Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes

Britta Bade1,2, Heidrun Elise Boettcher3, Jens Lohrmann4, Clara Hink-Schauer5, Kai Bratke1,2, Dieter E. Jenne5, J. Christian Virchow, Jr1 and Werner Luttmann1

1 Department of Pneumology, University Medical Clinic Rostock, Schillingallee 35, D-18057 Rostock, Germany
2 Department of Zoophysiology, Carl von Ossietzky University, Oldenburg, Germany
3 Department of Pneumology, University Medical Clinic, Freiburg, Germany
4 GENOVAC GmbH, Company for Genetic Immunization, Freiburg, Germany
5 Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Munich, Germany

Correspondence to: B. Bade; E-mail: britta.bade{at}gmx.de


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Granzymes (Gzm) are a group of serine proteases which are stored in the granules of cytotoxic lymphocytes. In humans, five granzymes have been characterized to date at the molecular level. While GzmA and GzmB have been extensively studied, little is known about GzmH, GzmK and GzmM. In this study, we describe the generation of mAbs against human GzmK and GzmM by genetic immunization. The obtained anti-GzmK and anti-GzmM mAbs are not cross-reactive with GzmA, GzmB, GzmM and GzmA, GzmB, GzmK, respectively, and show a granular staining pattern in human lymphocytes. Flow cytometric analysis of peripheral blood lymphocytes revealed that GzmA, GzmM and perforin show a similar distribution. They are expressed in almost all CD16+CD56+ NK cells, CD3+CD56+ NKT cells and {gamma}{delta} T cells as well as in 20–30% of all CD3+CD8+ TC cells. Surprisingly, GzmK was not detected in the highly cytotoxic CD16+CD56+ NK cells but was preferentially expressed in lymphocytes of the T cell lineage, staining 20% of CD3+CD8+ TC cells, 50% of CD3+CD56+ NKT cells and 40% of {gamma}{delta} T cells, as well as 60% of the small sub-population of CD56bright+ NK cells. Our data suggest that human granzymes are differentially expressed in distinct sub-populations of peripheral blood lymphocytes.

Keywords: antibodies, apoptosis, CTL, flow cytometry, NK cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocytes play a pivotal role in the regulation of immune responses. A key function of cytotoxic lymphocytes is the detection of potentially harmful cells and their elimination by the induction of apoptosis (1, 2). This is achieved through two principal pathways, the Fas/FasL pathway and the exocytosis of specialized granules. The latter pathway involves lytic granules containing several serine proteases termed granzymes (Gzm), as well as the pore-forming protein perforin, which delivers the granzymes into the cytoplasm of target cells by mechanisms currently unknown. To date, 11 murine (A–G and K–N) and 5 human (A, B, H, K and M) granzymes have been identified. While the expression and biological functions of human GzmA and GzmB have been well characterized, there is little information about GzmH, GzmK and GzmM.

Granzymes are a closely related family of serine proteases that are similar in structure but differ in their chromosomal location and substrate specificity. While GzmA and GzmK display tryptase activity and are clustered on human chromosome 5, GzmB and GzmH map to a gene cluster termed the ‘chymase locus’ on chromosome 14. The Met-ase GzmM is linked to the neutrophil elastase gene cluster on human chromosome 19.

The mechanisms of cell death induced by GzmA and GzmB have been extensively studied and many of the intracellular substrates as well as the activated apoptotic pathways have been identified (3). Induction of cell death in target cells has also been shown to occur through GzmK and GzmM (46), however, the mechanism of action as well as cellular substrates of these granzymes remain largely unknown. The potential of GzmH to induce cell death has not been studied to date.

The expression of human granzymes has mostly been observed in cytotoxic lymphocytes and thymocytes, although the expression of GzmA and GzmB in other cell types, which has been reported by some groups, is currently debated controversially (710). Until recently, most of the studies attempting to define the cellular expression patterns of orphan GzmH, GzmK and GzmM have relied on northern blots, reporter gene assays and reverse transcription (RT)–PCR analysis (11). Evidence of the expression at the protein level has come from Sedelies et al. (12) and Smyth et al. (13), who developed antibodies that could be used to detect denatured GzmH and GzmM, respectively, in western blots. Studies analyzing purified lymphocyte sub-populations with these antibodies reported the expression of GzmM in NK cells and {gamma}{delta} T cells but not in CD4+ and CD8+ T cells (14), while GzmH seemed to be highly enriched in NK cells but low in T lymphocytes (12).

