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H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells
Update
TRENDS in Immunology
Vol.28 No.9
Research Focus
H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells Nigel J. Waterhouse and Joseph A. Trapani Cancer Cell Death Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew’s Place, Melbourne, Victoria 3002, Australia
It is commonly held that the various granzymes (lethal proteases produced by cytotoxic lymphocytes) utilize their different substrate preferences to bring about various forms of target cell death. Although a considerable body of evidence supports this view, it has now become clear that human granzyme H could have evolved a proteolytic specificity that both interferes directly with adenovirus replication and prevents the virus from blocking the potent pro-apoptotic activity of granzyme B.
A variety of physiological roles for the different granzymes? Cytotoxic lymphocytes (CLs, comprising cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells) utilize granule exocytosis to protect mammals from virus infection. During granule exocytosis, CLs deliver a battery of lethal proteases (granzymes) with varying substrate preferences into the target cell cytoplasm via a perforin-dependent mechanism. The primary role so far attributed to granzymes has been to bring about the death of infected cells, and in support of this notion, granzymes A, B, C, H, K and M have all been reported to activate specific pathways to cell death [1–7]. However, there are several other granzymes (for example, rodent granzymes D, E, F, G, I and L), for which a pro-death role has not been demonstrated [8]. Humans and mice also differ significantly in their granzyme repertoires, as both species express granzymes A, B, K and M, whereas the other granzymes are restricted to one or other species. The importance of the granule pathway in protection against viruses is clearly evident from studies in gene-disrupted mice. Perforin-deficient mice are abnormally susceptible to various viruses [9]; however, the deficiency of one or a few granzymes results in more-focal gaps in the anti-viral response. For example, mice deficient in granzymes A and B are exquisitely (up to 100 000 times) more sensitive to the natural mouse poxvirus pathogen, ectromelia, than are wild-type mice [10]. Why mammals should require such a broad array of granzymes with varying proteolytic activity is debated. One possibility is that the many anti-apoptotic molecules expressed by various viruses need to be balanced by a whole family of proteases to circumvent these blocks and enforce target cell death. The recent finding in both mice Corresponding author: Waterhouse, N.J. ([email protected]); Trapani, J.A. ([email protected]). Available online 4 September 2007. www.sciencedirect.com
and humans that a given activated CTL might express only one granzyme or a small subset of granzymes argues against this possibility [11,12]. A fascinating recent paper from the laboratory of Felipe Andrade [13] raises the alternate possibility that at least some of the granzymes could have evolved to interfere more directly with the replication of certain pathogenic viruses. Andrade et al. reported that granzyme H (a protease of humans, with no direct orthologue in rodents [14]), cleaves and inactivates two adenoviral proteins, DNA binding protein (DBP) and the L4–100K assembly protein, thereby significantly restricting viral replication [13]. Crucially, the same group had previously showed that L4–100K, which is abundantly expressed in infected cells, acts as a ‘sink’ that binds to and inhibits granzyme B, preventing target cell death [15]. Andrade et al. now propose that by cleaving DBP and L4–100K, granzyme H participates in the anti-adenovirus response by both inhibiting virus replication directly and simultaneously re-sensitizing the infected cell to granzyme B-induced death (Figure 1). Direct cleavage of adenovirus proteins by granzymes B and H Adenoviral DNA replication begins with coating of the viral genome by DBP, which in turn has affinity for nuclear factor I (NFI), a cellular transcription factor that binds to the viral origin of replication. Following transcription, individual hexon and penton monomers form into capsomers in the cytoplasm and are assembled into immature capsids in the nucleus. L4–100K is an assembly protein involved in the transport of hexons from the cytoplasm to the nucleus. Virus particles accumulate in the nucleus and the infected cell remains intact; however, persistent infection can result in cell lysis, permitting virus escape and re-infection. Andrade et al. first noted that DBP (70 kDa) was cleaved to a 47 kDa product in adenovirus 5 (Ad5)infected K562 cells exposed to lymphokine activated killer (LAK) cells [13]. Cleavage of DBP was blocked by calcium chelation, suggesting the involvement of the calcium-dependent granule exocytosis pathway in this process. Recombinant DBP overexpressed in K562 cells was also cleaved to a predominant 47 kDa fragment, but less prevalent fragments of 50 kDa and 60 kDa were also observed. The addition of a granzyme B inhibitor abolished the 50 and 60 kDa forms, whereas caspase inhibition prevented appearance of the 60 kDa fragment. These data suggested that granzyme B cleaves DBP to the 50 kDa fragment and caspases activated as a consequence of granzyme
374
Update
TRENDS in Immunology Vol.28 No.9
Figure 1. CTL versus adenovirus. The final outcome, death of the infected cell or production of infectious virus, seems to involve a complex competition between granzymes and virus proteins. Granzyme B expressed by CLs induces death of an infected cell by apoptosis. Adenovirus L4–100K blocks granzyme B, thereby preventing apoptosis and permitting virus replication. CLs also express granzyme H, which cleaves L4–100K and prevents it from blocking granzyme B. This resensitizes the cell to granzyme B-induced apoptosis. Granzyme H also cleaves DBP, inhibiting efficient replication of virus DNA, and limiting the production of infectious virus particles.
