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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 1044-1054
Entry and Trafficking of Granzyme B in Target Cells During Granzyme
B-Perforin-Mediated Apoptosis
By
Michael J. Pinkoski,
Marita Hobman,
Jeffrey A. Heibein,
Kevin Tomaselli,
Feng Li,
Prem Seth,
Christopher J. Froelich, and
R.
Chris Bleackley
From the Department of Biochemistry, University of Alberta, Edmonton,
Alberta, Canada; IDUN Pharmaceuticals, La Jolla, CA; Medicine Branch,
National Cancer Institute, National Institutes of Health,
Bethesda, MD; the Department of Medicine, Evanston Hospital,
Northwestern University, Evanston, IL.
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ABSTRACT |
In the widely accepted model of granule-mediated killing by
cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate
internalization in the absence of perforin. Induction of the lytic
pathway, however, required a second signal that was provided by
perforin or adenovirus (Ad2). The combination of agents also resulted
in a dramatic relocalization of the granzyme. Microinjection of
granzyme B directly into the cytoplasm of target cells resulted in
apoptosis without the necessity of a second stimulus. This suggested
that the key event is the presence of granzyme B in the cytoplasm, and
that when the enzyme is internalized by a target cell, it trafficks to
an intracellular compartment and accumulates until release is
stimulated by the addition of perforin. We found that the proteinase
passed through rab5-positive vesicles and then accumulated within a
novel compartment. On the basis of these results, we propose a new
model for granzyme-perforin-induced target cell lysis in which
granzyme B is subjected to trafficking events in the target cell that
control and contribute to cell death.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CYTOTOXIC T LYMPHOCYTES (CTL) are the
effectors in cell-mediated immunity that destroy virus-infected and
neoplastically transformed cells.1-4 After adhesion of CTL
and target cell, the plasma membranes interdigitate and cytolytic
granules migrate toward the site of contact, releasing their contents
into the apposed space. This secretory process forms the basis of the
lethal hit model of granule-mediated cytotoxicity in which it is
postulated that the molecules contained within the cytoplasmic granules
are the lytic effectors that act in concert to bring about target cell
apoptosis.5
A number of proteins have been isolated and characterized from
cytolytic granules. The pore-forming protein, perforin,6 is
a granule protein that is believed to insert into the membrane of the
target cell, providing a channel that facilitates the entry of other
granule components into the cytoplasm of the target cell.7 Granzyme B, also known as cytotoxic cell proteinase 1 (CCP18) or fragmentin 2,9 is the prototypic
member of the family of serine proteinases that reside in the cytolytic
granules of CTL, natural killer (NK), and lymphokine-activated killer
(LAK) cells,8,10 collectively termed granzymes. Although
perforin alone causes necrotic cell death by osmolysis,
apoptosis is mediated by the concerted actions of perforin and the
granzymes.
Several laboratories have provided evidence for the synergistic
requirement of granule proteins in stimulating apoptosis in target
cells. Granzyme B and perforin have been shown to bring about rapid DNA
fragmentation in target cells by a mechanism that is attributed to
granzyme activity.9,11 Similarly, transfection of the rat
basophilic cell line, RBL, with perforin and granzyme B renders the RBL
capable of cytolytic activity when directed against a number of target
cell lines.12,13 Conclusive evidence for a role of
granzymes in target cell apoptosis has come from the generation of
granzyme B-deficient mice. The CTL of granyzme B / mice
display a severely reduced ability to induce rapid DNA fragmentation in
target cells.14,15
In support of the notion that the granzymes induce apoptosis, in part,
by cleaving cytosolic proteins, granzyme B has been reported to
activate certain ICE/CED-3 cysteine proteinases, or caspases,16 that are closely linked to apoptotic cell death (reviewed in Martin and Green17 and Nagata18),
including caspases 319 and 7.20,21 Caspase-3 is
processed in murine targets treated with CTL,19 and targets
exposed to granzyme B and perforin contain processed caspases
1,22 3, and 7.21 Thus, it is implied that granzyme B gains access to the interior of the target cell where it can
cleave and activate these substrates. Activation of the caspases has
been shown to be essential in DNA fragmentation,23 for
externalization of phosphatidylserine in the plasma membrane of cells
undergoing apoptosis,24 and for degradation of actin via
gelsolin activation.25 In addition to the cytoplasmic
substrates of granzyme B, there is an increasing body of evidence
suggesting that granzyme B may act in the nucleus. In two independent
studies, granzyme B was shown to bind to nuclear
proteins.26,27 At least one of these potential substrates
is associated with heterochromatin and the perinucleolar region of the
nucleus.26
Despite major advances in our understanding of the mechanisms
of granule-mediated CTL killing, there has been no direct demonstration of transfer of molecules from cytolytic granules to target cells and no
information on the resultant trafficking of CTL proteins once they have
gained entry into the target cell. Previously, we have shown that
granzyme B binds specifically to the surface of Jurkat cells, but
apoptosis did not occur until the subsequent addition of perforin. The
absolute requirement for the latter could be circumvented by the
treatment of the cells with a replication-deficient adenovirus (Ad2).
We hypothesized that granzyme B was taken into cells by endocytosis and
that the Ad2 or perforin caused release of granzyme into the
cytoplasm.28
To test our hypothesis, we designed experiments to directly visualize
granzyme B as it is internalized into the cell and elucidate its
intracellular trafficking. Using confocal laser scanning microscopy (CLSM), we demonstrate that granzyme B does indeed enter the cell independently of perforin. It appears to be transiently associated with
rab5+ vesicles, but ultimately accumulates in a novel
compartment. After treatment with perforin or Ad2, the granzyme B is
released into the cytoplasm, translocates rapidly to the nucleus, and
apoptosis ensues. In contrast, direct injection of granzyme B into the
cytoplasm induces death directly, without the requirement for perforin.
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MATERIALS AND METHODS |
Antisera and fluorescent markers.
Antisera was raised against residues 9-16 of granzyme B (provided by
Dorothy Hudig, University of Nevada, Reno) and used as previously
described.26 Secondary antibodies goat
antirabbit-fluorescein isothiocyanate (FITC), goat anti-rabbit-Texas
Red, donkey anti-mouse-Texas Red, and mouse anti-rabbit rhodamine were
purchased from Jackson Immunoresearch (West Grove, PA),
rab5 (S-19) and rab4 (D-20) polyclonal antisera from Santa Cruz
Biotechnology (Santa Cruz, CA), rab5 monoclonal antibody (clone #15)
from Transduction Laboratories (Lexington, KY), cathepsin D antisera
from Upstate Biotechnology, Inc (Lake Placid, NY), and cytochrome c
monoclonal antibody from Pharmingen (San Diego, CA). All antisera were
used at dilutions of 1:50 to 1:100 unless otherwise noted. Granzyme B
was fluoresceinated according to the protocol described by Jans et
al.29
Induction and measurement of apoptosis.
