Granzyme B-mediated Apoptosis Proceeds Predominantly through aBcl-2-inhibitable Mitochondrial Pathway*□S

Received for publication, October 3, 2000, and in revised form, January 12, 2001Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M009038200

Michael J. Pinkoski‡§, Nigel J. Waterhouse‡, Jeffrey A. Heibein¶i, Beni B. Wolf‡,Tomomi Kuwana‡, Joshua C. Goldstein‡, Donald D. Newmeyer‡, R. Chris Bleackley¶**, andDouglas R. Green‡ ‡‡

From the ‡Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121and the ¶Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Cytotoxic T lymphocytes kill virus-infected and tumorcell targets through the concerted action of proteinscontained in cytolytic granules, primarily granzyme Band perforin. Granzyme B, a serine proteinase with sub-strate specificity similar to the caspase family of apop-totic cysteine proteinases, is capable of cleaving andactivating a number of death proteins in target cells.Despite the ability to engage the death pathway at mul-tiple entry points, the preferred mechanism for rapidinduction of apoptosis by granzyme B has yet to beclearly established. Here we use time lapse confocal mi-croscopy to demonstrate that mitochondrial cyto-chrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor ofthis pivotal event. Caspase activation is not required forcytochrome c release, an activity that correlates withcleavage and activation of Bid, which we have found tobe cleaved more readily by granzyme B than eithercaspase-3 or caspase-8. Bcl-2 blocks the rapid destruc-tion of targets by granzyme B by blocking mitochondrialinvolvement in the process.

Cytotoxic T lymphocytes (CTL)1 and natural killer cells in-duce apoptosis in their targets through the concerted action ofeffector molecules contained in cytolytic granules that engagethe death pathway (1, 2). Granzyme B enters the target celland, with another granule protein, perforin, triggers all of thecharacteristic manifestations of apoptosis, providing the prin-cipal mechanism of killing by CD81 CTL and natural killer

cells (3). Granzyme B is a serine protease that shares substratespecificity with many members of the caspase family of cys-teine proteases (4). In fact, granzyme B cleaves and activatesthe apical caspase, caspase-8, as well as caspases-3 (5, 6), -6,and -7 (7, 8). Granzyme B can directly activate caspase-3 and iscapable of triggering apoptosis at multiple points of the caspase-dependent pathway (9, 10) and therefore is not absolutelydependent on caspase-8 cleavage. This pathway differs fromanother common death pathway utilized by CTL, signalingthrough the Fas surface receptor by Fas ligand expressed onthe surface of the CTL. Apoptotic signaling through Fas re-quires an obligate activation of caspase-8 (11) and can proceedvia mitochondria-dependent or -independent pathways (12).The mitochondrial pathway involves the release of cytochromec for caspase activation and apoptosis (13, 14).

Release of mitochondrial cytochrome c is a pivotal event inthe apoptosis of many cell types induced by many stimuli (15,16). Upon release, cytochrome c binds Apaf-1 and promotes theformation of an oligomeric Apaf-1 apoptosome that recruits andactivates the effector caspase, caspase-9 (17–19). In receptor-mediated apoptosis the requirement for cytochrome c release isdependent on the type of cell triggered to die, and the decisionto utilize the mitochondrial route appears to rely primarily onthe concentration of caspase-8 (20). Although the same path-ways are used in granule-mediated apoptosis, the conditionsgoverning the premitochondrial events remain to be clearlyestablished.

Granule-driven CTL killing represents a very effectivemeans to induce apoptosis largely because of the ability ofgranzyme B to engage the death pathway at multiple entrypoints. Despite recent advances outlining the mechanism(s) ofaction of granzyme B within the target cell, there are stillnumerous conflicting reports regarding the requirements anddependence of granzyme B-mediated apoptosis on caspases andthe potential for inhibition by the anti-apoptotic proto-onco-gene, Bcl-2. It is clear that granzyme B is capable of cleavingand activating a number of key enzymes in the caspase cas-cade, notably caspases-8 and -3 (10, 21–23), but it is unclearwhat preference, if any, exists for each potential event. In thisreport we address the contribution of some of these aspects ofgranzyme-mediated apoptosis. Also, because granzyme B canactivate the effector caspases directly, we sought to determinethe necessity for cytochrome c release and its contribution tothe efficient death in target cell apoptosis.

