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*
Evanston Northwestern Healthcare Research Institute, Northwestern University, Evanston, IL 60201;
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and the Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Amsterdam, The Netherlands; and
Medicine Branch, National Cancer Institute, National Institute of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have proposed that polymerized PFN pores and GrB are internalized into endosomes of the target cell during granule-mediated cytotoxicity. PFN then permeabilizes the vesicles delivering the granzyme to the cytosol. Subsequently, GrB induces cell death by activating the caspase cascade (4). Experimental models designed to characterize the biochemical events that occur during granule-mediated apoptosis have relied on the free granzyme. However, the cationic nature of the serine protease may lead to nonspecific interactions with both intercellular and intracellular anionic membranes as well as artifactual vesicular trafficking. As a consequence, results obtained from studies using isolated granzymes may not provide a valid view of granule-mediated apoptosis in vivo. We describe here a model system consisting of CS-GrB complexes. This system has been used to study the capacity of the neutral, high m.w. form of the granzyme to induce apoptosis after delivery by PFN or replication-deficient adenovirus type 2 (AD) as well as process the preferred caspase subtrates. In addition, we report preliminary evidence that a high m.w. form of GrB secreted by cytotoxic cells also has potent apoptotic activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat cells were maintained in RPMI 1640/10% heat-inactivated FCS supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 50 µg/ml streptomycin, and MCF-7 stably expressing procaspase-3 (5) were cultured in Iscove’s modified DMEM/10% heat-inactivated FCS plus antibiotics.
Reagents
Human GrB and perforin were purified to homogeneity from a human NK cell line (YT) (6), and a nonreplicating strain of adenovirus type 2 (AD) was cultured and isolated as described (7). The peptidic substrate, carbobenzoxy-DEVD-methylcoumarin (ac-DEVD-afc), was supplied by Kamiya Biomedical (Spokane, WA).
Generation of CS-GrB complexes
A neutral form of GrB was produced by mixing desalted CS
(Calbiocem, San Diego, CA) and GrB (3:1 w/w) in hypotonic Tris buffer
(25 mM NaCl, pH 7.4). Thereafter, the mixture was applied to a
cation-exchange column (Mono S, Pharmacia, Uppsala, Sweden) followed by
a continuous NaCl gradient (0–2.0 M). The amount of GrB in the
flow-through (electrochemically neutral GrB, hereafter called CS-GrB)
and in the eluate (free cationic GrB) was determined by enzyme
immunoassay (EIA) (8). When titrated with a specific active site
inhibitor (anti-GraB), 
80% of free GrB represents active enzyme
(9). Using an esterolytic assay (see below), the sp. act. of CS-GrB
recovered in the flow-through was found to be comparable to the free
form of the granzyme.
GrB EIA
Purified mAb GB11 was incubated at 2 µg/ml in 0.1 M sodium carbonate/bicarbonate buffer, pH 9.6, for 16 h at 4°C in microtiter plates (100 µl/well; Nunc Maxisorb Immunoplate, Roskilde, Denmark). The plates were then washed with PBS/0.02% (w/v) Tween 20. An identical washing procedure was performed after each incubation step, which consisted of 100 µl, except for the blocking step (150 µl). After coating, plates were blocked with PBS/2% (v/v) cow milk for 45 min. Samples and standards (free GrB at different concentrations) were diluted in proprietary ELISA buffer (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands) and incubated for 1 h. Next, the plates were incubated with an excess of biotinylated GB10 mAb (0.5 µg/ml) together with 1% (v/v) normal mouse serum for 1 h. The plates were incubated for 30 min with streptavidin-polymerized horseradish peroxidase (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service), after which bound peroxidase was visualized by incubation with a solution of 100 µg/ml 3,3',5,5'-tetramethyl-benzidine (Merck, Darmstadt, Germany) and 0.003% (v/v) H2O2 in 0.11 M sodium acetate buffer, pH 5.5. The reaction was stopped by the addition of an equal volume of 2 M H2SO4 to the wells. Finally, the absorbance at 450 nm was read on a Titertek Multiscan plate reader (Labsystems, Helsinki, Finland). Duplicate samples were measured, and the mean value is reported where SDs did not exceed 10%.