B-induced death cleave some of the remaining full-length DBP to the 60 kDa fragment. It remained unclear how DBP was cleaved to the 47 kDa fragment, but Andrade et al. made the key finding that purified granzyme H (but not granzyme A, K and M) could cleave in vitro transcribed and translated DBP, or DBP in lysates from Ad5-infected K562 cells. Cleavage occurred either at Met118 or Phe121, or possibly at a unique site that spans these two residues. Significantly higher viral titres resulted when K562 cells infected for 12 h with adenovirus expressing DBP that was mutated at Met118 and Phe121 were exposed to LAK cells, compared with K562 cells infected with wild-type adenovirus. This strongly suggested an essential role for granzyme H-mediated cleavage of DBP in restricting adenovirus replication. As DBP can be cleaved by both granzyme B and granzyme H, Andrade et al. next tested whether both proteases could also cleave L4–100K. Crucially, they found that L4– 100K protein was cleaved by granzyme H to a 90 kDa fragment that rendered it unable to block granzyme B. This was demonstrated by the observation that granzyme B-mediated cleavage of caspase-3 was delayed by the addition of L4–100K but restored if granzyme H was also added, despite the fact that granzyme H was incapable of directly processing pro-caspase-3. The findings strongly suggested that a crucial role for granzyme H is to rescue the pro-apoptotic activity of granzyme B in Ad5-infected cells, while co-incidentally having a direct negative effect on capsomer assembly and viral titre. Of the 49 adenovirus serotypes that infect humans, subgroup A induces tumours in rodents with high frequency and short latency, subgroup B is weakly oncogenic and subgroup C, D, E and F are non-oncogenic. By contrast, adenovirus-induced oncogenesis has never been observed in humans. Among the many possible mechanisms that might explain this difference, the low oncogenic potential of adenoviruses in humans could at least partly reflect the more efficient limitation of viral replication and spread www.sciencedirect.com
imposed by the human immune system. Therefore, it would be interesting to know whether DBP can be cleaved to the 47 kDa form when adenovirus-infected mouse cells are exposed to mouse LAK cells, resulting in a direct negative effect or virus replication. As there is no direct mouse orthologue of granzyme H, this function would presumably rely on an alternative granzyme(s) that, like granzyme H, has chymotrypsin-like cleavage specificity [16]. Interestingly, the DBP cleavage sites for granzymes B and H are not conserved in all adenovirus subgroups. The P1 residue for the principal predicted granzyme H site, Phe121, seems to be well conserved in human and simian isolates, and the granzyme B site is also partly conserved (Table 1); however, Met118 and the recognition site for caspases are not well conserved. It is therefore likely that the cleavage of DBP is restricted to specific adenovirus subgroups, particularly for granzyme B. Generalizing from this observation, it is possible that the diverse cleavage specificities of the granzymes might reflect the species-specific co-evolution of cytotoxic effector mechanisms and strategies used by many viruses for immune escape. Consistent with this possibility, Andrade et al. have also previously shown that the L4–100K protein from Ad5 fails to be cleaved efficiently by, or to inhibit, mouse granzyme B [17]. In turn, this finding is consistent with several recent independent reports showing that the substrate preferences (and associated kinetics) of mouse and human granzyme B are significantly different [18–20]. Therefore, even clearly orthologous granzymes seem to have evolved species-specific functions in response to relevant and dangerous infectious agents. This principle is probably not limited to granzymes and viruses, as a bacterial virulence factor can also be inactivated by neutrophil elastase [21]. Although the study by Andrade et al. makes a strong case for a