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Correspondence to M.M. Simon: simon{at}immunbio.mpg.de
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Abstract |
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Introduction |
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Significant advances in our understanding of the molecular basis for gzmA- and gzmB-induced cell death are based on experiments using the mast-cell exocytosis model (Nakajima et al., 1995) and from in vitro studies using purified effector molecules. It was concluded that gzmB initiates cell death through various pathways, either involving activation of caspases, directly or indirectly, and resulting in disruption of mitochondrial integrity (Barry et al., 2000; Heibein et al., 2000; Sutton et al., 2000, 2003; Alimonti et al., 2001; Goping et al., 2003; Metkar et al., 2003), by derepressing the endonuclease CAD (Thomas et al., 2000; Sharif-Askari et al., 2001) or by cleaving key structural proteins in the nuclear membrane or cytoskeleton (Browne et al., 2000; Zhang et al., 2001a,b). In contrast, gzmA seems to induce apoptosis by caspase-independent pathways (Beresford et al., 1999; Shresta et al., 1999). Besides other substrates of gzmA, like lamins (Zhang et al., 2001a) and histones (Zhang et al., 2001b), a special target seem to be the endoplasmic reticulumassociated SET complex (Fan et al., 2002). This complex contains three gzmA substrates, the nucleosome assembly protein SET, the DNA bending protein HMG-2, and the base excision repair endonuclease Ape1. The SET complex also contains the tumor suppressor protein pp32 and the DNase NM23-H1, the latter of which is released from inhibition by gzmA cleavage of SET and translocates to the nucleus (Fan et al., 2003a). Furthermore gzmA-mediated proteolysis of Ape1 interferes with its known oxidative repair function for DNA (Fan et al., 2003b).
These findings, principally obtained with purified effector molecules, emphasize the complexity of intracellular events triggered by gzmA and gzmB. However, it is unclear to what extent these in vitro observations reflect the biologically relevant interaction of NK and CTL with target cells in vivo. In particular, factors such as the quality of the proteins involved, the way and context of their delivery, and/or their accessibility to cellular compartments may play a crucial role in the outcome of cytolytic function. The present work uses ex vivoderived virus-immune CD8+ T cells from mice with targeted gene defects in perf or gzmA and/or gzmB to examine for the first time the apoptotic pathways activated by CTL via the two gzms in an in vitro cytotoxic assay using Fas-resistant tumor target cells.
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Results |
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Induction of loss of plasma membrane integrity and mitochondrial membrane potential elicited by CTL via gzmA or gzmB is dependent on perf
To determine if loss of plasma membrane and mitochondrial integrity in EL4.F15 target cells induced by gzmA- or gzmB-deficient CTL is dependent on perf, ex vivoderived (day eight after infection) and CD8-enriched LCMV-immune splenic T cells from B6, perf/, and gzmAxB/ mice were tested under the same conditions (2 h) for their potential to induce the same apoptotic parameters described in the previous paragraph. Effector cell populations from all three mouse strains contained similar numbers of gp33/D btetramer+/CD8+ T cells (10, 11, and 9% for B6, perf/, and gzmAxB/ T cells, respectively; Fig. 1 Ba). As previously found (Simon et al., 1997; Pardo et al., 2002), only CD8+ T cells from B6, but not from perf/ or gzmAxB/, mice were able to induce specific nucleolysis in EL4.F15 target cells (Fig. 1 Bb, 4-h assay). Therefore, target cell survival upon interaction with either B6-, perf/-, or gzmAxB/-derived CTL was inversely proportional to nucleolysis (Fig. 1 Bc). From the three effector cell populations, only CD8+ T cells from B6 mice readily induced loss of plasma membrane integrity in EL4.F15 target cells, whereas CD8+ T cells from perf/ or gzmAxB/ mice were, if at all, only marginally effective (Fig. 1 Bd). Similarly, only CD8+ T cells from B6 mice were able to optimally induce m loss (from 82 to 59%) and ROS generation (from 8 to 25%, in the absence vs. presence of gp33 peptide). In contrast,
m loss and ROS generation was much lower when CD8+ T cells from gzmAxB/ mice were used (
m: 83 to 77%, ROS: 7 to 13%; in the absence vs. presence of gp33 peptide) and not detectable with CTL from perf/ mice (Fig. 1 Be). These data demonstrate that for gzm-facilitated loss of plasma membrane and mitochondrial integrity in EL4.F15 target cells during specific CTL attack, the presence of perf is critical. In addition, in the absence of gzmA and gzmB, perf is able on its own or together with as yet unknown factors to induce, though only slightly,
m loss and ROS generation, however without affecting the survival of target cells (Fig. 1 B, e vs. c).