Measurement of GrB esterolytic activity
Using Boc-Ala-Ala-Asp-thiobenzyl ester (0.1 mM) diluted in reaction buffer (0.2 M HEPES, 0.3 M NaCl, 1 mM EDTA, 0.5% Triton X-100 (v/v), pH 7.0) (6), GrB activity was measured by colorimetric absorbance changes using a UVmax microplate reader (Molecular Device, Menlo Park, CA). GrB activity was defined as units where 1 U of esterolytic activity equaled the amount of enzyme that hydrolyzed 1 nmol of substrate per minute.
Delivery of free and CS-GrB to target cells with AD or PFN
Jurkat cells (1 x 106/ml) were treated with an equivalent amount of active free GrB or CS-GrB (1 µg/ml) and AD (100 pfu) in 1 ml microfuge tubes containing RMPI 1640 supplemented with 0.5% BSA (9). For PFN delivery, target cells were treated with either free GrB or CS-GrB for 15 min and then exposed to a predetermined sublytic concentration of the pore-forming protein (9).
Apoptosis: morphologic analysis
Apoptosis was assessed at 4 h with Hoescht stain and defined as either nuclear condensation or fragmentation. Cells were fixed with 0.5% paraformaldehyde for 15 min, cytospun to microscope slides, and stained with Hoescht 33342 (1 µg/ml) for 15 min. Using a Zeiss Fluorescent microscope, the percentage apoptotic cells among 300 cells was determined in duplicate samples. Mean values were reported where SDs did not exceed 10%.
Apoptosis: measurement of caspase-3-like fluorogenic activity in whole cells
Following delivery of free or CS-GrB, Jurkat cells were washed (RPMI 1640/0.5% BSA) and resuspended in new microfuge tubes. Anti-GraB, an anti-chymotrypsinogen engineered to react specifically with the granzyme (5 x 105 molecules/cell), was added to each tube to avoid artifactual in vitro cleavage of caspases after lysis. Lysates (100 µl) were added, in duplicate, to each microwell (Nunc Maxisorb Immunoplate) followed by a solution of ac-DEVD-afc (100 µl). Fluorescent values were determined in duplicate every 10 min over a defined period using a Fluorimeter plate reader (Cambridge Technologies, Cambridge, MA).
Cleavage of procaspases-3 and -7 by free and CS-GrB
Caspase-3 and -7 were encoded on vectors under the control of a T7 RNA polymerase promoter (10, 11, 12, 13). The [35S]methionine-labeled proteins were prepared from these vectors using a T7-coupled reticulocyte lysate transcription translation (TnT) system (Promega, Madison, WI). The cleavage assay consisted of 20 µl of TnT reaction mix and a volume of reaction buffer (100 mM HEPES, pH 7.5, 20% glycerol, 0.5 mM EDTA, 5 mM DTT) that contained equivalent amounts of active free or CS-GrB (10 nM). Samples were incubated at 37°C, and 30-µl aliquots were removed at various times between 0 and 90 min. Proteolysis was terminated by adding SDS buffer (5 µl) to the samples and heating to 100°C for 5 min. Samples were then submitted to SDS-PAGE using 15% Tris-Tricine minigels (Bio-Rad, Hercules, CA). Gels were then dried and imaged on film.
Degranulation and enrichment of GrB complexes from lymphocyte-activated killer cells (LAK)
IL-2-stimulated PBMC were harvested after 6 days of stimulation. The harvested cells, lymphocyte activated killer cells (LAK), were washed in Iscove’s modified DMEM supplemented with BSA (0.02%) and resuspended at 1 x 106 cells/ml in Iscove’s modified DMEM plus BSA (0.02%) containing three mAbs directed against CD2 (4B2, 6G4, and Hic 27, 1 µg/ml per mAb) and phorbol myristic acetate (1 ng/ml). Cell were then incubated in tissue culture flasks for 5 h. Supernatants were collected, centrifuged for 8 min at 1500 rpm to remove LAK cells and debris, and stored at -20°C until GrB levels were measured by EIA and esterolytic assay.
Western Blotting of procaspase-3 in cell lysates
Detection of processed procaspase-3 in MCF-7 cell lysates was performed as previously described (5). After treatment with either free or CS-GrB, the lysates (106 cell equivalents) were resolved by SDS-PAGE (15%) and transferred to nitrocellulose. Rabbit anti-caspase-3 (supplied by V. Dixit, Genentech, San Francisco, CA) was used at a dilution of 1:2500 followed by incubation with anti-rabbit Ig-horseradish peroxidase (1:10,000) (Amersham, Arlington Heights, IL), and signals were visualized with an enhanced chemiluminescence kit (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A nonionic form of GrB was produced by complexing the granzyme
with CS glycosaminoglycan. Commercially available CS was first
extensively dialyzed against water to remove sodium ions. Then a
threefold excess of CS was added to isolated GrB (3:1 w/w).
