Immunology and Cell Biology

(2004)

82

, 161–169 doi:10.1046/j.0818-9641.2004.01225.x

© 2004 Australasian Society for Immunology Inc.

Special Feature

Granzymes in cytolytic lymphocytes – to kill a killer?

M A T T H I A S R E G N E R a n d A R N O M Ü L L B A C H E R

Molecular Immunology and Immunopathology, John Curtin School of Medical Research, Australian National University, Canberra, Australia

Summary

Granzymes (gzm) are major components of the granules of cytolytic lymphocytes, natural killer andcytotoxic T cells. Their generally accepted mode of action consists of their directed secretion towards a virus-infected or neoplastic target cell and perforin-dependent delivery to the target cell cytosol, where they engage invarious actions resulting in target cell apoptosis. Here, based on observations of infection of gzmAxB

–/–

mice withectromelia virus, mousepox, we propose an additional – and distinct – function for gzmA and B. In this model, gzmconstitute one of the first lines of defence of immune cells against virus infection of immune cells themselves.Accordingly, endogenous gzm interfere with viral replication in cytolytic lymphocytes either directly, as a result oftheir proteolytic activity, leading to destruction of viral proteins, or indirectly, via: (i) processes akin to the caspasecascade when acting as effector molecules in the induction of target cell apoptosis; or (ii) their capacity to induceearly inflammatory mediators. We discuss the predictions of the model in the light of available data.

Key words

:

cytolytic granule, cytotoxic T cell, ectromelia, granzymes, innate, natural killer cell.

Background

Antiviral cytolytic mechanisms

Cytolytic lymphocytes (CL), NK and cytolytic CD8

+

T (Tc)cells, play a crucial role in the recovery from primary viralinfections in vertebrates. NK and Tc cells, upon encounterwith cognate target cells, exert their biological activity viatwo quite distinct means. One is the synthesis and release ofcytokines such as IFN-

γ

,

1

TNF-

α

,

2

and interleukins,

3

whichact proximal and distal to the effector cell. The other is directcytolysis and induction of apoptosis of the recognized targetcell. These two effector mechanisms are not necessarilyexecuted by the same cell

4

and are governed by distinctavidity thresholds.

5

Induction of apoptosis and lysis of target cells by CL canbe executed via at least two pathways. One is the exocytosis-mediated pathway executed by perforin (perf) and granzymes(gzm); the other is the Fas pathway involving the Fas ligand(FasL) on the effector cell engaging the Fas receptor (Fas/CD95) on the target cell.

6–13

It was generally believed that theexocytosis pathway is primarily involved in the eliminationand/or control of intracellular pathogens such as viruses,whereas the Fas pathway of cytotoxicity was thought to be inessence immunoregulatory.

11,13,14

However, recent evidencesuggests that in certain viral infections both pathways areinvolved in recovery or immunopathology.

15–20

Granzymes A and B

The three most abundant components present in cytolyticgranules and released by effector cells during degranulationare perf and the two gzm, gzmA and gzmB.

7,21

The gzm are afamily of serine proteinases found in cytoplasmic granules ofCL.

22–24

The role of none of the three principal molecules incytolysis and viral clearance is yet completely understood.

25,26

Perf was initially thought to act primarily on the cell surfaceof target cells facilitating the entry of the other components ofcytolytic granules.

7

Some recent evidence suggests that gzmenter the target cell independently of perf, but that perf isessential for both their release from endosomes as well astheir activation and nuclear translocation.

26–28

Evidence thatgzm may function even in the absence of perf is documentedin this issue.

20

The function of the two gzm in cytolysis iseven less clearly understood. Their different substrate specif-icity,

29,30

chromosomal gene location,

31,32

and structure,

29,30

strongly suggests that these two enzymes have evolved sepa-rately, and one would anticipate their functions not to bemutually redundant. In fact, recent studies demonstrate thatboth gzm are involved in apoptotic processes leading to DNAdamage and/or fragmentation, albeit by employing alternativepathways (see following).

