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The Journal of Immunology, 2005, 175: 3235-3243.
Copyright © 2005 by The American Association of Immunologists

Functional Analysis of Granzyme M and Its Role in Immunity to Infection1

Lily I. Pao2,§, Nital Sumaria2,{dagger},{ddagger}, Janice M. Kelly2,*, Serani van Dommelen{dagger},{ddagger}, Erika Cretney*, Morgan E. Wallace*, Desiree A. Anthony*, Adam P. Uldrich, Dale I. Godfrey, John M. Papadimitriou||, Arno Mullbacher#, Mariapia A. Degli-Esposti{dagger},{ddagger} and Mark J. Smyth3,*

* Cancer Immunology Program, Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; {dagger} Immunology and Virology Program, Centre for Opthalmology and Visual Science, The University of Western Australia, Crawley, Western Australia, Australia; {ddagger} Centre for Experimental Immunology, Lions Eye Institute, Nedlands, Western Australia, Australia; § Cancer Biology Program, Division of Hematology-Oncology, Beth Israel-Deaconess Medical Center and Havard Medical School, Boston, MA 02215; Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; || Department of Pathology, University of Western Australia, Crawley, Western Australia, Australia; and # Division of Immunology and Genetics, John Curtain School of Medical Research, Australian National University, Canberra, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytotoxic lymphocytes express a large family of granule serine proteases, including one member, granzyme (Grz)M, with a unique protease activity, restricted expression, and distinct gene locus. Although a number of Grzs, including GrzM, have been shown to mediate target cell apoptosis in the presence of perforin, the biological activity of Grz has been restricted to control of a number of viral pathogens, including two natural mouse pathogens, ectromelia, and murine CMV (MCMV). In this article, we describe the first reported gene targeting of GrzM in mice. GrzM-deficient mice display normal NK cell/T cell development and homeostasis and intact NK cell-mediated cytotoxicity of tumor targets as measured by membrane damage and DNA fragmentation. GrzM-deficient mice demonstrated increased susceptibility to MCMV infection typified by the presence of more viral inclusions and transiently higher viral burden in the visceral organs of GrzM-deficient mice compared with wild-type (WT) mice. The cytotoxicity of NK cells from MCMV-infected GrzM-deficient mice remained unchanged and, like WT control mice, GrzM-deficient mice eventually effectively cleared MCMV infection from the visceral organs. In contrast, GrzM-deficient mice were as resistant as WT control mice to mouse pox ectromelia infection, as well as challenge with a number of NK cell-sensitive tumors. These data confirm a role for GrzM in the host response to MCMV infection, but suggest that GrzM is not critical for NK cell-mediated cytotoxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytotoxic lymphocytes, including NK cells, recognize and kill tumor cells and host cells infected with intracellular pathogens such as viruses and certain types of bacteria (1). Death of target cells is predominantly mediated through granule exocytosis (2, 3). In this process, lysosome-like vesicles, whose principal components are perforin and a family of serine proteases known as the granzymes (Grz),4 are secreted toward the target cell. Perforin released from cytotoxic granules facilitates the entry of Grz into the target cell, probably through a process of endosomal disruption (3). NK cells also control viral infections in large part via their secretion of cytokines, including IFN-{gamma} (4).

The five known human Grz have varied primary substrate specificities. GrzA and K cleave after basic residues (5, 6), GrzB cleaves after aspartic acid (7), GrzH cleaves after aromatic residues (8), and GrzM cleaves after long aliphatic residues such as methionine, norleucine, and leucine (9, 10). A number of these Grz, including A, B, K, and M have been shown to mediate apoptosis in the presence of perforin (11, 12), and each induces a unique cell death pathway. Data from these cell-free apoptosis assays have only been supported in one instance by the observation that effector lymphocytes from GrzB-deficient (GrzB–/–) mice display delayed oligonucleosomal fragmentation of DNA in target cells (13). Intriguingly, lymphocytes from mice deficient in GrzA, GrzB, and GrzC retain the capacity to kill tumor targets (14, 15, 16), suggesting that additional Grz or granule components may contribute to cell death. The involvement of Grz in antiviral immune responses has been analyzed, and Grz are clearly essential for the control and latency of some, but not all, viral pathogens (2, 3, 17, 18). For example, GrzA and GrzB are essential for the efficient control of ectromelia virus (EV) (15), but are dispensable for the control of lymphocytic choriomeningitis virus infection (19). The role of other members of the Grz family in viral immunity remains to be defined.