In early studies involving Bcl-2 in CTL-mediated apoptosis,it appeared that target cell expression of the proto-oncogenecould confer some protection from CTL (24–26). Cytochrome crelease in target cells has been shown to occur, but it is notclear whether it is a requirement for death. In this report we

* This research was supported in part by National Institutes ofHealth Grants GM52735 and AI40646 and a Canadian Institute forHealth Research (CIHR) grant (to D. R. G.) and by CIHR and NationalCancer Institute of Canada grants (to R. C. B.). This is La Jolla Insti-tute for Allergy and Immunology Paper 373. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Quicktime movies for Figs. 2 and 3.

§ Supported by a postdoctoral fellowship from the CIHR.i Supported by a studentship from the CIHR.** Medical scientist of the Alberta Heritage Foundation for Medical

Research, CIHR distinguished scientist, and an international scholar ofthe Howard Hughes Medical Institute.

‡‡ To whom correspondence should be addressed: Division of CellularImmunology, La Jolla Institute for Allergy and Immunology, 10355Science Center Dr., San Diego, CA, 92121. E-mail: dgreen5240@aol.com.

1 The abbreviations used are: CTL, cytotoxic T lymphocytes; GFP,green fluorescent protein; PS, phosphatidylserine; FACS, fluorescence-activated cell sorter; CAD, caspase-activated DNase; ICAD, inhibitor ofcaspase-activated DNase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 15, Issue of April 13, pp. 12060–12067, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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follow single cells within populations to ascertain the order andextent of cytochrome c release in the context of its (in)depend-ence on caspase activation. We have also utilized our experi-mental system to assess the effects of Bcl-2 on both cytochromec release and subsequent apoptosis. In this way we determinedthat: 1) Bcl-2 inhibits cytochrome c release and the ultimatedeath of the target cell; and 2) granzyme B induces target cellcytochrome c release in a caspase-independent manner via Bidproteolysis. Because apoptotic Bcl-2-expressing cells do notrelease cytochrome c, we also conclude that when necessary,granzyme B bypasses the mitochondria via direct activation ofeffector caspases rather than overriding Bcl-2 by proteolyticdegradation or some other mechanism.

EXPERIMENTAL PROCEDURES

Cell Culture and Reagents—Jurkat and Jurkat-Bcl-2 were culturedin RPMI with 10% fetal calf serum, penicillin, and streptomycin. Wealso employed an HeLa cell line stably transfected with cytochromec-GFP (denoted 2H18) as described previously (27). Cytochrome c-GFPwas shown to behave identically to cytochrome c in 2H18 in apoptosisassays including side by side immunoblots for cytochrome c and cyto-chrome c-GFP in subcellular fractions as well as immunofluoresenceassays. A subpopulation of these cells was transfected with the Bcl-2gene in the plasmid pEFpGKpuro. The transfected cells were selectedfor resistance to puromycin, and surviving clones resistant to UV- andactinomycin D-induced apoptosis were analyzed for Bcl-2 expression byWestern blot analysis (Bcl-2 antibody 65111A; Pharmingen, La Jolla,CA). The data from one clone (2H18-Bcl-2) are reported here. Both2H18 and 2H18-Bcl-2 were maintained in Dulbecco’s modified Eagle’smedium with 10% fetal calf serum. All cells were grown at 37 °C under5% CO2. Granzyme B was purified from the human natural killer cellline YT-Indy as described previously (28). The broad spectrum caspaseinhibitor zVAD-fmk was purchased from Enzyme Systems Products(Dublin, CA). Antibodies against cytochrome c (1H5.2C12) were pur-chased from Pharmingen. Rabbit anti-Bid was raised against the pep-tide N-RDVFHTTVNFINQNLRTYVRSLARNGMD-C corresponding toa sequence in the C terminus of Bid. Specificity of the Bid antiserumwas verified by Western blot analysis of purified recombinant Bid andGST-Bid protein. N-terminal protein sequencing was performed by coreprotein facility at the Scripps Research Institute on an excised fragmenttransferred onto sequencing polyvinylidene difluoride (AmershamPharmacia Biotech) stained in Coomassie Blue in the absence of aceticacid.

Apoptosis and Cytochrome c Release Assays—CTL-free apoptosis wasinduced as described previously (28, 29). Briefly, target cells wereincubated in medium containing 0.5% serum with either 1 mg/ml gran-zyme B and 10 plaque-forming units/ml replication-deficient adenovi-rus type V or 100 ng/ml anti-Fas (CH-11) for the times indicated.Phosphatidylserine (PS) externalization was assayed with annexin V-fluorescein isothiocyante (CLONTECH; Palo Alto, CA) labeling andassessment with a Becton-Dickinson FACScan flow cytometer. Proteinlysates from apoptotic cells were produced by direct lysis into gel load-ing buffer containing SDS and subjected to examination by standardSDS-PAGE techniques and immunoblot analysis. Measurement of thereduction of full-length Bid protein was performed by densitometricanalysis of Western blots. Mitochondria were isolated from Xenopuslaevis according to Kluck et al. (30). After incubation at 30 °C underexperimental conditions noted in the text, mitochondria were removedfrom solution by centrifugation at 14,000 rpm for 10 min at 4 °C. Pelletscontaining mitochondria were resuspended in gel sample buffer con-taining SDS and 2-mercaptoethanol. Mitochondrial and supernatantprotein samples were analyzed for cytochrome c by standard immuno-blotting techniques after denaturing PAGE.