Gzm are post-translationally targeted as pro-proteases tothe secretory granules by the mannose-6-phosphate-receptor(M6PR).

33

Dipeptidyl peptidase I is then required for thecleavage of the pro-enzymes into active proteases.

34

However,in the acidic environment of the cytolytic granule, gzm arestored in an inactive form, in a supramolecular complex withperf and the proteoglycan, serglycin.

35

This has been sug-gested to protect CL from the potentially disastrous effects ofharbouring the free active effector molecules.

36

Upon degran-ulation and consequent elevation of pH in the extracellularspace between effector and target cell, gzm may dissociatefrom serglycin and gzmB can then gain access to the cytosol

Correspondence: Matthias Regner, Molecular Immunology andImmunopathology, Division of Immunology and Genetics, JohnCurtin School of Medical Research, Australian National University,GPO Box 334, Canberra, ACT 2601, Australia. Email: Matthias.Regner@anu.edu.au

Received 5 December 2003; accepted 5 December 2003.

162

M Regner and A Müllbacher

by M6PR-mediated endocytosis.

37

However, other workershave recently shown that M6PR is not essential for gzmBuptake.

38,39

Rather, the entire complex of gzm/serglycin, pos-sibly with bound perforin, is endocytosed, whereupon gzmgain access to the cytosol via a perforin-dependent, but yetunelucidated, mechanism.

35

Some of the gzm also locaterapidly to the nucleus, by a yet undefined mechanism. Oncein the cytosol, gzmA and B target distinct pathways leading totarget cell apoptosis.

GzmB can initiate apoptosis by at least two pathways,resulting in either caspase-dependent or independent apopto-sis. One triggers a caspase cascade, similar to the one result-ing from Fas ligation, that leads to the classical apoptotichallmarks of chromatin condensation, membrane blebbing,nuclear fragmentation and oliconucleosomal DNA fragmenta-tion (reviewed in Young

et al

.

25

and Trapani

et al

.

40

).

In vitro

,gzmB can target a number of different caspases, but cleavageof caspase-3 seems to take a predominant role, which thenleads to activation of downstream caspases, ultimatelyleading to apoptosis.

41

In addition to the generation of pro-apoptotic molecules, there also seems to be a requirement forthe relief of constitutive caspase inhibition for apoptosis tooccur.

42

The alternative pathway centres on disruption of themitochondrial membrane integrity. GzmB-mediated cleavageof Bid

43

results in recruitment of the pro-apoptotic Bcl-2family member Bax to the mitochondrial membrane, disrupt-ing the membrane potential. This leads to release of a numberof stress factors from the mitochondria, including cytochromeC.

44

Ultimately, cytochrome C release leads to cell death bynecrosis, but mitochondrial damage may also amplify thecaspase-dependent pathway by releasing caspase-9, itself anactivator of caspase-3, from a complex termed apoptosome(reviewed in Cain

et al

.

45

).Whichever pathway dominates

in vivo

, gzmB is clearly apluripotent effector molecule. Its multipronged action islikely to have evolved in response to strategies of pathogensto evade one or the other pathway.

The existence of virally encoded specific inhibitors dem-onstrates the relevance of gzmB in the immune response tocertain virus infections. Adenovirus encodes a highly specificgzmB pseudo-substrate, L4 100 kDa protein.

46

The cowpoxserin protease inhibitor (serpin) SPI-2, inhibits gzmB

invitro

,

47

although its

in vivo

relevance for inhibition of thiseffector pathway is questionable because it does not inhibittarget cell lysis by MHC-restricted murine effector Tc cells.

48

SPI-2 more likely targets the Fas pathway, which relies exclu-sively on the caspase cascade for initiation of apoptosis.

48–52

GzmB’s physiological importance is also supported by theexpression of endogenous inhibitors in human CL and DC,such as PI-9.