Human perforin and granzyme B were purified as previously
described.28,30 Type 2 adenovirus was purified according to
Seth et al.31 Granzyme B was added directly to Jurkat
target cells at 1 µg/mL in RPMI supplemented with bovine serum
albumin (BSA) (0.05% wt/vol). Activity of granzyme B was
120 U/µg where 1 U is the activity of enzyme required to hydrolyze 1 nmol/minute of the BAADT substrate. Sublytic doses of
perforin were used at 90 U/mL, where 1 U is defined in the standard
sheep red blood cell hemolytic assay. Cells were washed and resuspended
four times in medium before further treatment.28 Volumes
equivalent to the incubation volume were used for each wash. Infection
with adenovirus (Type 2) was performed in RPMI with BSA (0.05%), as noted above for granzyme B at a multiplicity of infection of 10 plaque-forming units per cell. COS M5 cells were transfected to express
granzyme B in the pAX142 cloning vector32 as previously described.33 Transiently transfected COS M5 cells were
treated with adenovirus or perforin as described above without prior
treatment of granzyme B. All cells were incubated at 37°C unless
otherwise noted.
Apoptosis of target cells was assessed by terminal deoxytransferase
nick end labeling (TUNEL) reaction to measure DNA
fragmentation.34 Fluorescein-deoxyuridine triphosphate
(dUTP) was incorporated at the sites of DNA nicks. The
DNA-binding dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI;
Sigma Chemical Corp, St Louis, MO) was added to saponin
permeabilized cells at 1 µg/mL for at least 15 minutes before viewing
by fluorescence microscopy. DAPI was used as a label for nuclear
morphology.
Microinjection of granzyme B into target cells.
Cell microinjection was performed on the stage of a Nikon Diaphot
inverted microscope (Nikon, Melville, NY) using an Eppendorf pressure
injector (Model 5246; Brinkmann Instruments, Inc, Westbury, NY) and
micromanipulator (Model 5171). Microinjection needles (approximately 0.1 µm inner diameter) were pulled from glass
capillaries using a horizontal electrode puller (Model P-97; Sutter
Instrument Co, Novato, CA) and loaded using Eppendorf microloaders.
Enzymatically active granzyme B and granzyme B inactivated with
antigranzyme B were diluted in 0.15 mol/L NaCl. To identify injected
cells, the injectate contained 0.3% Texas Red Lysine Fixable
(Molecular Probes, Eugene, OR). The concentration of
purified human granzyme B was 3.23 mg/mL, which corresponds to 119.4 U/µL. Solutions were injected into the cytoplasm of MCF-7 cells,
which were plated on glass cellocate cover slips (Eppendorf) 18 hours
before injection. Medium (RPMI 1640, 10% fetal bovine serum [FBS],
200 µg/mL G418, and 100 µg/mL hygromycin) was changed before and
after injection. Minaschek et al,35 using similar
equipment, demonstrated that the injection parameters used in the
present study (pressure, 100 hecto Pascal; time 0.5 seconds) delivers approximately 0.05 pL into the cytosol of 3T3 cells.
We estimate the volume of MCF-7 cells to be 5 pL, similar to that of
3T3 cells. Thus, intracellular concentrations of the injectate (Table
1) are estimated to be 1% of the pipette concentration, reflecting a
100-fold dilution in the cell.
Immunocytochemistry, confocal laser scanning microscopy, and flow
cytometry.
Jurkat cells were attached directly to glass microscope slides by
centrifugation for 15 seconds at 600g. COS cells were grown directly onto 0.5-mm glass coverslips. All cells were fixed immediately in paraformaldehyde (2% wt/vol in phosphate-buffered saline [PBS]) and washed in PBS before permeabilization and immunolabeling. Cells
were labeled as previously described26 and viewed with a
Zeiss fluorescence microscope (Carl Zeiss, Inc,
Thornwood, NY) or by confocal laser scanning microscopy and analyzed
with the accompanying CLSM software (CLSM; Leica, Heidelberg, Germany). Images were acquired by 32 or 64 line scan averaging using
100×/1.32N.A. objective under oil immersion.
Immunoelectronmicroscopy.
HeLa cells were incubated in medium containing granzyme B (1 µg/mL)
for 60 minutes at 4°C, washed on ice, and placed at 20°C or
37°C for 45 minutes. Immediately after incubations, cells were fixed in 4% paraformaldehyde at 4°C for 5 minutes, then 60 minutes at room temperature. Cells were washed in PBS and dehydrated in increasing concentrations of ethanol. Dehydrated pellets were infiltrated with Lowicryl K4M:ethanol at 20°C followed by
embedding at 20°C according to manufacturer's directions
(Polysciences Inc, Warrington, PA).
Ultrathin sections (40 to 70 nm) were placed on carbon/parlodion-coated
copper grids. Grids were blocked sequentially with double distilled
water (1 minute), 0.01 mol/L glycine (5 minutes), and PBS containing
1% BSA and 0.1% gelatin (20 minutes). Labeling was performed at room
temperature for 2 hours with antibody diluted in final blocking
solution. After washing in blocking solution, grids were incubated in
gold-conjugated secondary antisera (Jackson Immunoresearch) for 45 minutes at room temperature. After thorough washing in blocking
solution, grids were rinsed in distilled water, counterstained in
uranyl acetate and lead citrate, and viewed with a Philips 420 electron
microscope (Philips Electron Optics, Eindhoven, The Netherlands).
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RESULTS |
Autonomous entry of granzyme B into target cells.
Perforin has long been thought to be absolutely required to facilitate
the entry of granzyme B into target cells. To test this hypothesis, we
used direct and indirect fluorescence labeling techniques and confocal
microscopy to visualize granzyme B when target cells were treated with
the proteinase in the absence of perforin. Jurkat cells were incubated
in medium containing soluble granzyme B at 37°C for up to 120 minutes. After washing and permeabilization, they were treated with
antigranzyme antiserum, followed by a Texas Red-conjugated secondary
antibody and analyzed by CLSM. Figure 1
shows the accumulation of soluble granzyme B inside target cells. Within 60 minutes, there was a significant staining for granzyme B in a
distinct intracellular pattern compared with immunolabeled control
cells not exposed to granzyme B (Fig 1A).
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| Fig 1.