As studying the expression of the orphan granzymes at the single-cell level has in the past been difficult due to the lack of appropriate antibodies, we attempted to raise antibodies against these granzymes using genetic immunization. In this paper we report the successful generation of mAbs against human GzmA, GzmK and GzmM. All these antibodies specifically recognize the native antigen and therefore allow for the first time the flow cytometric detection of GzmK and GzmM. The development and characterization of these antibodies as well as the comparison of GzmK and GzmM expression with that of the well-characterized tryptase GzmA and perforin in lymphocytes of the peripheral blood are the subjects of the present study.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and recombinant proteins
Antibodies against the cell-surface markers CD3 (UCHT1), CD4 (MT310), CD8 (DK 25), CD16 (DJ130c) and CD19 (HD 37) conjugated to either PE, PE–Cy5 or allophycocyanin (APC) as well as FITC- and PE-labeled anti-mouse Ig antibodies were purchased from Dako (Hamburg, Germany). Anti-CD56 (B-A19) was obtained from Diaclone (Besancon, France) and anti-{gamma}{delta}-TCR from Becton Dickinson (Heidelberg, Germany). FITC-labeled anti-perforin mAb ({delta}G9) was purchased from Hölzel Diagnostica (Cologne, Germany). Mouse gamma globulins were obtained from Jackson ImmunoResearch Inc. (Cambridgeshire, UK). Antibodies against human GzmA (GM1H11), GzmK (GM24C3) and GzmM (GM2B4) were raised in our laboratory by genetic immunization of BALB/c mice (see below). All antibodies were titrated for optimal dilutions. Recombinant granzymes as zymogens (ZyA, ZyB, ZyK and ZyM) and active proteases (ActA, ActB and ActK) expressed in Escherichia coli (15) were kindly provided by D. Jenne (Max Planck Institute for Neurobiology, Martinsried, Munich, Germany).

Isolation and stimulation of human PBMCs
Venous blood was collected from healthy volunteers using EDTA vacutainers and diluted 1:3 in PBS. PBMCs were isolated by Ficoll-Hypaque centrifugation as described previously (16). Isolated cells were washed twice with PBS, counted and suspended in PBS containing 2% FCS for flow cytometry and confocal microscopy. For the extraction of total RNA, isolated cells were stimulated to induce the expression of granzyme mRNA. The wells of a 12-well cell culture plate were coated with anti-CD3 mAb at a concentration of 5 µg ml–1. After 2 h, the supernatant was removed and replaced by 1 x 106 PBMCs suspended in 3 ml RPMI-1640 medium supplemented with 10% (v/v) FCS, 100 U ml–1 penicillin/streptomycin and 300 U ml–1 recombinant IL-2. Cells were incubated for 24 h at 37°C in 5% CO2.

RT–PCR and cloning
Total RNA was isolated from stimulated PBMCs using the RNeasy RNA-isolation kit from Qiagen (Hilden, Germany) according to the instructions of the manufacturer. RT–PCR was performed using a reverse transcriptase, random primer hexamers and deoxythymidine triphosphate oligonucleotides from Stratagene (La Jolla, CA, USA) according to the manufacturer's protocol. Double-stranded cDNA templates were PCR amplified using a Taq DNA polymerase (AGS Gold, Hybaid, Heidelberg, Germany) together with the following primers: 5'-AGACCCAAGCTTGAAAATTATTGGAGGAAATGAAGTAAC-3' and 5'-GTCGACCTCGAGCGATAGTCATAATTATCCAGTTGAGGTG-3' (GzmA), 5'-AGATCTGGATCCAGAGATCATCGGGGGACATG-3' and 5'-GTCGACCTCGAGCGTAGCGTTTCATGGTTTTCTTTATC-3' (GzmB), 5'-AGATCTGGATCCAATTATTGGAGGGAAAGAAGTGTCAC-3' and 5'-GTCGACCTCGAGCATTTGTATGAGGCGGGACAA-3' (GzmK), 5'-AGACCCAAGCTTGATCATCGGGGGCCGGGAG-3' and 5'-GTCGACCTCGAGCTCGGCCGGTGACCTTCCTG-3' (GzmM). In addition to the granzyme-cDNA binding region, primers contained an overlap encoding recognition sequences for the digestion with HindIII and XhoI (GzmA and GzmM) or BamHI and XhoI (GzmB and GzmK). The resulting PCR fragments were digested and ligated into a specialized expression vector provided by GENOVAC GmbH (Freiburg, Germany). This immunization vector contained an ampicillin resistance gene as well as an open reading frame with the coding sequence for a marker peptide and a leader routing the expression of the cloned proteins to the cell surface. The ligation products were used to transform competent E. coli, which were plated onto agar plates and incubated overnight under selection of 50 µg ml–1 ampicillin. Several colonies were picked to generate overnight liquid cultures that were analyzed for the presence of the insert by enzymatic digestion and subsequent agarose gel electrophoresis. For large-scale production, positive clones were selected and grown overnight in LB medium supplemented with 50 µg ml–1 ampicillin. Plasmid DNA was extracted using the QIAfilter Plasmid Maxi Kit (Qiagen) according to the company's protocol. Cloned granzyme cDNA was shown by sequencing to be 100% identical to the corresponding sequence published in PubMed in all four cases (accession no. P12544, P10144, P49863 and P51124 for GzmA, GzmB, GzmK and GzmM, respectively).

