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Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo
Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo Joseph A Trapani, Joanne Davis, Vivien R Sutton and Mark J Smyth Recent advances in our understanding of cytolytic effector mechanisms include the partial characterization of caspaseindependent apoptotic pathways triggered by granzymes, a realization of the vital importance of perforin and granzymes in the defence against certain virus infections in vivo and the first description of hereditary immunodeficiency due to disordered perforin expression in humans. Addresses The Research Division, Peter MacCallum Cancer Institute, St. Andrew’s Place, East Melbourne 3002, Australia; and The John Connell Laboratory, Austin Research Institute, Studley Road, Heidelberg 3084, Australia Correspondence: Joseph A Trapani; e-mail: [email protected] Current Opinion in Immunology 2000, 12:323–329 0952-7915/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CTL cytotoxic T lymphocyte FasL Fas ligand IFN-γ interferon-γ LCMV lymphocytic choriomeningitis virus
Introduction This review describes recent advances in our understanding of the molecular and cellular pathways underpinning the cytotoxic effector function of cytotoxic T lymphocytes (CTLs) and NK cells, with particular emphasis on granulemediated cell death. That this form of ‘imposed’ cell death is central to our defences against viruses and intracellular bacteria had long been suspected and was proven definitively by the suppressed cellular immunity observed in perforin-deficient mice [1]. The principal molecular players involved in this death process, perforin (ostensibly a pore-forming protein) and the granzymes (a family of serine proteases), have been identified for well over a decade [2]. Nevertheless, assisted by the recent revolution in our understanding of generic cell death pathways, we are now coming to grips with the remarkable complexity and redundancy of the death pathways triggered when a target cell is exposed to a quantal barrage of granule-bound toxins. The imparted death stimulus is remarkably powerful and swift compared with apoptosis in response to toxins, irradiation or factor withdrawl: signs of target cell damage often appear within one or two minutes of the attack. This is because lymphocyte-mediated death involves a multifaceted assault on the target cell that is designed to limit the spread of an infectious agent. By absolute necessity, such death strategies have to account for viral defences against apoptotic death that have long evolved with and adapted to the immune system of higher organisms. Recent insights into the molecular means by which perforin and the granzymes interact to bring about target cell
death are discussed first below, setting the scene for a broader consideration of the pathophysiological significance of granule-mediated cytolysis in vivo and the indispensable role played by perforin in that process. Although the evidence for the involvement of perforindependent pathways in defence against viruses is overwhelming, it cannot be overlooked that direct killing of infected cells represents only one facet of an integrated immune response to intracellular pathogens. A cognate antiviral response takes some days to reach its peak and, in the interim, innate responses are of central importance — especially for containing cytopathic pathogens [3]. The importance of secretion of the key cytokines interferon-γ (IFN-γ) and TNF-α by NK cells is now becoming plain. A major emerging theme is that the balance between innate and adaptive responses is of paramount importance for maintaining lymphoid and more general homeostasis and that the pathophysiological outcomes of viral infection are influenced profoundly by both types of response. The power of perforin- and cytokine-gene-knockout mice is now becoming apparent in providing insights into these problems; meanwhile mice with defects in death receptor mediated killing (especially gld and lpr strains, representing deficiencies of Fas ligand [FasL] and its receptor, respectively) can also permit the influence of the alternative death pathway of cytotoxic lymphocytes to be gauged. Attempts to determine the importance of granzymes in vivo have been less fruitful in the short term but interesting findings are also finally emerging, particularly in mice deficient in more than one granzyme. Although both granzyme A deficient [4] and granzyme B deficient [5] mice have been available for some years, evidence has only recently emerged for an absolute requirement for either or both in the defence against certain specific pathogens.
The molecular role of perforin in apoptosis induction: where are the pores? The initial hypothesis of perforin’s molecular function was founded on the observation that polymerized perforin could form transmembrane channels in vitro and lead to osmotic lysis [6]. However, perhaps with the exception of one report [7], perforin ring lesions have been difficult to demonstrate in vivo. It would also seem undesirable to lyse cells that might be loaded with viable virus particles, particularly if high titer neutralizing antibodies had not yet developed in the host. Perforin is essential for the induction of apoptotic death in response to granzymes [8,9] but — although perforin membrane pores could in principle also account for granzyme B accessing intracellular substrates such as caspases — several groups have shown that granzyme entry can be mediated through receptordependent endocytosis and does not require perforin
Figure 1 Granzyme B
Target cell plasma membrane
R
Sequestered granzyme B Perforin Unsequestered granzyme B
R
Caspase activation
Nuclear damage
Nonnuclear damage Current Opinion in Immunology
A model to explain the combined actions of perforin and granzyme B in inducing apoptotic death of the target cell. Granzyme B released from an effector lymphocyte enters a target cell by receptor (R)-mediated endocytosis. The role of perforin is to induce the release of this sequestered granzyme B into the cytosol where it is free to cleave its substrates, including a proapoptotic cascade of caspases that also normally mediate programmed cell death in response to a variety of apoptotic stimuli. Nuclear damage in response to granzyme B is largely dependent on caspase activation; however cell death can be triggered directly by granzyme B independently of caspase activation, principally through non-nuclear means.
cannot account for the effects of other granule constituents; these may have proapoptotic or antimicrobial activity in their own right, depending on the type of intracellular pathogen encountered [2,14••]. It has been pointed out that granzyme B, which bears a strongly positive electrostatic charge at neutral pH, may normally be co-delivered as part of a high molecular weight complex in tight association with the proteoglycan serglycin and possibly other similarly charged granzymes [15•]. Neither can it be assumed that perforin is always (if, indeed, ever) delivered in sublytic quantities. The situation is further complicated in that most cells respond vigorously to osmotic stress by repairing membrane lesions through endocytosis or membrane budding, a fact that might be exploited by a CTL in order to ‘load’ the target with even more granule toxins. Although no-one has yet estimated the quantity of perforin delivered onto the surface of a target cell in the context of a bona fide conjugate, cells treated with perforin concentrations that permit near maximal release of 51Cr in vitro can still exclude proteins much smaller than granzymes for as long as 90 minutes, while facilitating apoptosis through granzyme B [13••]. Given that granzymes may be taken up as part of a macromolecular complex [15•], we believe that endosomal disruption may often supplant the need for transmembrane pore formation in order for proapoptotic factors to access their ligands in the target cell.
Caspase-independent cell death mediated by granzymes
[10,11,12•]. Addition of minute (‘sublytic’) perforin well after granzyme B can reconstitute the death signal ([11]; JA Trapani, unpublished data), strongly arguing that perforin may somehow permit granzymes to be liberated from endosomes to access death substrates (Figure 1). The ability of certain endosomolytic agents, such as adenovirus [11] and purified listeriolysin [13••], to substitute for sublytic perforin and the inhibition of perforin’s proapoptotic function by agents that dislocate vesicular trafficking (e.g. brefeldin A) [13••] support the feasibility of this hypothesis; however direct evidence for the mechanism is still lacking.
Having gained access to the cell’s interior, granzymes recruit ‘every available means’ of rapidly killing that cell. We strongly contend that in addition to powerfully activating generic cell death through caspases, granzymes must be capable of killing cells in which viral apoptotic inhibitors have stalled the intrinsic death program [16]. The first indication of these additional pathways was provided by Sarin et al. [17], who demonstrated that cells in which caspases were inactivated either by chemical inhibitors or by the baculovirus inhibitor p35 were still efficiently killed by CTLs acting in a perforin-dependent manner [17]. Surprisingly, it was next shown that inhibiting caspases markedly diminished nuclear damage in response to granzyme B but cell death still occurred due to direct granzyme-mediated damage to the remainder of the cell [18••,19•] (see Figure 1). This finding had significance for our understanding of the death process but it also raised the importance of measuring appropriate parameters of cell death when considering CTL/NK-mediated killing, a theme taken up again below.