Granzyme B is internalized independently, but requires
perforin to induce apoptosis. Jurkat target cells were incubated in medium containing granzyme B for 0 and 60 minutes at 37°C (A). Cells were fixed in 2% paraformaldehyde and immunolabeled with antigranzyme antiserum and staining with Texas Red-conjugated goat
antirabbit antibody. Cells were viewed by CLSM. Granzyme B label was
not present at 0 minutes, but appeared in a distinct punctate pattern
by 60 minutes. Despite the uptake of granzyme B, there were no
detectable signs of cell death. (B) Jurkat target cells were treated
with granzyme B, washed to remove soluble granzyme, and incubated in
sublytic levels of perforin at 37°C for the times indicated. Cells
were labeled for DNA fragmentation by the TUNEL protocol with
dUTP-FITC. Granzyme B+ targets showing the progression of
DNA fragmentation observed in apoptotic death induced by granzyme B and
perforin from a healthy cell (0 minutes); early DNA fragmentation
observed at early time points (30 minutes); an apoptotic cell with
considerable DNA fragmentation and reduced nuclear size (60 minutes);
and advanced DNA fragmentation and severe nuclear condensation at 120 minutes. (C) Yac-1 targets were treated with granzyme B and buffer or
in combination with perforin. Cells were labeled with the DNA-binding
dye DAPI and viewed by fluorescence microscopy. Examples of condensed
nuclei of apoptotic cells are indicated by arrows. Scale bar is 10 µm.
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Target cells that have internalized granzyme B undergo apoptosis only
after exposure to perforin.
We also studied the ability of granzyme B alone to trigger apoptosis in
target cells. Jurkat, treated with granzyme B, were labeled by the
TUNEL technique to assess DNA fragmentation and analyzed by CLSM. These
experiments were also performed in YAC-1 cells with analysis of nuclear
morphology, after DAPI staining, by fluorescence microscopy (Fig 1B and
C). Cells labeled by either TUNEL or DAPI were also immunolabeled to
detect granzyme B. No DNA fragmentation or nuclear condensation was
observed under these conditions and the cells appeared entirely normal
(Fig 1B and C). Despite the accumulation of granzyme B within target
cells, we did not observe DNA fragmentation or nuclear condensation, two hallmarks of apoptosis, even upon prolonged incubation.
Jurkat and YAC-1 target cells were treated concurrently with granzyme B
and sublytic doses of perforin. As shown by TUNEL label and DAPI stain,
this resulted in rapid DNA fragmentation and nuclear condensation
within 30 minutes. Targets were also incubated in medium containing
granzyme B for 60 minutes and washed extensively before being treated
with sublytic doses of perforin. Figure 1B (TUNEL label) and C (DAPI
stain) show that cells treated in this two-step sequential manner also
showed significant DNA fragmentation, severe nuclear condensation, and
a drastic reduction in size. This was in stark contrast to the results
seen with cells exposed to granzyme B alone. We conclude that although
perforin is dispensable for the internalization of granzyme B, it is
absolutely required to deliver a death signal to target cells
containing granzyme B.
Perforin/Ad2 functions to redistribute granzyme B from its
intracellular compartment.
Internalized granzyme B showed a punctate cytoplasmic staining, but the
cells were clearly not apoptotic. We therefore asked if either perforin
or Ad2 influenced the intracellular distribution of granzyme. Jurkat
were preloaded with granzyme B for 60 minutes at 37°C, and then
perforin or Ad2 was added. The targets were fixed and immunolabeled for
granzyme B. Simultaneous TUNEL label with dUTP-FITC was used to assay
apoptosis and cells were viewed by CLSM.
As shown in Fig 2A (see page 1048), when
perforin was added to granzyme-containing targets, we no longer
observed a punctate granzyme B pattern. Rather, granzyme was detected
in the nucleus concurrent with the onset of DNA fragmentation in
TUNEL-positive cells. A similar result was obtained when granzyme
B-containing Jurkat were subsequently treated with Ad2. In both of
these systems, we observed localization of granzyme B label in the
nucleus at the sites of TUNEL-positive DNA fragmentation (Fig 2A).
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| Fig 2.
Perforin and Ad2 cause an intracellular redistribution of
granzyme B. (A) Jurkat cells were pretreated with granzyme B at 37°C for 60 minutes to allow uptake of granzyme. Cells were washed and incubated in either perforin or Ad2. Cells were immunolabeled for
granzyme B and Texas Red (TR)-conjugated secondary antibody (red) and
TUNEL labeled with dUTP-FITC (green). Regions of colocalization appear
yellow. The punctate cytoplasmic labeling pattern of granzyme B alone
(see Fig 1 A) was disrupted and nuclear accumulation of granzyme B was
evident. The presence of granzyme B in the nucleus coincided with sites
of DNA fragmentation detected by the TUNEL protocol. (B) COS M5 cells
were transiently transfected to express granzyme B. After 48 hours,
cells were treated with buffer as a negative control or perforin and
labeled for granzyme B (TR) and TUNEL (dUTP-FITC) as in (A). In the
absence of perforin, granzyme B appeared to be contained in a
cytoplasmic structure, but was redistributed in the presence of
perforin. As with Jurkat targets, COS cells containing granzyme B
underwent rapid apoptosis when treated with perforin and nuclear
granzyme B was colocalized with sites of TUNEL-labeled DNA
fragmentation. Scale bar is 10 µm.
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We have shown that granzyme B can enter the target cell unassisted by
perforin or Ad2, but the possibility remained that sufficient residual
granzyme B was bound to the outside of cells and that this could
"piggyback" inside with perforin or Ad2 resulting in death. We
took advantage of the fact that COS cells transfected with the granzyme
B cDNA store the active enzyme in cytoplasmic vesicles. COS M5 cells
expressing granzyme B were treated with perforin or Ad2, as described
earlier for Jurkat, and analyzed by fluorescent TUNEL assay in
conjunction with immunolabeling with antigranzyme antiserum to identify
positively transfected cells. Perforin treatment of granzyme B positive
cells exhibited rapid DNA fragmentation, while cells not expressing
granzyme B were unaffected (Fig 2B). As seen with Jurkat cells, this
system also resulted in a relocation of the granzyme B from the
cytoplasm to the nucleus.
Despite extensive attempts, we have not been able to detect granzyme B
activity in the supernatant or granzyme B protein on the surface of
transfected COS cells. Thus, we believe that the ectopic protein is
retained within a cytoplasmic vesicle and not secreted for reuptake in
the presence of perforin or Ad2. In addition, COS cells appear to be
resistant to lysis by treatment with externally added granzyme B. They
can be infected with the virus, but do not appear to take up the
granzyme.