Transfection of BOSC23 cells with immunization vectors
To test the expression of the granzyme fusion proteins, the human cell line BOSC23, an ecotrophic envelope expressing packaging line derived from the ANJOU 65 (American Type Tissue Collection no. CLR-1269) cell line, was transiently transfected with the immunization vectors for GzmA, GzmB, GzmK and GzmM. DNA was delivered into the cells by lipofection using the cationic lipid reagent Lipofectamin 2000 (Gibco BRL, Karlsruhe, Germany) according to the instructions of the manufacturer. The accumulation of the fusion protein on the cell surface was assessed after 24 h by flow cytometric detection of the aminoterminal marker peptide. To test for the presence of granzyme-specific antibodies, mouse sera were diluted 1:100 in PBS supplemented with 3% (v/v) FCS and incubated with transfected BOSC23 cells for 30 min. Subsequently, cells were washed twice with PBS, incubated for 30 min with PE-labeled anti-mouse Ig antibodies and after two additional washing steps analyzed by flow cytometry. Similarly, transfected BOSC23 cells were used to detect antibodies in undiluted hybridoma supernatants either by flow cytometry or cell-based ELISA.

Genetic immunization
For genetic immunization, groups of five BALB/c mice were immunized by several intradermal applications of the immunization vectors for GzmA, GzmK and GzmM using DNA-coated gold particles, which were delivered by particle bombardment (gene gun, BioRad, USA). After 3 weeks, mice were routinely bled and immune sera were tested for the presence of anti-granzyme antibodies using transiently transfected BOSC23 cells. Mice showing an immune response against granzymes were sacrificed and the isolated lymphocytes fused with SP2/0-Ag14 mouse myeloma cells using polyethylene glycol. After 2 weeks, hybridoma supernatants were tested for the presence of granzyme-specific antibodies using transiently transfected BOSC23 cells in a cell-based ELISA and flow cytometry. Selected hybridomas were subjected to limiting dilutions and the clones were expanded and cryopreserved in liquid nitrogen.

Purification and biotinylation of mAbs
mAbs were purified from cell culture supernatants by protein G affinity chromatography (Pharmacia, Uppsala, Sweden). Purified mAbs were biotinylated using long-chain biotinyl-N-hydroxysuccinimide ester sulfonic acid (Pierce Chemical Co., Rockford, IL, USA) according to the instructions of the manufacturer.

Specificity testing of the generated antibodies
The specificity of the generated antibodies was tested by analyzing the recognition of different granzyme transfectants. In addition, the recognition of the inactive versus mature conformations was tested in a dot blot assay using recombinant granzymes expressed in E. coli. Zymogen and the active protease of GzmA, GzmB and GzmK as well as the zymogen of GzmM (200 ng, except for ZyA: 100 ng) were transferred onto nitrocellulose and stained with antibodies against the different granzymes as described previously (17). Lysates of mock-transfected BOSC23 cells as well as BSA immobilized on nitrocellulose served as negative controls, while a mAb directed against the signal peptide encoded in the immunization vector was used as irrelevant antibody. Binding of the antibodies to denatured granzymes was also assessed by heating the enzymes to 95°C for 5 min in SDS prior to the transfer.