Granzyme B Substrate Assays—The cDNA encoding full-length hu-man Bid was expressed as a GST fusion protein in the vector pGEX4T-1in BL21(DE3) cells as described earlier (31). Bid was used as a GSTfusion protein or on its own after GST was removed by thrombindigestion followed by purification over GSH-Sepharose (AmershamPharmacia Biotech). Radiolabeled Bid, caspase-3, and caspase-8 wereproduced by coupled in vitro transcription and translation (PromegaLife Science, Madison, WI) from full-length clones in the vectorpcDNA-3. Specific activity of each translation product was calculatedaccording to the amount of incorporated [35S]Met radiolabel relative tothe number of methiothionine residues in each polypeptide. GranzymeB digests were performed at 30 °C under conditions described previ-ously (32). Fragments were analyzed by SDS-PAGE, fixed in gel, dried,

and exposed to Kodak X-Omat radiography film. All reactions werecarried out with concentrations of substrate (3 nM) well below the Km,and first-order kinetics were assumed. kcat/Km values were calculatedfrom the linear relation between log(St/S0) versus granzyme B concen-tration where St is the concentration of substrate at time t, and S0

represents the initial substrate concentration.FACS Analyses—2H18 and 2H18-Bcl-2 cells cultured in flat-bot-

tomed 96-well plates were harvested at the times indicated using 0.25%trypsin. Floating cells and trypsin-treated cells were combined andpelleted in a round bottomed 96-well plate. The medium was aspirated,and the cells were resuspended in 100 ml of ice-cold CLAMI buffer (80mM KCl and 25 mg/ml digitonin in phosphate-buffered saline). The cellswere incubated on ice for 5 min and analyzed directly for GFP contentby FACS analysis. Because cytosolic cytochrome c-GFP is released intothe CLAMI buffer, cells in which mitochondria had not released cyto-chrome c-GFP were ;0.5–1 log brighter in FL-1 than cells in which themitochondria had released their cytochrome c-GFP. PS exposure wasdetected by staining the cells with 10 ml/ml v/v annexin V-Alexa 568(red) (CLONTECH). Cells with PS exposed on the outside were two logsbrighter than control cells when analyzed by FACS using the FL-1(green) detector.

Confocal Microscopy and Supplemental Material—Real time apopto-sis assays were performed on 2H18 and 2H18-Bcl-2 cells with a Bio-RadMRC1024ES confocal laser scanning microscope. During the killingassays, cells were cultured in phenol red-free medium under a layer ofmineral oil to prevent evaporation. Individual frames and QuicktimeTM

movies were analyzed for cytochrome c release according to Goldstein etal. (27). zVAD-fmk, when used, was added to a final concentration of100 mM 15 min prior to the addition of granzyme B and maintained inthe culture for the duration of the killing assays.

RESULTS

The Primary Granzyme B-mediated Death Pathway InvolvesMitochondrial Cytochrome c Release—To study the molecularevents during target cell apoptosis, we utilized an experimentalsystem of granule-mediated killing wherein purified granzymeB is added directly to the medium of target cells in the presenceof a replication-deficient adenovirus (28, 33, 34). Althoughgranzyme B is capable of entering the target autonomously,apoptosis does not occur in the absence of virus. The granzymeB/adenovirus system has been shown to trigger all biochemicaland cellular manifestations of target cell apoptosis observedwhen target cells are treated with granzyme B and perforinand those treated with whole CTL (9, 28, 29, 33–36). Using2H18 and 2H18-Bcl-2, novel cell lines stably expressing cyto-chrome c-GFP 6 Bcl-2, we examined molecular events up-stream and downstream of mitochondrial cytochrome c releasein granzyme-mediated killing. Using this system we sought toestablish a preference, if any, given to Bcl-2-inhibitable eventsby delivering granzyme B at limiting doses. 2H18 and 2H18-Bcl-2 cells were treated with decreasing amounts of granzymeB along with a constant amount of adenovirus. After 2 and 4 h,cells were harvested and quantitatively assayed by flow cytom-etry for apoptosis by annexin V binding. At concentrations ofgranzyme B as low as 10 ng/ml we observed rapid release ofcytochrome c which corresponded with PS externalization (Fig.1). Bcl-2 was capable of inhibiting death at doses of granzymeB as high as 1 mg/ml. As observed earlier, the presence ofzVAD-fmk, which afforded no protection from cytochrome crelease, inhibited PS externalization, which presumably re-quires caspase activity in short term assays.