The GrzM gene is collocated with a family of neutrophil elastases on human chromosome 19p13.3 (20, 21) and syntenic mouse chromosome 10C. GrzM is highly expressed in NK cells, but is not expressed in activated primary T cells (21, 22), and thus may have evolved to serve a specialized function in innate immunity. Given the unique cellular expression and protease specificity of GrzM, we undertook to replace the coding region of mouse GrzM with the cre recombinase, thereby creating a mouse strain deficient for GrzM. Analysis of this gene-targeted mouse strain has revealed normal immune homeostasis, normal NK cell cytotoxicity, and a normal response to EV, but a partially impaired response to murine CMV (MCMV), suggesting a differential role for GrzM in controlling some viral infections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of GrzM-deficient mice

The targeting vector (Fig. 1A) was constructed by ligating 2.8-kb genomic DNA directly upstream of the initiating ATG in exon I of GrzM, with sequence encoding cre recombinase and neomycin resistance (neor) gene cassette flanked by frt sites. This is followed by 6.3 kb of GrzM locus, encompassing the last 9 bp of exon I, the entire GrzM locus, and 635 bp after exon V. The NotI linearized targeting vector was electroporated into Bruce 4 (C57BL/6) embryonic stem (ES) cells, and G418-resistant colonies were obtained. To screen for homologous recombinants, G418-resistant colonies were digested with EcoRV, and the targeted allele was identified with a NsiI-HindIII fragment 3' of the targeting vector (data not shown). Two clones were injected into BALB/c-derived blastocysts to create chimeric mice and bred to C57BL/6 to obtain germline transmission. The targeted allele was identified by PCR using primers NKcre4 (within 5' UTR; 5'-ACACTTGTGCAAGGTGGG), Gz14 (within intron 1; 5'-GACGCGCGCCACCGC), and NKcre3 (within cre recombinase; 5'-AGCATGTTTAGCTGGCCC). NKcre4 and Gz14 amplified a 286 bp from the wild-type (WT) allele, whereas NKcre4 and NKcre3 amplified a 404 bp from the targeted allele. Mice with the targeted allele were then crossed to C57BL/6 WT mice. In addition, mice were crossed to a Flp deleter strain (three times backcrossed to C57BL/6 from original 129 mouse) (23) to remove frt-flanked neor cassette in vivo. Mice with one GrzM targeted allele and flp allele were screened by PCR, using primers NKcre8 (5'-TGGAAGATGGCGATTAGCC) and NKcre6 (5'-GGCCATTCGAATTCGACGT) that produced a 366-bp band, upon neor excision. Germline transmission of the neor deleted-targeted allele was confirmed by crossing to C57BL/6 WT mice and screening the progeny for the presence of cre, and the absence of both neor and flp recombinase. These mice were then further backcrossed nine generations to C57BL/6 to create the inbred C57BL/6 GrzM-cre homozygous gene-targeted mice that have been used in all of the experiments presented. Mice were bred to homozygosity and maintained on the C57BL/6 background.



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  		FIGURE 1. The gene targeting of GrzM in mice. A, The strategy for targeting the GrzM locus. Displayed sequentially are the targeting vector, targeted locus, and targeted locus without neo. The sequence encoding the cre recombinase and the neor gene cassette flanked by frt sites was inserted directly upstream of the initiating ATG in exon I of GrzM. Mice with the targeted allele were further propagated by crossing to C57BL/6 mice. In addition, mice were crossed to a Flp deleter strain to remove frt-flanked neor cassette in vivo. Germline transmission of the neor deleted-targeted allele was confirmed by crossing to C57BL/6 mice and screening the progeny for the presence of cre, and the absence of both neor and flp recombinase. All mice were further backcrossed to C57BL/6. B, Southern analysis of EcoRV-digested tail genomic DNA from C57BL/6 WT and GrzM–/– mice demonstrating the diagnostic 15- and 8.5-kb bands, respectively. C, RT-PCR analysis using primers and cDNA as described in Materials and Methods: lane 1, GeneRuler 100 bp DNA LadderPlus; lane 2, the expression of the cre transgene in the GrzM–/– mice; lane 3, the absence of cre transgene in the WT mice; lane 4, the absence of the mRNA message for full-length GrzM in GrzM–/– mice; lane 5, the presence of the mRNA message for full-length GrzM in WT mice; and lanes 6 and 7, the equivalent expression of mRNA for {beta}-actin in the WT and GrzM–/– mice.

 
Southern analysis

Tail genomic DNA was digested with EcoRV restriction enzyme, and the resulting fragments were separated on a 0.9% agarose gel at 15 V overnight. After electrophoresis, the DNA was transferred to a nitrocellulose filter and then X-linked by UV to the membrane. The DNA was hybridized to a 32P-labeled NsiI-HindIII fragment of genomic GrzM DNA overnight at 65°C. The membrane was washed in 2x SSC with 0.1% SDS at 65°C for 15 min, followed by several washes in 0.5x SSC with 0.1% SDS at 65°C. The blot was exposed to x-ray film.