What then, is the role for cell membrane perforation in the death process? Much of the recent work on the molecular aspects of granule-mediated death have utilized a ‘minimalist’ reconstituted cell free system incorporating only sublytic perforin and granzyme B, as first described by Shi et al. [8]. Although this model has proven valuable in many ways, its limitations need to be recognized. The model
The non-caspase targets of granzymes that can enforce cell death are now starting to be elucidated. The ability of granzyme B to mimic the Asp-ase activity of caspases (particularly effector caspases) allows it to cleave some downstream caspase targets, as pointed out recently by Andrade et al. [20•]. An alternative means of inducing single stranded breaks in DNA in response to granzyme A, which
was unimpaired by caspase blockade, was also recently identified in cells loaded with recombinant granzyme A [21••]. As granzymes have no direct nuclease activity, this pathway must involve activation of an unidentified nuclease. Although damage to nuclear structures no doubt contributes to dismantling a cell, the long known observation that cells lacking a nucleus can still die an apoptotic death in response to cytotoxic lymphocytes [22] suggests to us that truly essential granzyme substrates will be found to reside outside the nucleus. One focus of recent interest has been the mitochondrion. Exogenously added mitochondria greatly amplify granzyme-B-mediated cell death in a cell free system although the authors of one report [23] felt that nonmitochondrial pathways were also contributing to apoptosis and that caspase-3 activation could occur upstream of mitochondrial damage. Consistent with our own findings (J Davis, VR Sutton, JA Trapani, unpublished data), Helbein et al. [24•] recently showed that mitochondrial damage in response to granzyme B occurs without a requirement for caspase activation. The importance of the role played by mitochondria in granzyme-mediated death has come under scrutiny, as has the regulation of this pathway by pro-survival molecules such as Bcl-2 that operate by stabilising mitochondria. We have recently observed that overexpression of Bcl-2-like viral proteins such as BHRF1 of Epstein–Barr virus rigorously controls death mediated by granzyme B [25] and prevents both intracellular granzyme trafficking and accelerated granzyme uptake in response to perforin [26]. Furthermore this regulation is lost when the inhibitor is targeted to an alternative subcellular location such as the cell membrane (J Davis, JA Trapani, unpublished data), pointing to the central importance of a mitochondrial pathway activated by granzyme B. As normal endogenous levels of Bcl-2 cannot block this cell death, it follows that granzyme B must either inactivate Bcl-2 directly or alternatively activate a Bcl-2 antagonist, perhaps a proapoptotic Bcl-2 family member. We believe that the next few years will see a proliferation of granzyme ligands and substrates identified through a variety of biochemical and genetic approaches. We predict that these substrates will be cleaved exclusively by granzymes, or at least far more efficiently by granzymes than caspases. After many years of frustration, several groups have now expressed and purified proteolytically active recombinant granzymes A, B and H in bacteria, baculovirus and yeast; the proteolytic specificity of granzyme H, a chymotrypsin-like enzyme, has also been assigned [27•]. These developments should greatly assist researchers to define granzyme functions at the molecular level and the search for new apoptotic substrates in the cytosol and the cytoskeleton should be greatly facilitated. Insights into burning issues such as the nature of granzyme receptors on cells, and how granzymes traffic within target cells to gain access to their substrates, may open up new ways of regulating CTL and NK cell function.
New insights into granzyme functions in vivo Although it is indisputable that perforin deficiency is associated with increased mortality of mice from a number of natural pathogens, evidence of the importance of granzymes in vivo has been slower to become apparent. Much of the important progress in this area has come from the joint work of Mullbacher, Simon and co-workers [28,29••,30,31•]. Following on from previous observations that mice deficient in granzyme A are abnormally susceptible to the poxvirus ectromelia [28], these investigators have found that mice lacking both granzymes A and B are virtually as susceptible to primary ectromelia infection as perforin-deficient mice [29••]. This was despite the absence of any intrinsic inability of these mice to mount a CTL response to other pathogens [29••] or any additional susceptibility to related poxviruses [30]. A further important finding of this group was that serpins elaborated by this family of viruses are far less efficient at blocking the granule-mediated, perforin-dependent cell death imparted by MHC-restricted antiviral CTLs than the purely caspase-dependent death following Fas ligation [31•]. These findings both reiterate the relative importance of the granule pathway in defence against viruses in vivo and point to the lack of dependence by granzyme pathways solely on their capacity to activate caspases [16]. Further evidence of the requirement for granzymes for the normal cytolytic function of CTLs was gleaned from the studies of Shresta et al. [32•], who showed that the lethality of graft-versus-host disease imparted by transferred CD8+ alloreactive T cells was significantly reduced in the absence of both granzymes A and B. Granzyme deficiency states have not been identified in humans; however the identification of high levels of circulating soluble granzymes A and B in patients with active rheumatoid arthritis [33] may point to the active participation of CTLs in the pathogenesis of the inflammatory arthritides, providing another way of monitoring disease activity.
Granzyme regulation by endogenous serpins Pathways regulating granzyme function are also now under close scrutiny, as investigators search for ways of manipulating the cytotoxic response in vivo. It has recently become apparent that CTLs and NK cells express high levels of an intracellular endogenous serpin designated PI-9, which is capable of efficiently complexing and irreversibly inactivating granzyme B [34]. PI-9 is absent from cytolytic granules but is thought to be present in the cytosol to protect effector cells from granzyme molecules that undergo incorrect trafficking during their synthesis, processing, storage or exocytic release. An important feature of the inhibitory loop of PI-9 is the P1 Glu (not Asp) residue essential for binding and inhibiting granzyme B. Substitution of Asp into the P1 position of recombinant PI-9 resulted in a far reduced ability to interact with granzyme B and, surprisingly, the acquisition of inhibitory function to a variety of caspases [34]. Expression
Table 1 Death of Jurkat cells following exposure to mouse lymphokine-activated killer lymphocytes operating through the granule or FasL pathways, as measured in parallel by various parameters. Effector population* Perforin-deficient gld (FasL-deficient) None
Colony count† +VAD +FA 105+/–4 38+/–5 98+/–3
28+/–5 23+/–2 99+/–7
% specific 51Cr release‡ +VAD +FA 3+/–1 60+/–3 0
24+/–2 51+/–1 0
% specific 125I-DNA release§ +VAD +FA 2+/–2 12+/–7 0
48+/–6 39+/–14 0
*Effector cells were mouse splenocytes pre-incubated in vitro in medium supplemented with 100 units/ml IL-2 for four days (lymphokine-activated killer [LAK] cells). The effector : target cell ratio in the current experiment was 25:1. Prior to exposure to activated splenocytes, Jurkat cells were pre-incubated for 30 minutes in medium containing either z-VAD-fmk or z-FA-fmk (50 µM). † Clonogenic survival of Jurkat cells plated in soft agar, estimated by colony counting after seven days. LAK cells alone yielded no colonies.
All values shown are the mean of triplicate assays +/– standard error of the mean. Each fmk derivative was also included in the four hour cytotoxicity assays shown. Standard four hour standard cytotoxicity assays were performed in round-bottom 96-well plates. Values stated for the release of 51Cr‡ and 125I-DNA§ are relative to spontaneous release, measured in the absence of effector cells. For 51Cr, spontaneous release was <6% of total release; for 125I-DNA, spontaneous release was 10—30% of total release.
of this PI-9 mutant in Jurkat cells resulted in resistance of the transfectant cells to death following Fas ligation [34]. Thus the presence of Glu in the PI-9 inhibitory loop provides for specific protection against granzyme B, without unduly interfering with eventual clearing of the cytolytic cell mediated through the Fas pathway. We note that the inhibitory loop of PI-9 bears striking resemblance to that of viral serpins such as crmA and speculate that the gene encoding PI-9 may have been ‘hijacked’ by a virus, with subsequent mutation of the P1 Glu to Asp resulting in its use to inhibit generic caspase-mediated cell death. Another salutary lesson is that an optimal cleavage motif does not necessarily form the ideal core of an inhibitory oligopeptide for that protease.
through the granule pathway) was totally resistant to z-VAD-fmk and correlated well with reduced clonogenic survival of the treated cells. Notably, 125I-DNA release was markedly inhibited by z-VAD-fmk pretreatment. As several groups have shown that cell membrane damage as measured by 51Cr release occurs independently of caspases, we favor the use of this assay over estimates of nuclear damage if short term assays are to be relied on.