To determine if granzyme B can overcome the Bcl-2 block atlonger time points, we treated 2H18-Bcl-2 cells with granzymeand adenovirus and followed the target cells in culture by timelapse video confocal microscopy. As shown in Fig. 2, when2H18-Bcl-2 cells were incubated in the presence of granzyme Band adenovirus, there was no release of cytochrome c. After anextensive incubation (24 h), we observed only a small numberof apoptotic cells, and we did not observe cytochrome c releasein the apoptotic cells. This suggests that granzyme B treatmentcannot override the Bcl-2 blockade of cytochrome c release and

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is consistent with the model in which granzyme B is capable ofactivating effector caspases directly (10, 21, 22). However, it isclear from our observations that the most efficient pathway todeath in targets of granzyme B involves includes release ofcytochrome c from the mitochondria.

Cytochrome c Release by Granzyme Is Caspase-independ-ent—To characterize further the events leading to cytochromec release during target cell apoptosis, we utilized target cellsexpressing cytochrome c-GFP. HeLa has proven to be an excel-lent target for killing by granzyme B in combination withadenovirus (28, 29), and therefore we utilized 2H18, the HeLasublines expressing cytochrome c-GFP described above to studycytochrome c release during granzyme B-mediated apoptosis.The top series of panels in Fig. 3 shows a representative field of2H18 cells incubated in the presence of granzyme B and ade-novirus in which cytochrome c release occurs within 60 min asindicated by the diffuse cytoplasmic pattern of cytochrome c-GFP. As shown in the bottom series of panels, the addition ofthe broad spectrum caspase inhibitor zVAD-fmk did not dimin-

ish the time required to achieve comparable levels of cyto-chrome c release. Therefore, caspase-8 is not an obligate re-quirement for cytochrome c release in granzyme B-treatedtarget cells. These data are consistent with the observationthat granzyme B/adenovirus induces a drop in the mitochon-drial transmembrane potential which does not require activecaspases (36).

In a similar set of experiments we treated Jurkat targetswith granzyme B and adenovirus in the presence and absenceof zVAD-fmk. Fig. 4A shows an immunoblot time course ofcytosol isolated from treated Jurkat. In granzyme B/adenovi-rus-treated cells cytosolic cytochrome c appeared within 15–30min, whereas no cytosolic cytochrome c was observed in cellstreated with adenovirus alone. In Jurkat incubated in 100 mM

zVAD-fmk prior to the addition of granzyme and adenovirus,there was no reduction in the appearance of cytosolic cyto-chrome c, thus demonstrating further that caspases are notrequired for this phenomenon to occur. Similarly, experimentsin Jurkat-Bcl-2 targets showed that expression of Bcl-2 com-

FIG. 1. Quantitation of PS flip and cytochrome c release in 2H18 6 Bcl-2. Rapid cell death is associated with cytochrome c release. 2H18and 2H18-Bcl-2 targets were incubated in the presence of 10 plaque-forming units/ml granzyme B and adenovirus (Ad). The broad spectrumcaspase inhibitor zVAD-fmk was added to a final concentration of 100 mM 15–30 min prior to the addition of granzyme and adenovirus. Cells wereharvested at 2 and 4 h to measure cytoplasmic cytochrome c and annexin V binding. In the bottom panel, 2H18-Bcl-2 were incubated at 37 °C for12 h in 0.5 mg/ml granzyme B and 10 plaque-forming units/ml adenovirus. PS externalization was assayed by annexin V-alexa 568 binding as ameasure of apoptosis. Cytochrome c release was quantitated by measurement of loss of cytochrome c-GFP from the mitochondria by flow cytometryas described under “Experimental Procedures.”

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pletely prevented granzyme B-mediated cytochrome c releaseas noted by the absence of cytosolic cytochrome c. Analysis ofthe mitochondrial pellets (Fig. 4A, bottom panel) showed thatcytochrome c was maintained in its original cellular fraction.