RT-PCR

Total RNA was purified from 1 x 107 cells of an IL-2-activated splenocyte culture from WT and GrzM–/– mice in TRIzol reagent (Invitrogen Life Technologies). sscDNA synthesis was performed on 2 µg of DNase I-treated total RNA using random hexamers (0.1 µg) and M-MLV Reverse Transcriptase (Promega), in a total volume of 20 µl. PCR amplification reaction used 5 µl of the cDNA reaction volume, 20 pmol of primers, 0.2 µM dNTPs, 1.25 mM MgCl2, and 1.5 U Taq polymerase (Fisher Biotech). For the cre recombinase transgene, the forward primer sequence was 5'-GGAAATGGTTTCCCGCAGAAC-3', and the reverse primer sequence was 5'-ACGGAAATCCATCGCTCGACC-3'. For full-length mRNA expression of GrzM, the forward primer sequence was 5'-ATGGAGGTCTGCTGGTCC-3', and the reverse primer sequence was 5'-TCCAGGAGCTGTAGGGGG-3'. For mRNA expression of actin, the forward primer sequence was 5'-AGGCGGTGCTGTCCTTGTAT-3', and the reverse primer sequence was 5'-GGAAGGAAGGCTGGAAGAGT-3'. Amplification conditions for the cre transgene were 95°C, 5', and 35 cycles of 94°C, 40 s/60°C, 40 s/72°C, 40 s, then 72°C, 5'. For the actin and GrzM PCR, the amplification conditions involved 95°C, 5', 30 cycles of 95°C 1', 55°C 1', 72°C 2', followed by a single cycle of 95°C 1', 55°C 1', and 72°C 7'.

Mice

Inbred C57BL/6 WT mice were purchased from the Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia) or the Animal Resources Centre (Perth, Australia). The following gene-targeted mice were bred at the Peter MacCallum Cancer Centre: C57BL/6 perforin-deficient (B6 pfp–/–) (targeted in C57BL/6 ES cells (24)); C57BL/6J GrzA-deficient (B6 GrzA–/–) (25); C57BL/6J GrzB cluster-deficient (B6 GrzB–/–) (13); GrzA and -B cluster-deficient (B6 GrzAB–/–) mice and C57BL/6 RAG-1-deficient (B6 RAG-1–/–) mice were bred at the Peter MacCallum Cancer Centre; and all such Grz-deficient mice used were genotyped using the PCR screening protocol described previously (14). All other pfp-, Grz-, and RAG-1-gene-targeted mice were derived from C57BL/6 ES cells or have been backcrossed >10 generations to C57BL/6. Mice 6–20 wk of age were used in all of the experiments that were performed according to the guidelines of the relevant institution animal experimental ethics committees and the National Health and Medical Research Council of Australia. For the MCMV studies, C57BL/6 and GrzM-deficient mice (GrzM–/–) (adult females 8–18 wk old) were housed at the University of Western Australia under specific pathogen-free conditions. For the ectromelia studies, C57BL/6 and GrzM-deficient mice (GrzM–/–) were bred and maintained at the John Curtin School of Medical Research (Canberra City, Australia) under specific pathogen-free conditions. Only mice of the same sex were used in individual experiments at 12–20 wk of age.

Cell suspensions

Mice were killed by CO2 asphyxiation, and organs were removed for analysis. To obtain a single-cell suspension, organs were gently pressed between the frosted ends of two glass slides into cold PBS-FCS (2%)-sodium azide (0.02%) (FACS buffer) and filtered through 100-mm nylon mesh. Spleen samples were RBC lysed by incubation with RBC lysis buffer (Sigma-Aldrich). Cell concentration and viability were determined using an automated cell counter (Corixa). The cell suspensions were then washed by centrifugation (390 x g, 5 min, 4°C), and the cell pellets were resuspended in FACS buffer. Peripheral bloods were collected, and differential white blood cell counts were performed using an Advia 120 Hematology System (Bayer Diagnostics).

Flow cytometric analysis

Cell suspensions were stained in 96-well U-bottom plates (3 x 106 cells/test) by gently resuspending in 30 µl of appropriate mAb or secondary conjugate and incubating for 25 min at 4°C in the dark. Between incubations, cells were washed twice by the addition of 200 µl of FACS buffer and recovered by centrifugation (290 x g, 3 min, 4°C). Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences), and typically data were collected on at least 1 x 105 viable cells for each sample. Nonviable lymphocytes were excluded on the basis of forward light scatter vs side light scatter. Analysis was performed using CellQuest software (BD Biosciences).

Abs and reagents

For flow cytometric analysis, FITC-conjugated anti-CD8 (clone 53-6.7), CD4 (RM4-5), TCR-{alpha}{beta} (H57-597); PE-conjugated anti-NK-1.1 (PK136), CD8 (53-6.7); PerCP-conjugated anti-CD4 (RM4-5); and allophycocyanin-conjugated anti-CD8 (53-6.7), TCR-{alpha}{beta} (H57-597) Abs were purchased from BD Pharmingen. Biotinylated Abs were detected using streptavidin-PerCP or allophycocyanin (BD Pharmingen). In all of the experiments, Fc{gamma}II/III receptor block (anti-CD16/32, clone 2.4G2, generated in-house) was used to minimize nonspecific binding of Abs.

Primary cells and cell lines

Primary mouse embryo fibroblasts were cultured in MEM (Invitrogen Life Technologies) supplemented with 10% neonatal calf serum (Invitrogen Life Technologies) and antibiotics: 100 µg/ml penicillin (CSL) and 40 µg/ml gentamicin (Pharmacia and Upjohn). Mouse YAC-1 target cells were grown in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). The spontaneous B cell lymphomas {beta}2µNPN-2 and {beta}2µNPN-8 were originally derived from B6.pfp–/– {beta}2µ–/– mice and are deficient for MHC class I, CD1d, and NKG2D ligands (26). Mock-infected mouse RMA and RMA-S (H-2b) mutant lymphomas (derived from the Raucher virus-induced murine cell line RBL-5), RMA and RMA-S expressing Rae-1{beta} (RMA-S-Rae-1{beta}, RMA-Rae-1{beta}), and B16F10 melanoma cells have been described previously (27). All of the cells were grown in DMEM or RPMI 1640 supplemented with 10% (v/v) FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies).