Surface plasmon resonance analysis
The surface plasmon resonance (Biacore®) analysis was performed by Biaffin (Kassel, Germany). Anti-mouse Ig antibodies were immobilized by amine coupling to the dextran surface of the biochip, whereafter the anti-GzmK mAb GM24C3 was bound to the immobilized anti-mouse antibodies. Binding of active GzmK as well as the zymogen was assessed at a constant flow rate of 10 µl min–1 by monitoring the association phase for 3 min and the dissociation phase for 15 min. An irrelevant antibody served as negative control. Binding signals of this antibody were subtracted from the signal seen with anti-GzmK GM24C3.

Confocal microscopy
Isolated PBMCs were incubated for 20 min on adhesion slides at 1 x 106 cells ml–1. After adhesion, cells were fixed with PBS containing 4% (w/v) formaldehyde and permeabilized with PBS containing 0.1% (w/v) saponin. Staining of the cells was performed by incubation with anti-granzyme mAbs followed by two washing steps with PBS/2% FCS and incubation with Alexa 546-labeled anti-mouse Ig antibodies. Before staining with FITC-labeled anti-perforin mAb, the residual binding sites of the anti-mouse Ig antibodies were saturated by incubation with purified mouse IgG. After two final washing steps, slides were mounted using the ProLong® Antifade kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. Confocal microscopy was performed using a Leica TCS fluorescence microscope together with the appropriate software (Leica Microsystems, Heidelberg, Germany).

Granzyme staining and flow cytometric detection in PBMCs
Isolated PBMCs were washed twice in PBS, fixed with PBS containing 4% (w/v) formaldehyde for 10 min on ice and permeabilized with PBS/2% FCS containing 0.1% (w/v) saponin. Subsequently, 5 x 105 cells per well were stained by incubation with anti-granzyme mAbs followed by incubation with FITC-labeled anti-mouse Ig antibodies or stained with directly FITC-labeled anti-perforin mAb. After blocking of residual binding sites on the anti-mouse antibodies with mouse gamma globulins, cell-surface markers were stained using directly PE-, PE–Cy5- and/or APC-labeled mAbs. For intracellular double staining, cells were incubated after granzyme staining and blocking with either FITC-labeled anti-perforin mAb ({delta}G9) or biotinylated anti-GzmK mAb (GM24C3) followed by streptavidin–PerCP. Subsequently, cells were measured in a FACS-Calibur counting 15 000 cells per well. Four fluorescences were detected and the corresponding dot plots analyzed using the Cell QuestTM software from Becton Dickinson (San Jose, CA, USA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Specificity of mAbs
To determine the cellular distribution of human granzymes at the single-cell level, antibodies that recognize the native protein had to be raised. We therefore immunized BALB/c mice by genetic immunization with expression vectors for GzmK and GzmM. In addition, we also raised mAb against GzmA to compare the expression of the second human tryptase with that of GzmK. To test the specificity of the generated antibodies, we used transiently transfected BOSC23 cells that expressed human GzmA, GzmB, GzmK or GzmM on the cell surface. Expression of the fusion protein was assessed by detecting the aminoterminal marker peptide (Fig. 1B). To evaluate binding of the generated antibodies and at the same time test for potential cross-reactivity, each mAb was incubated with the four different granzyme transfectants as well as a mock-transfected control (Fig. 1C). Recognition was shown to be specific for each of the tested mAbs, and no cross-reactivity with other granzymes was observed.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. Specificity of the granzyme mAbs. To test whether the generated antibodies specifically react with their antigens, BOSC23 cells were transiently transfected with the expression vectors encoding GzmA, GzmB, GzmK and GzmM as well as an irrelevant protein (mock transfectant). To selectively analyze viable cells, a gate was set in the forward scatter/side scatter (FSC/SSC) plot (R1, A). Expression was confirmed by detection of the aminoterminal tag (broken lines); an irrelevant mAb served as negative control (intact lines, B). Hybridoma supernatants directed against GzmA (GM1H11), GzmK (GM24C3) and GzmM (GM2B4) were each incubated with the five different transfectants indicated by the broken lines (C). The mAbs showed no cross-reactivity with the other granzymes but reacted specifically with their respective antigens (BOSC tt GzmA: BOSC cells transiently transfected with a GzmA expression vector).