direct anti-viral role for granzyme H, it was recently reported that granzyme H could also be directly cytotoxic to target cells when delivered with perforin [22]. Recombinant granzyme H was able to induce mitochondrial depolarization, reactive oxygen species production, chromatin condensation and DNA fragmentation but did not involve cleavage of the pro-apoptotic Bcl-2 family member Bid or inhibitor of caspase-activated death (ICAD), release of cytochrome c from the mitochondrial intermembrane space or activation of caspases. The data suggest that in addition to having a constitutive anti-viral role, granzyme H might also be capable of killing certain target cells directly. A pro-death role for granzyme H was not uncovered by Andrade et al.; therefore, it would be intriguing to know whether granzyme H can kill adenovirus infected cells in the absence of granzyme B. It is also conceivable that the direct anti-viral and pro-apoptotic activities of granzyme H might require significantly different concentrations of intracellular granzyme H, so that a hierarchy of effector mechanisms might operate, depending on how much granzyme H activity can be delivered into an infected cell. Future studies in viral immunology The findings of Andrade’s group should provide a strong impetus for further identification of viral substrates for the granzymes. Crucially for future studies, granzymes B and H share significant structural similarity, and some of the
Update
TRENDS in Immunology
Vol.28 No.9
375
Table 1. Potential cleavage sites for granzyme B and granzyme H in DBP from various adenovirusesa Host species Human
Serotype 1
DBP sequence
Accession No AP 000514
Human
2
AP 000177
Human
5
AP 000213
Human
3
ABB 17782
Human
7
AP 000550
Human
11
AP 000454
Human
35
AP 000587
Monkey
21
AP 000277
Human
52
ABK 35046
Human
46
AAX 70936
Dog
1
AP 000061
a
DBP protein sequences from various selected adenoviruses that specifically infect humans, monkeys or dogs were obtained from Pubmed (http://www.ncbi.nlm.nih.gov/ sites/entrez?db=PubMed) and potential cleavage sites for granzyme H (after f or m) and granzyme B (after d) are highlighted. The single amino acid code is used, and residue numbers for each of the DBP sequences are indicated.
antibodies used to detect granzyme B cross-react with granzyme H [23]. Careful studies with monospecific antibodies have shown that circulating, unstimulated NK cells that have relatively low cytotoxic capacity express high levels of granzyme H but relatively little granzyme B [23]. These data might predict a role for granzyme H in the immediate innate immune response to virus, independent of or complementary to NK-induced killing. In concert with cytokines such as interferon-g, a role for granzyme H in limiting early viral replication would be consistent with the acute need to minimize viral load until a cognate CTL response can be raised. It would therefore be intriguing to know if the yield of productive virus is reduced in adenovirus-infected cells exposed to naive NK cells, and whether DBP is cleaved in infected cells that survive NK cell cytotoxic attack. It is interesting that NK cells stimulated in vitro with interleukin-2 express progressively greater amounts of perforin and granzyme B but reduce their granzyme H levels over 3–4 days [23]. This pattern could be consistent with a change in emphasis from viral containment to cytotoxicity against infected cells (possibly mediated by both NK cells and CTLs) in the days following infection. In conclusion, the study of Andrade et al. presents compelling evidence that granzyme B and granzyme H can directly and cooperatively cleave virus proteins that are essential for virus replication, and that granzyme H participates in the anti-virus response by neutralizing an inhibitor of granzyme B. Characterizing such roles for granzymes at the molecular level and demonstrating that different granzymes can act cooperatively to thwart viruses is sure to have a renewed focus in future immunology and virology research.