Tumor control in vivo

Five different experimental systems were used. The B cell lymphomas {beta}2µNPN-2 and {beta}2µNPN-8 were transplanted directly from B6 pfp–/– RAG-1–/– mice into B6 WT mice or B6 GrzM–/– mice as described previously (26). Groups of five B6 WT or B6 GzM–/– mice received injections i.p. with increasing numbers of {beta}2µNPN-2, {beta}2µNPN-8, or RMA-S lymphoma cells and monitored daily for tumor growth for 150 days. Some mice were depleted of NK cells using anti-NK1.1 mAb (PK136) as described previously (28). Mice were sacrificed when abdominal swelling was noted, and disseminated lymphoma development was confirmed. Groups of five untreated B6 WT or gene-targeted mice received injections s.c. with vector alone-infected or Rae-1{beta}-infected RMA tumor cells (5 x 104 cells) in 0.2 ml of PBS. Mice were checked every 2 days for tumor growth using a caliper square measuring along the perpendicular axes of the tumors (the product of two diameters ± SE) and sacrificed when tumors reached a size >12 mm in diameter. Mice without any signs of tumor growth were monitored for at least 100 days. B16F10 melanoma cells were inoculated i.v. at a dose of between 1 x 104 and 5 x 105 cells into groups of five WT or GrzM–/– mice, as described previously (29). Mice were sacrificed 14 days later, the lungs were removed, and surface metastases were counted with the aid of a dissecting microscope. In all metastasis models, the data were recorded as the mean number of metastases ± SEM. Significance was determined by a Mann-Whitney rank sum U test.

Cytotoxicity assays

Assays measuring the release of 51Cr and 125I-DNA from apoptotic YAC-1 and RMA-S-Rae-1{beta} target cells were performed as described previously (16). To measure membrane perturbation, target cells were radiolabeled with 51Cr for 1 h, then washed thoroughly to remove free label, and incubated with effector cells in 96-well round-bottom plates for various periods of time, as indicated. The supernatants were harvested using a Skatron Cell Harvester (Skatron), and the specific release of 51Cr into the supernatant was measured using a Wallac Wizard 1470 automatic gamma counter (PerkinElmer). To additionally measure 125I release, target cells were colabeled with 51Cr and 125IUdR. After harvesting manually for 51Cr release, cells were treated with 1% Triton X-100 for 10 min, as described previously, and the cells were centrifuged. The spontaneous release was determined by incubating target cells with buffer alone. The maximum release was determined by adding SDS to a final concentration of 5%. The percentage specific lysis was calculated as follows: 100 x ((experimental release – spontaneous release)/(maximum release – spontaneous release)).

Treatment of mice with MCMV

For the pathogenesis and histological studies, mice were infected i.p. with 5 x 104 or 1 x 105 PFU of salivary gland-propagated stocks of the virulent MCMV strain K181-Perth diluted in PBS-0.5% FCS. At designated times postinfection (PI), mice were sacrificed, and spleens, livers, lungs, and salivary glands were removed. All organs were individually weighed, homogenized in cold MEM-2% neonatal calf serum, and centrifuged at 3000 x g for 15 min at 4°C. The supernatants were stored at –80°C, and virus titers were subsequently quantified on mouse embryo fibroblasts by standard plaque assay (30).

Preparation of tissue sections for histological analysis

Mice were euthanized by cervical dislocation, and tissues (spleen and liver) were covered with Cryo-embed OCT (Tissue Tek), snap-frozen in liquid nitrogen, and stored at –80°C. Six-micrometer sections were cut on a cryostat and mounted onto silane (Valeant Pharmaceuticals)-coated slides. Sections were stained with hematoxylin, counterstained with eosin, and analyzed under a light microscope.

EV and immunization

The EV Moscow strain (ECTV) was prepared from mouse spleens and titrated on CV-1 cell monolayers as described previously (31). Mice were infected with 1 x 102 PFU ECTV into the hind footpads (fp) unless stated otherwise. The methods for virus titration in organs, histological evaluation, and liver enzyme levels in serum have been documented previously (31, 32, 33).

