Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin: Involvement of...

Granzyme B Induces Smooth Muscle Cell Apoptosis in theAbsence of Perforin

Involvement of Extracellular Matrix Degradation

Jonathan C. Choy, Vivian H.Y. Hung, Arwen L. Hunter, Paul K. Cheung, Bruce Motyka,Ing Swie Goping, Tracy Sawchuk, R. Chris Bleackley, Thomas J. Podor,

Bruce M. McManus, David J. Granville

Objective—T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to thepathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-inducedsmooth muscle cell (SMC) death.

Methods and Results—The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cellviability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed,indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes fromperforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolyticactivities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme Binternalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMCextracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation.

Conclusions—These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleavingextracellular proteins, such as fibronectin. (Arterioscler Thromb Vasc Biol. 2004;24:2245-2250.)

Key Words: granzyme B � perforin � smooth muscle cell � apoptosis � extracellular

The granule exocytosis pathway is one of the main mecha-nisms through which T cells kill pathogenic cells. Gran-

zyme B is a cytotoxic serine protease contained within cytotoxicT cell granules that induces apoptosis of target cells. Onactivation, granzyme B is exocytosed from T cells and isendocytosed through a receptor-dependent mechanism into in-tact vesicles of target cells.1 In the presence of perforin, gran-zyme B is released into the cytoplasm of target cells, where itinduces apoptosis through the indirect or direct activation ofcaspases.2 Because granzyme B requires perforin to be deliveredto the cytosol of target cells, induction of cell death by this serineprotease is believed to require perforin.

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Vascular cell death induced by activated T cells contributes tothe pathogenesis of several inflammatory vascular diseases.Cytotoxic T cells are observed in Takayasu arteritis and giantcell arteritis, and cytotoxic granule components derived fromthese lymphocytes localize to medial smooth muscle cells(SMC) in these diseases, suggesting that granule-mediated SMCdeath may contribute to vascular damage.3 Similar localization

of T cell cytotoxic granule components to medial SMCs isobserved in aortic aneurysms, suggesting that this type of SMCdeath may contribute to weakening of the vessel wall andeventual rupture of the aorta.4 Both granzyme B and perforin arealso abundant in allograft arteries, whereupon perforin-containing T cells localize to the immediate subendothelial spaceand granzyme B localizes to terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-positivemacrophages and SMCs in both the intima and media.5,6 Inallograft arteries, there is significant medial SMC death that ismediated by cytotoxic T cells.7 We have recently shown, usinga rat heterotopic heart transplant model, that immune-mediatedmedial degradation and medial cell apoptosis is associated withsevere defects in SMC constriction of allograft arteries.8 Finally,granule-mediated SMC death may also play a role in thepathogenesis of atherosclerosis as SMC apoptosis contributes toplaque destabilization and rupture, which is the leading cause ofmyocardial infarction.9 Granzyme B is observed in and aroundTUNEL-positive SMCs in both the intima and media of athero-sclerotic arteries, suggesting that the granule pathway may bemediating SMC death in this disease.6

Original received July 29, 2004; final version accepted September 27, 2004.From The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research (J.C.C., V.H.Y.H., A.L.H., P.K.C., T.J.P., B.M.M., D.J.G.),

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada; Department of Biochemistry(B.M., I.S.G., T.S., R.C.B.), University of Alberta, Edmonton, Alberta, Canada.

Correspondence to David J. Granville, PhD, The iCAPTURE Centre, Room 166, Burrard Building, St. Paul’s Hospital, 1081 Burrard St, Vancouver,BC, V6Z 1Y6 Canada. E-mail [email protected]

© 2004 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000147162.51930.b7

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Although T cell-induced SMC death contributes to a numberof vascular pathologies and affects vasomotor function in allo-graft arteries, the mechanisms through which this event ismediated has not been investigated. Because immunohistologi-cal experiments have implicated the granule pathway in theinduction of SMC death during autoimmunity, vascularizedorgan transplant rejection, and atherosclerosis, we have investi-gated the mechanisms through which granzyme B induces SMCdeath in vitro. Surprisingly, granzyme B can induce SMC deathin the absence of perforin. We provide evidence indicating thatthe mechanism through which granzyme B induces SMC apo-ptosis in the absence of perforin is mediated by the proteolysis ofextracellular proteins.

Materials and Methods

ReagentsPlease refer to online supplementary data available at http://atvb.ahajournals.org for details.

Quantitation of Cell ViabilityHuman coronary artery SMCs and HeLa cells were grown to 70%confluence in 96-well plates, maintained in medium �0.2% fetal calfserum for 48 hours, and treated with either granzyme B or granzymeB plus Ad5 (10 to 100 pfu/cell). In experiments using inhibitors andmannose-6-phosphate (M6P), SMCs were incubated withz-VAD.fmk (100 �mol/L), z-AAD.cmk (25 �mol/L), or solubleM6P (20 mmol/L) for 30 minutes before the addition of granzyme B.Cell viability was quantitated with an MTS assay as per themanufacturer’s instructions (Promega, Madison, Wis).