7
8 9
10
11
12
13
14
15
16
17
18 19
References 1 Johnson, H. et al. (2003) Cell death induced by granzyme C. Blood 101, 3093–3101 2 Kelly, J.M. et al. (2004) Granzyme M mediates a novel form of perforindependent cell death. J. Biol. Chem. 279, 22236–22242 3 MacDonald, G. et al. (1999) Mitochondria-dependent and -independent regulation of granzyme B-induced apoptosis. J. Exp. Med. 189, 131–144 4 Martinvalet, D. et al. (2005) Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis. Immunity 22, 355–370 5 Voskoboinik, I. et al. (2006) Perforin-mediated target-cell death and immune homeostasis. Nat. Rev. Immunol. 6, 940–952 6 Waterhouse, N.J. et al. (2006) Functional dissociation of DCm and cytochrome c release defines the contribution of mitochondria www.sciencedirect.com
20 21 22
23
upstream of caspase activation during granzyme B-induced apoptosis. Cell Death Differ. 13, 607–618 Zhao, T. et al. (2007) Granzyme K directly processes bid to release cytochrome c and endonuclease G leading to mitochondria-dependent cell death. J. Biol. Chem. 282, 12104–12111 Grossman, W.J. et al. (2003) The orphan granzymes of humans and mice. Curr. Opin. Immunol. 15, 544–552 Kagi, D. et al. (1994) Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 31–37 Mullbacher, A. et al. (1999) Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes. Proc. Natl. Acad. Sci. U. S. A. 96, 13950–13955 Grossman, W.J. et al. (2004) Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood 104, 2840–2848 Johnson, B.J. et al. (2003) Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice. Proc. Natl. Acad. Sci. U. S. A. 100, 2657–2662 Andrade, F. et al. (2007) Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition. EMBO J. 26, 2148–2157 Klein, J.L. et al. (1990) Characterization of a novel, human cytotoxic lymphocyte-specific serine protease cDNA clone (CSP-C). Tissue Antigens 35, 220–228 Andrade, F. et al. (2001) Adenovirus L4-100K assembly protein is a granzyme B substrate that potently inhibits granzyme B-mediated cell death. Immunity 14, 751–761 Edwards, K.M. et al. (1999) The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme Bcontaining endosomes. J. Biol. Chem. 274, 30468–30473 Andrade, F. et al. (2003) A novel domain in adenovirus L4-100K is required for stable binding and efficient inhibition of human granzyme B: possible interaction with a species-specific exosite. Mol. Cell. Biol. 23, 6315–6326 Kaiserman, D. et al. (2007) The major human and mouse granzymes are structurally and functionally divergent. J. Cell Biol. 175, 619–630 Casciola-Rosen, L. et al. (2007) Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway. J. Biol. Chem. 282, 4545–4554 Cullen, S. et al. (2007) Human and murine granzyme B exhibit divergent substrate preferences. J. Biol. Chem. 176, 435–444 Weinrauch, Y. et al. (2002) Neutrophil elastase targets virulence factors of enterobacteria. Nature 417, 91–94 Fellows, E. et al. (2007) Natural killer cell-derived human granzyme H induces an alternative, caspase-independent cell death program. Blood 110, 544–552 Sedelies, K.A. et al. (2004) Discordant regulation of granzyme H and granzyme B expression in human lymphocytes. J. Biol. Chem. 279, 26581–26587
1471-4906/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2007.08.001
TRENDS in Immunology
Vol.28 No.9
Research Focus
H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells Nigel J. Waterhouse and Joseph A. Trapani Cancer Cell Death Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew’s Place, Melbourne, Victoria 3002, Australia
It is commonly held that the various granzymes (lethal proteases produced by cytotoxic lymphocytes) utilize their different substrate preferences to bring about various forms of target cell death. Although a considerable body of evidence supports this view, it has now become clear that human granzyme H could have evolved a proteolytic specificity that both interferes directly with adenovirus replication and prevents the virus from blocking the potent pro-apoptotic activity of granzyme B.
