caspase dependent in
Jurkat targets.
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m loss suggesting
possible effects of granzyme B on mitochondria (Fig. 2), we were
interested to know whether release of cytochrome c was also
occurring in granzyme B-treated Jurkat targets. Cytochrome c
release from the membrane fraction (containing mitochondria) to the
cytosol did occur in Jurkats in response to granzyme B and adenovirus
(Fig. 7B, lanes 13–17). Cytochrome c
was detected in the cytosol as early as 30 min following administration
of granzyme B and adenovirus (Fig. 7B, lanes 15
and 22). Cytochrome c was not released in response to
granzyme B or adenovirus alone (Fig. 7B, lanes 18
and 19, respectively). Importantly, in contrast to
anti-Fas-treated cells, granzyme B/adenovirus-mediated cytochrome
c release also occurred in the presence of 100 µM zVAD-fmk
(Fig. 7B, lanes 20–24). Identical results were
observed in HeLa cells treated with granzyme B and adenovirus (Fig. 7C). Cytochrome c release did not occur when
granzyme B enzymatic activity was inhibited with DCI (data not shown).
Therefore, the granzyme B-dependent pathway can induce cytochrome
c loss from mitochondria through a mechanism that requires
granzyme B enzymatic activity but does not rely upon caspases. Granzyme B-mediated membrane damage is caspase independent
Jurkats treated with granzyme B and adenovirus release cytochrome
c from their mitochondria and lose
m in a caspase-independent fashion despite
the absence of an apoptotic morphology at early times following
treatment (Figs. 3B and 7B). To investigate the
long-term consequences of caspase inhibition following granzyme B
treatment, Jurkats were treated with granzyme B and adenovirus for
24 h in the presence or absence of either zVAD-fmk or zDEVD-fmk
(Fig. 8). At the indicated times, DNA
fragmentation was assessed by [3H]thymidine
release, and membrane damage was measured by 51Cr
release. In the presence of zVAD-fmk or zDEVD-fmk, granzyme B-mediated
DNA fragmentation was blocked (0%) even at 24 h following
initial treatment (Fig. 8A). However, membrane damage, while
initially diminished in the presence of the caspase inhibitors, reached
similar levels by 24 h posttreatment (30% in the absence and
presence of zVAD-fmk and 20% in the presence of zDEVD-fmk),
indicating that caspases are not required to elicit membrane damage.
Importantly, in the Fas system on the other hand, both DNA
fragmentation and membrane damage were blocked in the presence of the
caspase inhibitors and were therefore dependent upon caspases (Fig. 8B). Thus, cells treated with granzyme B and adenovirus for
longer periods of time suffered membrane damage that was induced
through a caspase-independent mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We confirmed that apoptosis was occurring in our in vitro system of
CTL-mediated killing by measuring DNA fragmentation, a hallmark of
apoptosis (46), using TUNEL. Jurkat targets treated with
granzyme B and perforin or granzyme B and adenovirus for 2 h
showed a 5- to 10-fold increase in DNA fragmentation over untreated
cells or cells treated with granzyme B, perforin, or adenovirus alone
(Fig. 1). Granzyme B-mediated DNA fragmentation was absolutely
dependent upon caspases since fragmentation was abrogated in the
presence of either the pan-specific caspase inhibitor zVAD-fmk or the
inhibitor of caspase-3-like caspases, zDEVD-fmk (Fig. 3B).
Either inhibitor at concentrations as low as 20 µM was sufficient to
completely block DNA fragmentation. The ability of such low inhibitor
concentrations to mediate such profound inhibition was similar to the
effects of these inhibitors on DNA fragmentation seen when cells were
treated with anti-Fas Abs (Fig. 3A); the Fas pathway is
known to be absolutely dependent upon caspases (50).