Cytochrome c Release by Granzyme B-activated Bid—Be-cause granzyme B directly cleaves Bid and cytochrome c re-lease can be achieved without caspase activation, we reasonedthat granzyme B might mediate cellular cytochrome c releasein a caspase-independent manner through Bid. To test thismodel, we treated isolated mitochondria with purified Bid pro-cessed by granzyme B. Fig. 4B shows an immunoblot of super-natants from mitochondria treated with increasing concentra-tions of granzyme B-cleaved and uncleaved Bid. At the highestconcentrations of untreated Bid tested (100 ng/ml) we observedsome cytochrome c release, but Bid that had been activated byprior treatment with granzyme B was significantly more potent

with respect to its cytochrome c releasing activity. Full-lengthGST-Bid, which possesses no cytochrome c releasing activity,became active after treatment with granzyme B. When GST-Bid was processed by granzyme B, the cytochrome c releasingactivity was comparable to that observed with processed wildtype Bid. It is important to note that mitochondria treated withgranzyme B alone did not release cytochrome c, indicating thatgranzyme B does not directly cleave any mitochondria-associ-ated proteins that result in cytochrome c release. Althoughgranzyme B cleaves Bid at only one of the caspase-8 sites, Bidprocessed by granzyme B is active in releasing mitochondrialcytochrome c.

Granzyme B cleaves and activates Bid in vitro, so we next setout to ascertain the pattern and requirements for Bid cleavagein target cells treated with granzyme B. Jurkat targets weretreated with granzyme B and adenovirus for 3 h at 37 °C.

FIG. 2. Bcl-2 inhibits cytochrome c release and rapid apoptosis in target cells. 2H18-Bcl-2 cells were treated with 1 mg/ml granzyme Band 10 plaque-forming units/ml adenovirus in the presence of annexin V-alexa 568 (red). Confocal images were captured at 2-min intervals toassess cytochrome c release and PS externalization. Cytochrome release was not observed at any point over the duration of these assays, but somecells eventually became annexin V-positive after .6 h, blebbed, and rounded up (see Fig. 2 movie in Supplemental Material). The time for inductionof apoptosis was significantly longer than in cells not expressing Bcl-2 (see Fig. 3).

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Whole cell lysates from these cells were analyzed for the pres-ence and cleavage of Bid protein. As shown in Fig. 4C, therewas significant cleavage of Bid in granzyme B/adenovirus-treated cells, which corresponds with cytosolic cytochrome crelease (Fig. 4A). The reduction in full-length Bid protein ingranzyme-treated targets was not inhibited in the presence ofthe broad spectrum caspase inhibitor zVAD-fmk. Whereasgranzyme-mediated Bid cleavage was independent of caspaseactivation, Jurkat treated with anti-Fas required activecaspases for Bid cleavage to occur because cleavage of Bid inanti-Fas-treated Jurkat, which proceeds via caspase-8 activa-tion, was inhibited by zVAD-fmk. These data are in agreementwith our previous findings that cleavage of Bid in target cellsstill occurs in the presence of the viral serpin caspase-8 inhib-itor, SPI-2 (9). Similar treatment of targets overexpressingBcl-2 did not block cleavage of Bid by death stimulus, granzymeB/adenovirus, or anti-Fas, suggesting that Bid processing oc-curs upstream of the apoptotic inhibition imparted by Bcl-2.

Kinetics of Bid Cleavage by Granzyme B—Granzyme Bcleaves and activates Bid, thus providing a mechanism bywhich granzyme B can cause caspase-independent cytochromec release. In light of these observations we analyzed Bid as asubstrate for granzyme B. To facilitate purification of the C-terminal cleavage product away from the similar sized N-ter-minal fragment, we used purified GST-Bid fusion protein. Asshown in Fig. 5A, granzyme B cleaves GST-Bid in a dose-de-pendent manner. After resolution by SDS-PAGE and transferto polyvinylidene difluoride, the C-terminal 12-kDa cleavagefragment was subjected to N-terminal sequencing, and thescissile bond in Bid was identified as C-terminal to aspartate75 (Fig. 5A). Asp-75 corresponds to the IEAD75SESQED se-quence that can also be recognized by caspase-8. Even at thehighest doses of granzyme B there were no detectable frag-ments corresponding to cleavage at Asp-59, the preferredcaspase-8 cleavage site. This confirms the earlier observation ofLi et al. (37) using Bid mutants, suggesting that granzyme Bcleaves at Asp-75, whereas caspase-8 prefers Asp-59 and isconsistent with the pattern of Bid cleavage products observedduring granzyme B-mediated events reported recently (9). It isimportant to note that although the granzyme B and caspase-8cleavage sites differ within Bid, both cleavage events are capa-ble of activating Bid.