CTL-mediated activation of caspase 3 and 9 is dependent on gzmB but not gzmA
LCMV-immune CD8+ T cells from B6, gzmA/, and gzmB/ mice were incubated with gp33-pulsed or mock-treated EL4.F15 target cells (2 h), and activation of intracellular caspase 3 was assessed using the mAb C92-605. Fig. 2 A shows that effector cells from B6 and gzmA/ mice specifically and comparably induced activation of caspase 3 in 30% of target cells, whereas those from gzmB/ mice did not (Fig. 2 Aa, top). The presence of blocking anti-FasL mAb did not alter activation of caspase 3 by the two CD8+ T cell populations (Fig. 2 Aa, bottom). This excludes a contribution of the Fas pathway and suggests that the CTL-mediated effect is solely elicited via the exocytosis pathway (Pardo et al., 2002). When caspase activation in EL4.F15 cells was tested under similar conditions by applying intracellular staining with the caspase 3specific agent SRH-DEVD-fmk, comparable results were obtained (Fig. 2 Ab, top). Gp33-pulsed but not mock-treated target cells incubated with either B6- or gzmA/-derived LCMV-immune CD8+ T cells stained positive for active caspase 3 (44 vs. 6% and 60 vs. 15%, respectively). Again, the presence of blocking anti-FasL mAb did not alter activation of caspase 3 by B6 and gzmA/ CTL (Fig. 2 Ab, middle, + gp33 peptide). In both cases, pretreatment of targets with the specific inhibitor Z-DEVD-fmk (Fig. 2 Ab, orange) leads to a drastic reduction in positively staining cells (Fig. 2 Ab, bottom). Surprisingly and in contrast to results obtained using the specific mAb for active caspase 3, a small fraction of gp33-pulsed EL4.F15 cells (30 vs. 17% for mock-treated cells) also stained positive with SRH-DEVD-fmk after incubation with gzmB/ LCMV-immune CD8+ T cells (Fig. 2 Ab, top). However, the number of EL4.F15 cells staining for active caspase 3 were only marginally reduced by the specific inhibitor Z-DEVD-fmk (Fig. 2 Ab, bottom). This finding indicates that the apparent caspase 3 activation observed by using the fluorescent substrate (but not the mAb) is due to unspecific binding of the substrate to a molecule distinct from caspase 3 (Amstad et al., 2001).
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CTL-mediated activation of caspase 3 and 9 by gzmB is strictly dependent on perf
To determine whether or not CTL-induced and gzmB-facilitated activation of caspase 3 is also dependent on perf, EL4.F15 cells were cocultured with LCMV-immune CD8+ T cells from B6, perf/, and gzmAxB/ mice. Fig. 2 Ba shows that only CTL from B6 mice were able to specifically induce activation of caspase 3, as monitored with the mAb C92-605 in a fraction of EL4.F15 cells (23 vs. 2% for mock-treated cells). Similar results were obtained when SRH-DEVD-fmk was used to detect active caspase 3 (Fig. 2 Bb, top). As expected, the number of cells staining positive with SRH-DEVD-fmk, upon incubation with B6 CTL, was drastically reduced after preincubation of EL4.F15 cells with Z-DEVD-fmk (Fig. 2 Bb, bottom, orange). A considerable increase in positive staining cells for SRH-DEVD-fmk was also seen after incubation with LCMV-immune gzmAxB/-derived CD8+ T cells (Fig. 2 Bb, top). However, fluorescence was not altered after preincubation of cells with Z-DEVD-fmk (Fig. 2 Bb, bottom), indicating that SRH-DEVD-fmk binds to a protein(s) distinct from caspase 3.
Similarly, only effector cells from B6 mice were able to specifically induce activation of caspase 9, as observed with FAM-LEHD-fmk (52 vs. 15% for mock-treated cells; Fig. 2 Bc, top), which was inhibited in the presence of Z-LEHD-fmk (52 to 36%; Fig. 2 Bc, bottom, orange). In contrast, LCMV-immune CD8+ T cells from perf/ mice did not induce activation of caspase 9. Furthermore, the increase in intracellular staining of EL4.F15 cells for FAM-LEHD-fmk after incubation with gzmAxB/-derived LCMV-immune CD8+ T cells (47 vs. 15% for mock-treated cells) was not inhibited by Z-LEHD-fmk (47 vs. 53%; Fig. 2 Bc, bottom).