A variety of physiological roles for the different granzymes? Cytotoxic lymphocytes (CLs, comprising cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells) utilize granule exocytosis to protect mammals from virus infection. During granule exocytosis, CLs deliver a battery of lethal proteases (granzymes) with varying substrate preferences into the target cell cytoplasm via a perforin-dependent mechanism. The primary role so far attributed to granzymes has been to bring about the death of infected cells, and in support of this notion, granzymes A, B, C, H, K and M have all been reported to activate specific pathways to cell death [1–7]. However, there are several other granzymes (for example, rodent granzymes D, E, F, G, I and L), for which a pro-death role has not been demonstrated [8]. Humans and mice also differ significantly in their granzyme repertoires, as both species express granzymes A, B, K and M, whereas the other granzymes are restricted to one or other species. The importance of the granule pathway in protection against viruses is clearly evident from studies in gene-disrupted mice. Perforin-deficient mice are abnormally susceptible to various viruses [9]; however, the deficiency of one or a few granzymes results in more-focal gaps in the anti-viral response. For example, mice deficient in granzymes A and B are exquisitely (up to 100 000 times) more sensitive to the natural mouse poxvirus pathogen, ectromelia, than are wild-type mice [10]. Why mammals should require such a broad array of granzymes with varying proteolytic activity is debated. One possibility is that the many anti-apoptotic molecules expressed by various viruses need to be balanced by a whole family of proteases to circumvent these blocks and enforce target cell death. The recent finding in both mice Corresponding author: Waterhouse, N.J. ([email protected]); Trapani, J.A. ([email protected]). Available online 4 September 2007. www.sciencedirect.com
and humans that a given activated CTL might express only one granzyme or a small subset of granzymes argues against this possibility [11,12]. A fascinating recent paper from the laboratory of Felipe Andrade [13] raises the alternate possibility that at least some of the granzymes could have evolved to interfere more directly with the replication of certain pathogenic viruses. Andrade et al. reported that granzyme H (a protease of humans, with no direct orthologue in rodents [14]), cleaves and inactivates two adenoviral proteins, DNA binding protein (DBP) and the L4–100K assembly protein, thereby significantly restricting viral replication [13]. Crucially, the same group had previously showed that L4–100K, which is abundantly expressed in infected cells, acts as a ‘sink’ that binds to and inhibits granzyme B, preventing target cell death [15]. Andrade et al. now propose that by cleaving DBP and L4–100K, granzyme H participates in the anti-adenovirus response by both inhibiting virus replication directly and simultaneously re-sensitizing the infected cell to granzyme B-induced death (Figure 1). Direct cleavage of adenovirus proteins by granzymes B and H Adenoviral DNA replication begins with coating of the viral genome by DBP, which in turn has affinity for nuclear factor I (NFI), a cellular transcription factor that binds to the viral origin of replication. Following transcription, individual hexon and penton monomers form into capsomers in the cytoplasm and are assembled into immature capsids in the nucleus. L4–100K is an assembly protein involved in the transport of hexons from the cytoplasm to the nucleus. Virus particles accumulate in the nucleus and the infected cell remains intact; however, persistent infection can result in cell lysis, permitting virus escape and re-infection. Andrade et al. first noted that DBP (70 kDa) was cleaved to a 47 kDa product in adenovirus 5 (Ad5)infected K562 cells exposed to lymphokine activated killer (LAK) cells [13]. Cleavage of DBP was blocked by calcium chelation, suggesting the involvement of the calcium-dependent granule exocytosis pathway in this process. Recombinant DBP overexpressed in K562 cells was also cleaved to a predominant 47 kDa fragment, but less prevalent fragments of 50 kDa and 60 kDa were also observed. The addition of a granzyme B inhibitor abolished the 50 and 60 kDa forms, whereas caspase inhibition prevented appearance of the 60 kDa fragment. These data suggested that granzyme B cleaves DBP to the 50 kDa fragment and caspases activated as a consequence of granzyme
374
Update
TRENDS in Immunology Vol.28 No.9
Figure 1. CTL versus adenovirus. The final outcome, death of the infected cell or production of infectious virus, seems to involve a complex competition between granzymes and virus proteins. Granzyme B expressed by CLs induces death of an infected cell by apoptosis. Adenovirus L4–100K blocks granzyme B, thereby preventing apoptosis and permitting virus replication. CLs also express granzyme H, which cleaves L4–100K and prevents it from blocking granzyme B. This resensitizes the cell to granzyme B-induced apoptosis. Granzyme H also cleaves DBP, inhibiting efficient replication of virus DNA, and limiting the production of infectious virus particles.