In Fas-mediated apoptosis the apical death-inducing signal-ing complex-associated caspase, caspase-8, has been shown toactivate Bid to effect cytochrome c release (37, 38). We recentlydemonstrated that granzyme B can bypass caspase-8 activa-tion (9). We sought to clarify the relevance of granzyme B-de-pendent Bid cleavage by comparing the preference of granzyme

B for Bid and caspase-8 as substrates. We performed in vitrocleavage assays to compare the rates of cleavage of Bid andcaspases. Equimolar amounts of radiolabeled in vitro tran-scribed and translated substrate were digested with increasingconcentrations of granzyme B. As shown in Fig. 5, B and C,granzyme B cleaved Bid more efficiently than caspase-8 orcaspase-3. Granzyme B at concentrations as low as 3.75 nM

cleaved Bid, whereas these concentrations induced little or nocaspase-3 or caspase-8 cleavage. Based on the publishedkcat/Km value for cleavage of caspase-3 by granzyme B (3.6 3104 M21 s21) using similar measurements (22) we calculatedrelative kcat/Km values for granzyme B cleavage of Bid (6.0 3105 M21 s21) and caspase-8 (2.4 3 104 M21 s21). These datasuggest that Bid is a better substrate for granzyme B thancaspase-8 and caspase-3 by more than 10-fold and support thenotion that Bid represents a target protein sensitive to activa-tion by granzyme B. They also support our contention that theBcl-2-nhibitable mitochondrial pathway is utilized more effi-ciently by granzyme B than that involving direct activation ofcaspases.

DISCUSSION

Granzyme B is a critical mediator of target cell death. This isdemonstrated by the inability of CTL and natural killer cellsfrom granzyme B2/2 mice to induce rapid DNA fragmentationand subsequent apoptosis in allogeneic target cells (39, 40).These knockout mice also have severely depressed ability toovercome infection by cytomegalovirus (39, 41) and ectromelia(42). There is some low efficiency residual killing activity in thegranzyme B2/2 mice which is attributed to another granuleprotease, granzyme A, in a mechanism that is thought to pro-vide a backup to the principal mechanism involving granzymeB (43). Double knockout of granzymes B and A results in aphenotype similar to that observed in the perforin knockoutmouse (42, 44). The mechanism by which granzyme B activateapoptosis is therefore of significant interest.

Granzyme B cleaves and activates Bid, which provides acaspase-independent means of releasing cytochrome c duringCTL granule-mediated apoptosis. In physiological settings it ispossible that granzyme B also utilizes caspase-8 activation as ameans to bring about the rapid destruction of its targets. How-ever, direct activation of Bid by granzyme B provides a caspase-independent pathway to mitochondria. Thus, caspase-8 maycontribute but is not required for cytochrome c release in gran-ule-mediated apoptosis. It is also clear from our data andothers (45) that regardless of the availability of caspase-8, themost efficient pathway to target cell apoptosis is via the mito-chondria. As one might expect, redundance is built into the

FIG. 3. Cytochrome c release is not dependent on active caspases in granzyme B-treated cells. 2H18 targets were incubated with 0.5mg/ml granzyme B and adenovirus and observed by confocal video microscopy. The top series of panels shows the progression of apoptosis asindicated by cytochrome c release. As cytochrome c is released during apoptosis the fluorescent label changes from a punctate pattern of healthymitochondria to a diffuse cytoplasmic pattern. As cells condense, bleb, and round up, cytochrome c-GFP resumes a granular pattern in late stageapoptotic cells. The bottom series of panels shows 2H18 treated with 100 mM zVAD-fmk prior to the addition of granzyme and adenovirus. Thesepanels demonstrate that cytochrome c release proceeds in a caspase-independent manner in these cells but that other indices of apoptosis, suchas rounding up and blebbing, require active caspases (see Fig. 3 movies in Supplemental Material).

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CTL arsenal, and granzyme B is capable of initiating apoptoticevents through a mitochondrial bypass route by direct activa-tion of downstream caspases, but with greatly reducedefficiency.

It was shown previously that Bid is cleaved by granzyme B(37), but it remained to be demonstrated that this cleavageevent resulted in active Bid with respect to its cytochrome c

releasing activity. However, it is abundantly evident from ourobservations that although the preferred caspase-8 and gra-nzyme B cleavage sites in Bid differ, cleavage of Bid by gra-nzyme B results in productive activation and potent cytoc-