On measuring death imparted by CTLs and NK cells or granule components As alluded to above, cell death in response to granule components may occur in the absence of gross nuclear damage. This places a special onus on investigators to interpret data arising from cytotoxicity assays with care, especially when caspases are not optimally activated. Purely nuclear readouts such as DNA end-labelling or 125I-DNA release assays can be particularly misleading, as depicted in the experiment shown in Table 1 (J Davis, unpublished data). As all short-term assays are merely surrogate measures of cell death or survival, our group is increasingly turning to clonogenic survival assays to determine true life/death outcomes. In our experiment, the ‘death’ of Jurkat cells was measured in various ways following four-hour exposure to intact mouse lymphokine-activated killer cells. Target cells were also incubated before and throughout the assay with the caspase inhibitor z-VAD-fmk or with the control inhibitor zFA-fmk, which does not block caspases. Perforin-deficient effector cells (operating through Fas ligation) caused specific 51Cr and 125I-DNA release, which measure cell membrane damage and nuclear damage, respectively. Both parameters were inhibitable by z-VAD-fmk, consistent with the total reliance on caspases of this pathway. In contrast, the 51Cr release in response to gld effectors (operating
The respective roles of perforin and IFN-γ secretion in defence against viruses in vivo: an immunoregulatory role for perforin There is now no doubt that direct cytotoxicity mediated by lymphocyte perforin is essential for the control of both non-cytopathic [1,35] and cytopathic viruses [30]. More recently, it has become apparent that a critical balance exists between direct cytotoxicity mediated by perforin and IFN-γ secretion, and that this balance controls the outcome of viral infection and dictates immune homeostasis. Augmented perforin-mediated immunopathology is observed in IFN-γ-deficient mice infected with lymphocytic choriomeningitis virus (LCMV) [36••]. Conversely, an overproduction of inflammatory cytokines is observed in perforin-deficient mice infected with LCMV. Furthermore a recent study suggests that T cells can turn off their cytokine production while retaining intracellular stores of perforin, thereby potentially maintaining effective immune surveillance while minimizing systemic immunopathology [37••]. In addition to the description of perforin-deficient gld mice [38•], further evidence supports a pivotal role played by perforin in the regulation of immune responses. Several studies have recently suggested that perforin-dependent cytotoxicity regulates the elimination of CD8+ T cells following an acute exposure to foreign antigen. Notably, perforin-deficient mice showed an increased expansion and persistence of superantigen- and virus-specific T cells that was not explained by a failure to deplete antigen-presenting cells in wild-type mice [39•]. Similarly, an increased
expansion of alloreactive perforin-deficient T cells following transfer into irradiated scid/scid mice (these lack T and B cells) further supported a role for perforin in activationinduced cell death [40•]. Following on from previous studies by Binder et al. [41•], Matloubian et al. [42•] demonstrated that perforin was involved in downregulating T cell responses during chronic LCMV infection. An excessive accumulation of CD8+ T cells resulting in immune-mediated damage in perforin-deficient mice mimics the clinical picture in perforin-deficient children suffering familial hemophagocytic lymphohistiocytosis (FHL) [43••]. Although a causative infectious agent for this infantile disease has not yet been defined, it is likely to be a microbe whose control is strictly perforin-dependent. Perforin-based effector pathways appear to be involved in the death of abnormal cells that accumulate in FHL; it is these cells and the cytokines they produce (TNF-α, IFN-γ) that are ultimately lethal.
A role for perforin in immune surveillance against cancer We feel that the ability of perforin-secreting killer cells to eliminate transformed cells in vivo has been overlooked as researchers have emphasized perforin’s importance in T cell homeostasis. Recent studies in our laboratory (J Davis, VR Sutton, JA Trapani, unpublished data) have indicated that perforin-deficient mice are more susceptible to spontaneous lymphoma; malignancy in the lymphoid compartment is particularly consistent with dysregulated lymphocyte activation. Previous experiments with murine-leukaemia-virusinduced lymphomas have also indicated an important role for perforin [44]. It remains to be assessed whether perforinmediated immune surveillance controls nonlymphoid malignancies; however past studies would suggest that at least some chemically induced sarcomas are controlled in a perforin-dependent manner [45•]. Furthermore the natural [45•] and IL-12-induced [46] antimetastatic activities of NK cells against a variety of nonlymphoid tumors including melanomas, and prostate and breast carcinomas have been shown to be perforin-mediated. In each of these tumor models, FasL mutant mice demonstrated normal protection from tumor growth and metastasis. Nonetheless these models strictly involved innate immune responses and two recent experimental studies have suggested that Fas [47] or alternative pathways [48] may also contribute to T-celldependent tumor regression. Strikingly, malignant transformation of keratinocytes appears to be regulated locally by FasL–Fas interactions [49••]; however immune surveillance by unknown mechanisms may still possibly protect these nonlymphoid tissues. We therefore postulate that rapid and direct perforin-mediated cytotoxicity of microbes or the cells they infect prevents prolonged activation of the lymphoid compartment. Perforin is also responsible, at least in part, for the elimination of activated CD8+ T cells following such an immune response. A potential by-product of aberrant and
sustained proliferation of hematopoietic tissue is the increased propensity to oncogenic transformation; however perforin-expressing killer lymphocytes can also eliminate these transformed cells through their role in providing immune surveillance. Since perforin clearly evolved to control microbial challenge, it will be fascinating to examine the importance of perforin-mediated cytotoxicity in the surveillance of cancers of viral and non-viral aetiology.
Conclusions The past decade has seen a revolution in our understanding of the molecular pathways underpinning the death signal imparted by cytotoxic lymphocytes. Although some of these findings might have been predicted by like findings made in other types of apoptotic death, it is clear that perforin-dependent, granule-mediated apoptosis represents a specialised and complex death process with many unusual features. The apoptotic attack by cytolytic lymphocytes is highly efficient, very rapid and multilayered at the molecular level, without absolute reliance on caspases — normally the enzymatic 'engine-room' of generic apoptosis. Beyond cursory consideration, this is hardly surprising — the killer lymphocyte must be capable of destroying cells in which the proapoptotic program has been corrupted by a virus or by coincident cellular transformation. Death imposed on such a recalcitrant cell thus differs appreciably from cell suicide or programmed death initiated and accomplished intrinsically by the dying cell itself. There are many questions that remain regarding apoptotic pathways induced by cytotoxic granules. The precise function of perforin, an indispensable keystone in the whole process, remains unclear and a comprehensive structure/function analysis of this molecule is sadly lacking. The nature of granzyme receptors and their uptake into normal cells remain unclear. Although a few granzyme substrates (other than caspases) have also been identified, those that deliver caspase-independent death remain elusive. Elucidating such pathways may provide us with novel ways to kill malignant or parastised cells that have lost their ability to undergo programmed cell death.
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Sarin A, Williams MS, Alexander-Miller MA, Berzofsky JA, Zacharchuk CM, Henkart PA: Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 1997, 6:209-215.