Together the results obtained from visual assessment of granzyme B in
Jurkat and COS M5 cells suggest that both perforin and Ad2 affect the
intracellular compartment occupied by granzyme B. The enzyme is taken
up independently of perforin, but is sequestered so that apoptosis does
not occur. Upon exposure to perforin, or Ad2, the granzyme is released
into the cytoplasm and then rapidly translocates to the nucleus.
However, as soon as it is liberated, the granzyme B can interact with
its substrates and hence initiate the cell death cascade.
Entry of granzyme B is via receptor-mediated endocytosis.
Macromolecules can be internalized into cells by a variety of
mechanisms, such as fluid phase engulfment and receptor-mediated endocytosis. Incubation of cells at 4°C prevents internalization of
molecules due to a lack of fluidity of the plasma membrane, but does
not prevent binding to surface receptors.36 Therefore, treatment at this temperature can be an effective means to
differentiate between nonspecific fluid phase engulfment and
receptor-mediated uptake. Accordingly, we treated Jurkat targets with
soluble granzyme for 60 minutes at 4°C. The cells were then washed
extensively with medium (pH 7.4) or citrate buffer (pH 3.0) and
incubated for an additional 60 minutes at 37°C. Both treatment
groups were then resuspended in medium (pH 7.4) and incubated in the
presence of Ad2 for 60 minutes at 37°C. Aliquots of cells were
labeled by TUNEL or annexin V-FITC and analyzed by
fluorescence-activated cell sorting (FACS).
Cells treated at 4°C and washed in a neutral conditions exhibited
similar levels of apoptosis to cells treated at 37°C
(Fig 3). Those exposed to
granzyme B at 4°C and washed under acidic conditions did not
undergo apoptosis. Acid treatment of cells incubated at 37°C had no
adverse effect on killing, suggesting that sufficient granzyme B had
gained access to the cell before the acid wash.
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| Fig 3.
Uptake of granzyme B is mediated through specific
interactions at the cell surface. Jurkat cells were incubated in
granyzme B at 4°C or 37°C for 60 minutes, washed in citrate (pH
3.0) or medium pH 7.4. Cell death was assayed by TUNEL and annexin
V-FITC and analyzed by flow cytometry. Percentage of specific killing by granzyme B and Ad2 at 4°C was calculated relative to that
observed at 37°C. Sufficient granzyme B remained bound to targets
at 4°C after a neutral wash to induce apoptosis after addition of
Ad2 at 37°C, whereas acid wash of granzyme B-treated cells at
4°C significantly reduced the level of cell death.
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If entry of granzyme B into the target cell is required to induce
apoptosis, internalization must have occurred in order for killing to
occur. Therefore, from these data we conclude that granzyme B interacts
specifically with a surface receptor with sufficient affinity so as to
withstand removal during washes, and that these interactions are
susceptible to disruption by acid treatment. These data are consistent
with the notion that internalization of granzyme B in the absence of
perforin occurs via a receptor-mediated process and not passive
engulfment of the fluid phase.
Microinjection of granzyme B into the cytoplasm of target cells
results in cell death.
Granzyme B appeared to enter target cells autonomously, but required a
second signal to induce apoptosis. This correlated with a
redistribution of the granzyme within the target cell and suggested the
possibility that perforin and Ad2 function to liberate granzyme B into
the cytoplasm. To test this hypothesis, we used microinjection to
introduce granzyme B directly into the cytoplasm of MCF-7 target cells.
Granzyme B induced apoptosis in MCF-7 cells in a dose-dependent fashion
(Table 1). Introduction of approximately 80 fg/cell (intracellular concentration of approximately 500 nmol/L) granzyme B led to apoptosis in 40% of cells in 2 hours. Staining of
injected cells with Hoechst showed condensed nuclei, a hallmark of
apoptotic cell death (data not shown). Apoptosis was due to the
enzymatic activity of granzyme B, as an equivalent amount of granzyme
that had been inactivated with antigranzyme B did not induce apoptosis.
Microinjection of granzyme B into the cytoplasm of target resulted in
apoptosis, but granzyme internalized by the target did not. This
confirms our hypothesis that the key step is release of granzyme B into
the cytoplasm. The target cell appears to traffick granzyme B and
sequester it away from its cellular substrates. Perforin treatment then
effects the release of the enzyme where it can activate its substrates.
Microinjection likely bypasses this trafficking control to cause
apoptosis independently of the second signal.
Granzyme B is transported through an endocytic pathway.
Our results thus far suggest that internalized granzyme B is
sequestered within a cytoplasmic vesicle. Because granzyme B appeared
to be taken up by a receptor-mediated mechanism, we investigated the
possibility of transport through the endocytic pathway by performing
double label experiments with endosomal markers.
Small Ras-like proteins of the Rab family are known to be involved in
vesicular trafficking.37,38 Rab5 is known to control early
endocytic processes and is found in vesicles at the plasma membrane and
in newly formed early endosomes and perinuclear recycling endosomes,39 and rab4 has been identified on early
endosomes and endocytic vesicles that recycle plasma membrane
constituents from early endosomes.40-43 To determine
whether granzyme B was internalized into such a vesicle, colocalization
studies were performed with antibodies to granzyme and rab4 or rab5.
Due to the difficulty in ascertaining distinct cellular morphology in
Jurkat, we used HeLa for intracellular localization studies. We
assessed the early trafficking of granzyme B by incubation of cells in
the presence of granzyme B at 20°C, which prevents membrane fusion
events required for endosomal maturation.36 HeLa, grown on
glass coverslips, were incubated in medium containing 1 µg/mL
FITC-conjugated granzyme B at 20°C. Cells were fixed, permeabilized, and immunolabeled with antisera against rab4 or antibody
against rab5 and corresponding Texas Red-conjugated antisera. Immunolabeled cells treated with granzyme B-FITC were analyzed by CLSM
(Fig 4A, see page
1048). We observed accumulation of
granzyme B in early endosomes and perinuclear recycling vesicles
characterized by colocalization with rab5. A significant, but lesser
amount, of colocalization of granzyme B with rab4 was also seen,
consistent with the indication that granzyme B is contained in an early
endosome at 20°C.
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| Fig 4.