53

PI-9 is a highly specific inhibitor of gzmB andlikely serves to protect effector CL from misdirected gzmBduring degranulation,

54

but may also protect antigen-presentingcells such as DC from immune elimination before they haveserved their task of antigen presentation to T cells. There arealso several murine homologues of PI-9.

55,56

GzmA-induced apoptosis is entirely independent ofcaspase activation.

57

Also, the DNA laddering characteristicof apoptotic pathways, executed by gzmB or Fas-mediated, isabsent. Other apoptotic features, such as membrane blebbing,chromatin condensation and nuclear fragmentation, areobserved in apoptosis mediated by either gzm. This distinct

pathway of apoptosis results from gzmA disrupting the so-called SET complex, an assembly containing oxidativestress repair enzymes.

58,59

Thus, gzmA may block cellularrepair in response to stress factors. In parallel, gzmA cleaveshistones and other proteins with nucleosome assembly func-tion.

60

This leads to chromatin unwinding and rendersgenomic DNA susceptible to cleavage by exogenous nucle-ases.

61

Mitochondrial damage has also been suggested tooccur, but without release of cytochrome C or other apoptoticdeath mediators.

62

No viral inhibitors of gzmA have been described to date,but the discovery of an endogenous inhibitor, pancreaticsecretory trypsin inhibitor (PSTI), expressed in the gut,

63

suggests that gzmA action in the intestine needs to beregulated to avoid unwarranted tissue damage.

Granzymes as effector molecules in ectromelia virus infection

Considering these diverse modes for induction of apoptosis, itis surprising that CL from gzmAxB

–/–

mice are still able to killtarget cells

in vitro

.

12,64,65

Furthermore, gzmAxB

–/–

mice haveno deficiency in controlling selected tumours that are sensi-tive to CD8

+

T cell surveillance.

66

The most important physi-ological role for gzm is probably their role in protection of thehost from viral infections.

18,20,64,67–69

Ectromelia virus (EV) is a natural mouse pathogen,causing mousepox, and provides a small animal model forhuman variola virus infection.

70,71

EV also provides an excel-lent animal model for a cytopathic viral infection.

72,73

Forrecovery from EV, both perf and the gzm are absolutelyessential. C57BL/6 (B6) mice, while highly resistant to EV

74

(LD50 > 10

6

plaque forming units (PFU) subcutaneously),succumb to EV infection with >10

1

PFU in the absence ofeither perf or both gzmA and B.

48,75,76

Absence of only one ofthe two gzm increases susceptibility, but to a lesser extent:gzmA deficiency makes them less susceptible than gzmBdeficiency.

64,69

The very clear phenotype observed with perf-and gzm-deficient mice with mousepox contrasts with otherviral models. This most likely reflects the inhibition of the Faspathway of cytotoxicity by the EV encoded serpin SPI-2,

48,77

which is operative in other viral models and, at least partially,may compensate for lack of exocytosis-mediated cytolysisand apoptosis of virally infected cells by Tc and NKcells.

15–18,20

Consistent with the notion that gzm are effector moleculesof cytolytic lymphocytes, expression of gzmA and B has beenshown to be restricted almost exclusively to activated T andNK cells, with gzmA also being expressed during T celldevelopment

78,79

and in long-term memory T cells.

79–81

Norole other than effector molecules initiating apoptotic targetcell death mediated by effector cytolytic cells has yet beenproposed for gzm.

However, in light of the kinetics of EV replication in gzm-deficient mice

64

and the ability of gzmAxB

–/–

CL to kill targetcells,

64,65

we postulate that gzm have an additional function tothat of effector molecules in CL.

Hypothesis

We hypothesize that endogenous gzm in lymphocytes, eitherpresent or induced as a result of viral infection, provide one of

Granzymes – to kill a killer?

163

the first lines of defence to immune cells against viraldissemination. These gzm interfere with viral replicationeither directly as a result of their proteolytic activity, leadingto destruction of viral proteins, or indirectly via: (i) processesakin to the caspase cascade when acting as effector moleculesin the induction of target cell apoptosis; or (ii) their capacityto induce early inflammatory mediators essential in an anti-viral response.