Assessment of ApoptosisChromatin condensation was assessed by staining SMC nuclei withthe DNA dye Hoechst 33342 and phosphatidylserine outer mem-brane localization determined by staining unfixed cells with Alexa488-labeled Annexin V. Please see online supplementary data fordetails.

Silver StainingPlease refer to supplementary data for details.

Splenocyte Killing AssaysSplenocyte killing assays were performed using calcein-loadedFisher rat aortic SMCs or concanavalin A (Con A)-treated ratsplenocytes as targets, and Con A-activated C57BL/6 (wild-type) orperforin-knockout splenocytes as effectors. Calcein release was usedto quantitate SMC death. The following formula was used tocalculate percent specific calcein release:

% specific calcein release � (calcein release in sample �spontaneous release) / (total calcein release � spontaneous release)

Please refer to supplementary data for details.

Cell Lysis and Western BlotSMCs were grown to 70% confluence on 60 mm tissue culture (TC)dishes, maintained in SmBM �0.2% fetal calf serum for 48 hours,and treated with granzyme B (75 nM). After 24 hours, cells werelysed and Western blot analysis was performed as describedpreviously.10

Granzyme B Uptake AssaySee supplementary data for details.

Assessment of Proteolysis of Extracellular ProteinsSMCs were grown to 70% confluence in a 24-well TC plate andmaintained in SmBM �0.2% fetal calf serum for 48 hours. SMCswere then washed 3 times with cold PBS, and lysed with 0.25 mol/LNH4OH for 30 minutes. Biotinylation of the extracellular proteinswas performed using a biotinylation kit as per the manufacturer’sinstructions (Pierce Chemicals). At the indicated time points, sam-ples of the supernatant were collected. At 24 hours after treatment,supernatant was collected, and total extracellular proteins collectedin SDS lysis buffer and Western blot performed to assess the proteinprofile.

StatisticsAn ANOVA was performed to determine significance betweengranzyme B-treated and control groups in the dose response exper-iments. In all other experiments, a Student t test was performed. Pvalue (�-error) of �0.05 was considered significant.

ResultsGranzyme B Alone Induces Apoptosis of SMCsGranzyme B alone reduced SMC viability (Figure 1A). Therewas a significant decrease in cell viability beginning at 37.5

Figure 1. Granzyme B induces SMCapoptosis in the absence of perforin. A,SMCs were treated with varying concen-trations of granzyme B (black bars) orgranzyme B with Ad5 (gray bars) for 24hours, and cell death was quantitatedusing an MTS assay. (*P�0.01 as com-pared with control). B, SMCs weretreated with granzyme B for 24 hours,and chromatin condensation and phos-phatidylserine outer membrane localiza-tion assessed by staining with Hoechst33342 or Annexin V–Alexa 488 (greencolor), respectively. Arrows denote apo-ptotic SMCs (320X). C, Activated spleno-cytes from wild-type and perforin-knockout mice were cocultured withSMCs at an E:T ratio of 12:1 and specificcalcein release quantitated at 48 hours(*P�0.05).

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nM, and this reached a minimum of 56.5�5.9% of control at150 nM. The addition of Ad5, which mimics the granzymeB-delivery actions of perforin in vitro through its endosomo-lytic properties,2 did not affect the granzyme B-induceddecrease in SMC viability, indicating that the addition of anendosomolytic agent may not potentiate granzyme B-inducedcytotoxicity toward SMCs. By transfecting SMCs with Ad5containing a GFP vector and assessing GFP-positive SMCs,we have ensured that Ad5 was capable of transfecting �50%of SMCs, indicating that Ad5 is an effective protein deliveryagent in SMCs (data not shown). As a control, HeLa cellswere also treated with granzyme B and consistent withprevious reports granzyme B and Ad5 acted in concert toinduce a dramatic and significant reduction in HeLa cellviability (Figure IA, available online at http://atvb.ahajournals.org).11 Importantly, granzyme B alone did not affectHeLa cell viability, suggesting that perforin-independent gran-zyme B-induced cell death is cell-type specific.

To ensure that the cytotoxicity of purified granzyme Btoward SMCs was not a result of perforin contamination,silver staining and Western blotting for perforin was per-formed on purified granzyme B preparations. These prepara-tions contained a single band at 32 kDa, which is themolecular weight of granzyme B (Figure IB and IC). Theabsence of other bands in either assay indicates that perforin(molecular weight �66 kDa) was not present in thepreparations.

Assessment of SMC morphology after 24 hours of treat-ment with granzyme B (75 nM) indicated that this proteaseinduced morphological alterations such as cell rounding andsurface detachment (Figure IIA, available online at http://atvb.ahajournals.org). Granzyme B did not have any effect onHeLa cells in the absence of an endosomolytic agent. Becausethe observed morphological changes in granzyme B-treatedSMCs are indicative of apoptosis, SMCs were stained withHoechst 33342 and Annexin V to visualize chromatin con-densation and the externalization of phosphatidylserine, re-spectively, to further characterize apoptosis. Granzyme Binduced marked chromatin and nuclear condensation (Figure1B), as well as phosphatidylserine outer membrane localiza-tion in several SMCs (Figure 1B), indicating that granzyme Bwas inducing SMC apoptosis. Quantitation of AnnexinV-positive cells 24 hours after treatment with granzyme Brevealed a significant increase in SMC apoptosis (19.0�2.6%in granzyme B-treated SMCs as compared with 10.3�0.7%in untreated SMCs, P�0.04).