As a second confirmation of apoptosis in our system, we examined the
extent of phosphatidylserine externalization from the inner to the
outer leaflet of the plasma membrane. As seen for DNA fragmentation,
granzyme B in combination with perforin or adenovirus induced an
10-fold increase in phosphatidylserine externalization over cells
treated with any of the components alone (Fig. 1). Similar to their
effects on granzyme B-induced DNA fragmentation, the caspase inhibitors
zVAD-fmk and zDEVD-fmk were also able to prevent phosphatidylserine
externalization in response to granzyme B and adenovirus (Fig. 3B) or granzyme B and perforin (data not shown). However,
concentrations up to 100 µM of either inhibitor were required to
mediate this inhibition completely. This contrasts with the Fas system,
in which lower concentrations of either zVAD-fmk (5 µM) or zDEVD-fmk
(20 µM) were sufficient to block phosphatidylserine externalization
(Fig. 3A). These differences may be a reflection of the
dependence of the Fas system on caspase-8 at the most proximal step of
activation (50) where zVAD-fmk would block apoptotic
signaling at the plasma membrane. Peptide inhibitors containing the
sequence DEVD are good inhibitors of caspase-3-like enzymes but are
also reasonably good at inhibiting caspase-8 (51). Thus
the substantial inhibition of DNA fragmentation and phosphatidylserine
externalization observed in the presence of zVAD-fmk likely reflects
caspase-8 inhibition at the initiation stage. In contrast, the results
with zDEVD-fmk may represent inhibition of caspase-3-like enzymes in
the effector stages, where more zDEVD-fmk may be required to achieve
comparable inhibition to zVAD-fmk.
In the granzyme B pathway, the pronounced inhibition of DNA
fragmentation by either zVAD-fmk or zDEVD-fmk, even at times up to
24 h following treatment (Figs. 3B and 8), points
toward the dependence of DNA fragmentation in this system on
caspase-3-like enzymes. Since higher concentrations of either inhibitor
are required to block phosphatidylserine externalization in the
granzyme B pathway, it is possible that a wider variety of caspases are
required to induce membrane alterations through granzyme B-dependent
events or that caspase-independent mechanisms are initiated.
Caspase-independent phosphatidylserine externalization and 
-fodrin
cleavage in response to TNF have been described (58).
Indeed, the fact that cells appear to have lost membrane integrity
24 h following granzyme B treatment in the presence of either
zVAD-fmk or zDEVD-fmk (Fig. 8A) suggests that caspases are
not required for the ultimate demise of the cell. Our results support
the view of Henkart et al. that killing by the granule pathway occurs
through caspase-dependent and -independent pathways (26, 27). It appears that, in particular, DNA fragmentation is
sensitive to caspase inhibition. In contrast, membrane damage slows but
still continues in the presence of either zVAD-fmk or zDEVD-fmk. It is
tempting to speculate that the caspase-independent mitochondrial
effects observed explain this necrotic form of death and may be
responsible for cell death even in the presence of "natural"
caspase inhibitors such as the inhibitors of apoptosis (IAP) family of
proteins (59).
Events such as the loss of the m, the
release of mitochondrial cytochrome c, and, in some cases,
the production of ROS, have been linked with the onset of apoptosis
induced by a variety of stimuli. These stimuli include Fas
cross-linking, TNF, glucocorticoids, and a number of chemotherapeutic
agents (28, 29, 30, 55). We investigated the possible role of
mitochondria in apoptosis induced through the granzyme B pathway, and
we observed that granzyme B induced a decrease in the
m only when administered in conjunction
with either perforin or adenovirus (Fig. 2). When Jurkats were treated
with hCTL or with granzyme B and adenovirus in the presence of either
zVAD-fmk or zDEVD-fmk, m loss was not
prevented (Figs. 3B and 6), suggesting that granzyme
B-mediated events at the mitochondria occur through a
caspase-independent mechanism. In this respect, our data support those
recently published by MacDonald et al. (60). This latter
study used a single in vitro system based on perforin and granzyme B.
Our present data extend this work to include a second in vitro system
and most importantly, intact CTL. Thus the results of our in vitro
studies were confirmed in a physiologically relevant model of killing
where CTL induced death in targets at low E:T ratios. There are,
however, some differences between our conclusions and those of the
earlier publication.