Based on this hypothesis, several testable predictions canbe made (Table 1).

Predictions

Virus dissemination by CL

Antiviral CL are uniquely at risk of infection, given that theyactively search out virus-infected loci and stay for prolongedtimes within infected tissues while delivering effector func-tions. Their high mobility would seem to make them a highlydesirable vehicle for viruses to ascertain rapid spread through-out the host. Viruses that do not infect endothelial cells, thusbeing unable to enter or leave the bloodstream to infectperipheral tissues, would benefit enormously from the abilityof activated CL to extravasate and infiltrate virtually anyorgan. The utility of lymphocyte trafficking for viral spreadmay be one reason why HIV, via infection of CD4

+

Tlymphocytes, is successful in reaching reservoirs wherelatency may prevail, before the immune response can mounteffective control measures. Recent findings of membrane –and perhaps cytoplasmic – exchange between CL and targetcells,

82–84

strengthen the notion that CL are at high risk ofpassively acquiring viruses themselves. Indeed, EV spreadduring primary viremia has been found to be mainly leucocyte-associated,

85–87

although the phenotype of the cell(s) responsiblehas not been determined.

Residual presence of gzm in the CL after the lethal hit

For the reasons outlined above, it is likely that CL would haveevolved strategies to avoid infection and/or combat passivelyacquired virus, by enforcing suicide on themselves withoutrequiring fratricide, to be committed by a bystander virus-immune cell. If indeed gzm turn out to be one such safetydevice, ensuring that a virus-infected CL does not turn into aTrojan horse, one would expect that endogenous gzm in CLare produced in excess to the quantities required as effectormolecules during target cell killing. Indeed, it has beendemonstrated that substantial amounts of gzm remain in theCL after extensive cytolytic effector function.

88,89

Isaaz

et al

.

88

and others interpreted this as a mechanism enabling the CL toengage in ‘serial killing’ of successive target cells, whichdoes occur, but it is also consistent with the idea that somecytolytic effector molecules are retained as a contingency incase of infection. Intriguingly, the same study showed that alarge proportion of newly, in response to initial TCR trigger-ing, synthesized gzm, instead of being trafficked for storageto the granules, are secreted via the constitutive secretorypathway.

88

Granule exocytosis was not required for thissecretion to occur and it was not observed for perforin. Thisfinding could be explained if the observed de novo gzmsynthesis were primarily a mechanism of self-defence of the

CL. In this scenario, while in apposition to the antigen-presenting cell, flooding the secretory pathway and the extra-cellular space, as well as the granules, with gzm, couldprovide a means by which the CL could surround itself by acloud of ‘friendly fire’. Due to the lack of perforin co-secretedwith the gzm, these would not gain access to the CL cytosol.However, if cell membrane integrity were to be disturbed –for example, by fusion/endocytosis of virions (see following)– these gzm could enter the cytoplasm and mediate killing ofthe killer (Fig. 1). Once in the cytosol, two effector mechan-isms could be envisaged, by which gzm could counter viralreplication. One involves the regular functions that gzmperform in target cells, thus inducing caspase-dependent orindependent apoptosis of the CL. Alternatively, gzm couldcleave viral proteins synthesized de novo.

Cytosolic presence of granzymes

In order to initiate their respective apoptosis pathways, gzmwould therefore be required to gain access to the cytosol ofthe recently infected cell. This could be achieved by either anactive release/transport across the granule membrane or byinadvertent leakage due to processes or signals associatedwith viral entry.

To date, there is no evidence for an active relocation ofgranule enzymes into the cytosol. However, the granulesof NK cells are an intriguing hybrid of vesicles that arenormally considered to belong to the endosomal/lysosomal orthe exocytic lineage.