Internalized granzyme B colocalizes with the endosomal
markers, rab4 and rab5 at 20°C. (A) HeLa target cells were
incubated for 60 minutes at 20°C to allow uptake of soluble
granzyme B-FITC, immunolabeled for rab5 or rab4 with corresponding
TR-conjugated secondary antisera and viewed by CLSM.
rab5+ and rab4+ endosomes appear red and
sites of granzyme B-FITC appear green. Colocalization of granzyme B and
rab5 or rab4 is indicated by the appearance of yellow. Scale bar is
10 µm. (B) Ultrathin sections of Lowicryl embedded
HeLa cells treated with granzyme B at 20°C were immunolabeled with
polyclonal antisera against rab4 (10 nm gold) and monoclonal antibody
against granzyme B (18 nm gold). Endosomal compartments labeled with
rab4 (arrowheads) occasionally contain granzyme B (arrows). Some rab4
is seen in the cytoplasm, but the general background is low, as
illustrated by the lack of gold particles in the nucleus (N). Scale bar
is 1.0 µm.
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To obtain a more detailed characterization of early trafficking of
granzyme B, we performed immunoelectron microscopic analyses on targets
treated with granzyme B at 20°C. HeLa were allowed to bind soluble
granzyme at 4°C for 60 minutes, washed, then shifted to 20°C
for 45 minutes to allow uptake. Ultrathin sections of fixed and
embedded cells were labeled with antibodies against granzyme B and rab4
with corresponding gold-conjugated secondary antisera, 18 nm particle
for granzyme B and 10 nm particle for rab4. As shown in Fig 4B, we
observed colocalization of granzyme B with rab4 in targets treated at
20°C. Our observation of granzyme B in rab4+ early or
recycling endosome is consistent with the colocalization of granzyme B
with rab4 and rab5 by CLSM (Fig 4A).
Granzyme B accumulates in a novel intracellular compartment.
Although granzyme B appeared to be internalized via receptor-mediated
uptake and to follow an endocytic route, the further trafficking
pathway remained to be established. To investigate the downstream
events, we performed double label studies with granzyme B and markers
of other vesicles and organelles after continuous granzyme B uptake at
37°C. HeLa were incubated for 60 minutes at 37°C in the
presence of soluble granzyme B-FITC, fixed in paraformaldehyde, and
immunolabeled for rab5 and rab4, mannose 6-phosphate receptor (MPR), a
marker of late endosomes and the Golgi apparatus, cathepsin D and
lgp120, both lysosomal markers, and cytochrome c, a mitochondrial
protein. Figure 5 (see page 1048) shows
representative cells labeled as described above, demonstrating that the
site of granzyme B accumulation is not in vesicles or organelles
characterized by any of these markers. Target cells incubated in medium
containing granzyme B-FITC at 37°C for 60 minutes, or
greater, no longer displayed dominant colocalization with either rab5
or rab4. This suggested that although granzyme B appeared to be
transported through early endosomes, this was clearly not the
site of granzyme B accumulation.
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| Fig 5.
Granzyme B accumulates in an uncharacterized
intracellular vesicle. HeLa target cells were incubated for 60 minutes
at 37°C to allow uptake of soluble granzyme B-FITC. Cells were then
immunolabeled for markers of intracellular compartments: rab5 and rab4
for early endosomes, cathepsin D and lgp120 for lysosomes, MRP for late endosomes and the Golgi apparatus, and cytochrome c for mitochondria, each with corresponding TR-conjugated secondary antisera. Labeled cells
were viewed by CLSM. Scale bar is 10 µm.
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In an attempt to better define the site of granzyme B accumulation, we
performed uptake experiments at 37°C for analysis by electron
microscopy. HeLa were incubated at 4°C for 60 minutes with
granzyme, washed, and shifted to 37°C to allow uptake and trafficking. Cells were fixed, embedded, and sectioned for
immunolabeling. Figure
6A shows the intracellular localization
of granzyme B (10 nm gold) in HeLa treated at 37°C as a distinct
compartment that is morphologically discernible from rab4+
vesicles that contained granzyme B at 20°C (Fig 4B). Double label of granzyme B (10 nm gold) and rab5 (18 nm gold) showed some
colocalization, but that the predominant granzyme label was distinct
from rab5-positive vesicles (Fig 6B).
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| Fig 6.
Immunoelectron microscopy of granzyme B in its
intracellular compartment. (A) HeLa cells were treated with granzyme B
as in Fig 4B, except that after binding of granzyme B to the cell
surface at 4°C, cells were moved to 37°C to allow transport to
its retention compartment. Labeling with antibody against granzyme B
followed by 10 nm gold-conjugated secondary antibody demonstrates that granzyme B is retained in a compartment with morphologic
characteristics different from rab4+ endosomes shown in
Fig 4B. (B) HeLa cells were treated with granzyme B as
in (A). Sections were incubated with antibodies to granzyme B and
antisera against rab5 followed by corresponding secondary antibodies
conjugated to 10 nm and 18 nm gold particles for granyzme B and rab5,
respectively. At 37°C, granzyme B, denoted by arrows, was found
primarily in a compartment not containing rab5. However, the more
electron dense endosomal vesicles containing rab5 (denoted by filled
arrows) are only occasionally labeled with granzyme B (open arrowhead).
The morphology of the compartment containing granzyme B is distinct
from the rab5 compartment. As a negative control in both panels,
sections were labeled with each gold-conjugated secondary antibody in
the absence of the corresponding primary antibody or antiserum.
Sections treated in this manner did not display any labeling
(data not shown). Scale bars are 0.1 µm.
|
|
Taken together with colocalization of granzyme B with rab5 and rab4 at
20°C, these data suggest that granzyme B is transported through the
early stages of the rab5/rab4 endosomal pathway, but that it
accumulates in a cellular compartment that is not characterized by
rab5, rab4, or any of the other vesicle markers tested.
| |
DISCUSSION |
As originally formulated, the granule exocytosis model of CTL-mediated
cytotoxicity envisaged lethal lytic effector molecules being delivered
to target cells by vectoral exocytosis of CTL granules. Initially it
was believed that perforin was sufficient to induce target cell lysis,
but then it became apparent that the granzymes (in particular granzyme
B) were required to bring about physiologically relevant apoptotic
death. The model was modified to include granzymes passing through
perforin channels into the cytoplasm of the condemned cell. The
formation of a complete macromolecular channel was not clearly
demonstrated, but perforin damage to the membrane was a key factor in
granzyme uptake. Despite the fact that many laboratories have been
unable to replace perforin with a variety of channel forming and
membranolytic agents, this model has become the widely accepted
paradigm.
Autonomous entry of granzyme B and requirement of perforin for
apoptosis.