To assess the timing of granzyme B-induced SMC apopto-sis, we temporally characterized the morphological changesinduced by this protease. Assessment of dark and light fieldphotomicrographs of SMCs labeled with Alexa 488-conjugated Annexin V indicated that SMCs displaying An-nexin V-positivity were also undergoing morphologicalchanges of apoptosis (Figure IIB). Granzyme B-inducedSMC apoptosis was a slow event. Morphological changes didnot begin until at least 16 hours after treatment and were notabundant until 20 hours after treatment. At 24 hours and 48hours after treatment, several SMCs were undergoing cellrounding, surface detachment, and membrane blebbing (Fig-ure IIC).

Splenocytes Induce SMC Death Through aPerforin-Independent GranzymeB-Mediated PathwayLimited SMC death was induced by activated splenocytes attime points at or before 24 hours, and splenocyte-inducedSMC death was not optimal until 48 hours. At this time point,splenocytes induced SMC death in a dose-dependent manner,reaching a maximum of 41.4�3.0% specific calcein releaseat an effector:target (E:T) of 25:1 (Figure IIIA, availableonline at http://atvb.ahajournals.org). There was no SMCdeath induced by inactivated splenocytes or the supernatantfrom activated splenocytes at 48 hours, indicating that SMCdeath was mediated by activated T cells and was not causedby the passive release of granule components at 48 hours(data not shown). In addition, Con A-activated mouse spleno-cytes induced cell death of isolated rat splenocytes by 6hours, indicating that the slow time course of SMC death isnot a result of the assay system but represents a reducedsensitivity of SMCs to immune-mediated cell death as com-pared with certain other cell types (Figure IIIB). Also,activated syngeneic splenocytes did not induce cell death.

SMCs were incubated with wild-type or perforin-knockoutCon A-activated splenocytes at an E:T of 12:1 in the presenceor absence of z-AAD.cmk (a synthetic granzyme B inhibitor)to determine the contribution of granzyme B to splenocyte-induced SMC death in the absence of perforin. We havepreviously shown that z-AAD.cmk does not inhibit apoptosisinduced by caspases10 and this inhibitor does not preventTRAIL-induced apoptosis (data not shown), indicating thatz-AAD.cmk does not block receptor-mediated apoptosis.There was a reduction in SMC death induced by perforin-knockout splenocytes as compared with wild-type spleno-cytes, indicating that SMCs are sensitive to cell death inducedby a perforin pathway. However, the addition of z-AAD.cmkto perforin-knockout splenocytes further reduced SMC death,indicating that activated T cells can kill SMCs through aperforin-independent granzyme B-mediated pathway (Figure1C). Finally, because we have shown that purified granzymeB alone induces SMC apoptosis, the calcein release measuredin these experiments represents membrane damage that oc-curs during late-stage apoptosis.

Granzyme B–Induced SMC Apoptosis IsDependent on Caspase and Granzyme BProteolytic ActivityGranzyme B treatment resulted in the processing ofprocaspase-3 to its p17 fragment, suggesting that the caspasecascade was being activated in SMCs (Figure IV, availableonline at http://atvb.ahajournals.org). z-VAD.fmk completelyinhibited the granzyme B-mediated loss in cell viability,indicating that caspase activation is required for perforin-independent SMC death (Figure 2). SMCs were also treatedwith the granzyme B inhibitor z-AAD.cmk (25 �mol/L) for30 minutes before the addition of granzyme B to assess thecontribution of granzyme B proteolysis to SMC death.Similar to z-VAD.fmk, z-AAD.cmk completely inhibited thereduction in SMC viability (Figure 2).

Choy et al Granzyme B–Induced Smooth Muscle Cell Death 2247



Granzyme B Acts Extracellularly to InduceSMC ApoptosisAlthough granzyme B-induced SMC death was not apparentuntil 16 hours after treatment, GrB-488 was associated withSMCs as early as 2 hours, and this interaction was morepronounced at 24 hours (Figure V, available online athttp://atvb.ahajournals.org). The pattern of GrB-488 localiza-tion with SMCs appeared to be very punctate and perinuclear,which is similar to that observed in other cell types and issuggestive of its localization to intact vesicles.2 To inhibitgranzyme B uptake, SMCs were incubated with 20 mmol/Lsoluble mannose-6-phosphate (M6P) before treatment withGrB-488. M6P has been shown to inhibit granzyme Binternalization in other cell types.12 At 2 hours and 24 hours,M6P attenuated GrB-488 association with SMCs, indicatingthat M6P can attenuate granzyme B internalization intoSMCs (Figure V).