The generated CS-GrB complexes were separated from residual free
granzyme by cation-exchange chromatography where neutral complexes
resided in the flow-through and free GrB bound to the matrix and was
eluted during performance of linear NaCl gradient. The amount of free
and CS-GrB was then determined by a recently described capture EIA (8).
Using this strategy, the influence of pH on the formation of CS-GrB
complexes was determined (Table I
). When
dialyzed CS and free GrB were mixed at pH 5.5 and 7.4 (NaCl 150 mM),
85% and 63% of the granzyme, respectively, associated with the
glycosaminoglycan.
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590 mM NaCl (6). On the basis of
this observation, it was predicted that once GrB and CS associated,
NaCl concentrations below mM 590 would be unable to disrupt the ionic
interaction. To examine the strength of this ionic interaction, CS-GrB
complexes were mixed in NaCl solutions ranging from 200 to 500 mM
Tris-HCl, pH 7.4, and the amount of dissociated GrB was measured after
elution from a cation-exchange column. Regardless of the salt
concentration, >99% of GrB remained associated with the
glycosaminoglycan (Table II). This result
demonstrated the remarkable stability of the ionic interaction and
suggests that once the granzyme binds to granule-associated anionic CS
side chains of serglycin a stable interaction would be maintained in
vivo.
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Caspase-3 and -7 are both efficiently processed by GrB, where
cleavage occurs at the IXXD sequence connecting the large and small
subunits (14, 15). To determine whether the cationic charge might
influence the rate of cleavage of the zymogens, procaspase-3 and -7
were expressed by TnT and rates of proteolysis were determined in the
presence of equal amounts of active free and CS-GrB. The time required
to consume the zymogen provides a qualitative estimate of the Vmax
(16). Caspase-7 was almost completely processed by free GrB at 30 min
while the CS-GrB complex processed caspase-7 to the same extent after
60 min (Fig. 1a). Caspase-3,
on the other hand, was minimally cleaved at 90 min by CS-GrB, while
free GrB processed about 50% of the zymogen by this time (Fig. 1b). Clearly, processing of both procaspase-3 and -7 is
diminished significantly when GrB is complexed with CS.
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We have shown previously that AD effectively delivers GrB because
the granzyme appears to bind to the target cell in a specific,
saturable fashion (9). This interaction allows the granzyme to be
internalized and delivered to the cytosol by the endosomolytic action
of AD. However, because the studies were performed with the cationic
granzyme, it was impossible to exclude the possibility that a portion
of the observed binding to the plasma membrane was charge dependent. To
address this issue, we asked whether equivalent amounts of active free
and CS-GrB could induce similar levels of apoptosis after delivery to
Jurkat cells by AD. Jurkat cells were treated with either free or
CS-GrB plus AD, and the percentage of cells undergoing apoptosis was
visualized by Hoescht stain and fluorescent microscopy. Both forms of
the granzyme produced comparable levels of apoptosis, suggesting CS-GrB
does not have a reduced capacity to bind and be internalized by the
target cell. (Fig. 2a). To
ensure that the apoptotic activity of CS-GrB was not unique to the AD
delivery system, Jurkat cells were pulsed with either free GrB or
CS-GrB complexes and treated with a sublytic concentration of PFN (9).
Both free GrB and CS-GrB induced similar levels of cell death when
delivered by PFN (Fig. 2b).
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Assuming similar amounts of free GrB and CS-GrB were bound and
delivered by AD and on the basis of the reduced rate of proteolysis
that CS-GrB displayed against caspase-3, it could be predicted that
CS-GrB should generate lower levels of caspase-3-like activity in whole
cells with delayed kinetics. Using the fluorogenic substrate,
ac-DEVD-afc, to quantify levels of caspase-3-like activity, a
dose-response analysis was performed comparing delivery of equivalent
amounts of active free and CS-GrB. As shown in Fig. 3a, the levels of ac-DEVD-afc
activity were similar for both forms of the granzyme. Furthermore, the
delivery of equivalent amounts of free and CS-GrB complexes resulted in
similar time-dependent increases in DEVD-afc activity (Fig. 3b).