GzmB-activated caspases contribute to loss of plasma membrane integrity and mitochondrial membrane potential
We tested if in the course of CTL-mediated cytolysis, activation of caspases is essential for gzm-induced PS exposure on plasma membrane, cell death (PI incorporation), m loss, and ROS generation. For this purpose, LCMV-immune CD8+ T cells from B6, gzmA/, or gzmB/ mice were incubated with EL4.F15 cells in the presence of the universal caspase inhibitor Z-VAD-fmk or with the caspase 3 inhibitor Z-DEVD-fmk. As shown in Fig. 3 (A and B; mean of three independent experiments), the number of annexinV+ and PI+ cells was drastically reduced by either Z-DEVD-fmk or Z-VAD-fmk when gzmA/-derived CTL were used, only partially in the presence of B6 CTL, and not at all with gzmB/ CTL. Furthermore,
m loss and ROS generation induced by B6- and gzmA/- but not gzmB/-derived CTL were also reduced, though only partially, in the presence of Z-DEVD-fmk or Z-VAD-fmk (Fig. 3, C and D). These data suggest that CTL-mediated induction of plasma membrane perturbation is critically dependent on functional active caspases when elicited by gzmB but not by gzmA and reveals caspase-dependent and -independent mechanisms of gzmB-induced mitochondrial depolarization. In both cases, a DEVD-sensitive caspase, in particular caspase 3, may be the main executor of these caspase-dependent processes.
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To see whether or not the generation of functional active caspase 9 as monitored with the specific substrate FAM-LEHD-fmk was dependent on previous activation of caspase 3, aliquots of the cytotoxicity assay (CML) cultures derived from B6 and gzmA/ mice were incubated in the presence of inhibitors for either caspase 3 (Z-DEVD-fmk, gray) or caspase 9 (Z-LEHD-fmk, orange). Fig. 3 F shows that specific induction of caspase 9 activity by both CTL populations (top) is completely inhibited by Z-LEHD-fmk (from 36 to 13% for B6 CTL and from 36 to 14% for gzmA/ CTL; Fig. 3 F, middle) and also by Z-DEVD-fmk (from 36 to 16% for B6 CTL and from 36 to 17% for gzmA/ CTL; Fig. 3 F, bottom). Together, these results suggest that in CTL-mediated cytolysis activation of caspase 3 is a prerequisite for caspase 9 activation and that the generation of the former caspase is amplified by the latter.
Effect of oxidative stress on gzmA- and gzmB-induced loss of plasma membrane integrity and mitochondrial membrane potential
As shown in the first paragraph of Results, incubation of EL4.F15 cells with either B6, gzmA/, or gzmB/ LCMV-immune CD8+ T cells leads to ROS generation. To evaluate the requirement for ROS generation in gzmA- and gzmB-mediated apoptotic processes, the respective CML cultures were incubated with targets (2 h) previously treated with the antioxidant N-acetylcysteine (NAC) and tested for the annexinV±/PI± phenotype, m loss, and presence of active caspases 3 and 9. As expected, ROS formation is similarly suppressed after pretreatment of EL4.F15 cells with NAC, irrespective of the source of CTL (Fig. 4 D). The number of cells with the annexinV+/PI+ phenotype was significantly reduced in NAC-treated as compared with mock-treated EL4.F15 cells after culture with B6- and gzmA/-derived CTL and reduced to near background level when cultured with gzmB/-derived CTL (Fig. 4, A and B). NAC protected EL4.F15 cells only partially from
m loss, independent of the effector cell population (Fig. 4 C). Pretreatment of EL4.F15 cells with NAC had no effect on the activation of caspase 3, as monitored by mAb C92-605 fluorescence (Fig. 4 E, left), but resulted in considerably reduced activation of caspase 9 after incubation with either B6- or gzmA/-derived CTL, as determined by staining with FAM-LEHD-fmk (Fig. 4 E, right). Upon addition of both NAC and LEHD-fmk to cultures of B6 CTL and EL4.F15 target cells, inhibition of caspase 3 activation did not exceed that obtained with LEHD-fmk alone (Fig. 3 E and not depicted). The fact that suppression of plasma membrane perturbation (annexin+/PI± phenotype) by NAC is more pronounced when gzmB/- compared with gzmA/-derived CTL are used suggests that the generation of ROS is critical in gzmA-induced cell surface membrane perturbation, but less so in gzmB-mediated but caspase-dependent processes.