Because soluble M6P attenuates granzyme B uptake intoSMCs, these cells were incubated with 20 mmol/L solubleM6P for 30 minutes before the addition of granzyme B. At 24hours after treatment, MTS assay was performed. SolubleM6P did not inhibit SMC death, indicating that granzyme Binternalization is not required for SMC death and thatgranzyme B is acting extracellularly to induce cell death(Figure 3).

Granzyme B Induces the Cleavage of MultipleExtracellular ProteinsTreatment of SMC matrix with granzyme B induced thecleavage of a number of extracellular proteins (Figure 4). Inthe collected insoluble proteins, 4 protein bands between �50to 70 kDa and �236 kDa disappeared 24 hours aftertreatment with granzyme B and cleavage fragments of �25 to39 kDa were evident in the matrix at this same time point.Further, at least 6 proteins and/or cleavage fragments rangingin molecular weight from �29 to 148 kDa were eluted into

the supernatant as early as 2 hours after granzyme B treat-ment. To ensure that the SMC extracellular protein prepara-tions used were devoid of intracellular proteins, Westernblotting for �-actin was performed on the collected superna-tant and extracellular proteins. �-actin was apparent in SMClysates (positive control) but was absent from matrix andsupernatant preparations.

To identify extracellular proteins that are cleaved bygranzyme B, we performed Western blots for fibronectin,collagen, and vitronectin on lysates from untreated andgranzyme B-treated SMCs. In SMCs treated with granzyme Bfor 24 hours, there was a reduction in the total amount offibronectin in lysates collected from SMCs. In the superna-tants of granzyme B-treated SMCs at 24 hours, a fibronectincleavage product was detected (Figure 4). No cleavage ofcollagen IV or vitronectin was observed (Figure VIA, avail-able online at http://atvb.ahajournals.org). Therefore, gran-zyme B induces the cleavage of fibronectin in SMC extra-cellular matrices but does not affect collagen IV orvitronectin.

To determine whether granzyme B was inducing SMCapoptosis through the generation of soluble pro-apoptoticfragments from extracellular proteins or through the disrup-tion of established cell–matrix interactions, we collected thesupernatant from granzyme B-treated SMC extracellular pro-teins (which contain granzyme B-generated cleavage prod-ucts) and inhibited granzyme B activity with the addition ofz-AAD.cmk before the addition of these fragments to SMCsfor 24 hours. The granzyme B-generated SMC extracellularprotein fragments were not capable of inducing SMC death(Figure VIB). Because these results suggest that granzyme Bis disrupting cell–matrix interactions, we evaluated the integ-rity of extracellular matrix-derived intracellular signals byassessing focal adhesion kinase (FAK) phosphorylation byWestern blot analysis. In adherent cell types, FAK is acti-vated by phosphorylation in response to cellular interactionswith extracellular components and this kinase regulates sur-vival signals. Disruption of FAK activation through elimina-tion of extracellular-derived signals can induce apoptosis.13

Treatment of SMCs with granzyme B for 24 hours reduced

Figure 3. Internalization is not required for granzyme B-inducedSMC death. SMCs were incubated with soluble M6P for 30 min-utes (to attenuate granzyme B uptake) before the addition ofgranzyme B. At 24 hours after treatment, an MTS assay wasperformed to assess cell viability.Figure 2. Granzyme B-induced SMC death is dependent on

caspase and granzyme B proteolytic activity. SMCs were incu-bated with z-VAD.fmk or z-AAD.cmk for 30 minutes before theaddition of granzyme B. After 24 hours, an MTS assay was per-formed to assess cell death (*P�0.02 as compared withcontrol).




FAK phosphorylation but did not alter the levels of total FAKprotein (Figure 4). These results indicate that granzyme B isinducing SMC death by disrupting extracellular SMC signalsthrough the proteolysis of extracellular proteins.

DiscussionIn the current study, we demonstrate that granzyme B caninduce SMC apoptosis in the absence of perforin. Perforin-independent granzyme B-induced SMC death was dependenton granzyme B and caspase proteolytic activity, and did notrequire granzyme B internalization. Further, granzyme Binduced the cleavage of multiple extracellular proteins, in-cluding fibronectin, and disrupted extracellular SMC interac-tions. These data suggest that the mechanism of perforin-independent granzyme B-induced cell death involves theproteolysis of extracellular proteins.

Recent investigations suggest that granzyme B can be inter-nalized in a M6PR-independent manner in vitro.14 However,granzyme B is secreted from cytotoxic granules as a complexwith proteoglycan,15 and Veugelers et al16 have demonstrated

that the M6PR is critical for granzyme B/proteoglycan-mediatedapoptosis. We have treated SMCs with proteoglycan-boundgranzyme B, and this induces SMC apoptosis with similarefficiency as purified granzyme B and is similarly not inhibitedby M6P (data not shown). Although studies in certain types ofcells from M6PR knockout mice have suggested that thisreceptor is not required for granzyme B internalization, M6Pclearly attenuates SMC uptake of granzyme B because itattenuated SMC association with GrB-488.17 Recently, Gross etal18 have suggested that granzyme B can also be internalized intocertain tumor cells by plasma membrane-bound Hsp70, and thatthis induces apoptosis in a perforin-independent manner. How-ever, Hsp70 localizes to the cell surface only in certain tumorcells and minimal levels are present only within the cytosol ofSMCs.19,20 Combined, these results strongly support the notionthat granzyme B is acting extracellularly to induce SMC death.In addition, the long time point required for splenocyte-inducedSMC death further supports the notion that the mechanism ofSMC apoptosis involves the degradation of extracellular proteinsand is not a mitochondrion-mediated process.