Statistical analysis

A Mann-Whitney rank sum U test was performed on relevant data to determine statistical significance. For analyses conducted on smaller group sizes, a nonparametric unpaired, two-tailed t test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The generation of GrzM-deficient mice

To determine the role of GrzM in NK cell-mediated cytotoxicity, we generated a strain of mice deficient in GrzM. The targeting vector was constructed, as described in Materials and Methods, by inserting sequence encoding cre recombinase and neor gene cassette flanked by frt sites directly upstream of the initiating ATG in exon I of GrzM (Fig. 1A). Mice with the targeted allele were propagated by crossing to C57BL/6 mice, and these were designated as the GrzM neo.cre strain. In addition, these mice were crossed to a Flp deleter strain (23) to remove the frt-flanked neor cassette in vivo. Mice with one GrzM-targeted allele and a flp allele were screened by PCR, using primers NKcre8 (5'-TGGAAGATGGCGATTAGCC) and NKcre6 (5'-GGCCATTCGAATTCGACGT) that produced a 366-bp band, upon neor excision. Germline transmission of the neor deleted-targeted allele was confirmed by crossing to C57BL/6 mice and screening the progeny for the presence of cre, and the absence of both neor and flp recombinase. These mice, designated GrzM cre or GrzM–/– were inbred and used for all of the experimental studies described below. Southern analysis of tail DNA from the GrzM neo.cre and GrzM cre strains digested with EcoRV revealed the expected alleles for each (Fig. 1B). No differences in immune status between the mice with or without the neomycin cassette were observed (data not shown). A rabbit polyclonal Ab generated against a peptide of mouse GrzM could detect recombinant mouse GrzM, but not endogenous protein in cell lysates, presumably because of the low abundance of the endogenous GrzM protein. Therefore, RT-PCR analysis was performed to determine whether GrzM was indeed absent in GrzM–/– mice. The absence of full-length GrzM mRNA and presence of cre mRNA in GrzM cre mice was demonstrated by RT-PCR (Fig. 1C). GrzM–/– mice were born at the expected Mendelian ratios and showed no gross developmental abnormalities (data not shown).

GrzM is not essential for NK and T cell development and homeostasis

We first compared the immune repertoire of GrzM–/– mice, with particular focus on the homeostasis of the NK cell and T cell compartments given the expression profile of GrzM (22). A broad assessment of NK cells, NKT cells, and T cell subsets in bone marrow, thymus, spleen, liver, lymph nodes, and peripheral blood did not reveal any major perturbances in these populations in GrzM–/– mice (Fig. 2). An analysis of spleen B cells (B220 vs IgM), myeloid and dendritic cell subsets (data not shown), and differential peripheral blood count analysis including monocytes, lymphocytes, neutrophils, eosinophils, and basophils in peripheral blood (Table I) also did not reveal any variations in number and proportion between WT and GrzM–/– mice. The unchanged lymphocyte differentiation and maturation are consistent with the unaltered immunophenotypes described previously in GrzA–/– and GrzB–/– mice (13, 25).



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  		FIGURE 2. GrzM was not essential for NK cell/T cell development and homeostasis. Liver, spleen, bone marrow, and thymus mononuclear cells isolated from 8-wk-old C57BL/6 and C57BL/6 GrzM–/– mice were stained with Abs to NK1.1, TCR-{alpha}{beta}, CD8, and CD4. Representative NK1.1 vs TCR-{alpha}{beta} and CD8 vs CD4 FACS profiles are shown. Data are representative of the analysis of five independent experiments, each containing three C57BL/6 and three C57BL/6 GrzM–/– mice.

 

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  		Table I. Differential leukocyte counts in GrzM–/– micea

 
GrzM is not essential for NK cell-mediated cytotoxicity

The mouse lymphoma cell lines Yac-1 and RMA-S-Rae-1{beta} both express NKG2D ligands and are both very sensitive targets of naive splenic NK cells. To examine the two major death parameters of these target cells, they were colabeled with 51Cr (to measure membrane integrity) and 125IUdR (to measure DNA fragmentation) and then exposed to naive spleen NK cells from WT, GrzM–/–, and GrzB–/– mice. It has been shown previously that GrzB mediates DNA fragmentation in the presence of perforin (11) and GrzB in NK cells, and CTL is an important early effector of target cell DNA fragmentation (13). On the other hand, deficiency of GrzA does not influence target cell death by splenic NK cells (25). By contrast, we have shown recently that GrzM mediates rapid 51Cr release, but not DNA fragmentation, in the presence of recombinant perforin (12). Clearly, however, intact NK cells do not require GrzM to mediate effective membrane or nuclear damage of RMA-S-Rae-1{beta} (Fig. 3) or YAC-1 (data not shown) target cells. In accordance with previous reports (14), NK cells from GrzB cluster-deficient mice induced target cell membrane damage, but were relatively inefficient at inducing DNA fragmentation (Fig. 3). Additional experiments using CD3–DX5+ NK cells sorted from the spleens of WT and GrzM–/– mice and cultured in IL-2 or IL-15 did not reveal any defects in NK cell proliferation or differentiation in the absence of GrzM–/– (data not shown).



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  		FIGURE 3. GrzM was not essential for NK cell-mediated cytotoxicity. Splenocytes derived from WT, GrzB–/–, and GrzM–/– mice were incubated with RMA-S-Rae-1{beta} target cells for 6 h at various E:T ratios as indicated. Cell membrane damage (51Cr release) (A) and DNA fragmentation (125I-DNA release) (B) were measured as described previously. Spontaneous release was routinely between 5 and 10% of total release. Results are expressed as mean ± SE of triplicate samples, and data shown are representative of two separate experiments.