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Discussion |
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The findings that during CTL-target cell killing, perf-induced plasma membrane disturbance in EL4.F15 target cells, as revealed by PS exposure, occurs faster with gzmB as compared with gzmA and that in the case of gzmB annexinV reactivity precedes membrane permeabilization are new. Together with the fact that this proapoptotic program was greatly repressed by universal and specific caspase inhibitors only in case of gzmA/, but not gzmB/, CTL supports recent findings with purified perf and gzms (Sarin et al., 1998; Heibein et al., 1999) and suggests that activation of caspase(s) contributes, together with other pathways (i.e., mitochondria; see below in Discussion), to the accelerated process by gzmB. This finding also emphasizes the notion that CTL-mediated apoptosis elicited in the presence of both gzms is greatly influenced by the quality of the individual target cell (Pardo et al., 2002).
The result that PS exposure on EL4.F15 cells was only marginally increased upon incubation with gzmAxB/ CTL makes it unlikely that additional molecules, besides gzmA and gzmB, contribute to plasma membrane perturbation. However, because the targeted null mutation of gzmB also affects other downstream genes in the domain, including gzmC, -D, -E, -F, and -G (Pham et al., 1996), their involvement in this CTL-mediated pathway cannot be excluded. Especially as it was recently shown that purified gzmC together with perf induces PS exposure on plasma membrane in a process distinct from gzmB (Johnson et al., 2003). In addition, other gzms, termed orphan gzms (Grossman et al., 2003), such as gzmK and gzmM, with cellular expression patterns distinct from gzmA and gzmB may also facilitate this proapoptotic process (Shresta et al., 1997; MacDonald et al., 1999; Kelly et al., 2004).
The finding that the ROS scavenger NAC completely blocks the induction of PS exposure on plasma membrane of EL4.F15 cells by gzmB/ but not gzmA/ CTL is novel and indicates that ROS synergize with gzmA but not gzmB in CTL-induced plasma membrane perturbation. This finding is remarkable in the context of a recent paper demonstrating the coupling of actin cytoskeleton dynamics and the production of ROS in cell death of yeast and most probably also mammalian cells (Gourlay et al., 2004). Thus, we speculate that gzmA is destabilizing the actin cytoskeleton in target cells by cleaving a yet unknown substrate, leading to depolarization of mitochondrial membrane and ROS generation, and that the latter amplifies PS exposure at the plasma membrane, thereby accelerating cell death and/or uptake of targets by phagocytes via the PS receptor (Savill and Fadok, 2000; Danial and Korsmeyer, 2004). Alternatively, as the SET complex, a major substrate of gzmA (Fan et al., 2002, 2003a,b), is associated with the ER, gzmA may initiate apoptotic processes by perturbing ER membrane and inducing Ca2+ release from ER stores. This cell death pathway is elicited by interorganelle flow of Ca2+ and facilitated by direct physical connections between ER and mitochondria (Newmeyer and Ferguson-Miller, 2003), leading to caspase-independent m loss and ROS generation (Demaurex and Distelhorst, 2003; Orrenius et al., 2003).
Mitochondria are implicated in CTL-mediated pathways of apoptosis, but the contribution of m loss and ROS generation to this process is still controversial (MacDonald et al., 1999; Barry et al., 2000; Heibein et al., 2000; Danial and Korsmeyer, 2004). The data that gzmA/ and gzmB/, but not perf/, CTL induce
m reduction and ROS generation in EL4.F15 target cells to a similar extent indicates that both processes are dependent on perf, but are similarly and independently elicited by gzmA or gzmB. In addition, the finding that activation of the mitochondrial pathway is inhibited, at least partially, by Z-VAD-fmk and DEVD-fmk in the case of gzmA/ but not gzmB/ CTL argues that gzmA and gzmB induce
m loss via distinct molecular pathways. Furthermore, it indicates that gzmA induces mitochondrial depolarization in a caspase-independent manner, whereas gzmB may use both caspase-dependent and -independent pathways and supports previous findings (Heibein et al., 1999; Barry et al., 2000; Alimonti et al., 2001; Pinkoski et al., 2001; Goping et al., 2003; Sutton et al., 2003). Understanding how these complex processes induced by gzmA and gzmB interrelate and facilitate target cell lysis by CTLs requires further analysis.