B-induced death cleave some of the remaining full-length DBP to the 60 kDa fragment. It remained unclear how DBP was cleaved to the 47 kDa fragment, but Andrade et al. made the key finding that purified granzyme H (but not granzyme A, K and M) could cleave in vitro transcribed and translated DBP, or DBP in lysates from Ad5-infected K562 cells. Cleavage occurred either at Met118 or Phe121, or possibly at a unique site that spans these two residues. Significantly higher viral titres resulted when K562 cells infected for 12 h with adenovirus expressing DBP that was mutated at Met118 and Phe121 were exposed to LAK cells, compared with K562 cells infected with wild-type adenovirus. This strongly suggested an essential role for granzyme H-mediated cleavage of DBP in restricting adenovirus replication. As DBP can be cleaved by both granzyme B and granzyme H, Andrade et al. next tested whether both proteases could also cleave L4–100K. Crucially, they found that L4– 100K protein was cleaved by granzyme H to a 90 kDa fragment that rendered it unable to block granzyme B. This was demonstrated by the observation that granzyme B-mediated cleavage of caspase-3 was delayed by the addition of L4–100K but restored if granzyme H was also added, despite the fact that granzyme H was incapable of directly processing pro-caspase-3. The findings strongly suggested that a crucial role for granzyme H is to rescue the pro-apoptotic activity of granzyme B in Ad5-infected cells, while co-incidentally having a direct negative effect on capsomer assembly and viral titre. Of the 49 adenovirus serotypes that infect humans, subgroup A induces tumours in rodents with high frequency and short latency, subgroup B is weakly oncogenic and subgroup C, D, E and F are non-oncogenic. By contrast, adenovirus-induced oncogenesis has never been observed in humans. Among the many possible mechanisms that might explain this difference, the low oncogenic potential of adenoviruses in humans could at least partly reflect the more efficient limitation of viral replication and spread www.sciencedirect.com
imposed by the human immune system. Therefore, it would be interesting to know whether DBP can be cleaved to the 47 kDa form when adenovirus-infected mouse cells are exposed to mouse LAK cells, resulting in a direct negative effect or virus replication. As there is no direct mouse orthologue of granzyme H, this function would presumably rely on an alternative granzyme(s) that, like granzyme H, has chymotrypsin-like cleavage specificity [16]. Interestingly, the DBP cleavage sites for granzymes B and H are not conserved in all adenovirus subgroups. The P1 residue for the principal predicted granzyme H site, Phe121, seems to be well conserved in human and simian isolates, and the granzyme B site is also partly conserved (Table 1); however, Met118 and the recognition site for caspases are not well conserved. It is therefore likely that the cleavage of DBP is restricted to specific adenovirus subgroups, particularly for granzyme B. Generalizing from this observation, it is possible that the diverse cleavage specificities of the granzymes might reflect the species-specific co-evolution of cytotoxic effector mechanisms and strategies used by many viruses for immune escape. Consistent with this possibility, Andrade et al. have also previously shown that the L4–100K protein from Ad5 fails to be cleaved efficiently by, or to inhibit, mouse granzyme B [17]. In turn, this finding is consistent with several recent independent reports showing that the substrate preferences (and associated kinetics) of mouse and human granzyme B are significantly different [18–20]. Therefore, even clearly orthologous granzymes seem to have evolved species-specific functions in response to relevant and dangerous infectious agents. This principle is probably not limited to granzymes and viruses, as a bacterial virulence factor can also be inactivated by neutrophil elastase [21]. Although the study by Andrade et al. makes a strong case for a direct anti-viral role for granzyme H, it was recently reported that granzyme H could also be directly cytotoxic to target cells when delivered with perforin [22]. Recombinant granzyme H was able to induce mitochondrial depolarization, reactive oxygen species production, chromatin condensation and DNA fragmentation but did not involve cleavage of the pro-apoptotic Bcl-2 family member Bid or inhibitor of caspase-activated