Although we are not aware of any reports implicating gran-zyme B proteolysis of extracellular substrates in the induction ofapoptosis, certain reports demonstrate that granzyme B candegrade extracellular matrices synthesized by certain cells.Specifically, Sayers et al21 showed that the addition of purifiedrat granzyme B to certain tumor cell lines resulted in similarmorphological changes, as we have described in this report, andthat this led to a decrease in tumor cell proliferation. CytotoxicT cells can also degrade the microenvironment of hematopoieticstromal cells.22 In addition to these existing findings, we havedemonstrated that granzyme B induces the cleavage of multipleSMC extracellular proteins and have identified fibronectin asone of the granzyme B substrates. Fibronectin is important incell survival of SMCs.23 Cleavage of fibronectin by mast cellchymase induces SMC apoptosis.24 Therefore, although theidentity of other granzyme B extracellular substrates remains tobe determined, cleavage of fibronectin likely contributes togranzyme B-induced SMC death. Interestingly, addition ofgranzyme B-induced SMC extracellular protein cleavage prod-ucts does not induce SMC apoptosis. This is in contrast to thedeath-inducing properties of chymase-cleaved fibronectin.24 Thereasons for this are unclear but may involve differences in thetypes of fibronectin fragments generated. Fibronectin fragmentsgenerated by granzyme B may not be capable of interfering withestablished SMC–extracellular protein interactions.

Induction of SMC death by granzyme B may have impli-cations for vasomotor dysfunction and intimal remodeling ininflammatory vascular diseases. In arteritic diseases andallograft arteries, there is T cell-mediated medial SMC deaththat is accompanied by defects in SMC constriction.3,8 Inatherosclerosis, both T cells and granzyme B localize toTUNEL-positive SMCs.6 The induction of SMC death inatherosclerosis contributes to plaque destabilization and rup-ture.9 Granzyme B may also play a role in arterial damageinduced by neutrophils, because these leukocytes also expressgranzyme B.25 Also, mast cell chymase (which is secretedfrom granules in this cell type) has recently been implicatedas a mediator of arterial damage in atherosclerosis.24 Becausegranzyme B is secreted from T cell granules, and because

Figure 4. Granzyme B induces cleavage of a number of extracel-lular proteins. Extracellular proteins from SMC cultures were biotin-ylated and incubated with granzyme B. The supernatant was col-lected at 2, 4, and 24 hours after treatment, and the entireinsoluble extracellular protein preparation collected at 24 hours.Extracellular proteins were visualized by Western blot for biotin.Clear arrowheads denote cleaved proteins in the insoluble extra-cellular proteins, solid arrows denote cleavage fragments in eitherthe insoluble extracellular proteins or the supernatant. Western blotfor �-actin confirmed that the extracellular protein preparation wasdevoid of intracellular proteins. Western blots for fibronectin, phos-phorylated FAK (P-FAK), and total FAK were also performed onlysates of SMC treated with granzyme B.

Choy et al Granzyme B–Induced Smooth Muscle Cell Death 2249



chymase is secreted by mast cell granules, SMC apoptosisinduced by the extracellular activity of granzyme B and mastcell chymase may represent a common mechanism throughwhich granule-associated serine proteases contribute to arte-rial damage during inflammatory vascular diseases. Althougha role for granzyme B in the induction of SMC anoikis andthe resultant pathogenesis of vascular diseases has beensuggested,26 to our knowledge, our current findings are thefirst to directly show that granzyme B can induce SMC deaththrough the cleavage of extracellular proteins.

In summary, we have identified a perforin-independentmechanism of granzyme B-mediated cell death through thecleavage of extracellular proteins. This novel mechanism ofgranzyme B-induced apoptosis accounts for �30% of SMCdeath induced by T cells, whereas 62% of T cell-inducedSMC death is perforin-dependent. Granzyme B-induced celldeath in the absence of perforin may be cell type–dependent,which has implications for the regulation of the granzyme-induced apoptotic pathways in vascular diseases and suggeststhat granzyme B may not be completely dependent onperforin for cytotoxicity. The latter may have implications formatrix remodeling that could effect SMC migration and/oratherosclerotic plaque stability.