18. Sarin A, Haddad EK, Henkart PA: Caspase dependence of target •• cell damage induced by cytotoxic lymphocytes. J Immunol 1998, 161:2810-2816. The first paper to point out that nuclear damage, particularly DNA release, inflicted by cytotoxic lymphocytes requires caspase activation whereas nonnuclear parameters of cell death are largely independent of caspases. 19. Trapani JA, Jans DA, Browne KA, Smyth MJ, Jans PJ, Sutton VR: • Efficient nuclear targeting of granzyme B, the nuclear consequences of apoptosis induced by granzyme B and perforin
are caspase-dependent, but cell death is caspase-independent. J Biol Chem 1998, 273:27934-27938. This paper first localised the essential proapoptotic effect of granzyme B as residing outside the nucleus. Cell death in response to granzyme B and perforin occurs irrespective of the apparent inhibition of nuclear damage by caspase inhibitors. 20. Andrade F, Roy S, Thornberry N, Rosen A, Casciola-Rosen L: • Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity 1998, 8:451-460. This elegant and comprehensive paper shows that downstream caspase substrates can be targeted directly by granzyme B when caspases are inhibited. 21. Beresford PJ, Xia Z, Greenberg AH, Lieberman J: Granzyme A •• induces rapid cytolysis, a novel form of DNA damage independently of caspases. Immunity 1999, 10:585-594. Granzyme A loading into target cells was used to demonstrate for the first time that granzyme A can induce single-stranded DNA breaks in a caspaseindependent manner. The significance of this finding is yet to be reported when using granzyme A deficient effector cells. 22. Nakajima H, Golstein P, Henkart PA: The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity. J Exp Med 1995, 181:1905-1909. 23. MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg A: Mitochondria-dependent and -independent regulation of granzyme B-induced apoptosis. J Exp Med 1999, 189:131-144. 24. Helbein JA, Barry M, Motyka B, Bleackley RC: Granzyme B-induced • loss of mitochondrial inner membrane potential (Delta Psi m) and cytochrome c release are caspase independent. J Immunol 1999, 163:4683-4693. This elegant study documented non-nuclear events that occur independently of caspases and that may influence cell survival. 25. Sutton VR, Vaux DA, Trapani JA: Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes. J Immunol 1997, 158:5783-5790. 26. Jans DJ, Sutton VR, Jans PJ, Froelich CJ, Trapani JA: BCL-2 blocks perforin-induced nuclear translocation of granzymes, concomitant with protection against the nuclear events of apoptosis. J Biol Chem 1999, 274:3953-3961. 27. •
Edwards KM, Kam C-M, Powers J, Trapani JA: The human cytotoxic T cell granule protease granzyme H has chymotrypsin-like (chymase) activity, is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes. J Biol Chem 1999, 274:30468-30473. The first characterization of a chymase granzyme in human CTLs. This study utilized recombinant granzyme H expressed in insect Sf9 cells. 28. Mullbacher A, Ebnet K, Blanden RV, Hla RT, Stehle T, Museteanu C, Simon MM: Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia. Proc Natl Acad Sci USA 1996, 93:5783-5787. 29. Mullbacher A, Waring P, Tha Hla R, Tran T, Chin S, Stehle T, •• Museteanu C, Simon MM: Granzymes are essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes. Proc Natl Acad Sci USA 1999, 96:13950-13955. This is the first reported instance of a pathophysiological consequence of a granzyme deficiency. The investigators make the observation that CTLs equipped with normal levels of perforin but lacking granzymes A and B are specifically deficient in protecting the host from a natural mouse pathogen, the poxvirus ectromelia. Despite this, mice lacking granzymes A and B have no intrinsic problem with raising CTL responses. The defective response to ectromelia is virtually as profound as perforin-deficiency per se. 30. Mullbacher A, Hla RT, Museteanu C, Simon MM: Perforin is essential for control of ectromelia virus but not related poxviruses in mice. J Virol 1999, 73:1665-1667. 31. Mullbacher A, Wallich R, Moyer RW, Simon MM: Poxvirus-encoded • serpins do not prevent cytolytic T cell-mediated recovery from primary infections. J Immunol 1999, 162:7315-7321. This elegant in vivo study reinforces the notion that CTLs recruited in the context of most antiviral responses can bypass antiapoptotic mechanisms erected by the pathogen. 32. Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ: Granzyme A • initiates an alternative pathway for granule-mediated apoptosis. Immunity 1999, 10:595-605. + CD8 CTLs mediating graft-versus-host disease inflict less potent tissue damage in the recipient if they are deficient in granzyme expression, especially granzyme A.
33. Tak PP, Spaeny-Dekking L, Kraan MC, Breedveld FC, Froelich CJ, Hack CE: The levels of soluble granzyme A and B are elevated in plasma and synovial fluid of patients with rheumatoid arthritis. Clin Exp Immunol 1999, 116:366-370.
infection with lymphocytic choriomeningitis virus. J Exp Med 1998, 187:1903-1920. Perturbation of the normal immune response to LCMV due to perforin deficiency results in bone marrow hypoplasia secondary to excessive secretion of cytokines.
34. Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong TA, Trapani JA, Bird PI: A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. J Biol Chem 1996, 271:27802-27809.
42. Matloubian M, Suresh M, Glass A, Galvan M, Chow K, Whitmire JK, • Walsh CM, Clark WR, Ahmed R: A role for perforin in downregulating T-cell responses during chronic viral infection. J Virol 1999, 73:2527-2536. This paper demonstrates an accumulation of CD8+ T cells resulting in immune-mediated damage in perforin-deficient mice that are incapable of clearing LCMV infection.
35. Kagi D, Seiler P, Pavlovic J, Ledermann B, Burki K, Zinkernagel RM, Hengartner H: The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses. Eur J Immunol 1995, 25:3256-3262. 36. Nansen A, Jensen T, Christensen JP, Andreasen SO, Ropke C, •• Marker O, Thomsen AR: Compromised virus control and augmented perforin-mediated immunopathology in IFN-gammadeficient mice infected with lymphocytic choriomeningitis virus. J Immunol 1999, 163:6114-6122. Increased immunopathology mediated by perforin is observed when IFN-γdeficient mice are infected with LCMV, indicating for the first time a role for both cytokine secretion and direct cytotoxicity in the control of this pathogen. 37. Slifka MK, Rodriguez F, Whitton JL: Rapid on/off cycling of cytokine •• production by virus-specific CD8+ T cells. Nature 1999, 401:76-79. This elegant and comprehensive paper points out that the balance between direct cytotoxicity and release of inflammatory cytokines can be regulated at the single-cell level. 38. Spielman J, Lee RK, Podack ER: Perforin/Fas-ligand double • deficiency is associated with macrophage expansion and severe pancreatitis. J Immunol 1998, 161:7063-7070. The first paper that pointed out a role for perforin other than in direct cytotoxicity and indicated a role in modulating the immune response. 39. Kagi D, Odermatt B, Mak TW: Homeostatic regulation of CD8+ • T cells by perforin. Eur J Immunol 1999, 29:3262-3272. Perforin-deficient mice show an increased number and persistence of superantigen- and virus-specific T cells: further evidence (with [40•]) of a role for perforin in immune regulation. 40. Spaner D, Raju K, Rabinovich B, Miller RG: A role for perforin in • activation-induced T cell death in vivo: increased expansion of allogeneic perforin-deficient T cells in SCID mice. J Immunol 1999, 162:1192-1199. See annotation to [39•]. 41. Binder D, van den Broek MF, Kagi D, Bluethmann H, Fehr J, • Hengartner H, Zinkernagel RM: Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent
43. Stepp SE, Dufourcq-Lagelouse R, Le Deist F, Bhawan S, Certain S, •• Mathew PA, Henter JI, Bennett M, Fischer A, de Saint Basile G, Kumar V: Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science 1999, 286:1957-1959. The first description of the clinical consequences of perforin deficiency in humans. FHL patients present with a syndrome that is highly reminiscent of perforin-deficient mice chronically infected with a viral pathogen that cannot be cleared, for example LCMV. 44. van den Broek ME, Kagi D, Ossendorp F, Toes R, Vamvakas S, Lutz WK, Melief CJ, Zinkernagel RM, Hengartner H: Decreased tumor surveillance in perforin-deficient mice. J Exp Med 1996, 184:1781-1790. 45. Smyth MJ, Thia KY, Cretney E, Kelly JM, Snook MB, Forbes CA, • Scalzo AA: Perforin is a major contributor to NK cell control of tumor metastasis. J Immunol 1999, 162:6658-6662. The first demonstration that innate immune mechanisms can control tumor metastasis in a perforin-dependent manner. 46. Kodama T, Takeda K, Shimozato O, Hayakawa Y, Atsuta M, Kobayashi K, Ito M, Yagita H, Okumura K: Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12. Eur J Immunol 1999, 29:1390-1396. 47.
Medema JP, de Jong J, van Hall T, Melief CJ, Offringa R: Immune escape of tumors in vivo by expression of cellular FLICEinhibitory protein. J Exp Med 1999, 190:1033-1038.
48. Winter H, Hu HM, Urba WJ, Fox BA: Tumor regression after adoptive transfer of effector T cells is independent of perforin or Fas ligand (APO-1L/CD95L). J Immunol 1999, 163:4462-4472. 49. Hill LL, Ouhtit A, Loughlin SM, Kripke ML, Ananthaswamy HN, Owen •• Schaub LB: Fas ligand: a sensor for DNA damage critical in skin cancer etiology. Science 1999, 285:898-900. Immune surveillance of cellular transformation in the skin involves local expression of FasL and deletion of cells with DNA damage through a Fasdependent mechanism.