Although there is a wealth of indirect evidence to suggest that
granzyme B enters the target, this key experimental fact has not been
established. We therefore decided to look directly at whether granzyme
B gains access to the target cell in the presence and absence of
perforin. Using direct and indirect immunocytochemistry and CLSM, we
found that granzyme became internalized into an early endosome and
finally accumulated in a larger, uncharacterized vesicle. Importantly,
we saw no evidence for the induction of apoptosis in these cells. In
contrast, when granzyme B and perforin were added together, we observed
rapid fragmentation of target cell DNA. Indeed, target cells could also
be preloaded with granzyme B and then induced to die by subsequent
addition of perforin. Thus, granzyme B alone can be taken up into a
target cell but, for death to occur, perforin must also be present.
Perforin and Ad2 have been shown to stimulate granzyme-mediated
apoptosis in targets loaded with granzyme B.28 Here we show that the granzyme is sequestered in cytoplasmic vesicles where it
appears to be innocuous to the target. In addition, it appears that
perforin and Ad2 caused a redistribution of the granzyme, which allowed
the protease access to substrates and resulted in nuclear accumulation
of granzyme B.
Ad2 is known to cause a disruption in endocytic
vesicles,31,44,45 and perforin is also known to possess
membrane-disrupting properties6 and also affects endocytic
processes.46 The delivery of granzyme into the cytoplasm
may be mediated directly by insertion of perforin channels in endosomal
membranes or by stimulating a signal that ultimately results in release
of granzyme within the target cell. At present, we cannot rule out that
a signal triggered by perforin may be entirely independent of its
pore-forming activity.
COS M5 cells transfected with granzyme B provided a system that allowed
us to bypass the internalization process. Perforin and Ad2 both induced
rapid DNA fragmentation in granzyme B-expressing COS cells and neither
of these stimuli had adverse effects on mock transfected cells. Because
the normal pathway targeting granzyme B to cytolytic granules of
CTL47,48is not present in COS cells, the granzyme may be
targeted to a vesicle similar to that observed in target cells. It is
unlikely that granzyme B is secreted by COS cells and reinternalized,
as exhaustive efforts to detect granzyme B activity or protein in the
supernatants of these cultures have been negative (data not shown).
Thus, we believe that the perforin/Ad2 is acting not to increase uptake
of granzyme B, but rather to stimulate the release of the granzyme into
the cytoplasm. The induction of the apoptotic program could then be
induced by the cleavage of key substrates, such as caspase-3. The
importance of "free granzyme B" was confirmed by our
demonstration that directly microinjected enzyme was able to induce
death.
The granzyme does not seem to linger in the cytoplasm, as we observe a
rapid translocation into the nucleus. The importance of this nuclear
accumulation is unclear, but it has also been shown in an in vitro
system described by Jans et al.29 It is intriguing that the
site of granzyme buildup in the nucleus corresponds to the early
regions detected by the TUNEL assay. Perhaps, as previously
suggested,26 nuclear substrates for granzyme B exist and
play a role in apoptotic events.
Receptor-mediated endocytosis of granzyme B.
We have previously demonstrated that Jurkat target cells possess on
their surface saturable binding sites for granzyme B (3 × 104 sites per cell; kd  10 nmol/L) and
suggested the likelihood of a granzyme B receptor. In this report, we
expand the previous study by demonstrating that granzyme B binds to
cells via high-affinity interactions at the cell surface. By
incubating target cells in granzyme B at 4°C, we allowed binding,
but inhibited its uptake. Washing the cells at pH 3.0 disrupts
protein-protein interactions and dissociated bound granzyme B from the
cell surface, thus preventing the induction of cell death after the
addition of perforin or Ad2. Incubation of targets in granzyme B at
4°C followed by acid wash reduced killing to approximately 25% of
that observed when targets were washed under neutral conditions.
Residual killing of acid-washed cells at 4°C may be due to entry of
granzyme during handling and processing.
These data lend further support to the notion of a specific granzyme B
receptor. We have also ruled out the previous suggestion that granzyme
B remained bound to its putative receptor and required perforin or Ad2
to stimulate entry,28 as acid treatment of cells incubated
in granzyme B at 37°C did not show any reduction in apoptosis
compared with cells washed under neutral conditions. These data
strongly suggest that granzyme B binds to target cells as proposed by
Froelich et al,28 but that the granzyme B is internalized
by the target in the absence of additional exogenous factors, namely
perforin or Ad2.
Endocytic internalization of granzyme B.
The likelihood of a granzyme B receptor and the involvement of
endocytosis and trafficking of Ad2 led us to investigate the possibility that granzyme B follows a similar route. Using double label
studies with markers of intracellular compartments, we found granzyme B
label associated with early endosomal markers rab5 and rab4 in cells
treated at 20°C to inhibit endosomal trafficking. Uptake of
granzyme B by endocytosis is consistent with earlier reports that
showed that the microtubule inhibitor, cytochalasin B, effectively
inhibited granzyme B-induced killing.9
Our results appear to be in direct contradiction to the recent report
of Shi et al,49 who reported internalization of granzyme B
directly into the cytoplasm. However, in their study, it was very
difficult to discern the morphologic features represented in the
electron micrographs. In addition, endocytic compartmentalization is
generally believed to be the primary route of entry taken by the
majority of molecules after receptor binding.50 In our
study, we clearly show granzyme B in a distinct cytoplasmic
compartment.
Our results, indicating the regulated uptake and trafficking of
granzyme B, are supported by the observation that cytoplasmic microinjection of granzyme resulted in apoptosis. This result also
appears to be contrary to a recent report in which microinjected granzyme B did not induce death.49 The apparent difference
may be explained by the dose-dependent nature of the apoptotic response that we report in the present study. We observed apoptosis when granzyme B was injected at 54 fg/cell, which represents a ninefold higher cellular concentration than in the previous study. Our data
support a model in which granzyme B enters the target via receptor-mediated endocytosis and is transported to a cytoplasmic compartment, where it is unable to access substrates. Transport to this
compartment is not associated with cell death; however, release is
vital for initiation of the apoptotic program.
Although granzyme B appears to be transported through early endosomes,
these vesicles are clearly not the site of accumulation of granzyme. In
target cells that were allowed to take up granzyme B at 37°C, the
protease was found in a cellular compartment that is not characterized
by the presence of rab5 or rab4 as seen at 20°C. Interestingly,
Ad2-induced redistribution of granzyme B from its intracellular
compartment did not occur at 20°C (data not shown). Additionally,
we performed immunofluorescent labeling of other cellular markers on
targets containing granzyme B-FITC and did not find granzyme B
associated with markers of lysosomes, mitochondria, or structures
containing MPR. Thus, it appears that granzyme B is internalized via a
receptor-mediated process into an early endosome, but by further
trafficking, it continues on to a compartment that is not characterized
by any of our cellular markers.