90

They display an intermediate morphol-ogy, consisting of domains featuring a homogenous, round,electron-dense core containing proteins destined for secretion,encapsulated by lamellar, multivesicular structures that harbourmainly lysosomal proteins. Burkhard

et al

. termed these gran-ules ‘secretory prelysosomes’.

90

The two suborganelle struc-tures were sometimes, but not always, found to be separatedby a thin cortex surrounding the electron-dense part of theorganelle. Furthermore, some interchange between the pro-teins predominant in these suborganelles was detected.

90

Tccell granules have essentially the same morphology.

90–93

Thus,presence of granule enzymes at a key point in the endocyticpathway may allow them to escape their secretory destiny.

Indeed, Brunk

et al.

while studying responses of fibro-blasts to stress, observed partial relocalization of lysosomalenzymes into the cytosol, due to disrupture of lysosomalmembrane integrity.

94,95

Interestingly, while high levels ofUV-induced stress lead to necrosis, moderate irradiation

Table 1

Predictions by the hypothesis

Obligatory

GzmAxB

–/–

CL are an important vehicle for virus dissemination.Residual presence of gzm in the CL after the lethal hit.Access of gzm to the cytosol of the CL.Higher viral replication in the absence of gzmA and gzmB.

Facultative

Reduced apoptosis in gzmAxB

–/–

infected cells.Gzm-mediated activation of IL-1

β

and/or IL-18.Gzm induction upon virus infection in cells other than CL.

Granzymes (gzm) A and B; cytolytic lymphocytes (CL).

164

M Regner and A Müllbacher

resulted in apoptosis. The authors proposed that ‘proteasesand endonucleases of lysosomal origin may induce apoptosisif relocalized from the acidic vacuolar compartment into thecytosol’.

94

Similar investigations have not been conducted onCL, but it is tempting to speculate that leakage of granulecontents would not be limited to lysosomal proteins if suchcells were exposed to stress. Entry of viral particles, bywhatever pathway, might provide the necessary physical orbiochemical stress signals.

Even in non-stressed CL there is recent evidence ofextragranular presence of gzmB, as demonstrated in the NK-like cell line YT,

54

suggesting that some gzm leakage may be

common. In that report, cytoplasmic gzmB was observedcomplexed with PI-9, which was demonstrated to effectivelydegrade gzmB, thus preventing initiation of the death path-way. In CL that are subjected to stress factors, this presum-ably constitutive marginal leakage might reach levels wherethe protective effect of PI-9 may be exceeded. Alternatively,under inhibition of host protein synthesis, such as during poxvirus infection, sufficient supply of an inhibitor could bejeopardized, leaving gzmB activity unchecked.

The very nature of viral entry into cells seems to ensurean intersection with gzm (Figs 1 and 2). Pox viruses spreadbetween cells as extracellular, enveloped virions (EEV;

Figure 1

Death by ‘friendly fire’. Rapidly after granule exocytosis, de novo granzyme (gzm) synthesis is initiated. While some of thisnewly synthesized gzm is trafficked to granules for replenishment of the cytolytic lymphocyte’s (CL) cytolytic machinery, a proportion issecreted via the constitutive secretory pathway, independent of TCR triggering. This results in a ‘flooding’ of exocytic pathways and theimmediate extracellular space with gzm that could be re-internalized, particularly if endosomal uptake of virions were to occur.Perturbation of the lysosomal membrane during fusion with the virion membrane could result in leakage of internalized gzm that then carryout their effector function in the CL. TGN,

trans

-Golgi network; A, gzmA; B, gzmB; per, perforin; EEV, extracellular enveloped virion (ofectromelia).

Granzymes – to kill a killer?

165

reviewed in Smith

et al

.

96

), and may be cell-associated.

97

Viralentry involves endocytosis of the EEV particle, followed bylow pH-induced disruption of the wrapping membrane inan early lysosomal/late endosomal compartment.