The present demonstration that CTL from B6 and gzmA/, but not from gzmB/, perf/, or gzmAxB/, mice induced activation of caspase 3 and 9 in EL4.F15 cells corroborates and extends previous work with purified proteins and CTL on the differential effect of gzmA and gzmB in the perf-facilitated caspase pathway (Heibein et al., 1999; Pardo et al., 2002; Goping et al., 2003; Metkar et al., 2003; Sutton et al., 2003). However, because the targeted null mutation of gzmB also affects other downstream gzm genes in the domain (gzmCG; Pham et al., 1996), their putative effect on these processes cannot be assessed. Activation of caspase 3 and 9 by wild-type B6 and gzmA/ CTL was completely blocked by pretreatment of EL4.F15 cells with the caspase 3specific inhibitor Z-DEVD-fmk. In contrast, using similar conditions, the caspase 9specific inhibitor Z-LEHD-fmk completely inhibited activation of caspase 9, but only partially that of caspase 3. Thus we conclude that in gzmB-mediated cytolysis, activation of caspase 3 is an early event in target cell apoptosis and is a prerequisite for the generation of the caspase 9 apoptosomemost likely via mitochondrial permeabilizationwhich in turn cleaves additional procaspase 3. Such an amplification loop between caspase 3 and 9 has been proposed before with purified gzmB and perf (Metkar et al., 2003).
Ape-1, a multifunctional component of the SET complex (Fan et al., 2003b) expressing both endonuclease activity and the potential to repair oxidative damage to DNA and oxidative changes to transcription factors (Bennett et al., 1997; Hirota et al., 1997; Evans et al., 2000), is similarly cleaved in EL4.F15 cells, irrespective of whether CTL from either B6, gzmA/, or gzmB/ mice are used as effectors. Cleavage of Ape-1 was also observed, though at reduced levels, with gzmAxB/ CTL, but was absent in perf/ CTL. Fan et al. (2003b) have shown that gzmA specifically binds Ape-1 and destroys its known oxidative repair function by proteolytic cleavage. Our data indicate that CTL contain additional enzymatic activity(ies) distinct from gzmA and gzmBG, such as gzmK (Shresta et al., 1997), which is able to block cellular repair in a perf-dependent manner. However, this gzmA/gzmB-independent process does not seem to be relevant for the induction of cell death, as demonstrated by survival of EL4.F15 cells after their incubation with gzmAxB/ CTL (Fig. 2 C).
The lack of inhibition of CTL-mediated cell death of EL4.F15 cells, as monitored by target cell survival, by either the universal caspase inhibitor Z-VAD-fmk, the caspase 3 inhibitor DEVD-fmk, or the antioxidant agent NAC when effector cells from B6, gzmA/, or gzmB/ mice were used emphasizes the multitude of gzmA- and gzmB-facilitated effector pathways elicited by CTL. Moreover, the data reiterate the notion that gzmB-induced cell death similarly occurs via caspase-dependent and -independent intracellular processes, whereas all gzmA-induced cell death events are caspase independent and also occur in the absence of ROS generation. This enormous versatility of CTL probably evolved in response to the multiple strategies of pathogens and tumors to evade immune-mediated apoptosis (Benedict et al., 2002; Igney and Krammer, 2002; Trapani and Smyth, 2002).
In conclusion, the present study is the first account on gzmA- and/or gzmB-initiated intracellular processes during CTL-target cell interaction, a model of which is schematically represented in Fig. 7. Although the biological significance of the multiple overlapping and nonoverlapping proapoptotic pathways observed with ex vivoderived CTLs reported herein for their in vivo performance is still elusive, this type of in vitro cytotoxic assay should help to provide a physiologically relevant perspective for gzm-mediated control of infections and cancer.
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Materials and methods |
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Generation of ex-vivo CD8+ cells
Mice were infected with 105 pfu LCMV-WE i.p. according to established protocols (Balkow et al., 2001). On day eight after infection, CD8+ cells were positively selected from spleen using -CD8-MicroBeads (Miltenyi Biotec) with an autoMACS (Miltenyi Biotec) and resuspended in MEM/2 mg/ml BSA before use in cytotoxic assays. Purity of selected CD8+ cells was assessed by FACS staining and found to be between 9598%.
Target cells
The mouse tumor lines EL4.F15 and L1210.Fas were used as target cells (Pardo et al., 2002). Cell lines were cultured in MEM supplemented with 10% heat-inactivated FCS and 2-mercaptoethanol (105 M) at 7% CO2, as described previously (Pardo et al., 2002).
CMLs
The DNA release assay and the target cell survival assay was performed as described previously (Müllbacher et al., 1999). For LCMV-immune CTL-mediated lysis, target cells were pretreated with the synthetic peptide KAVYNTATC (gp33) as described previously (Balkow et al., 2001).