Because of a lack of colocalization with the cellular markers we
tested, we have not been able to better define the site to which
granzyme B trafficks. However, it appears to be a homogeneous, translucent structure that resembles immature cytolytic granules of
CTL.51 Although it remains to be conclusively established, it appears that trafficking of granzyme B to this novel compartment is
necessary for apoptosis, as granzyme B does not reach this compartment
at 20°C.
Conclusions.
In this study, we have provided further evidence to support the
existence of a granzyme B receptor and, more importantly, we have shown
that trafficking of granzyme B by the target is required for apoptosis.
This provides us with an additional level at which to direct drug
design to enhance or abrogate CTL- or NK-based immune responses.
Targeting the granzyme B receptor in transplants may provide a means to
suppress rejection due to infiltrating cytotoxic lymphocytes. Our
results suggest that a search for other granzyme B-binding molecules,
in addition to substrates, may show proteins that play an essential
role in the internalization and intracellular transport of this
important cytotoxic effector.
| |
FOOTNOTES |
Submitted February 17, 1998;
accepted April 1, 1998.
Supported by the National Cancer Institute of Canada and the
Medical Research Council of Canada (Ottawa, Canada). R.C.B. is a
Medical Scientist of the Alberta Heritage Foundation for Medical Research (Edmonton, Canada), a Distinguished Scientist of the Medical
Research Council of Canada, and a Howard Hughes
International Research Scholar. J.A.H. holds a
studentship from the Medical Research Council of Canada.
C.J.F. was supported by the Arthritis Foundation-Illinois
Chapter.
Address reprint requests to R. Chris Bleackley, PhD,
Department of Biochemistry, 4-63 Medical Sciences Bldg,
University of Alberta, Edmonton, Alberta, Canada T6G 2H7;
e-mail: Chris.Bleackley{at}UAlberta.Ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dorothy Hudig and Ulrike Winkler for antigranzyme
antiserum and Irene Shostak and Tracy Sawchuk for technical assistance.
| |
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J. Immunol.,
July 15, 2006;
177(2):
1171 - 1178.
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D.-M. Zhao, A. M. Thornton, R. J. DiPaolo, and E. M. Shevach
Activated CD4+CD25+ T cells selectively kill B lymphocytes
Blood,
May 15, 2006;
107(10):
3925 - 3932.
[Abstract]
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M. L. Janas, P. Groves, N. Kienzle, and A. Kelso
IL-2 Regulates Perforin and Granzyme Gene Expression in CD8+ T Cells Independently of Its Effects on Survival and Proliferation
J. Immunol.,
December 15, 2005;
175(12):
8003 - 8010.
[Abstract]
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C. H. Bird, J. Sun, K. Ung, D. Karambalis, J. C. Whisstock, J. A. Trapani, and P. I. Bird
Cationic Sites on Granzyme B Contribute to Cytotoxicity by Promoting Its Uptake into Target Cells
Mol. Cell. Biol.,
September 1, 2005;
25(17):
7854 - 7867.
[Abstract]
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S. M. Raja, S. S. Metkar, S. Honing, B. Wang, W. A. Russin, N. H. Pipalia, C. Menaa, M. Belting, X. Cao, R. Dressel, et al.
A Novel Mechanism for Protein Delivery: GRANZYME B UNDERGOES ELECTROSTATIC EXCHANGE FROM SERGLYCIN TO TARGET CELLS
J. Biol. Chem.,
May 27, 2005;
280(21):
20752 - 20761.
[Abstract]
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L. Shi, D. Keefe, E. Durand, H. Feng, D. Zhang, and J. Lieberman
Granzyme B Binds to Target Cells Mostly by Charge and Must Be Added at the Same Time as Perforin to Trigger Apoptosis
J. Immunol.,
May 1, 2005;
174(9):
5456 - 5461.
[Abstract]
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F. C. Kurschus, R. Bruno, E. Fellows, C. S. Falk, and D. E. Jenne
Membrane receptors are not required to deliver granzyme B during killer cell attack
Blood,
March 1, 2005;
105(5):
2049 - 2058.
[Abstract]
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J. C. Choy, V. H.Y. Hung, A. L. Hunter, P. K. Cheung, B. Motyka, I. S. Goping, T. Sawchuk, R. C. Bleackley, T. J. Podor, B. M. McManus, et al.
Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin: Involvement of Extracellular Matrix Degradation
Arterioscler Thromb Vasc Biol,
December 1, 2004;
24(12):
2245 - 2250.
[Abstract]
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K. Veugelers, B. Motyka, C. Frantz, I. Shostak, T. Sawchuk, and R. C. Bleackley
The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis
Blood,
May 15, 2004;
103(10):
3845 - 3853.
[Abstract]
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J. Zhao, L.-H. Zhang, L.-T. Jia, L. Zhang, Y.-M. Xu, Z. Wang, C.-J. Yu, W.-D. Peng, W.-H. Wen, C.-J. Wang, et al.
Secreted Antibody/Granzyme B Fusion Protein Stimulates Selective Killing of HER2-overexpressing Tumor Cells
J. Biol. Chem.,
May 14, 2004;
279(20):
21343 - 21348.
[Abstract]
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J. Sun, C. H. Bird, K. Y. Thia, A. Y. Matthews, J. A. Trapani, and P. I. Bird
Granzyme B Encoded by the Commonly Occurring Human RAH Allele Retains Pro-apoptotic Activity
J. Biol. Chem.,
April 23, 2004;
279(17):
16907 - 16911.
[Abstract]
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Y. Liu, L. H. Cheung, W. N. Hittelman, and M. G. Rosenblum
Targeted delivery of human pro-apoptotic enzymes to tumor cells: In vitro studies describing a novel class of recombinant highly cytotoxic agents
Mol. Cancer Ther.,
December 1, 2003;
2(12):
1341 - 1350.
[Abstract]
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E. Ramsburg, R. Tigelaar, J. Craft, and A. Hayday
Age-dependent Requirement for {gamma}{delta} T Cells in the Primary but Not Secondary Protective Immune Response against an Intestinal Parasite
J. Exp. Med.,
November 3, 2003;
198(9):
1403 - 1414.
[Abstract]
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C. Gross, W. Koelch, A. DeMaio, N. Arispe, and G. Multhoff
Cell Surface-bound Heat Shock Protein 70 (Hsp70) Mediates Perforin-independent Apoptosis by Specific Binding and Uptake of Granzyme B
J. Biol. Chem.,
October 17, 2003;
278(42):
41173 - 41181.