 
GrzM-deficient mice display altered MCMV pathology

MCMV is a natural pathogen of mice that has been used widely to study the nature of the immune effectors and the molecular mechanisms involved in controlling infection. In mouse strains such as B6, MCMV infection is effectively controlled in the spleen, but the virus replicates to high titers in the liver during the acute stage of infection (days 2–6) (34). In B6 mice, viral replication during the acute stage of infection is controlled by antiviral activities mediated by NK cells (35). To determine whether GrzM contributes to the antiviral activity of NK cells and/or the control of MCMV replication, B6 WT and GrzM-deficient mice were infected with MCMV and viral pathogenesis assessed by measuring viral titers in visceral organs (spleen and liver) at various times PI. As shown in Fig. 4, viral titers in the spleen and liver were significantly higher in GrzM–/– mice at days 3 (spleen, p = 0.006 and p = 0.025; liver, p = 0.027 and p = 0.006) (Fig. 4A) and 4 (spleen, p = 0.035; liver, p = 0.061) (Fig. 4B) PI. These increases, however, were transient, and by days 5 and 6 PI, viral titers were equivalent in GrzM and WT mice (Fig. 4B).



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  		FIGURE 4. Kinetics of MCMV replication in the visceral organs of GrzM-deficient mice. Groups of at least three mice were infected i.p. with MCMV, and organs were harvested at days 3, 4, 5, and 6 PI. A, Viral titers at day 3 PI in spleen (filled symbols) and liver (open symbols) of GrzM–/– (diamonds) and B6 WT (circles) mice are shown. Two independent experiments are represented. Individual data points and SEM are shown. B, Viral titers in spleen and liver of GrzM–/– ({cjs2108}) and B6 WT ({square}) mice are shown as log mean and SEM. The results are representative of those from independent experiments performed with either 1 x 104 or 1 x 105 PFU of MCMV-K181-Perth.

 
Viral replication and pathology were further assessed by histological analysis of the spleens and livers of infected GrzM–/– and WT mice. The number of cells showing signs of cytomegaly and the frequency of viral inclusions were significantly increased in GrzM-deficient mice compared with B6 WT mice at both day 3 (Fig. 5) and 4 PI. In addition, differences in the number of viral foci were also observed when the livers of GrzM–/– and B6 WT mice were compared (Fig. 5). The extent of leukocyte infiltration to the foci of infection was equivalent in both GrzM and B6 WT mice (Fig. 5). A slight but significant increase in tissue damage, characterized by an increase in nonspecific hemorrhage, was observed in both the spleens and the livers of GrzM–/– mice (Fig. 5, and data not shown).



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  		FIGURE 5. Histological assessment of MCMV replication and tissue pathology in the livers of GrzM-deficient mice. Groups of three mice were infected i.p. with 105 PFU MCMV, and the livers were harvested at day 3 PI for histological analysis. Six-micrometer sections stained with H&E were analyzed. The number of inflammatory foci and their size were determined from >10 fields of at least five different tissue sections. The frequency of cytomegalic cells and viral inclusions was derived by examining at least 50 randomly selected foci from each tissue section. Representative fields show the difference in the number of foci of infection (characteristic focus marked by arrow) observed in B6 WT (top left panel) and GrzM–/– (bottom left panel) livers at day 3 PI. Middle panels show the size of representative foci of infection. The right hand panels show representative foci at a magnification that allows the visualization of cells showing cytomegaly and viral inclusions (indicated by arrows). The results are representative of those from three independent experiments, each using three mice per group. HP {emptyset}, high-power field diameter. The differences in liver inflammatory foci between WT and GrzM–/– mice were statistically significant (p < 0.0001).

 
The cytotoxic activities of NK cells from MCMV-infected GrzM and WT mice were compared. Splenocytes from both strains were harvested at days 2, 4, and 6 PI and used as effectors against a panel of NK cell-sensitive targets. The level of YAC-1, RMA, and RMA-S lysis was equivalent for both GrzM–/– and WT effectors (data not shown). These results clearly show that GrzM-deficient NK cells retain their cytotoxic capacity, and suggest that the phenotype we observed in MCMV-infected GrzM-deficient mice was independent of a defect in the killing function of NK cells.

GrzM-deficient mice display normal clearance of EV

Like MCMV, EV, causing mouse pox, is a natural mouse pathogen and represents one of the best-characterized viral disease models available (36, 37, 38). The EV model has been used very successfully in elucidating the role of specific effector molecules of the cytolytic pathways of NK cells and cytolytic T cells, especially in identifying the GrzA and GrzB as principal mediators of resistance to EV infections (15, 31, 32). Thus, we investigated whether GrzM-deficient mice were differentially resistant to EV infections as compared with B6 WT mice. B6 mice are highly resistant to EV infections (>2 x 106 PFU) (36) when infected via the hind fp, mimicking natural infections (37). Similarly, GrzM–/– mice did not succumb to EV infections when given doses of up to 2 x 106 PFU fp (data not shown). We then investigated the kinetics of viral replication and histology in liver and spleen, the two principal organs involved in mouse pox, and liver enzyme levels in serum after low-dose (102 PFU) infection via fp. Individual mice (n = 5/strain/time point) were sacrificed 4, 6, and 8 days PI, and virus titers were estimated in liver and spleen (Fig. 6). No significant differences in virus burden between WT and GrzM–/– mice were observed. Histological examination of spleen and liver of the same mice did not show differences in tissue damage (necrosis) in liver and spleen or leukocyte infiltration in liver between WT and GrzM–/– mice (data not shown). Similarly, no significant differences were found in liver enzyme levels (aspartate aminotransferase and alanine aminotransferase) in serum (data not shown). These results suggest that GrzM does not play a crucial role in the recovery of mice from primary EV infections.