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We have recently provided unequivocal evidence that GrB initiates
the caspase cascade in whole cells by activating caspase-3 (5). Because
CS-GrB processed isolated caspase-3 less efficiently than free GrB, the
similar time-dependent increase in ac-DEVD-afc activity mediated
by the two forms of the granzyme was unexpected. To account for this
discrepancy, cationic GrB, once delivered intracellularly, might bind
to negatively charged cytosolic proteins, reducing the amount of
granzyme able to process procaspase-3. To partially mimic conditions in
whole cells, we compared the rates of proteolysis of procaspase-3 that
free and CS-GrB produced after the addition to MCF-7 cytosolic
extracts. In comparison to the results that showed free GrB cleaved
isolated procaspase-3 more rapidly than CS-GrB (Fig. 2
a),
the capacity of free GrB to process the caspase in cell lysates was at
least similar if not somewhat less than the rate observed for CS-GrB
(Fig. 4). Therefore, the equivalent rates
of ac-DEVD-afc activity generated by delivery of free and CS-GrB to
intact cells may be, in part, attributed to binding of the cationic
free granzyme to cytosolic proteins, leading to a reduction in the
amount of protease available for processing the endogenous
procaspase-3.
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Because GrB appears to tightly bind to CS, the prediction
was made that GrB exocytosed by stimulated cytotoxic cells should be
released in a high m.w. form. Following stimulation with IL-2 for 5
days, LAK cells were induced to undergo granule exocytosis by exposure
to phorbol myristic acetate and anti-CD2 mAbs in media supplemented
with BSA. Combining two approaches, we determined whether the granzyme
was secreted as a neutral, high m.w. complex. First, no cationic free
GrB was detected in the supernatant analyzed by cationic-exchange
chromatography. Then the levels of free and complexed GrB were
estimated by EIA after filtration of the supernatant through a
100,000-kDa membrane. The vast majority of the granzyme was retained by
the membrane, suggesting the presence of a macromolecular complex
(>99%) (Fig. 5). Having shown the
supernatant from stimulated LAK cells contained only a form of granzyme
that exceed 100,000 kDa, we then compared the apoptotic potential of
the GrB derived from the LAK cells to free GrB. Jurkat cells were
pulsed with equivalent amounts of active free and LAK cell-derived GrB,
washed, and treated with AD. As shown in Fig. 6, the free and the high m.w. form of GrB
were able to induce similar levels of cell death after delivery by AD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results extend studies described for the tryptase-like activity identified in the granules, which is primarily attributable to GrA. The tryptase activity of cloned NK cells was found to associate with a proteoglycan, presumably serglycin. On the basis of esterolytic activity, murine granzyme A, likewise, has been shown to remain complexed upon exocytosis from cytotoxic cells (17, 18, 19). We show here: 1) GrB remains tightly bound to CS glycosaminoglycans under physiologic conditions; 2) while the cationic charge of GrB apparently enhances the proteolytic efficiency of GrB against isolated caspases, complex formation with CS protects the granzyme from nonspecific inhibition in the presence of complex cytosolic extracts; 3) CS-GrB has the capacity to induce cell death when delivered by AD or PFN generating levels of caspase-3-like activity similar to equivalent amounts of free GrB; and 4) GrB is secreted from stimulated LAK cells in a macromolecular form (>100 kDa), which also induces apoptosis after intracellular delivery. Taken together, the evidence suggests that a neutral, high m.w. complex form of GrB induces apoptosis in vivo.
Numerous reports have described the biochemical and biologic effects of isolated GrB (1, 4, 20). However, little attention has been given to the concept that the physiologically relevant form may represent a neutral macromolecular complex. Although the final outcome, cell death, occurs regardless of the form of GrB delivered to the target cell, studies performed with the cationic granzyme may not provide insights to the mechanism of apoptosis that occurs in vivo. In this regard, it has been reported that GrB readily binds and enters the nucleus of the target cell after delivery by PFN (21, 22). Because nuclear translocation appears to require the interaction of free GrB with an endogenous cytosolic factor (23), this process may not occur when complexed GrB is delivered intracellularly.