AcknowledgmentsThis work was generously supported by operating grants from theCanadian Institutes of Health Research (CIHR; D.J.G, B.M.M,R.C.B), Michael Smith Foundation for Health Research (MSFHR;D.J.G), Canadian Foundation for Innovation (D.J.G, B.M.M), St.Paul’s Hospital Foundation (D.J.G), British Columbia TransplantFoundation (D.J.G.), and the National Cancer Institute of Canada(R.C.B). J.C.C is a recipient of a CIHR Doctoral Research Award,Michael Smith Foundation for Health Research Traineeship, andHonorary Killam Fellowship. A.L.H. is grateful for support from aCIHR/MSFHR Transplant Traineeship and a MSFHR Junior Grad-uate Studentship. D.J.G is a Canada Research Chair in Cardiovas-cular Biochemistry and a MSFHR Research Scholar. R.C.B is aCIHR Distinguished Scientist, a Medical Scientist of the AlbertaHeritage Foundation for Medical Research, a Howard HughesInternational Scholar, and a Canada Research Chair.

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18. Gross C, Koelch W, DeMaio A, Arispe N, Multhoff G. Cellsurface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B.J Biol Chem. 2003;278:41173–41181.

19. Multhoff G, Botzler C, Wiesnet M, Muller E, Meier T, Wilmanns W,Issels RD. A stress-inducible 72-kDa heat-shock protein (HSP72) isexpressed on the surface of human tumor cells, but not on normal cells.Int J Cancer. 1995;61:272–279.

20. Zhu WM, Roma P, Pirillo A, Pellegatta F, Catapano AL. Oxidized LDLinduce hsp70 expression in human smooth muscle cells. FEBS Lett.1995;372:1–5.

21. Sayers TJ, Wiltrout TA, Sowder R, Munger WL, Smyth MJ, HendersonLE. Purification of a factor from the granules of a rat natural killer cellline (RNK) that reduces tumor cell growth and changes tumor mor-phology. Molecular identity with a granule serine protease (RNKP-1).J Immunol. 1992;148:292–300.

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Supplementary Data – Granzyme B induces smooth muscle cell apoptosis in the absence of perforin: Involvement of extracellular matrix degradation. JC Choy, VH Hung, AL Hunter, PK Cheung, B Motyka, IS Goping, T Sawchuk, RC Bleackley, TJ Podor, BM McManus, DJ Granville

MATERIALS AND METHODS

Reagents

Human coronary artery SMC (Clonetics, San Diego, CA) were cultured in SmBM

+ 5% FCS and used up to the eighth passage. HeLa cells (ATCC) were cultured in

DMEM + 10% FCS. Granzyme B was purified from the cytotoxic granules of YT Indy

cells as described previously 1. Monoclonal caspase-3 antibody was purchased from

Santa Cruz Biotechnologies (Santa Cruz, CA), biotin and FAK antibodies from New

England BioLabs (Beverly, MA), β-actin and fibronectin antibodies from Sigma, and

collagen IV antibody from Weislab (Lund, Sweden). The inhibitors z-VAD.fmk and z-

AAD.cmk were purchased from Enzyme Systems Products (Livermore, CA), and M6P

purchased from Sigma.

Assessment of apoptosis

Chromatin condensation was assessed by staining SMC nuclei with the DNA dye

Hoechst 33342. SMCs were treated with granzyme B for 24h and fixed in 10% neutral

buffered formalin for 10 min. After washing gently in PBS, SMCs were permeabilized

with 1% Triton X-100 for 15 min., incubated in a 1/1000 dilution of Hoechst 33342, and

coverslipped in an aqueous mounting medium. Gentle washings were performed

between each step. Chromatin condensation was visualized with fluorescence

microscopy.

Phosphatidylserine (PS) outer membrane localization was determined by staining

unfixed cells with Alexa 488-labeled Annexin V. An Annexin V-labeling kit (Molecular

Probes, Eugene, OR) was used as per the manufacturer’s instructions, and Annexin V-


positivity was visualized with fluorescence microscopy. All digital photomicrographs

were obtained on a Spot camera. Quantitation of Annexin V-positive cells was

performed in a blinded manner by counting the number of Annexin V-positive cells in

three low power (80X) photomicrographs and then dividing by the total number of cells.

Data is presented as an average ± SEM of three independent experiments.

Silver staining

Purified granzyme B (0.1 µg/mL and 0.2 µg/mL) was electrophoresed in a 4-12%

bis-tris acylamide gel at 200V for 35min in MES buffer (Invitrogen, Burlington, ON,

Canada) as per manufacturer’s instruction. After rinsing the gel in water for 1 min., it

was fixed in 40% methanol, 10% acetic acid and 50% deionized water for 30min. Silver

staining was performed with the SilverQuest staining kit (Invitrogen) under standard

conditions. In brief, the fixed gel was sensitized for 10 min., rinsed in 30% ethanol for

10 min., washed with deionized water for 10 min., stained for 30 min. and then finally

washed with deionized water for 1 min. The gel was then immersed in developing

solution for approximately 7 min. (until the appropriate band intensities were observed),

at which point the stopping solution was added. The stained gel was washed with

deionized water and imaged.