Introduction This review describes recent advances in our understanding of the molecular and cellular pathways underpinning the cytotoxic effector function of cytotoxic T lymphocytes (CTLs) and NK cells, with particular emphasis on granulemediated cell death. That this form of ‘imposed’ cell death is central to our defences against viruses and intracellular bacteria had long been suspected and was proven definitively by the suppressed cellular immunity observed in perforin-deficient mice [1]. The principal molecular players involved in this death process, perforin (ostensibly a pore-forming protein) and the granzymes (a family of serine proteases), have been identified for well over a decade [2]. Nevertheless, assisted by the recent revolution in our understanding of generic cell death pathways, we are now coming to grips with the remarkable complexity and redundancy of the death pathways triggered when a target cell is exposed to a quantal barrage of granule-bound toxins. The imparted death stimulus is remarkably powerful and swift compared with apoptosis in response to toxins, irradiation or factor withdrawl: signs of target cell damage often appear within one or two minutes of the attack. This is because lymphocyte-mediated death involves a multifaceted assault on the target cell that is designed to limit the spread of an infectious agent. By absolute necessity, such death strategies have to account for viral defences against apoptotic death that have long evolved with and adapted to the immune system of higher organisms. Recent insights into the molecular means by which perforin and the granzymes interact to bring about target cell
death are discussed first below, setting the scene for a broader consideration of the pathophysiological significance of granule-mediated cytolysis in vivo and the indispensable role played by perforin in that process. Although the evidence for the involvement of perforindependent pathways in defence against viruses is overwhelming, it cannot be overlooked that direct killing of infected cells represents only one facet of an integrated immune response to intracellular pathogens. A cognate antiviral response takes some days to reach its peak and, in the interim, innate responses are of central importance — especially for containing cytopathic pathogens [3]. The importance of secretion of the key cytokines interferon-γ (IFN-γ) and TNF-α by NK cells is now becoming plain. A major emerging theme is that the balance between innate and adaptive responses is of paramount importance for maintaining lymphoid and more general homeostasis and that the pathophysiological outcomes of viral infection are influenced profoundly by both types of response. The power of perforin- and cytokine-gene-knockout mice is now becoming apparent in providing insights into these problems; meanwhile mice with defects in death receptor mediated killing (especially gld and lpr strains, representing deficiencies of Fas ligand [FasL] and its receptor, respectively) can also permit the influence of the alternative death pathway of cytotoxic lymphocytes to be gauged. Attempts to determine the importance of granzymes in vivo have been less fruitful in the short term but interesting findings are also finally emerging, particularly in mice deficient in more than one granzyme. Although both granzyme A deficient [4] and granzyme B deficient [5] mice have been available for some years, evidence has only recently emerged for an absolute requirement for either or both in the defence against certain specific pathogens.
The molecular role of perforin in apoptosis induction: where are the pores? The initial hypothesis of perforin’s molecular function was founded on the observation that polymerized perforin could form transmembrane channels in vitro and lead to osmotic lysis [6]. However, perhaps with the exception of one report [7], perforin ring lesions have been difficult to demonstrate in vivo. It would also seem undesirable to lyse cells that might be loaded with viable virus particles, particularly if high titer neutralizing antibodies had not yet developed in the host. Perforin is essential for the induction of apoptotic death in response to granzymes [8,9] but — although perforin membrane pores could in principle also account for granzyme B accessing intracellular substrates such as caspases — several groups have shown that granzyme entry can be mediated through receptordependent endocytosis and does not require perforin
Figure 1 Granzyme B
Target cell plasma membrane
R
Sequestered granzyme B Perforin Unsequestered granzyme B
R
Caspase activation
Nuclear damage
Nonnuclear damage Current Opinion in Immunology
A model to explain the combined actions of perforin and granzyme B in inducing apoptotic death of the target cell. Granzyme B released from an effector lymphocyte enters a target cell by receptor (R)-mediated endocytosis. The role of perforin is to induce the release of this sequestered granzyme B into the cytosol where it is free to cleave its substrates, including a proapoptotic cascade of caspases that also normally mediate programmed cell death in response to a variety of apoptotic stimuli. Nuclear damage in response to granzyme B is largely dependent on caspase activation; however cell death can be triggered directly by granzyme B independently of caspase activation, principally through non-nuclear means.
cannot account for the effects of other granule constituents; these may have proapoptotic or antimicrobial activity in their own right, depending on the type of intracellular pathogen encountered [2,14••]. It has been pointed out that granzyme B, which bears a strongly positive electrostatic charge at neutral pH, may normally be co-delivered as part of a high molecular weight complex in tight association with the proteoglycan serglycin and possibly other similarly charged granzymes [15•]. Neither can it be assumed that perforin is always (if, indeed, ever) delivered in sublytic quantities. The situation is further complicated in that most cells respond vigorously to osmotic stress by repairing membrane lesions through endocytosis or membrane budding, a fact that might be exploited by a CTL in order to ‘load’ the target with even more granule toxins. Although no-one has yet estimated the quantity of perforin delivered onto the surface of a target cell in the context of a bona fide conjugate, cells treated with perforin concentrations that permit near maximal release of 51Cr in vitro can still exclude proteins much smaller than granzymes for as long as 90 minutes, while facilitating apoptosis through granzyme B [13••]. Given that granzymes may be taken up as part of a macromolecular complex [15•], we believe that endosomal disruption may often supplant the need for transmembrane pore formation in order for proapoptotic factors to access their ligands in the target cell.
Caspase-independent cell death mediated by granzymes
[10,11,12•]. Addition of minute (‘sublytic’) perforin well after granzyme B can reconstitute the death signal ([11]; JA Trapani, unpublished data), strongly arguing that perforin may somehow permit granzymes to be liberated from endosomes to access death substrates (Figure 1). The ability of certain endosomolytic agents, such as adenovirus [11] and purified listeriolysin [13••], to substitute for sublytic perforin and the inhibition of perforin’s proapoptotic function by agents that dislocate vesicular trafficking (e.g. brefeldin A) [13••] support the feasibility of this hypothesis; however direct evidence for the mechanism is still lacking.
Having gained access to the cell’s interior, granzymes recruit ‘every available means’ of rapidly killing that cell. We strongly contend that in addition to powerfully activating generic cell death through caspases, granzymes must be capable of killing cells in which viral apoptotic inhibitors have stalled the intrinsic death program [16]. The first indication of these additional pathways was provided by Sarin et al. [17], who demonstrated that cells in which caspases were inactivated either by chemical inhibitors or by the baculovirus inhibitor p35 were still efficiently killed by CTLs acting in a perforin-dependent manner [17]. Surprisingly, it was next shown that inhibiting caspases markedly diminished nuclear damage in response to granzyme B but cell death still occurred due to direct granzyme-mediated damage to the remainder of the cell [18••,19•] (see Figure 1). This finding had significance for our understanding of the death process but it also raised the importance of measuring appropriate parameters of cell death when considering CTL/NK-mediated killing, a theme taken up again below.
What then, is the role for cell membrane perforation in the death process? Much of the recent work on the molecular aspects of granule-mediated death have utilized a ‘minimalist’ reconstituted cell free system incorporating only sublytic perforin and granzyme B, as first described by Shi et al. [8]. Although this model has proven valuable in many ways, its limitations need to be recognized. The model
The non-caspase targets of granzymes that can enforce cell death are now starting to be elucidated. The ability of granzyme B to mimic the Asp-ase activity of caspases (particularly effector caspases) allows it to cleave some downstream caspase targets, as pointed out recently by Andrade et al. [20•]. An alternative means of inducing single stranded breaks in DNA in response to granzyme A, which
was unimpaired by caspase blockade, was also recently identified in cells loaded with recombinant granzyme A [21••]. As granzymes have no direct nuclease activity, this pathway must involve activation of an unidentified nuclease. Although damage to nuclear structures no doubt contributes to dismantling a cell, the long known observation that cells lacking a nucleus can still die an apoptotic death in response to cytotoxic lymphocytes [22] suggests to us that truly essential granzyme substrates will be found to reside outside the nucleus. One focus of recent interest has been the mitochondrion. Exogenously added mitochondria greatly amplify granzyme-B-mediated cell death in a cell free system although the authors of one report [23] felt that nonmitochondrial pathways were also contributing to apoptosis and that caspase-3 activation could occur upstream of mitochondrial damage. Consistent with our own findings (J Davis, VR Sutton, JA Trapani, unpublished data), Helbein et al. [24•] recently showed that mitochondrial damage in response to granzyme B occurs without a requirement for caspase activation. The importance of the role played by mitochondria in granzyme-mediated death has come under scrutiny, as has the regulation of this pathway by pro-survival molecules such as Bcl-2 that operate by stabilising mitochondria. We have recently observed that overexpression of Bcl-2-like viral proteins such as BHRF1 of Epstein–Barr virus rigorously controls death mediated by granzyme B [25] and prevents both intracellular granzyme trafficking and accelerated granzyme uptake in response to perforin [26]. Furthermore this regulation is lost when the inhibitor is targeted to an alternative subcellular location such as the cell membrane (J Davis, JA Trapani, unpublished data), pointing to the central importance of a mitochondrial pathway activated by granzyme B. As normal endogenous levels of Bcl-2 cannot block this cell death, it follows that granzyme B must either inactivate Bcl-2 directly or alternatively activate a Bcl-2 antagonist, perhaps a proapoptotic Bcl-2 family member. We believe that the next few years will see a proliferation of granzyme ligands and substrates identified through a variety of biochemical and genetic approaches. We predict that these substrates will be cleaved exclusively by granzymes, or at least far more efficiently by granzymes than caspases. After many years of frustration, several groups have now expressed and purified proteolytically active recombinant granzymes A, B and H in bacteria, baculovirus and yeast; the proteolytic specificity of granzyme H, a chymotrypsin-like enzyme, has also been assigned [27•]. These developments should greatly assist researchers to define granzyme functions at the molecular level and the search for new apoptotic substrates in the cytosol and the cytoskeleton should be greatly facilitated. Insights into burning issues such as the nature of granzyme receptors on cells, and how granzymes traffic within target cells to gain access to their substrates, may open up new ways of regulating CTL and NK cell function.