[Abstract]
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C. E. Mackewicz, B. Wang, S. Metkar, M. Richey, C. J. Froelich, and J. A. Levy
Lack of the CD8+ cell anti-HIV factor in CD8+ cell granules
Blood,
July 1, 2003;
102(1):
180 - 183.
[Abstract]
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D. McIlroy, P.-F. Cartron, P. Tuffery, Y. Dudoit, A. Samri, B. Autran, F. M. Vallette, P. Debre, and I. Theodorou
A triple-mutated allele of granzyme B incapable of inducing apoptosis
PNAS,
March 4, 2003;
100(5):
2562 - 2567.
[Abstract]
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J. A. Trapani, V. R. Sutton, K. Y.T. Thia, Y. Q. Li, C. J. Froelich, D. A. Jans, M. S. Sandrin, and K. A. Browne
A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death
J. Cell Biol.,
January 21, 2003;
160(2):
223 - 233.
[Abstract]
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E. Wieckowski, G.-Q. Wang, B. R. Gastman, L. A. Goldstein, and H. Rabinowich
Granzyme B-mediated Degradation of T-Cell Receptor {zeta} Chain
Cancer Res.,
September 1, 2002;
62(17):
4884 - 4889.
[Abstract]
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G.-Q. Wang, E. Wieckowski, L. A. Goldstein, B. R. Gastman, A. Rabinovitz, A. Gambotto, S. Li, B. Fang, X.-M. Yin, and H. Rabinowich
Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells
J. Exp. Med.,
November 5, 2001;
194(9):
1325 - 1338.
[Abstract]
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P. Gorak-Stolinska, J.-P. Truman, D. M. Kemeny, and A. Noble
Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-{alpha}
J. Leukoc. Biol.,
November 1, 2001;
70(5):
756 - 766.
[Abstract]
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R. G. Uzzo, V. Kolenko, C. J. Froelich, C. Tannenbaum, L. Molto, A. C. Novick, N. H. Bander, R. Bukowski, and J. H. Finke
The T Cell Death Knell: Immune-mediated Tumor Death in Renal Cell Carcinoma
Clin. Cancer Res.,
October 1, 2001;
7(10):
3276 - 3281.
[Abstract]
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R. D. Pettersen, G. Bernard, M. K. Olafsen, M. Pourtein, and S. O. Lie
CD99 Signals Caspase-Independent T Cell Death
J. Immunol.,
April 15, 2001;
166(8):
4931 - 4942.
[Abstract]
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M. Hayashida, H. Kawano, T. Nakano, K. Shiraki, and A. Suzuki
Cell Death Induction by CTL: Perforin/Granzyme B System Dominantly Acts for Cell Death Induction in Human Hepatocellular Carcinoma Cells
Experimental Biology and Medicine,
November 1, 2000;
225(2):
143 - 150.
[Abstract]
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Y. Kawasaki, T. Saito, Y. Shirota-Someya, Y. Ikegami, H. Komano, M.-H. Lee, C. J. Froelich, N. Shinohara, and H. Takayama
Cell Death-Associated Translocation of Plasma Membrane Components Induced by CTL
J. Immunol.,
May 1, 2000;
164(9):
4641 - 4648.
[Abstract]
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C. Berthou, J.-F. Bourge, Y. Zhang, A. Soulie, D. Geromin, Y. Denizot, F. Sigaux, and M. Sasportes
Interferon-gamma -induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis
Blood,
April 1, 2000;
95(7):
2329 - 2336.
[Abstract]
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E. H. A. Spaeny-Dekking, A. M. Kamp, C. J. Froelich, and C. E. Hack
Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors
Blood,
February 15, 2000;
95(4):
1465 - 1472.
[Abstract]
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K. A. Browne, E. Blink, V. R. Sutton, C. J. Froelich, D. A. Jans, and J. A. Trapani
Cytosolic Delivery of Granzyme B by Bacterial Toxins: Evidence that Endosomal Disruption, in Addition to Transmembrane Pore Formation, Is an Important Function of Perforin
Mol. Cell. Biol.,
December 1, 1999;
19(12):
8604 - 8615.
[Abstract]
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J. A. Heibein, M. Barry, B. Motyka, and R. C. Bleackley
Granzyme B-Induced Loss of Mitochondrial Inner Membrane Potential ({Delta}{Psi}m) and Cytochrome c Release Are Caspase Independent
J. Immunol.,
November 1, 1999;
163(9):
4683 - 4693.
[Abstract]
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K. M. Edwards, C.-M. Kam, J. C. Powers, and J. A. Trapani
The Human Cytotoxic T Cell Granule Serine Protease Granzyme H Has Chymotrypsin-like (Chymase) Activity and Is Taken Up into Cytoplasmic Vesicles Reminiscent of Granzyme B-containing Endosomes
J. Biol. Chem.,
October 22, 1999;
274(43):
30468 - 30473.
[Abstract]
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C. T. N. Pham and T. J. Ley
Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo
PNAS,
July 20, 1999;
96(15):
8627 - 8632.
[Abstract]
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J. P. Galvin, L. H. A. Spaeny-Dekking, B. Wang, P. Seth, C. E. Hack, and C. J. Froelich
Apoptosis Induced by Granzyme B-Glycosaminoglycan Complexes: Implications for Granule-Mediated Apoptosis In Vivo
J. Immunol.,
May 1, 1999;
162(9):
5345 - 5350.
[Abstract]
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J. Sun, J. C. Whisstock, P. Harriott, B. Walker, A. Novak, P. E. Thompson, A. I. Smith, and P. I. Bird
Importance of the P4' Residue in Human Granzyme B Inhibitors and Substrates Revealed by Scanning Mutagenesis of the Proteinase Inhibitor 9 Reactive Center Loop
J. Biol. Chem.,
April 27, 2001;
276(18):
15177 - 15184.
[Abstract]
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J. B. Alimonti, L. Shi, P. K. Baijal, and A. H. Greenberg
Granzyme B Induces BID-mediated Cytochrome c Release and Mitochondrial Permeability Transition
J. Biol. Chem.,
March 2, 2001;
276(10):
6974 - 6982.
[Abstract]
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M. J. Pinkoski, N. J. Waterhouse, J. A. Heibein, B. B. Wolf, T. Kuwana, J. C. Goldstein, D. D. Newmeyer, R. C. Bleackley, and D. R. Green
Granzyme B-mediated Apoptosis Proceeds Predominantly through a Bcl-2-inhibitable Mitochondrial Pathway
J. Biol. Chem.,
April 6, 2001;
276(15):
12060 - 12067.
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Abstract
Introduction
Abstract