Splenocyte killing assays

Splenocyte killing assays were performed using Fisher rat aortic SMCs or

concanavalin A (Con A)-treated rat splenocytes as targets and C57BL/6 (WT) or PKO

splenocytes as effectors. Calcein release was used to quantitate SMC death. This method


measures membrane permeability that occurs during necrotic cell death or late stage

apoptosis. Calcein is a cell membrane impermeable fluorescent dye. Conjugation of

calcein to acetoxymethyl (calcein, AM) facilitates membrane permeability of this dye.

The AM is cleaved from calcein by intracellular methyl esterases, resulting in the

intracellular localization of calcein subsequent to loading with calcein, AM.

Compromization of the plasma membrane integrity during cell death results in the release

of calcein into the supernatant and quantitation of this release by fluorometry was used to

measure cell death.

Rat SMCs were grown to 70% confluence in 24-well TC dishes, washed twice

with PBS, and loaded with 10 µg/mL calcein, AM in MCDB for 30 min. Calcein, AM

was then removed and SMCs washed twice with PBS before incubation overnight in

MCDB + 10% NCS. Activated splenocytes were added the following morning. The

number of cells/well was calculated to be approximately 5 X 104 in the first experiment

and this cell concentration/well was used in the subsequent experiments to calculate the

effector:target (E:T) ratio. In experiments using rat splenocytes as targets, 5 X 106 rat

splenocytes were treated with 5 µg/mL Con A for 24h prior to loading with calcein, AM

as described above. This treatment was required to reduce spontaneous calcein release

from the target cells.

To obtain effectors, WT or PKO mice were anesthetized with ketamine/xylene

and the spleen was removed aseptically. Splenocytes were obtained by disruption of the

spleen on a wire mesh with the blunt end of a 3 mL syringe in RPMI + 10% FCS. After

centrifugation for 5 min. at 1300 rpm to isolate cells, red blood cells (RBC) were lysed


with the addition of RBC lysis buffer (10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA

(pH 8.0)) for 3 min. 107 splenocytes were then reconstituted in 10 mL of medium and

stimulated with 5 µg/mL Con A for 72h. Con A is a plant sugar that cross-links the CD3

molecules associated with the T cell receptor (TCR) and in this way polyclonally

activates T cells. Activation of T cells through this mechanism has been shown to induce

activation to a cytotoxic phenotype in a subset of T cells 2. Activated T blasts were

morphologically identified and counted before addition of varying concentrations of

activated T cells to SMCs in order to set up the various E:T ratios. After incubation of

Con A-activated splenocytes with SMCs, the supernatant was removed, centrifuged for 5

min. at 1300 rpm to remove suspended cells, and calcein release quantitated with a Tecan

GENios fluorescence microplate reader at Ex = 485 nm and Em = 535 nm. Spontaneous

release was calculated on the supernatant of calcein, AM-loaded SMCs without the

addition of splenocytes and was always less than 35%. Total calcein release was

determined by lysis of SMCs with 0.1% Triton for 30 min. The following formula was

used to calculate % specific calcein release:

% Specific Calcein Release = (calcein release in sample – spontaneous release)/(total

calcein release – spontaneous release)

Finally, in experiments using zAAD.cmk to inhibit granzyme B activity in T cells, 50 µM

zAAD.cmk was added to Con A-activated splenocytes prior to incubation with rat SMCs.

Fresh zAAD.cmk was added to the co-culture culture after 24h to ensure the maintained

inhibition of granzyme B.


Cell lysis and Western blot

SMCs were grown to 70% confluence on 60mm TC dishes, maintained in SmBM

+ 0.2% FCS for 48h, and treated with granzyme B (75 nM). After 24h, cells were

washed 1X with cold PBS, and lysed in 400ul of SDS lysis/loading buffer. Western blot

was performed as described previously 3,4 using a 1:100 dilution of anti-caspase-3,

1:1000 dilution of anti-biotin, 1:200 dilution of anti-fibronectin, 1:500 dilution of anti-

collagen, 1:1000 dilution of anti-vitronectin, 1:1000 of anti-FAK or anti-P-FAK, and

1:1000 dilution of anti-β-actin. The gel was stained with Gelcode Blue (Pierce

Chemicals, Rockford, IL) to ensure equal loading.

Granzyme B uptake assay

Granzyme B was labeled with Alexa-488 (GrB-488)(Molecular Probes, Eugene,

OR) as per the manufacturer’s instructions and as described previously 5. SMCs grown

on 96-well TC dishes were treated with GrB-488 for either 2h or 24h. Live SMCs were

washed gently with PBS before visualization with fluorescence microscopy. Data are

representative of 3 independent experiments.


References

1. Darmon AJ, Nicholson DW, Bleackley RC. Activation of the apoptotic protease

CPP32 by cytotoxic T-cell-derived granzyme B. Nature. 1995;377:446-8.

2. Bevan MJ, Cohn M. Cytotoxic effects of antigen- and mitogen-induced T cells on

various targets. J Immunol. 1975;114:559-65.

3. Granville DJ, Shaw JR, Leong S, Carthy CM, Margaron P, Hunt DW, McManus

BM. Release of cytochrome c, Bax migration, Bid cleavage, and activation of

caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis. Am J Pathol.