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  		FIGURE 6. Kinetics of EV replication in spleen and liver. Mice were immunized with 102 PFU EV fp. Groups of five mice of each strain were sacrificed at 4, 6, and 8 days PIs, and virus titer was estimated by plaque assay on CV-1 cell monolayers. Virus titers are expressed as PFU per gram of tissue.

 
GrzM is not essential for NK cell-mediated tumor rejection

We have recently demonstrated the ability of NK cells to suppress or reject a number of different experimental tumors and tumor metastases (24, 26, 27). Although the cytotoxicity of GrzM-deficient NK cells appeared intact, the NK cell-mediated response to tumor cells in vivo may be impaired. To address this, we examined the ability of B6 WT, GrzM–/–, and pfp–/– mice to reject two different MHC class I-deficient B cell lymphomas (26). As described previously, at least 107 of these lymphomas cells are rejected in WT mice, whereas pfp–/– mice succumb to challenge with as few as 103 lymphoma cells. In this study, both GrzM–/– mice and WT mice rejected challenge with either tumor, whereas B6 pfp–/– mice died between 40 and 60 days (Fig. 7A). We next challenged B6 WT and GrzM–/– mice in the peritoneum with increasing doses of MHC class I-deficient RMA-S lymphoma cells (Fig. 7B). In accordance with the B cell lymphoma data, there was no difference between GrzM–/– and WT mice in their resistance to RMA-S challenge. Cell-mediated suppression of the s.c. growth of MHC class I and NKG2D ligand-expressing RMA tumor cells was also equivalent between WT and GrzM–/– mice, whereas mice lacking both NK cells and T cells were unable to respond (Fig. 7C). Lastly, we examined the ability of GrzM–/– mice to suppress B16F10 experimental metastases, a resistance normally mediated exclusively by NK cells (24). At a number of different cell doses, WT and GrzM–/– mice displayed similar numbers of B16 lung metastases (Fig. 7D). Overall, these data suggest that NK cell-mediated control of tumors is independent of GrzM function.



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  		FIGURE 7. GrzM was not essential for NK cell-mediated tumor rejection. A, Groups of five B6 WT, B6 Pfp–/–, or B6 GzM–/– mice received injections i.p. with 107 {beta}2µNPN-2 or {beta}2µNPN-8 B cell lymphoma cells and monitored daily for tumor growth for 150 days. Mice were sacrificed upon abdominal swelling, and disseminated lymphoma development was confirmed. B, Groups of five untreated B6 WT or B6 GzM–/– mice received injections i.p. with increasing doses of RMA-S lymphoma cells and monitored daily for tumor growth for 150 days. Mice were sacrificed upon abdominal swelling, and disseminated lymphoma development was confirmed. C, Groups of five untreated B6 WT or B6 GzM–/– mice received injections s.c. with vector alone-infected or Rae-1{beta}-infected RMA tumor cells (5 x 104 cells) in 0.2 ml of PBS. Some groups of RAG-1–/– were depleted of NK cells, using anti-NK1.1 mAb as described in Materials and Methods. Mice were checked every 2 days for tumor growth using a caliper square measuring along the perpendicular axes of the tumors (the product of two diameters ± SE) and sacrificed when tumors reached a size >12 mm in diameter. Mice without any signs of tumor growth were monitored for at least 100 days. D, B16F10 melanoma cells were inoculated i.v. at a dose of between 1 x 104 and 5 x 105 cells into groups of five WT or GrzM–/– mice, as described in Materials and Methods. Mice were sacrificed 14 days later, the lungs were removed, and surface metastases were counted with the aid of a dissecting microscope. In all metastasis models, the data were recorded as the mean number of metastases ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
GrzM-deficient mice displayed a transient but significant increase in susceptibility to infection with the herpesvirus MCMV, but not with the poxvirus EV. Although viral titers were only moderately increased in GrzM–/– mice compared with WT controls, the differences were statistically significant, observed after infection with different viral doses, and reproduced in independent experiments. Furthermore, histopathological analysis of the visceral organs of infected mice showed a clear difference in hepatotoxicity and spleen pathology. In the spleen, there was a greater loss of tissue architecture, and the red pulp was more hemorrhagic in GrzM-deficient mice than WT control mice. In the liver, a characteristic feature of infection in GrzM-deficient mice was the increased number of cells displaying viral inclusions.