 
In situ, granzymes are synthesized as inactive precursors that assume their active conformation after cleavage of the inhibiting pro-peptide. To assess whether the raised mAbs recognize the zymogen as well as the active enzyme or are specific for either one of these conformations, binding of the mAbs to recombinant granzymes was analyzed in a dot blot assay (Fig. 2A). While GzmM was only available as zymogen, which was recognized by the anti-GzmM mAb (GM2B4), mAbs against GzmA (GM1H11) and GzmK (GM24C3) were shown to recognize both conformations of their respective antigens. In addition, we could show that the anti-GzmK mAb GM24C3 also binds murine active GzmK (data not shown). Again, no cross-reactivity with other granzymes was observed. It was also shown that GM1H11 and GM2B4 recognize conformational epitopes, as they did not bind to the denatured antigen. However, as anti-GzmK mAb GM24C3 also partly recognized the denatured protein, binding could be directed against an epitope that was denatured as a consequence of immobilization of the protein on nitrocellulose rather than reflect recognition of the two different native GzmK conformations.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2. Recognition of zymogen and active enzyme. Recombinant proteins expressed in Escherichia coli were used to assess whether the antibodies recognize both the active and inactive conformations of the granzyme or are specific for either conformation. (A) The recombinant proteins were transferred onto nitrocellulose and stained using hybridoma supernatants of the four granzymes as well as the anti-tag mAb as irrelevant control. While GzmA, GzmB and GzmK were provided as zymogens and as active enzymes, GzmM was only available as zymogen. BSA and a lysate of mock-transfected BOSC23 cells were used as controls for background staining. To assess whether linear or conformational epitopes were recognized, proteins were also analyzed after denaturation with SDS at 95°C. Antibodies raised against GzmA (GM1H11) and GzmK (GM24C3) were shown to react specifically with both zymogen and active enzyme, while anti-GzmM GM2B4 recognized the GzmM zymogen. The denatured protein was partly recognized by anti-GzmK GM24C3, whereas the other antibodies failed to stain their antigens in the denatured conformation. (B) Binding of anti-GzmK GM24C3 to recombinant GzmK was also assessed by surface plasmon resonance analysis to assure the native conformation of zymogen and active enzyme. GM24C3 was immobilized on a biochip while the recombinant proteins were provided in solution at a constant flow rate. The antibody was shown to bind to both conformations with similar kinetics (ZyA: inactive conformation of GzmA, ActA: active conformation of GzmA).

 
As recognition of both zymogen and active enzyme could not be conclusively shown in the dot blot, we additionally performed a surface plasmon resonance analysis where the antibody was immobilized on the surface of a biochip and the different forms of GzmK were added in the fluid phase, thereby assuring their native conformation (Fig. 2B). Binding of GM24C3 to GzmK was observed with the inactive as well as active conformations. No quantitative analysis was performed to assess the binding affinity of GM24C3; however, the fact that no dissociation of the antigen was observed during the 15 min of the dissociation phase indicates that GM24C3 binds GzmK with high affinity (Kdiss ≤ 10–4 s–1).

Subcellular localization
Having shown that the generated antibodies react specifically with their antigens, we subsequently used these mAbs to assess the subcellular localization of the granzymes by intracellular staining of human PBMCs (Fig. 3, red fluorescence). As has been shown previously, GzmA and GzmM (14, 18, 19) were localized to granules in the cytoplasm of the stained cells. As expected, staining with the anti-GzmK mAb also revealed a granular staining pattern. In addition to the staining for granzymes, cells were also incubated with an FITC-labeled anti-perforin mAb (green fluorescence). While perforin staining was more frequent in PBMCs than GzmK, in double-positive cells, fluorescence signals were essentially co-localized (yellow), indicating co-expression of perforin and granzymes in cytoplasmic granules.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 3. Subcellular localization. PBMCs were isolated from venous blood of healthy volunteers, permeabilized and stained intracellularly for GzmA, GzmK and GzmM (red fluorescence, C–E, respectively). Simultaneously, all cells were stained with FITC-labeled anti-perforin mAb (green fluorescence) as well as Hoechst 33342 nuclear dye (blue fluorescence). Cells stained with Hoechst and irrelevant antibody in combination with Alexa 546-labeled anti-mouse antibody and irrelevant FITC-labeled antibody (A) as well as cells stained with Hoechst and anti-perforin mAb but no granzyme mAbs (B) served as negative controls. Images are shown as overlays of the three fluorescences (I) and with each individual channel as well as bright field in separate panels (II). Size is represented by white bars, with the length depicted in I being also valid for the bars in II. Granzymes all showed a granular staining pattern that essentially co-localized with perforin staining (I, yellow).