FIG. 4. Bid cleavage and cytochrome c release are caspase-independent in granzyme B-treated cells. Panel A, cytochrome crelease in Jurkat by granzyme B/adenovirus (Ad) is caspase-independ-ent but is inhibited by Bcl-2. Jurkat were treated with granzyme B andadenovirus for the times indicated in the presence or absence of thecaspase inhibitor zVAD-fmk or Bcl-2. Cytosol was isolated away frommitochondria and lysates analyzed by immunoblot. Bands correspond-ing to cytochrome c are marked by asterisks for each treatment. Cyto-chrome c release was detected in the presence of zVAD-fmk but wasinhibited by Bcl-2. Controls in which cells incubated for 120 min withgranzyme B or adenovirus alone were also included. Panel B, granzymeB-“activated” Bid causes cytochrome c release in isolated mitochondria.Isolated Xenopus mitochondria were treated with full-length Bid or Bidthat had been digested with 17.5 ng/ml granzyme B prior to the additionto mitochondria. After incubation of mitochondria with 1, 10, or 100ng/ml purified Bid or equimolar amounts of GST-Bid at 30 °C for 1 h,mitochondrial pellets were removed by centrifugation, and the super-natants were analyzed for the presence of cytochrome c by immunoblotanalysis. Untreated mitochondria incubated at 30 °C and 4 °C wereincluded as negative controls, and the supernatant from a NonidetP-40-treated mitochondria sample was included as a positive control forcytochrome c release. Panel C, cleavage of Bid is caspase-independentand upstream of Bcl-2. Jurkat and Jurkat stably expressing Bcl-2 weretreated with 1 mg/ml granzyme and adenovirus for 3 h in the absence orpresence of 100 mM zVAD-fmk. Cell lysates were collected, analyzed byimmunoblot for disappearance of the full-length Bid, and quantitatedwith NIH-Image software. The percentage of Bid cleavage was normal-ized according to the amount of full-length Bid in untreated cells.

FIG. 5. Kinetics of Bid cleavage by granzyme B. Purified GST-Bid was incubated in increasing doses of granzyme B (0.1, 0.25, 1, 2.5,10, and 25 ng/ml; 3.75 nM–0.94 mM), resolved on standard SDS-PAGE,transferred to sequencing polyvinylidene difluoride, and stained inCoomassie Brilliant Blue. To determine the site at which granzyme Bcleaves Bid, we subjected GST-Bid to complete digestion by granzymeB, and the band corresponding to the new C-terminal fragment(s) wasexcised and subjected to N-terminal sequencing. No other fragmentswere observed, even at high concentrations of granzyme B. For com-parison of Bid, caspase-3, and caspase-8 as substrates for granzyme B,equimolar amounts of in vitro transcribed and translated proteins weresubjected to digestion by increasing amounts of granzyme B for 30 minat 30 °C. The amount of granzyme B (0.1, 0.25, 1, 2.5, 10, and 25 ng/ml;3.75 nM–0.94 mM) was kept consistent between substrates. The cleavageproducts were analyzed by SDS-PAGE and autoradiography. Concen-trations of granzyme B required to cleave detectable amounts ofcaspase-3 and caspase-8 (lower panels) were capable of cleaving Bid tocompletion, suggesting that Bid is a better substrate than either ofthese caspases for granzyme B.

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hrome c releasing activity. At first glance this may appear to bea subtle distinction, but one must consider that cleavage bygranzymes or caspases does not always result in activation. Forexample, granzyme B cleaves ICAD (inhibitor of CAD, thecaspase-activated DNase) (46, 47) at a site different from thescissile bond for caspase-3. However, the primary cleavage bygranzyme does not result in productive activation of CAD andnucleolytic activity as a result of ICAD cleavage (46). A secondcleavage event occurs at significantly elevated granzyme conc-entrations and even then in a much less efficient manner thanthat observed in caspase-3-treated ICADzCAD (47). Whereas aconcentration of 4.7 mM granzyme B was required to activatethe ICADzCAD complex (47), as little as 3.75 nM (0.1 ng/ml)granzyme B was sufficient to cleave enough Bid to trigger thecytochrome c releasing activity of Bid (see Figs. 4 and 5). This3,000-fold difference in sensitivity strongly implicates the mit-ochondria/caspase-dependent pathway as the principal mecha-nism for granzyme B-mediated activation of CAD, with result-ant DNA fragmentation.

It has been observed that in some cells, Bcl-2 gives onlypartial or temporary protection from apoptosis and that effec-tor caspases may lead to proteolytic inactivation of Bcl-2 (48,49). In that model, removal of Bcl-2 then allows for cytochromec release and a full-blown apoptotic response. However, ourexperiments show that a small percentage of granzyme B-treated Bcl-2-expressing target cells eventually undergo apop-tosis, but they do so without the coordinate release of cyto-chrome c. This suggests that in this system Bcl-2 retains itsactivity to block cytochrome c release but does not prevent theend result of apoptosis. This is believed to be the result ofengaging the apoptotic machinery downstream of the mito-chondria, which is consistent with the ability of granzyme B toactivate caspases directly, albeit less efficiently than Bid.Therefore, granzyme B can circumvent the necessity, althoughinefficiently, for mitochondrial cytochrome c release, but it doesnot override the protection afforded by Bcl-2 to inhibit cyto-chrome c release.