97,98

Theexposed ‘intracellular mature virion’ (IMV) then fuses withthe endosomal membrane, releasing the viral core into thecytoplasm (Fig. 2). Intriguingly, this mode of virus entryappears to run almost parallel to the proposed entry of gzminto the target cell cytosol. This model suggests that EEVparticles travel along the endocytic pathway at least until theprelysosomal stage. In CL, this would suggest an intracellularcompartment where endocytosed virus and granular enzymesco-localize. It is therefore tempting to speculate that the finalIMV fusion perturbs the endosomal membrane in a way thatallows granule protein leakage into the cytosol beyond consti-tutive levels.

54

Alternatively, virus entry may trigger specific

stress signals that allow gzm leakage, or active transport,across the prelysosomal membrane.

Apart from experimental evidence that endosomolyticagents, such as listeriolysin, can deliver ‘exogenous’ gzmB tothe cytosol of cells,

99

adenovirus has clearly been demon-strated to be able to allow gzmB to co-translocate fromendosomes into the cytosol.

27

Higher viral replication in the absence of gzmA and gzmB

If CL lacking gzm are indeed susceptible to infection and amajor vehicle for viral dissemination, then one shouldexpect more rapid dissemination and increased rates of viralreplication in target organs. Experimental evidence supportsthis idea. In wild-type (wt) B6 mice, virus is undetectable inliver and spleen up to day 4 post-immunization (p.i.), and

Figure 2

The ‘secretory pre-lysosome’ intersection. Cross-traffic between the exocytic and the endocytic pathways permits granulecontents to enter the endocytic pathway. Thus, gzm intersect with a pathway of virus entry into the cell. Perturbation of the lysosomalmembrane during fusion with the virion membrane could result in leakage of internalized gzm that then carry out their effector function inthe CL. TGN,

trans

-Golgi network; A, gzmA; B, gzmB; per, perforin; EEV, extracellular enveloped virion (of ectromelia).

166

M Regner and A Müllbacher

peaks at d6 to d8, reaching titres of 10

5

–10

6

plaque-formingunit (pfu). In contrast, gzmAxB

–/–

mice have detectable virusin both organs already at 2–3 days p.i. At this time point

ex vivo

NK nor Tc cell reactivity is similar in these two strains(unpublished observation). Virus titres in spleen and liver ofgzmAxB

–/–

are 2–3 logs higher than wt at days 6–8 p.i.(Table 2). Wild-type mice clear the infection and recover,whereas gzmAxB

–/–

mice succumb to infection. Althoughother factors, such as impaired NK cell function, cannot beruled out, cytolytic function, as measured by

51

Cr-releaseassays, is not compromised in gzmAxB

–/–

mice.

65

Theseobservations are consistent with a role of gzm early ininfection when Tc cell activity is not yet measurable. Inaddition, we have preliminary evidence that splenocytes fromgzmAxB

–/–

mice support substantially higher replication ofEV

in vitro

(not shown). In this instance, it is unlikely thatinnate cytolytic effector function could account for thesedifferences.

Reduced apoptosis of infected gzmAxB

–/–

leucocytes

Pox virus infection is commonly associated with a block ofany cell-intrinsic apoptotic pathway. This is mediated by arange of pox virus-encoded antiapoptotic proteins, such asBcl-2 homologues and serpins,

51

and has evolved to ensurethe survival of the infected cell until successful viral replica-tion has occurred.

Higher viral replication in tissues and cells of gzmAxB

–/–

mice

in vivo

may be the consequence of either a lack of directinhibition of the EV replicative cycle by gzm, or due to

inability of normally gzm-expressing cells to commit suicide.In the latter case, gzm gaining access to the cytosol concomi-tantly with, or early after, virus entry may pre-empt thedisposition of the viral antiapoptotic machinery. Interestingly,the higher virus yields (mentioned previously) from infectedgzmAxB

–/–

mice correlate with a higher viability of spleno-cytes from these mice compared to infected wild-type cellsin vitro (not shown), a surprising finding in light of the factthat pox viruses are considered highly cytopathic. In fact,long-term cultures of infected gzmAxB–/– splenocytes (notshown) are readily established. We also have preliminaryevidence of a reduction of the number of apoptotic cells inspleen, liver and draining lymph nodes of gzmAxB–/– mice2 days after EV infection (not shown), although this does notseem to be restricted to T cells.