New insights into granzyme functions in vivo Although it is indisputable that perforin deficiency is associated with increased mortality of mice from a number of natural pathogens, evidence of the importance of granzymes in vivo has been slower to become apparent. Much of the important progress in this area has come from the joint work of Mullbacher, Simon and co-workers [28,29••,30,31•]. Following on from previous observations that mice deficient in granzyme A are abnormally susceptible to the poxvirus ectromelia [28], these investigators have found that mice lacking both granzymes A and B are virtually as susceptible to primary ectromelia infection as perforin-deficient mice [29••]. This was despite the absence of any intrinsic inability of these mice to mount a CTL response to other pathogens [29••] or any additional susceptibility to related poxviruses [30]. A further important finding of this group was that serpins elaborated by this family of viruses are far less efficient at blocking the granule-mediated, perforin-dependent cell death imparted by MHC-restricted antiviral CTLs than the purely caspase-dependent death following Fas ligation [31•]. These findings both reiterate the relative importance of the granule pathway in defence against viruses in vivo and point to the lack of dependence by granzyme pathways solely on their capacity to activate caspases [16]. Further evidence of the requirement for granzymes for the normal cytolytic function of CTLs was gleaned from the studies of Shresta et al. [32•], who showed that the lethality of graft-versus-host disease imparted by transferred CD8+ alloreactive T cells was significantly reduced in the absence of both granzymes A and B. Granzyme deficiency states have not been identified in humans; however the identification of high levels of circulating soluble granzymes A and B in patients with active rheumatoid arthritis [33] may point to the active participation of CTLs in the pathogenesis of the inflammatory arthritides, providing another way of monitoring disease activity.
Granzyme regulation by endogenous serpins Pathways regulating granzyme function are also now under close scrutiny, as investigators search for ways of manipulating the cytotoxic response in vivo. It has recently become apparent that CTLs and NK cells express high levels of an intracellular endogenous serpin designated PI-9, which is capable of efficiently complexing and irreversibly inactivating granzyme B [34]. PI-9 is absent from cytolytic granules but is thought to be present in the cytosol to protect effector cells from granzyme molecules that undergo incorrect trafficking during their synthesis, processing, storage or exocytic release. An important feature of the inhibitory loop of PI-9 is the P1 Glu (not Asp) residue essential for binding and inhibiting granzyme B. Substitution of Asp into the P1 position of recombinant PI-9 resulted in a far reduced ability to interact with granzyme B and, surprisingly, the acquisition of inhibitory function to a variety of caspases [34]. Expression
Table 1 Death of Jurkat cells following exposure to mouse lymphokine-activated killer lymphocytes operating through the granule or FasL pathways, as measured in parallel by various parameters. Effector population* Perforin-deficient gld (FasL-deficient) None
Colony count† +VAD +FA 105+/–4 38+/–5 98+/–3
28+/–5 23+/–2 99+/–7
% specific 51Cr release‡ +VAD +FA 3+/–1 60+/–3 0
24+/–2 51+/–1 0
% specific 125I-DNA release§ +VAD +FA 2+/–2 12+/–7 0
48+/–6 39+/–14 0
*Effector cells were mouse splenocytes pre-incubated in vitro in medium supplemented with 100 units/ml IL-2 for four days (lymphokine-activated killer [LAK] cells). The effector : target cell ratio in the current experiment was 25:1. Prior to exposure to activated splenocytes, Jurkat cells were pre-incubated for 30 minutes in medium containing either z-VAD-fmk or z-FA-fmk (50 µM). † Clonogenic survival of Jurkat cells plated in soft agar, estimated by colony counting after seven days. LAK cells alone yielded no colonies.
All values shown are the mean of triplicate assays +/– standard error of the mean. Each fmk derivative was also included in the four hour cytotoxicity assays shown. Standard four hour standard cytotoxicity assays were performed in round-bottom 96-well plates. Values stated for the release of 51Cr‡ and 125I-DNA§ are relative to spontaneous release, measured in the absence of effector cells. For 51Cr, spontaneous release was <6% of total release; for 125I-DNA, spontaneous release was 10—30% of total release.
of this PI-9 mutant in Jurkat cells resulted in resistance of the transfectant cells to death following Fas ligation [34]. Thus the presence of Glu in the PI-9 inhibitory loop provides for specific protection against granzyme B, without unduly interfering with eventual clearing of the cytolytic cell mediated through the Fas pathway. We note that the inhibitory loop of PI-9 bears striking resemblance to that of viral serpins such as crmA and speculate that the gene encoding PI-9 may have been ‘hijacked’ by a virus, with subsequent mutation of the P1 Glu to Asp resulting in its use to inhibit generic caspase-mediated cell death. Another salutary lesson is that an optimal cleavage motif does not necessarily form the ideal core of an inhibitory oligopeptide for that protease.
through the granule pathway) was totally resistant to z-VAD-fmk and correlated well with reduced clonogenic survival of the treated cells. Notably, 125I-DNA release was markedly inhibited by z-VAD-fmk pretreatment. As several groups have shown that cell membrane damage as measured by 51Cr release occurs independently of caspases, we favor the use of this assay over estimates of nuclear damage if short term assays are to be relied on.
On measuring death imparted by CTLs and NK cells or granule components As alluded to above, cell death in response to granule components may occur in the absence of gross nuclear damage. This places a special onus on investigators to interpret data arising from cytotoxicity assays with care, especially when caspases are not optimally activated. Purely nuclear readouts such as DNA end-labelling or 125I-DNA release assays can be particularly misleading, as depicted in the experiment shown in Table 1 (J Davis, unpublished data). As all short-term assays are merely surrogate measures of cell death or survival, our group is increasingly turning to clonogenic survival assays to determine true life/death outcomes. In our experiment, the ‘death’ of Jurkat cells was measured in various ways following four-hour exposure to intact mouse lymphokine-activated killer cells. Target cells were also incubated before and throughout the assay with the caspase inhibitor z-VAD-fmk or with the control inhibitor zFA-fmk, which does not block caspases. Perforin-deficient effector cells (operating through Fas ligation) caused specific 51Cr and 125I-DNA release, which measure cell membrane damage and nuclear damage, respectively. Both parameters were inhibitable by z-VAD-fmk, consistent with the total reliance on caspases of this pathway. In contrast, the 51Cr release in response to gld effectors (operating
The respective roles of perforin and IFN-γ secretion in defence against viruses in vivo: an immunoregulatory role for perforin There is now no doubt that direct cytotoxicity mediated by lymphocyte perforin is essential for the control of both non-cytopathic [1,35] and cytopathic viruses [30]. More recently, it has become apparent that a critical balance exists between direct cytotoxicity mediated by perforin and IFN-γ secretion, and that this balance controls the outcome of viral infection and dictates immune homeostasis. Augmented perforin-mediated immunopathology is observed in IFN-γ-deficient mice infected with lymphocytic choriomeningitis virus (LCMV) [36••]. Conversely, an overproduction of inflammatory cytokines is observed in perforin-deficient mice infected with LCMV. Furthermore a recent study suggests that T cells can turn off their cytokine production while retaining intracellular stores of perforin, thereby potentially maintaining effective immune surveillance while minimizing systemic immunopathology [37••]. In addition to the description of perforin-deficient gld mice [38•], further evidence supports a pivotal role played by perforin in the regulation of immune responses. Several studies have recently suggested that perforin-dependent cytotoxicity regulates the elimination of CD8+ T cells following an acute exposure to foreign antigen. Notably, perforin-deficient mice showed an increased expansion and persistence of superantigen- and virus-specific T cells that was not explained by a failure to deplete antigen-presenting cells in wild-type mice [39•]. Similarly, an increased
expansion of alloreactive perforin-deficient T cells following transfer into irradiated scid/scid mice (these lack T and B cells) further supported a role for perforin in activationinduced cell death [40•]. Following on from previous studies by Binder et al. [41•], Matloubian et al. [42•] demonstrated that perforin was involved in downregulating T cell responses during chronic LCMV infection. An excessive accumulation of CD8+ T cells resulting in immune-mediated damage in perforin-deficient mice mimics the clinical picture in perforin-deficient children suffering familial hemophagocytic lymphohistiocytosis (FHL) [43••]. Although a causative infectious agent for this infantile disease has not yet been defined, it is likely to be a microbe whose control is strictly perforin-dependent. Perforin-based effector pathways appear to be involved in the death of abnormal cells that accumulate in FHL; it is these cells and the cytokines they produce (TNF-α, IFN-γ) that are ultimately lethal.