Although a physiological relevance has yet to be establishedfor the nuclear translocation of granzyme B in target cell ap-optosis (29, 50–52), it has been shown that movement of gran-zyme B into the nucleus is inhibited by Bcl-2 (51). However, ifcytochrome c release is a key event controlling all of the down-stream events during target cell apoptosis, then it is likely thatthe Bcl-2 block of nuclear granzyme is due to the fact thatrelease of mitochondrial proteins, such as cytochrome c, is theswitch necessary for all downstream effects. This situation mayparallel that reported earlier in which it was found that themitochondrial components of the death machinery were neces-sary for caspase-8-activated cell free extracts to elicit all of thedownstream apoptotic events, notably nuclear membranechanges (53).

As demonstrated for death receptor-mediated apoptosis,some cell types are prone to death by a type I or type IIpathway, characterized by the independence or dependence,respectively, of mitochondrial events (20). For example, hepa-tocytes from Bid2/2 mice are resistant to killing by anti-Fas,suggesting that the preferred apoptotic pathway in these cellsinvolves the Bid-mediated, Bcl-2-inhibitable release of proteinsfrom the mitochondria (54). The preference for the Bcl-2-inhib-itable mitochondrial pathway as the primary apoptotic path-way utilized by granzyme B provides an explanation for theearlier observations that Bcl-2 offers protection from rapidapoptosis induced by granzyme B and perforin (24–26, 55). Ourmodel also accounts for the reduced levels of apoptosis observedin early studies, by a type I-like mitochondrial bypass pathway(20) engaged directly by granzyme B, which we found to be less

efficient in our experimental system. In the studies of Sutton etal. (25), the Bcl-2 block of apoptosis could be overcome at highereffector to target ratios and increased doses of granzyme B inCTL-free killing (25), which is consistent with the notion thatgranzyme B, like caspase-8, acts most efficiently via the releaseof cytochrome c (53). It is possible that there are some cell typesthat are more sensitive to direct activation of caspase-3 bygranzyme B, and in such cells Bcl-2 would not be expected tointerfere with this form of death. In cells lacking Bid, forexample, granzyme B may not be able to induce release ofproteins from the mitochondria; however, higher levels of gran-zyme would be necessary to induce apoptosis. It remains to bedetermined what effect the newly identified Smac/Diablo pro-tein plays in granzyme B-mediated apoptosis. It is possible thatrelease of mitochondrial Smac/Diablo (56, 57) also contributestoward the rapid destruction of target cells through repressionof the inhibitor XIAP in target cells, making caspase-3 morereadily activated directly by granzyme B. If the release ofSmac/Diablo from the mitochondria is inhibited by Bcl-2, thenit is reasonable to propose that there are important mitochon-drial events in granzyme-induced apoptosis in addition to therelease of cytochrome c.

In our studies we have demonstrated that although gran-zyme B can activate caspases directly, the preferred route toapoptosis under limiting conditions is via Bid cleavage, mito-chondrial outer membrane permeabilization, and cytochrome crelease. It is also likely that these events are accompanied bythe release of Smac/Diablo from the mitochondrial intermem-brane space, which functions to interfere with the inhibitoryactivity of inhibitor of apoptosis proteins. The latter may blockcaspases activated by granzyme B, and therefore the involve-ment of mitochondrial outer membrane permeabilization ingranzyme B-induced apoptosis might be through this molecule(or in conjunction with cytochrome c). In any case, the involve-ment of the mitochondria in granzyme B-induced apoptosisprovides an explanation for the ability of Bcl-2 to block CTL-induced apoptosis in many cases, depending on the cells beingtargeted and the amount of granzyme B that enters those cells.

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GreenKuwana, Joshua C. Goldstein, Donald D. Newmeyer, R. Chris Bleackley and Douglas R.

Michael J. Pinkoski, Nigel J. Waterhouse, Jeffrey A. Heibein, Beni B. Wolf, TomomiBcl-2-inhibitable Mitochondrial Pathway

Granzyme B-mediated Apoptosis Proceeds Predominantly through a

doi: 10.1074/jbc.M009038200 originally published online January 12, 20012001, 276:12060-12067.J. Biol. Chem. 

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