The GrzM-deficient mice displayed a normal NK cell cytotoxic response. GrzM–/– and WT mice equivalently rejected MHC class I-deficient or NKG2D ligand-expressing tumors in vivo, suggesting that GrzM was not required for tumor cell death in vivo, nor effective activation and recruitment of NK cells to the tumor site. Spleen NK cell cytotoxicity against P815 and YAC-1 NK cell-sensitive targets, induced by exposure of mice to Semliki Forest virus, one of the most potent stimulators of NK cell cytotoxicity, was not altered in GrzM-deficient mice compared with WT, Semliki Forest virus-infected mice (data not shown). Similarly, NK cells purified from the spleens of MCMV-infected GrzM-deficient and WT mice killed NK cell-sensitive target cells in vitro with equivalent efficiency, providing further evidence that GrzM is not critical for the cytotoxic capacity of NK cell effectors. These findings suggest that the differences in the manner in which GrzM-deficient and WT mice responded to MCMV challenge represent differences in other biological processes mediated by GrzM. Some of the NK cell-mediated antiviral activities involve the release of IFN-{gamma}, and indeed this cytokine is required for the control of MCMV infection (39, 40). Therefore, we considered the possibility that GrzM deficiency may alter the capacity of NK cells to release IFN-{gamma}. Our analysis of serum IFN-{gamma} levels, however, showed no defect in GrzM-deficient mice, with levels of this cytokine after MCMV infection being equivalent in GrzM–/– and WT mice (data not shown).

The increased number of MCMV inclusions observed in hepatocytes of GrzM–/– mice suggests that perhaps this Grz is involved in cellular processes that control effective viral replication. The unique protease specificity of GrzM may be important in proteolytic processes that might affect the virus life cycle. Alternatively, the phenotype of MCMV-infected GrzM-deficient mice may be due to effects of this Grz on a specific type of cell death. Our previous studies indicated that GrzM induced a novel type of cell death, which was not accompanied by DNA fragmentation, mitochondrial disruption, or annexin V display on the cell surface (12). Because cell death may be required for at least some of the release of viral progeny, in the absence of GrzM, infected cells would accumulate virus particles, a hypothesis consistent with the increased numbers of viral inclusions observed in the livers of GrzM-deficient mice. In the liver, hepatocytes and lymphocytes, including NK cells, are infected by MCMV (M. A. Degli-Esposti, unpublished observations). GrzM is expressed in NK cells (21, 22), but expression in hepatocytes is not known.

A number of Grz have now been gene targeted in mice, including GrzA (25), GrzB, and GrzB cluster (13, 41), and in this article, GrzM. Thus far, the granule exocytosis-mediated cytotoxicity of granulated NK cells and CTL from each of these strains of mice appears intact, and only the ability of GrzB cluster-deficient lymphocytes to mediate rapid DNA fragmentation in target cells is compromised. This data may be interpreted to suggest that Grz-mediated apoptosis of target cells is either redundant or Grz are not absolutely required to kill target cells in a perforin-dependent manner when perforin is delivered by a killer lymphocyte. The intact cell death mediated by GrzA-deficient lymphocytes may be explained by the presence of GrzK (42), but does not explain the increased sensitivity of GrzA-deficient mice to EV infection. In contrast, GrzM and GrzB display very unique serine protease specificities and are unlikely to be substituted functionally by other Grz family members. Very clearly from our study, GrzM is not essential for target tumor cell membrane perturbation or DNA fragmentation mediated by NK cells. Similarly, NK cell-mediated rejection of NK cell-sensitive tumors in vivo was GrzM independent.

The importance of Grz to lymphocyte-mediated cell death will not be definitely answered until all Grz are genetically silenced in mice. Thus far, we appreciate that killer lymphocytes lacking dipeptidyl peptidase I (DPPI) display defective DNA fragmentation of target cells, just like lymphocytes deficient for both GrzA and -B cluster (14, 43); however, it is not clear whether these target cells still die. DPPI is required for the activation of Grz and other bone marrow-derived serine proteases with an activation dipeptide sequence (43, 44, 45, 46). It is not clear whether DPPI is required for GrzM activation.

In conclusion, gene targeting has provided novel insight into the biological functions of GrzM, and these studies suggest additional complexities as to the role of members of the Grz family, which are important in controlling certain viral infections.


   Acknowledgments
 
We thank Dr. Klaus Rajewsky for his contribution to the generation of the GrzM gene-targeted mice at the University of Cologne (Cologne, Germany). We also thank Warren Alexander and Jason Corbin (Walter and Eliza Hall Institute) for white blood cell analysis, Aulikki Koskinen for excellent technical support, and Professor R.V. Blanden (John Curtain School of Medical Research) for advice on EV-induced pathology.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The work was supported by a Doherty Fellowship (to M.E.W.), Research Fellowship (to M.J.S.), Project and Program grants from the National Health and Medical Research Council of Australia, and a grant from Deutsche Forschungsgemeinschaft through SFB 243. We also thank the Australian Academy of Science for supporting the initiation of this project. Back

2 L.I.P., J.M.K., and N.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Mark J. Smyth, Cancer Immunology Program, Trescowthick Laboratories, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria 3002, Australia. E-mail address: mark.smyth{at}petermac.org Back

4 Abbreviations used in this paper: Grz, granzyme; EV, ectromelia virus; MCMV, murine CMV, ES, embryonic stem; WT, wild type; PI, postinfection; fp, footpad; DPPI, dipeptidyl peptidase I. Back

Received for publication January 14, 2005. Accepted for publication June 15, 2005.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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