A role for perforin in immune surveillance against cancer We feel that the ability of perforin-secreting killer cells to eliminate transformed cells in vivo has been overlooked as researchers have emphasized perforin’s importance in T cell homeostasis. Recent studies in our laboratory (J Davis, VR Sutton, JA Trapani, unpublished data) have indicated that perforin-deficient mice are more susceptible to spontaneous lymphoma; malignancy in the lymphoid compartment is particularly consistent with dysregulated lymphocyte activation. Previous experiments with murine-leukaemia-virusinduced lymphomas have also indicated an important role for perforin [44]. It remains to be assessed whether perforinmediated immune surveillance controls nonlymphoid malignancies; however past studies would suggest that at least some chemically induced sarcomas are controlled in a perforin-dependent manner [45•]. Furthermore the natural [45•] and IL-12-induced [46] antimetastatic activities of NK cells against a variety of nonlymphoid tumors including melanomas, and prostate and breast carcinomas have been shown to be perforin-mediated. In each of these tumor models, FasL mutant mice demonstrated normal protection from tumor growth and metastasis. Nonetheless these models strictly involved innate immune responses and two recent experimental studies have suggested that Fas [47] or alternative pathways [48] may also contribute to T-celldependent tumor regression. Strikingly, malignant transformation of keratinocytes appears to be regulated locally by FasL–Fas interactions [49••]; however immune surveillance by unknown mechanisms may still possibly protect these nonlymphoid tissues. We therefore postulate that rapid and direct perforin-mediated cytotoxicity of microbes or the cells they infect prevents prolonged activation of the lymphoid compartment. Perforin is also responsible, at least in part, for the elimination of activated CD8+ T cells following such an immune response. A potential by-product of aberrant and
sustained proliferation of hematopoietic tissue is the increased propensity to oncogenic transformation; however perforin-expressing killer lymphocytes can also eliminate these transformed cells through their role in providing immune surveillance. Since perforin clearly evolved to control microbial challenge, it will be fascinating to examine the importance of perforin-mediated cytotoxicity in the surveillance of cancers of viral and non-viral aetiology.
Conclusions The past decade has seen a revolution in our understanding of the molecular pathways underpinning the death signal imparted by cytotoxic lymphocytes. Although some of these findings might have been predicted by like findings made in other types of apoptotic death, it is clear that perforin-dependent, granule-mediated apoptosis represents a specialised and complex death process with many unusual features. The apoptotic attack by cytolytic lymphocytes is highly efficient, very rapid and multilayered at the molecular level, without absolute reliance on caspases — normally the enzymatic 'engine-room' of generic apoptosis. Beyond cursory consideration, this is hardly surprising — the killer lymphocyte must be capable of destroying cells in which the proapoptotic program has been corrupted by a virus or by coincident cellular transformation. Death imposed on such a recalcitrant cell thus differs appreciably from cell suicide or programmed death initiated and accomplished intrinsically by the dying cell itself. There are many questions that remain regarding apoptotic pathways induced by cytotoxic granules. The precise function of perforin, an indispensable keystone in the whole process, remains unclear and a comprehensive structure/function analysis of this molecule is sadly lacking. The nature of granzyme receptors and their uptake into normal cells remain unclear. Although a few granzyme substrates (other than caspases) have also been identified, those that deliver caspase-independent death remain elusive. Elucidating such pathways may provide us with novel ways to kill malignant or parastised cells that have lost their ability to undergo programmed cell death.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ••of outstanding interest 1.
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Smyth MJ, Trapani JA: The relative role of lymphocyte granule exocytosis versus death receptor-mediated cytotoxicity in viral pathophysiology. J Virol 1998, 72:1-9.
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10. Shi L, Mai S, Israels S, Browne K, Trapani JA, Greenberg AH: Granzyme B (GraB) autonomously crosses the cell membrane, perforin initiates apoptosis and GraB nuclear localization. J Exp Med 1997, 185:855-866. 11. Froelich CJ, Orth K, Turbov J, Seth P, Gottlieb R, Babior B, Shah GM, Bleackley RC, Dixit VM, Hanna W: New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind, internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. J Biol Chem 1996, 271:29073-29079. 12. Trapani JA, Jans P, Froelich CJ, Smyth MJ, Sutton VR, Jans D: • Perforin-dependent nuclear accumulation of granzyme B precedes apoptosis and is not a consequence of nuclear membrane dysfunction. Cell Death Differ 1998, 5:488-496. The first study to show that granzyme B entry to the nucleus precedes cell death and does not occur as a consequence of cell death. Granzyme B is targeted specifically and rapidly to the nucleus by an unknown mechanism, enabling it to access nuclear substrates directly. See also [20•]. 13. Browne KA, Blink E, Sutton VR, Froelich CJ, Jans DA, Trapani JA: •• Bacterial toxins facilitate intracellular delivery of granzyme B: evidence that endosomal disruption is an important function of perforin, in addition to transmembrane pore-formation. Mol Cell Biol 1999, 19:8604-8615. The first study providing quantitative and kinetic data challenging the notion that perforin functions through simple transmembrane pore formation. The role of perforin in facilitating apoptosis can be replicated by endosomolytic agents and, in the apparent absence of sizeable transmembrane lesions, even in the presence of ‘high’ perforin concentrations. 14. Stenger S, Hanson D, Teitelbaum R, Dewan P, Niazi K, Froelich CJ, •• Ganz T, Thoma-Uszynski S, Melian A, Bogdan C et al.: An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998, 282:121-125. The first instance of perforin-dependent killing, of an intracellular microbial pathogen, that does not appear to utilize granzymes. 15. Galvin JP, Spaeny-Dekking LH, Wang B, Seth P, Hack CE, • Froelich CJ: Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo. J Immunol 1999, 162:5345-5350. The first paper to point out that the co-delivery of proapoptotic agents to the target cell is probably achieved in the context of macromolecular complexes containing chondroitin sulfate proteoglycans. 16. Trapani JA, Sutton VR, Smyth MJ: Cytotoxic lymphocyte granules: evolution of vesicles essential for combating virus infections. Immunol Today 1999, 20:351-356. 17.
Sarin A, Williams MS, Alexander-Miller MA, Berzofsky JA, Zacharchuk CM, Henkart PA: Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 1997, 6:209-215.