 
Cellular distribution of granzymes and perforin
In addition to the subcellular localization, the cellular distribution of GzmA, GzmK and GzmM as well as perforin was studied at the single-cell level by flow cytometric analysis of unstimulated human PBMCs. The constitutive expression of these cytotoxic mediators was assessed in all lymphocytes from peripheral blood (Fig. 4B) as well as in seven distinct lymphocytic sub-populations characterized by the following cell-surface markers: CD19+ B cells, CD3+CD4+ Th cells, CD3+CD8+ TC cells, CD3+{gamma}{delta}-TCR+ {gamma}{delta} T cells, CD3+CD56+ NKT cells, CD3CD56+CD16+ NK cells and CD56bright+ NK cells (Fig. 4A and B). In addition, a summary of all CD3+ T cells is shown. Expression of all granzymes and perforin was mostly restricted to cytotoxic lymphocytes. As expected, granzyme and perforin expression was absent in B cells, and only a very small population of Th cells expressed detectable levels of GzmA, GzmK and GzmM.




View larger version (65K):
[in this window]
[in a new window]
 
Fig. 4. Expression pattern of granzymes in PBMCs. Human PBMCs were isolated from venous blood of healthy volunteers, permeabilized and stained for GzmA (GM1H11), GzmK (GM24C3) and GzmM (GM2B4) as well as perforin ({delta}G9). Different lymphocyte subsets were characterized by the expression of one to three cell-surface markers (B cells: CD19+; T cells: CD3+; Th cells: CD3+CD4+; TC cells: CD3+CD8+; {gamma}{delta} T cells: CD3+, {gamma}{delta}-TCR+; NKT cells: CD16, CD56+, CD3+; NK cells: CD16+, CD56+, CD3). (A) FACS analysis representative of six normal donor samples with similar expression patterns. A gate was set in the forward scatter/side scatter plot to selectively analyze the lymphocytes (data not shown). Of these lymphocytes, the characterized subset is depicted in red, while remaining cells are shown in gray. Data shown represent the percentage of granzyme/perforin-positive cells of the characterized subset (red cells). (B) Summarized data of granzyme and perforin expression in whole lymphocytes, T cells, {gamma}{delta} T cells, NKT cells and NK cells from six or more donors.

 
Perforin expression was detected in almost all NK, NKT, CD56bright+ and {gamma}{delta} T cells as well as in 20% of TC cells. Within the perforin-positive cell population, a different staining intensity was observed. While almost all NK cells were perforinbright+, cells of the T cell lineage were either perforindim+ or perforin negative. Interestingly, the CD3CD56bright+ population was not perforinbright+ as the NK cells but rather displayed a perforindim+ phenotype.

Of the granzymes expressed in human PBMCs, GzmA and GzmM were found to be the most abundant. Of 11 healthy donors tested, a median of 28 and 27% of all lymphocytes stained positive for GzmA and GzmM, respectively. Analyzing the expression in different lymphocyte subsets, GzmA and GzmM were found to be expressed in almost all NK, NKT and {gamma}{delta} T cells. Both granzymes were also detected in ~35% of TC cells. In contrast to GzmA and GzmM, GzmK was detected in only 8% of total peripheral blood lymphocytes. When analyzing different lymphocyte subsets, we found to our surprise that GzmK expression was almost completely absent in CD16+CD56+ NK cells, although these cells express abundant amounts of GzmA and GzmM as well as high levels of perforin. Rather than in CD16+CD56+ NK cells, GzmK expression was found in cells of the T cell lineage, namely, 45% of NKT cells, 40% of {gamma}{delta} T cells and 20% of TC cells, as well as in 60% of the small subset of CD56bright+ NK cells.

The mean expression of GzmA, GzmK and GzmM as well as perforin in PBMCs of six healthy volunteers is summarized in Table 1. While GzmA and GzmM are the most abundant granzymes and are mostly expressed in the same lymphocyte subsets, expression of GzmK is more restricted.