Whereas we find that both m collapse and
release of cytochrome c occur in the presence of zVAD-fmk
(Fig. 3A and Fig. 7, B and
C), MacDonald et al. reported that cytochrome c
release is caspase dependent. Our experiments were performed primarily
in Jurkats; however, we obtained identical results in HeLa, the same
cells used in the other study. It is possible that the explanation for
the discrepancy lies in a difference in experimental design. The
concentrations of granzyme B used were similar, but it is difficult to
rule out differences in the specific activities of the two enzymes. In
addition, we used human granzyme B on human targets whereas the other
group utilized a rat enzyme. To our knowledge, a direct comparison of
the caspases activated by granzyme B between species has not been
published. Nevertheless, it has been reported that murine granzyme B
cleaves only a limited number of caspases, and this contrasts with the
variety of caspases activated by human granzyme B (61). It
is noteworthy in this regard that MacDonald et al. were able to block
the initial cleavage of caspase-3 to p20 with zVAD-fmk in cells treated
with granzyme B and perforin. This suggests that caspase-3 activation
by granzyme B requires another caspase. In direct contradiction, we
have clear evidence that, in both our in vitro and whole cell killing
systems, this cleavage event is caspase independent
(22).
Another variable between the two experimental strategies could be the
role of perforin. In the other study, granzyme-independent effects were
clearly evident. In our experiments, we used sublytic doses of perforin
that gave no evidence of m collapse, ROS
production, or cytochrome c release in the absence of
enzymatically active granzyme B. Moreover, we have never observed
m loss in cells using concentrations of
adenovirus greater than ten times those used for the experiments
described here (Heibein et al., unpublished observations). It may be
that we are providing a milder, granzyme B-dependent stimulus to our
target cell mitochondria. Indeed, at 2 h, we still see a
considerable amount of cytochrome c in the mitochondria
whereas MacDonald et al. have completely depleted all mitochondrial
cytochrome c at this time point. Despite the fact that our
in vitro evidence strongly suggests that perforin by itself has no
effect on mitochondria, we cannot exclude the possibility that perforin
may be having some effect when Jurkat targets are exposed to hCTL. At
the sites of contact between target and CTL, the local concentration of
perforin may be sufficiently higher than perforin added exogenously to
induce mitochondrial alterations. The differences between our two
systems are intriguing, and further studies to establish the basis of
the discrepancies may be very informative for elucidating events
in vivo.
Cytochrome c, once released from mitochondria, is believed to form a complex with Apaf-1 and caspase-9. This "apoptosome" then mediates activation of caspase-3 in an ATP-dependent fashion. We do not have any direct evidence that cytochrome c released during granzyme B-mediated apoptosis forms a complex with Apaf-1 and caspase-9. Nevertheless, such an interaction has been described for Jurkats in response to the Fas pathway (56), and so it is not unreasonable to assume that such a process may occur in these cells in response to granzyme B. Since granzyme B is capable of directly activating caspase-3 in intact cells (10, 11, 12, 13, 22), one possible role for the Apaf-1/cytochrome c/caspase-9 complex is to amplify the cascade. Indeed, the role of the mitochondrion as an accelerator of the apoptotic response during granzyme B-dependent killing in a cell-free system has recently been described (60). The results reported here indicate that, in the granzyme pathway, only events downstream of mitochondria are sensitive to zVAD-fmk. This confirms our earlier report that initial caspase-3 activation is independent of any other caspase activity (22).
In some cases, m loss has been shown to
result from, or to induce, the opening of PT pores in the inner
mitochondrial membrane (32). PT pore opening induced by a
variety of chemical and physiological agents is inhibited in many, but
not all, instances, by the immunosuppressive agent CsA
(32). Jurkats were treated with PPIX, a ligand of the
benzodiazapine receptor (62). The benzodiazapine receptor
is a protein believed to be a constituent of the PT pore, and PPIX has
been shown to induce PT pore opening with the subsequent loss of
m in thymocytes (52). Our
results in Jurkats confirmed those previously observed in thymocytes in
that PPIX was able to induce m loss in
Jurkats in a CsA-inhibitable fashion (Fig. 4). By contrast,
m loss induced by granzyme B and adenovirus
was not inhibited in the presence of CsA. It is possible that the
granzyme B pathway induces PT pore opening through a CsA-insensitive
mechanism. Alternatively, it is likely that the
m loss observed results not from PT pore
opening but, rather, from some other mechanism, such as the loss of
cytochrome c interfering with electron transport and
oxidative phosphorylation, that would disrupt the ability of
mitochondria to maintain an intact m.