18. Sarin A, Haddad EK, Henkart PA: Caspase dependence of target •• cell damage induced by cytotoxic lymphocytes. J Immunol 1998, 161:2810-2816. The first paper to point out that nuclear damage, particularly DNA release, inflicted by cytotoxic lymphocytes requires caspase activation whereas nonnuclear parameters of cell death are largely independent of caspases. 19. Trapani JA, Jans DA, Browne KA, Smyth MJ, Jans PJ, Sutton VR: • Efficient nuclear targeting of granzyme B, the nuclear consequences of apoptosis induced by granzyme B and perforin
are caspase-dependent, but cell death is caspase-independent. J Biol Chem 1998, 273:27934-27938. This paper first localised the essential proapoptotic effect of granzyme B as residing outside the nucleus. Cell death in response to granzyme B and perforin occurs irrespective of the apparent inhibition of nuclear damage by caspase inhibitors. 20. Andrade F, Roy S, Thornberry N, Rosen A, Casciola-Rosen L: • Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity 1998, 8:451-460. This elegant and comprehensive paper shows that downstream caspase substrates can be targeted directly by granzyme B when caspases are inhibited. 21. Beresford PJ, Xia Z, Greenberg AH, Lieberman J: Granzyme A •• induces rapid cytolysis, a novel form of DNA damage independently of caspases. Immunity 1999, 10:585-594. Granzyme A loading into target cells was used to demonstrate for the first time that granzyme A can induce single-stranded DNA breaks in a caspaseindependent manner. The significance of this finding is yet to be reported when using granzyme A deficient effector cells. 22. Nakajima H, Golstein P, Henkart PA: The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity. J Exp Med 1995, 181:1905-1909. 23. MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg A: Mitochondria-dependent and -independent regulation of granzyme B-induced apoptosis. J Exp Med 1999, 189:131-144. 24. Helbein JA, Barry M, Motyka B, Bleackley RC: Granzyme B-induced • loss of mitochondrial inner membrane potential (Delta Psi m) and cytochrome c release are caspase independent. J Immunol 1999, 163:4683-4693. This elegant study documented non-nuclear events that occur independently of caspases and that may influence cell survival. 25. Sutton VR, Vaux DA, Trapani JA: Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes. J Immunol 1997, 158:5783-5790. 26. Jans DJ, Sutton VR, Jans PJ, Froelich CJ, Trapani JA: BCL-2 blocks perforin-induced nuclear translocation of granzymes, concomitant with protection against the nuclear events of apoptosis. J Biol Chem 1999, 274:3953-3961. 27. •
Edwards KM, Kam C-M, Powers J, Trapani JA: The human cytotoxic T cell granule protease granzyme H has chymotrypsin-like (chymase) activity, is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes. J Biol Chem 1999, 274:30468-30473. The first characterization of a chymase granzyme in human CTLs. This study utilized recombinant granzyme H expressed in insect Sf9 cells. 28. Mullbacher A, Ebnet K, Blanden RV, Hla RT, Stehle T, Museteanu C, Simon MM: Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia. Proc Natl Acad Sci USA 1996, 93:5783-5787. 29. Mullbacher A, Waring P, Tha Hla R, Tran T, Chin S, Stehle T, •• Museteanu C, Simon MM: Granzymes are essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes. Proc Natl Acad Sci USA 1999, 96:13950-13955. This is the first reported instance of a pathophysiological consequence of a granzyme deficiency. The investigators make the observation that CTLs equipped with normal levels of perforin but lacking granzymes A and B are specifically deficient in protecting the host from a natural mouse pathogen, the poxvirus ectromelia. Despite this, mice lacking granzymes A and B have no intrinsic problem with raising CTL responses. The defective response to ectromelia is virtually as profound as perforin-deficiency per se. 30. Mullbacher A, Hla RT, Museteanu C, Simon MM: Perforin is essential for control of ectromelia virus but not related poxviruses in mice. J Virol 1999, 73:1665-1667. 31. Mullbacher A, Wallich R, Moyer RW, Simon MM: Poxvirus-encoded • serpins do not prevent cytolytic T cell-mediated recovery from primary infections. J Immunol 1999, 162:7315-7321. This elegant in vivo study reinforces the notion that CTLs recruited in the context of most antiviral responses can bypass antiapoptotic mechanisms erected by the pathogen. 32. Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ: Granzyme A • initiates an alternative pathway for granule-mediated apoptosis. Immunity 1999, 10:595-605. + CD8 CTLs mediating graft-versus-host disease inflict less potent tissue damage in the recipient if they are deficient in granzyme expression, especially granzyme A.
33. Tak PP, Spaeny-Dekking L, Kraan MC, Breedveld FC, Froelich CJ, Hack CE: The levels of soluble granzyme A and B are elevated in plasma and synovial fluid of patients with rheumatoid arthritis. Clin Exp Immunol 1999, 116:366-370.
infection with lymphocytic choriomeningitis virus. J Exp Med 1998, 187:1903-1920. Perturbation of the normal immune response to LCMV due to perforin deficiency results in bone marrow hypoplasia secondary to excessive secretion of cytokines.
34. Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong TA, Trapani JA, Bird PI: A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. J Biol Chem 1996, 271:27802-27809.
42. Matloubian M, Suresh M, Glass A, Galvan M, Chow K, Whitmire JK, • Walsh CM, Clark WR, Ahmed R: A role for perforin in downregulating T-cell responses during chronic viral infection. J Virol 1999, 73:2527-2536. This paper demonstrates an accumulation of CD8+ T cells resulting in immune-mediated damage in perforin-deficient mice that are incapable of clearing LCMV infection.
35. Kagi D, Seiler P, Pavlovic J, Ledermann B, Burki K, Zinkernagel RM, Hengartner H: The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses. Eur J Immunol 1995, 25:3256-3262. 36. Nansen A, Jensen T, Christensen JP, Andreasen SO, Ropke C, •• Marker O, Thomsen AR: Compromised virus control and augmented perforin-mediated immunopathology in IFN-gammadeficient mice infected with lymphocytic choriomeningitis virus. J Immunol 1999, 163:6114-6122. Increased immunopathology mediated by perforin is observed when IFN-γdeficient mice are infected with LCMV, indicating for the first time a role for both cytokine secretion and direct cytotoxicity in the control of this pathogen. 37. Slifka MK, Rodriguez F, Whitton JL: Rapid on/off cycling of cytokine •• production by virus-specific CD8+ T cells. Nature 1999, 401:76-79. This elegant and comprehensive paper points out that the balance between direct cytotoxicity and release of inflammatory cytokines can be regulated at the single-cell level. 38. Spielman J, Lee RK, Podack ER: Perforin/Fas-ligand double • deficiency is associated with macrophage expansion and severe pancreatitis. J Immunol 1998, 161:7063-7070. The first paper that pointed out a role for perforin other than in direct cytotoxicity and indicated a role in modulating the immune response. 39. Kagi D, Odermatt B, Mak TW: Homeostatic regulation of CD8+ • T cells by perforin. Eur J Immunol 1999, 29:3262-3272. Perforin-deficient mice show an increased number and persistence of superantigen- and virus-specific T cells: further evidence (with [40•]) of a role for perforin in immune regulation. 40. Spaner D, Raju K, Rabinovich B, Miller RG: A role for perforin in • activation-induced T cell death in vivo: increased expansion of allogeneic perforin-deficient T cells in SCID mice. J Immunol 1999, 162:1192-1199. See annotation to [39•]. 41. Binder D, van den Broek MF, Kagi D, Bluethmann H, Fehr J, • Hengartner H, Zinkernagel RM: Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent
43. Stepp SE, Dufourcq-Lagelouse R, Le Deist F, Bhawan S, Certain S, •• Mathew PA, Henter JI, Bennett M, Fischer A, de Saint Basile G, Kumar V: Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science 1999, 286:1957-1959. The first description of the clinical consequences of perforin deficiency in humans. FHL patients present with a syndrome that is highly reminiscent of perforin-deficient mice chronically infected with a viral pathogen that cannot be cleared, for example LCMV. 44. van den Broek ME, Kagi D, Ossendorp F, Toes R, Vamvakas S, Lutz WK, Melief CJ, Zinkernagel RM, Hengartner H: Decreased tumor surveillance in perforin-deficient mice. J Exp Med 1996, 184:1781-1790. 45. Smyth MJ, Thia KY, Cretney E, Kelly JM, Snook MB, Forbes CA, • Scalzo AA: Perforin is a major contributor to NK cell control of tumor metastasis. J Immunol 1999, 162:6658-6662. The first demonstration that innate immune mechanisms can control tumor metastasis in a perforin-dependent manner. 46. Kodama T, Takeda K, Shimozato O, Hayakawa Y, Atsuta M, Kobayashi K, Ito M, Yagita H, Okumura K: Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12. Eur J Immunol 1999, 29:1390-1396. 47.
Medema JP, de Jong J, van Hall T, Melief CJ, Offringa R: Immune escape of tumors in vivo by expression of cellular FLICEinhibitory protein. J Exp Med 1999, 190:1033-1038.
48. Winter H, Hu HM, Urba WJ, Fox BA: Tumor regression after adoptive transfer of effector T cells is independent of perforin or Fas ligand (APO-1L/CD95L). J Immunol 1999, 163:4462-4472. 49. Hill LL, Ouhtit A, Loughlin SM, Kripke ML, Ananthaswamy HN, Owen •• Schaub LB: Fas ligand: a sensor for DNA damage critical in skin cancer etiology. Science 1999, 285:898-900. Immune surveillance of cellular transformation in the skin involves local expression of FasL and deletion of cells with DNA damage through a Fasdependent mechanism.