We have recently provided convincing evidence that GrB activates the executioner caspases-3 and -7 through a novel two-step process. After intracellular delivery, GrB first processes procaspase-3 even though this zymogen is not the most preferred caspase substrate (5). As verified here with CS-GrB, the granzyme cleaves caspase-7 more rapidly than caspase-3. However, during GrB-mediated apoptosis the caspase-7 propeptide is removed first, then cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB (5). Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to cleave the caspase-7 between the large and small subunits. Thus GrB initiates the death pathway by processing the accessible caspase-3, and caspase-7 propeptide regulates transactivation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7. It will be important to learn whether complexed GrB activates these executioner caspases in a similar manner.
In addition to influencing how free GrB processes caspases in vivo, studies that employ cationic granzyme may not accurately portray the kinetics of cell death. The addition of free GrB to cytosolic extracts appeared to decrease the rate of proteolysis of caspase-3 compared with CS-GrB. As a consequence, the Kcat that we have reported for free GrB against isolated caspase-3 (15) may be an overestimate of the reaction rate that is likely to occur in vivo. This finding could explain why both forms of the granzyme produced equivalent levels of caspase-3-like activity after intracellular delivery. The reduction in caspase activation by free GrB could be due to two mechanisms: the free GrB interacts with cytosolic membranes reducing the amount available for cleaving the caspases or soluble cytoplasmic proteins could bind the granzyme and act as nonspecific competitive inhibitors. The latter issue becomes important for the interpretation of studies where cells are transfected with anti-apoptotic proteins. For example, Bcl-2 has been reported to protect cells treated with GrB and PFN but not against the rapid cell death mediated by cytotoxic T cells. (24). The inability of free GrB to induce death might be due to a protein-protein interaction of the granzyme with overexpressed Bcl-2, resulting in a nonspecific reduction in GrB-mediated processing of caspase-3.
In comparison to free tryptase-like granzyme, GrA-proteoglycan complexes are relatively resistant to inactivation by antithrombin III, a major plasma inhibitor of this granzyme.4 The protease inhibitor-9, unlike extracellular serpins, has been reported to efficiently inactivate free GrB (25). Protease inhibitor-9 has been postulated to protect the cytotoxic cell from misdirected GrB released during granule exocytosis. However, protease inhibitor-9 is expressed at highest levels in the cytosol of cytotoxic cells. The location of protease inhibitor-9 suggest that the serpin might selectively inhibit free GrB that is inadvertently targeted and activated in the cytosol and not block granzyme complexed with serglycin. Therefore, GrB/serglycin complexes might offer a host advantage against cells infected with viruses that manufacture similar anti-granzyme serpins.
The cationic charge of free GrB would also favor nonspecific binding to the anionic plasma membrane. We reported that GrB appears to specifically interact with Jurkat cells (9). The work here found that CS-GrB as well as GrB secreted from LAK cells induced cell death after intracellular delivery. Because PFN- and AD-mediated delivery requires interaction of the granzyme with the plasma membrane, the results suggest that the nonionic form of GrB also binds to cells. This finding provides further evidence that the granzyme interaction with the cell membrane is not dependent on cationic charge. Although the stoichiometric interaction of GrB with serglycin remains to be determined, it is intriguing to speculate that a single serglycin protein might bind multiple granzyme molecules (26). Serglycin could then serve to focus GrB-receptor interactions as well as provide a multigranzyme proteolytic complex that enhances the processing of preferred caspase and noncaspase substrates after intracellular delivery.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher Froelich, Evanston Hospital, Research Department, WH Building, Room B643, 2650 Ridge Avenue, Evanston, IL 60201. E-mail address:
3 Abbreviations used in this paper: PFN, perforin; AD, replication-deficient adenovirus type 2; ac-DEVD-afc, carbobenzoxy-DEVD-methylcoumarin; CS, chondroitin sulfate; GrB, granzyme B; EIA, enzyme immunoassay.
4 E. H. A. Spaeny-Dekking, A. M. Kamp, Y. van Hensbergen, W. van Haren, C. J. Froelich, and C. E. Hack. Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors. Submitted for publication.
Received for publication September 29, 1998. Accepted for publication February 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by an ICE/CED 3-like protease induces characteristics of apoptosis. J. Exp. Med. 184:2399.This article has been cited by other articles:
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D. Zhang, M. S. Pasternack, P. J. Beresford, L. Wagner, A. H. Greenberg, and J. Lieberman Induction of Rapid Histone Degradation by the Cytotoxic T Lymphocyte Protease Granzyme A J. Biol. Chem., January 26, 2001; 276(5): 3683 - 3690. [Abstract] [Full Text] [PDF]
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