The production of ROS has been shown to accompany apoptosis-induced
m collapse (63) and, in some
instances, has been shown to facilitate PT pore opening
(29). In other cases, ROS production occurs well after
classical apoptotic morphology is evident, suggesting that ROS in these
systems may arise as a consequence of m
loss and the resultant uncoupling of electron transport and oxidative
phosphorylation (30). ROS production was observed in our
Jurkat system in response to granzyme B and perforin or granzyme B and
adenovirus (Fig. 2). However, ROS production decreased in the presence
of zVAD-fmk or zDEVD-fmk, suggesting that ROS production was in some
way dependent upon caspase activation (Fig. 3B). Since ROS
production was caspase dependent while m
loss was caspase independent, it is unlikely that ROS production in the
granzyme B system is the cause of m loss.
Rather, ROS production is likely a consequence of caspase activation
and the subsequent activation by caspases of downstream mediators. Over
the course of a 2-h assay in the presence of zVAD-fmk, ROS production
decreased despite the loss of cytochrome c (Figs. 3B and 7B). With the loss of cytochrome
c from mitochondria, uncoupling of electron transport and
oxidative phosphorylation would be expected to occur, which would lead
to ROS production at least in the long term. In our system, however,
unlike that of MacDonald et al., substantial cytochrome c
remains in mitochondria even at 2 h (Fig. 7B,
lanes 5 and 12), and such cytochrome c
levels may be sufficient to prevent complete uncoupling of electron
transport and oxidative phosphorylation. Other potential sources of ROS
production could be the endoplasmic reticulum and the cytoplasm
(64), and these sources could potentially be coupled to
caspase-dependent events.
Our results provide evidence for a two-pronged mechanism for the
destruction of target cells by CTL. The possibility of such a mechanism
was first revealed by Sarin et al. (26), and we now
suggest that the caspase-independent pathway may be mediated through
mitochondrial dysfunction. As depicted in Fig. 9, once inside the cell, granzyme B can
initiate apoptosis by the cleavage and activation of caspase-3. In
addition, we demonstrate that the protease can also mediate
caspase-independent effects on mitochondria that result in cytochrome
c release and loss of m. The
release of cytochrome c will likely activate the
Apaf-1/caspase-9 pathway, but our data indicate that initial caspase-3
activation is achieved through direct cleavage by granzyme B, resulting
in apoptosis (22). In parallel with caspase-3 activation,
cells with dysfunctional mitochondria are also destined to die by
necrosis. Such a two-pronged approach in vivo may serve to amplify
caspase activation. Importantly, both the necrotic and apoptotic
pathways are activated in response to granzyme B, clearly indicating
that both pathways will have to be considered in therapeutic
manipulations of the immune system. The next critical step in this
research will be the identification of the granzyme B substrate(s)
responsible for the observed effects on mitochondria.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. Chris Bleackley, Department of Biochemistry, Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7 Canada. E-mail address:
3 Abbreviations used in this paper: ROS, reactive oxygen species; m, mitochondrial inner membrane transmembrane potential; AIF, apoptosis-inducing factor; PT pore, permeability transition pore; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; HE, hydroethidine; mClCCP, carbonyl cyanide m-chlorophenyl hydrazone; PPIX, protoporphyrin IX; CsA, cyclosporine A; DASPEI, 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide; hCTL, human CTL; GFP, green fluorescent protein; JpEGFP, Jurkat stable transfectants containing the pEGFP-N1 plasmid; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; zDEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone; DCI, 3,4-dichloroisocoumarin; MOI, multiplicity of infection.
Received for publication March 25, 1999. Accepted for publication August 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are substrates for the apoptotic mediator CPP32. J. Biol. Chem. 271:27099.