Online supplemental material
For analysis of intracellular apoptotic processes in target cells, effector and target cells were incubated at a ratio of 10:1 (effector/target) and aliquots of CML cultures (105 targets/experimental point) were stained with anti-CD8 mAb. CD8-negative targets were subsequently analyzed for proapoptotic processes and cell death, including plasma membrane disintegration, mitochondrial perturbation, caspase activation, Ape-1 cleavage, and thymidine release/cell survival. Online supplemental material consists of the following methods: 3[H] DNA release and cell survival assays; analysis of inhibitor stability; flow cytometry, including cell surface phenotype analysis, exposure/incorporation of annexin-V/PI, and reduction of mitochondrial membrane potential/generation of ROS (i.e., m/ROS); activation of caspases; and proteolytic degradation of Ape-1. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200406115/DC1.
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Acknowledgments |
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Submitted: 18 June 2004
Accepted: 1 October 2004
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References |
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Alimonti, J.B., L. Shi, P.K. Baijal, and A.H. Greenberg. 2001. Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability transition. J. Biol. Chem. 276:69746982.
Amstad, P.A., G. Yu, G.L. Johnson, B.W. Lee, S. Dhawan, and D.J. Phelps. 2001. Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques. 31:608610, 612, 614, passim.[Medline]
Balkow, S., A. Kersten, T.T. Tran, T. Stehle, P. Grosse, C. Museteanu, O. Utermohlen, H. Pircher, F. von Weizsacker, R. Wallich, et al. 2001. Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection. J. Virol. 75:87818791.
Barry, M., J.A. Heibein, M.J. Pinkoski, S.F. Lee, R.W. Moyer, D.R. Green, and R.C. Bleackley. 2000. Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol. Cell. Biol. 20:37813794.
Benedict, C.A., P.S. Norris, and C.F. Ware. 2002. To kill or be killed: viral evasion of apoptosis. Nat. Immunol. 3:10131018.[CrossRef][Medline]
Bennett, R.A., D.M. Wilson III, D. Wong, and B. Demple. 1997. Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway. Proc. Natl. Acad. Sci. USA. 94:71667169.
Beresford, P.J., Z. Xia, A.H. Greenberg, and J. Lieberman. 1999. Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity. 10:585594.[Medline]
Biron, C.A. 1994. Cytokines in the generation of immune responses to, and resolution of, virus infection. Curr. Opin. Immunol. 6:530538.[CrossRef][Medline]
Boatright, K.M., M. Renatus, F.L. Scott, S. Sperandio, H. Shin, I.M. Pedersen, J.E. Ricci, W.A. Edris, D.P. Sutherlin, D.R. Green, and G.S. Salvesen. 2003. A unified model for apical caspase activation. Mol. Cell. 11:529541.[Medline]
Boehm, U., T. Klamp, M. Groot, and J.C. Howard. 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15:749795.[CrossRef][Medline]
Browne, K.A., R.W. Johnstone, D.A. Jans, and J.A. Trapani. 2000. Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis. J. Biol. Chem. 275:3926239266.
Danial, N.N., and S.J. Korsmeyer. 2004. Cell death: critical control points. Cell. 116:205219.[CrossRef][Medline]
Demaurex, N., and C. Distelhorst. 2003. Apoptosis: The calcium connection. Science. 300:6567.
Dressel, R., S.M. Raja, S. Honing, T. Seidler, C.J. Froelich, K. von Figura, and E. Gunther. 2004. Granzyme-mediated cytotoxicity does not involve the mannose 6-phosphate receptors on target cells. J. Biol. Chem. 279:2020020210.
Evans, A.R., M. Limp-Foster, and M.R. Kelley. 2000. Going APE over ref-1. Mutat. Res. 461:83108.[Medline]
Fan, Z., P.J. Beresford, D. Zhang, and J. Lieberman. 2002. HMG2 interacts with the nucleosome assembly protein SET and is a target of the cytotoxic T-lymphocyte protease granzyme A. Mol. Cell. Biol. 22:28102820.
Fan, Z., P.J. Beresford, D.Y. Oh, D. Zhang, and J. Lieberman. 2003a. Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell. 112:659672.[Medline]
Fan, Z., P.J. Beresford, D. Zhang, Z. Xu, C.D. Novina, A. Yoshida, Y. Pommier, and J. Lieberman. 2003b. Cleaving the oxidative repair protein Ape1 enhances cell death mediated by granzyme A. Nat. Immunol. 4:145153.[CrossRef][Medline]
Froelich, C.J., K. Orth, J. Turbov, P. Seth, R. Gottlieb, B. Babior, G.M. Shah, R.C. Bleackley, V.M. Dixit, and W. Hanna. 1996. New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. J. Biol. Chem. 271:2907329079.