death (ICAD), release of cytochrome c from the mitochondrial intermembrane space or activation of caspases. The data suggest that in addition to having a constitutive anti-viral role, granzyme H might also be capable of killing certain target cells directly. A pro-death role for granzyme H was not uncovered by Andrade et al.; therefore, it would be intriguing to know whether granzyme H can kill adenovirus infected cells in the absence of granzyme B. It is also conceivable that the direct anti-viral and pro-apoptotic activities of granzyme H might require significantly different concentrations of intracellular granzyme H, so that a hierarchy of effector mechanisms might operate, depending on how much granzyme H activity can be delivered into an infected cell. Future studies in viral immunology The findings of Andrade’s group should provide a strong impetus for further identification of viral substrates for the granzymes. Crucially for future studies, granzymes B and H share significant structural similarity, and some of the
Update
TRENDS in Immunology
Vol.28 No.9
375
Table 1. Potential cleavage sites for granzyme B and granzyme H in DBP from various adenovirusesa Host species Human
Serotype 1
DBP sequence
Accession No AP 000514
Human
2
AP 000177
Human
5
AP 000213
Human
3
ABB 17782
Human
7
AP 000550
Human
11
AP 000454
Human
35
AP 000587
Monkey
21
AP 000277
Human
52
ABK 35046
Human
46
AAX 70936
Dog
1
AP 000061
a
DBP protein sequences from various selected adenoviruses that specifically infect humans, monkeys or dogs were obtained from Pubmed (http://www.ncbi.nlm.nih.gov/ sites/entrez?db=PubMed) and potential cleavage sites for granzyme H (after f or m) and granzyme B (after d) are highlighted. The single amino acid code is used, and residue numbers for each of the DBP sequences are indicated.
antibodies used to detect granzyme B cross-react with granzyme H [23]. Careful studies with monospecific antibodies have shown that circulating, unstimulated NK cells that have relatively low cytotoxic capacity express high levels of granzyme H but relatively little granzyme B [23]. These data might predict a role for granzyme H in the immediate innate immune response to virus, independent of or complementary to NK-induced killing. In concert with cytokines such as interferon-g, a role for granzyme H in limiting early viral replication would be consistent with the acute need to minimize viral load until a cognate CTL response can be raised. It would therefore be intriguing to know if the yield of productive virus is reduced in adenovirus-infected cells exposed to naive NK cells, and whether DBP is cleaved in infected cells that survive NK cell cytotoxic attack. It is interesting that NK cells stimulated in vitro with interleukin-2 express progressively greater amounts of perforin and granzyme B but reduce their granzyme H levels over 3–4 days [23]. This pattern could be consistent with a change in emphasis from viral containment to cytotoxicity against infected cells (possibly mediated by both NK cells and CTLs) in the days following infection. In conclusion, the study of Andrade et al. presents compelling evidence that granzyme B and granzyme H can directly and cooperatively cleave virus proteins that are essential for virus replication, and that granzyme H participates in the anti-virus response by neutralizing an inhibitor of granzyme B. Characterizing such roles for granzymes at the molecular level and demonstrating that different granzymes can act cooperatively to thwart viruses is sure to have a renewed focus in future immunology and virology research.
7
8 9
10
11
12
13
14
15
16
17
18 19
References 1 Johnson, H. et al. (2003) Cell death induced by granzyme C. Blood 101, 3093–3101 2 Kelly, J.M. et al. (2004) Granzyme M mediates a novel form of perforindependent cell death. J. Biol. Chem. 279, 22236–22242 3 MacDonald, G. et al. (1999) Mitochondria-dependent and -independent regulation of granzyme B-induced apoptosis. J. Exp. Med. 189, 131–144 4 Martinvalet, D. et al. (2005) Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis. Immunity 22, 355–370 5 Voskoboinik, I. et al. (2006) Perforin-mediated target-cell death and immune homeostasis. Nat. Rev. Immunol. 6, 940–952 6 Waterhouse, N.J. et al. (2006) Functional dissociation of DCm and cytochrome c release defines the contribution of mitochondria www.sciencedirect.com
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