1999;155:1021-5.

4. Granville DJ, Cassidy BA, Ruehlmann DO, Choy JC, Brenner C, Kroemer G, van

Breemen C, Margaron P, Hunt DW, McManus BM. Mitochondrial release of

apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis.

Am J Pathol. 2001;159:305-11.

5. Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, Barry M,

Shostak I, Sawchuk T, Holmes CF, Gauldie J, Bleackley RC. Mannose 6-

phosphate/insulin-like growth factor II receptor is a death receptor for granzyme

B during cytotoxic T cell-induced apoptosis. Cell. 2000;103:491-500.

Supplementary Data – Granzyme B induces smooth muscle cell apoptosis in the absence of perforin: Involvement of extracellular matrix degradation.JC Choy, VH Hung, AL Hunter, PK Cheung, B Motyka, IS Goping, T Sawchuk, RC Bleackley, TJ Podor, BM McManus, DJ Granville

0.1 µg

0.2 µg

Ladd

erA. B.

0%

20%

40%

60%

80%

100%

120%

Untx Ad5 4.7 9.4 18.8 37.5 75.0Granzyme B (nM)

Cel

l Via

bilit

y (%

of C

ontro

l)

Without Ad5With Ad5

~32kDa -

YT

Indy

Gra

nzym

e B

pre

p

C.

Perforin

Figure I. A. HeLa cells were treated with varying concentrations of granzyme B with (gray bars) or without (black bars) Ad5 for 24 h, and cell death quantitated with an MTS assay. B. Granzyme B preparations were silver stained to ensure that they were not contaminated with perforin. C. Granzyme B preparations were Western blotted for perforin.

Figure I - Choy et al.


Darkfield Lightfield

Granzyme B

Untreated

B.Untreated Granzyme B

SMC

HeLa

A.

C. 12h 16h 20h 24h 48h

Untreated

Granzyme B

Figure II. A. The morphology of SMCs and HeLa cells treated with granzyme B (75 nM) for 24h was assessed with phase contrast microscopy. Arrows denote SMCs that are rounding and detaching from the culture plate (160X). B. Phase contrast photomicrographs of the same field of view as that stained with Annexin V indicated that Annexin V-positive cells were displaying membrane blebbing (arrows; 320X). C. SMCs were treated with granzyme B and photomicrographs obtained at 12, 16, 20, 24, and 48h. Arrows denote SMCs displaying morphological characteristics of apoptosis, such as cell rounding and membrane blebbing (160X).

Figure II – Choy et al.


A.

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

25 12 6 3

Effector:Target

% S

peci

fic C

alce

in R

elea

se

24h48h

B.

0%

5%

10%

15%

20%

25%

30%

6 3 1.5 0.75Effector:Target

% S

peci

fic C

alce

in R

elea

se

Rat Splenocytes

Syngeneic Splenocytes

Figure III. A. Activated mouse splenocytes were co-cultured with calcein-loaded rat SMCs and cell death assessed by specific calcein release. B. The kinetics of SMC death was then compared to that of splenocyte death. Activated mouse splencotyes (black bars) or activated syngeneic rat splenocytes (gray bars) were co-cultured with calcein-loaded rat splenocytes and calcein release quantitated at 6h.

Figure III – Choy et al.


Gra

nzym

e B

Unt

reat

ed

Procaspase-3

p17p19

Gelcode Blue

Figure IV. SMCs were treated for 24h with granzyme B, lysed in an SDS lysisbuffer, and Western blot performed for caspase-3.

Figure IV – Choy et al.


GrB-488 GrB-488 + M6PUntreated

2h

24h

Figure V. SMCs were incubated for 2h or 24h with GrB-488 in the absence or presence of M6P (20mM), and the localization of this protease visualized with fluorescence microscopy.

Figure V – Choy et al.


A.Collagen IV

GelCode Blue

Vitronectin

GelCode Blue

B.

0%

20%

40%

60%

80%

100%

120%

140%

Untreated zAAD.cmk zAAD.cmk + GrBfragments

Cel

l Via

bilit

y (%

of C

ontro

l)

Figure VI. A. Western blots for collagen and vitronectin were performed on lysatesand supernatants from untreated and granzyme B-treated SMCs. B. The supernatant from granzyme B-treated SMC extracellular proteins was then treated with z-AAD.cmk to block granzyme B activity, added to SMCs for 24 h, and cell viability quantitated with an MTS assay.

Figure VI – Choy et al.

David J. GranvilleSwie Goping, Tracy Sawchuk, R. Chris Bleackley, Thomas J. Podor, Bruce M. McManus and Jonathan C. Choy, Vivian H.Y. Hung, Arwen L. Hunter, Paul K. Cheung, Bruce Motyka, Ing

Involvement of Extracellular Matrix DegradationGranzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin:

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2004 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/01.ATV.0000147162.51930.b72004;

2004;24:2245-2250; originally published online October 7,Arterioscler Thromb Vasc Biol.

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