Goping, I.S., M. Barry, P. Liston, T. Sawchuk, G. Constantinescu, K.M. Michalak, I. Shostak, D.L. Roberts, A.M. Hunter, R. Korneluk, and R.C. Bleackley. 2003. Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity. 18:355365.[Medline]
Gourlay, C.W., L.N. Carpp, P. Timpson, S.J. Winder, and K.R. Ayscough. 2004. A role for the actin cytoskeleton in cell death and aging in yeast. J. Cell Biol. 164:803809.
Grossman, W.J., P.A. Revell, Z.H. Lu, H. Johnson, A.J. Bredemeyer, and T.J. Ley. 2003. The orphan granzymes of humans and mice. Curr. Opin. Immunol. 15:544552.[CrossRef][Medline]
Heibein, J.A., M. Barry, B. Motyka, and R.C. Bleackley. 1999. Granzyme B-induced loss of mitochondrial inner membrane potential (Delta Psi m) and cytochrome c release are caspase independent. J. Immunol. 163:46834693.
Heibein, J.A., I.S. Goping, M. Barry, M.J. Pinkoski, G.C. Shore, D.R. Green, and R.C. Bleackley. 2000. Granzyme Bmediated cytochrome c release is regulated by the Bcl-2 family members bid and Bax. J. Exp. Med. 192:13911402.
Henkart, P.A. 1994. Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity. 1:343346.[Medline]
Hirota, K., M. Matsui, S. Iwata, A. Nishiyama, K. Mori, and J. Yodoi. 1997. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc. Natl. Acad. Sci. USA. 94:36333638.
Igney, F.H., and P.H. Krammer. 2002. Immune escape of tumors: apoptosis resistance and tumor counterattack. J. Leukoc. Biol. 71:907920.
Johnson, H., L. Scorrano, S.J. Korsmeyer, and T.J. Ley. 2003. Cell death induced by granzyme C. Blood. 101:30933101.
Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, and P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science. 265:528530.[Medline]
Kelly, J.M., N.J. Waterhouse, E. Cretney, K.A. Browne, S. Ellis, J.A. Trapani, and M.J. Smyth. 2004. Granzyme M mediates a novel form of perforin-dependent cell death. J. Biol. Chem. 279:2223622242.
Krammer, P.H. 1999. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv. Immunol. 71:163210.[Medline]
Li, P., D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, and X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479489.[Medline]
Lowin, B., M. Hahne, C. Mattmann, and J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature. 370:650652.[CrossRef][Medline]
MacDonald, G., L. Shi, C. Vande Velde, J. Lieberman, and A.H. Greenberg. 1999. Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis. J. Exp. Med. 189:131144.
Metkar, S.S., B. Wang, M.L. Ebbs, J.H. Kim, Y.J. Lee, S.M. Raja, and C.J. Froelich. 2003. Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis. J. Cell Biol. 160:875885.
Motyka, B., G. Korbutt, M.J. Pinkoski, J.A. Heibein, A. Caputo, M. Hobman, M. Barry, I. Shostak, T. Sawchuk, C.F. Holmes, et al. 2000. Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell. 103:491500.[Medline]
Müllbacher, A., P. Waring, R.T. Hia, T. Tran, S. Chin, T. Stehle, C. Museteanu, and M.M. Simon. 1999. Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes. Proc. Natl. Acad. Sci. USA. 96:1395013955.
Nagata, S., and P. Golstein. 1995. The Fas death factor. Science. 267:14491456.[Medline]
Nakajima, H., H.L. Park, and P.A. Henkart. 1995. Synergistic roles of granzymes A and B in mediating target cell death by rat basophilic leukemia mast cell tumors also expressing cytolysin/perforin. J. Exp. Med. 181:10371046.[Abstract]
Newmeyer, D.D., and S. Ferguson-Miller. 2003. Mitochondria: releasing power for life and unleashing the machineries of death. Cell. 112:481490.[Medline]
Orrenius, S., B. Zhivotovsky, and P. Nicotera. 2003. Regulation of cell death: the calcium-apoptosis link. Nat. Rev. Mol. Cell Biol. 4:552565.[CrossRef][Medline]
Pardo, J., S. Balkow, A. Anel, and M.M. Simon. 2002. The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells. Eur. J. Immunol. 32:19801985.[CrossRef][Medline]
Pham, C.T., D.M. MacIvor, B.A. Hug, J.W. Heusel, and T.J. Ley. 1996. Long-range disruption of gene expression by a selectable marker cassette. Proc. Natl. Acad. Sci. USA. 93:1309013095.