A helper function in the initiation of the immune response?

Our discussion has so far concentrated on the role of gzmAand B in CL, but it is also possible that gzm are induced incells other than CL, as a consequence of viral infection.Although there is little experimental evidence for this to occurwidely, expression of gzmB and perforin mRNA and proteinby human keratinocytes in response to growth to confluencehas been reported.100 In any case, de novo gzm expressionwould have to occur very rapidly in order to pre-empt poxviral inhibition of host protein synthesis. Should this ‘ectopic’expression of gzm occur in cells such as macrophages, thiscould result in modulation of cytokine responses, since gzmAdisplays interleukin-1beta converting enzyme (ICE)-like

Table 2 Kinetics of viral replication in vivo (modified from Müllbacher et al.)64

Strain Mouse no. Day p.i.

Liver Spleen

Infiltration/necrosis Virus titre Congestion/necrosis Virus titre

B6 1 3 – < 200 – < 2002 ± < 200 – < 2003 – < 200 – < 2001 6 – 7 × 105 – 1.3 × 105

2 – 7.5 × 105 – 1.4 × 105

3 ± 9 × 105 – 5.3 × 104

1 8 ± 2.3 × 105 – 1 × 105

2 + + 4 × 104 ± 5 × 103

3 + 3 × 103 – 8 × 103

1 10 + < 200 + 1 × 105

2 + < 200 ± < 2003 ± < 200 + 7 × 103

GzmAxB–/– 1 3 – 1 × 103 – 1.7 × 103

2 ± 1.2 × 103 – 2.1 × 103

3 † †1 6 + 4 × 107 + + + 1.7 × 107

2 + + 2.4 × 107 + + + 2.5 × 107

3 † †1 8 + + + 1.3 × 108 + + + + 1.5 × 107

2 + + + 7 × 107 + + + + 6 × 106

3 + + + + 1.2 × 108 + + + + 5.5 × 107

1 10 † †2 † †3 † †

† Mouse died. Liver: increase in numbers of foci and/or areas affected by cellular infiltration and/or necrosis. Scoring from ±, very few scatteredfoci of cellular infiltrates and/or necrosis, to + + + +, confluence of necrosis in tissue specimens. Spleen: increase of congestion of sinuses in redpulp and/or of foci of necrosis of white pulp. Scoring from ±, little congestion/very few scattered foci of necrosis, to + + + +, extensive congestion/confluence of necrosis in white pulp. p.i, post-infection.

Granzymes – to kill a killer? 167

activity and can, in vitro, release bioactive IL-1β from itsinactive precursor.101 Because IL-18 is also activated by ICE,the same might also apply for this interleukin. By virtue of itsbroad substrate specificity, gzmB activity could also modu-late such mechanisms, for example, by activation of membersof the ICE/Ced3 family.41,102,103 Thus, their enzymatic activi-ties could be involved in the production of bioactive IL-1βand IL-18, early inflammatory cytokines that are importantantiviral mediators in response to pox virus infection ingeneral,104,105 and in EV infection in particular.106

Finally, two intriguing recent reports have clearly demon-strated a role for caspase-8 in T cell homeostasis and T cell-mediated immune responses.107,108 Caspase-8 is immediatelyupstream of caspase-3 in the caspase cascade initiated by Fassignalling. Based on their ability to modulate this cascade,gzm may have the potential to modulate T cell responsesand/or homeostasis.

Concluding remarks

In vivo evidence suggests a role for gzmA and gzmB in innatedefence against viral infection, distinct from their role incytolytic effector function. Available evidence is consistentwith a possible mechanism for apoptosis of virus-infectedcells induced by endogenous gzm.

Acknowledgement

We thank Markus Simon for valuable discussion.

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