Pinkoski, M.J., N.J. Waterhouse, J.A. Heibein, B.B. Wolf, T. Kuwana, J.C. Goldstein, D.D. Newmeyer, R.C. Bleackley, and D.R. Green. 2001. Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway. J. Biol. Chem. 276:1206012067.
Podack, E.R. 1986. Molecular mechanisms of cytolysis by complement and by cytolytic lymphocytes. J. Cell. Biochem. 30:133170.[CrossRef][Medline]
Rouvier, E., M.F. Luciani, and P. Golstein. 1993. Fas involvement in Ca2+-independent T cellmediated cytotoxicity. J. Exp. Med. 177:195200.[Abstract]
Sarin, A., E.K. Haddad, and P.A. Henkart. 1998. Caspase dependence of target cell damage induced by cytotoxic lymphocytes. J. Immunol. 161:28102816.
Savill, J., and V. Fadok. 2000. Corpse clearance defines the meaning of cell death. Nature. 407:784788.[CrossRef][Medline]
Sharif-Askari, E., A. Alam, E. Rhéaume, P.J. Beresford, C. Scotto, K. Sharma, D. Lee, W.E. DeWolf, M.E. Nuttall, J. Lieberman, and R.P. Sékaly. 2001. Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation. EMBO J. 20:31013113.
Shresta, S., P. Goda, R. Wesselschmidt, and T.J. Ley. 1997. Residual cytotoxicity and granzyme K expression in granzyme A-deficient cytotoxic lymphocytes. J. Biol. Chem. 272:2023620244.
Shresta, S., T.A. Graubert, D.A. Thomas, S.Z. Raptis, and T.J. Ley. 1999. Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity. 10:595605.[Medline]
Simon, M.M., and M.D. Kramer. 1994. Granzyme A. Methods Enzymol. 244:6879.[Medline]
Simon, M.M., M. Hausmann, T. Tran, K. Ebnet, J. Tschopp, R. ThaHla, and A. Mullbacher. 1997. In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells. J. Exp. Med. 186:17811786.
Stinchcombe, J.C., G. Bossi, S. Booth, and G.M. Griffiths. 2001. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity. 15:751761.[CrossRef][Medline]
Sutton, V.R., J.E. Davis, M. Cancilla, R.W. Johnstone, A.A. Ruefli, K. Sedelies, K.A. Browne, and J.A. Trapani. 2000. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme Bmediated caspase activation. J. Exp. Med. 192:14031414.
Sutton, V.R., M.E. Wowk, M. Cancilla, and J.A. Trapani. 2003. Caspase activation by granzyme B is indirect, and caspase autoprocessing requires the release of proapoptotic mitochondrial factors. Immunity. 18:319329.[CrossRef][Medline]
Thomas, D.A., C. Du, M. Xu, X. Wang, and T.J. Ley. 2000. DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity. 12:621632.[Medline]
Trapani, J.A., and M.J. Smyth. 2002. Functional significance of the perforin/granzyme cell death pathway. Nat. Rev. Immunol. 2:735747.[CrossRef][Medline]
Trapani, J.A., D.A. Jans, P.J. Jans, M.J. Smyth, K.A. Browne, and V.R. Sutton. 1998. Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspase-independent. J. Biol. Chem. 273:2793427938.
Trapani, J.A., V.R. Sutton, K.Y. Thia, Y.Q. Li, C.J. Froelich, D.A. Jans, M.S. Sandrin, and K.A. Browne. 2003. A clathrin/dynamin- and mannose-6-phosphate receptorindependent pathway for granzyme Binduced cell death. J. Cell Biol. 160:223233.
Tschopp, J. 1994. Granzyme B. Methods Enzymol. 244:8087.[Medline]
Vassalli, P. 1992. The pathophysiology of tumor necrosis factors. Annu. Rev. Immunol. 10:411452.[CrossRef][Medline]
Waring, P., D. Lambert, A. Sjaarda, A. Hurne, and J. Beaver. 1999. Increased cell surface exposure of phosphatidylserine on propidium iodide negative thymocytes undergoing death by necrosis. Cell Death Differ. 6:624637.[CrossRef][Medline]
Zhang, D., P.J. Beresford, A.H. Greenberg, and J. Lieberman. 2001a. Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis. Proc. Natl. Acad. Sci. USA. 98:57465751.
Zhang, D., M.S. Pasternack, P.J. Beresford, L. Wagner, A.H. Greenberg, and J. Lieberman. 2001b. Induction of rapid histone degradation by the cytotoxic T lymphocyte protease Granzyme A. J. Biol. Chem. 276:36833690.