View this table:
[in this window]
[in a new window]
 
Table 1. Granzyme/perforin expression in human PBMCs

 
Intracellular double staining
To test whether in CD3+ T cells, which were only partly positive for granzymes and perforin, these proteins were localized in the same subsets, intracellular double staining was performed (Fig. 5). Simultaneous staining of GzmK together with GzmA, GzmM and perforin as well as GzmA and GzmM together with perforin revealed that GzmK-expressing cells mostly co-expressed GzmA, GzmM and perforindim.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5. Intracellular double staining of granzymes and perforin. FACS analysis representative of four donors with similar expression patterns. A gate was set in the forward scatter/side scatter plot to selectively analyze the lymphocytes (data not shown). Data shown represent the percentage of cells in the respective quadrants. Cells were incubated with mAbs against GzmA (GM1H11) or GzmM (GM2B4) and stained using a PE-labeled secondary antibody. After blocking of residual binding sites, cells were incubated with either a FITC-labeled anti-perforin mAb ({delta}G9) or biotinylated anti-GzmK mAb (GM24C3) followed by streptavidin–PerCP.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previous works have studied the expression of GzmA and GzmB in human lymphocytes while the analysis of the orphan granzymes has been limited by the lack of appropriate antibodies. As genetic immunization enables the targeted production of antibodies recognizing the native antigen we chose this strategy to raise mAbs against human GzmA, GzmK and GzmM. The generated antibodies recognized their corresponding antigens specifically, showing no cross-reactivity with other granzymes. Furthermore, we demonstrated that mAbs against GzmA and GzmK recognize both the inactive zymogen as well as the mature enzyme expressed in E. coli (15), while anti-GzmM GM2B4 could only be shown to bind the zymogen as the mature enzyme was not available. As expected, all antibodies recognized the respective granzymes in their native conformations, which enabled us to assess cellular expression patterns by flow cytometry.

In PBMCs isolated from peripheral blood, GzmA and GzmM were most abundantly expressed. These findings are in accordance with previous studies analyzing the constitutive expression of GzmA in human lymphocytes (2023). For GzmM, however, this finding was unexpected as expression of the Met-ase has been reported in several studies to be restricted to lymphocytes involved in innate immunity (14, 2426). However, analyzing the expression of GzmA, GzmB and GzmM in PBMCs, Sayers et al. (14) also did not detect GzmA expression in unstimulated sorted T cells, while these cells have been shown to contain GzmA-positive sub-populations in our as well as in other studies (20, 21). This discrepancy could be explained by the purification procedure applied by Sayers et al. (14), favoring the accumulation of small T cells that had not been activated. Possibly, the GzmA- and GzmM-expressing subset was contained in a different fraction.

To our knowledge, GzmK expression in human lymphocytes has not been previously analyzed, except for studies which reported the isolation of human GzmK from human lymphokine-activated killer cells (2729). In the present study, we show that in contrast to the other granzymes, expression of GzmK was markedly absent in human CD16+CD56+ NK cells, whereas it is expressed in different subsets of TC, NKT and {gamma}{delta} T cells. Interestingly, it is also expressed in CD56bright+ cells, a small NK subset that has been shown to possess poor cytolytic potential (22).

Compared with GzmA, the second human granzyme with tryptase specificity, GzmK is expressed with markedly lower frequencies in PBMCs, suggesting both different functional requirements and expression control. Unlike GzmA, GzmK expression does not seem to correlate with the cytotoxic potential of the lymphocytes, although GzmK has been shown to introduce cell death in target cells when delivered with sublytic doses of perforin (4, 5). Cell death induced by GzmK involves the late release of DNA from target cells in a fashion similar to GzmA. This is accompanied by a loss of the mitochondrial transmembrane potential and the generation of reactive oxygen species but does not involve caspase activation or chromatin condensation. However, as no mouse strains defective in the expression of GzmK have been described to date, the physiological significance of cell death induced by GzmK has not been assessed.

Recently, it has also been shown that GzmM induces cell death in target cells in a unique manner that is more reminiscent of necrosis than apoptosis (6). Thus, cell death induced by GzmM is accompanied by rapid membrane disruption but does not involve changes in mitochondria, caspase activation or DNA release from the target cell. Recent evidence also implies that GzmM might assist GzmB-mediated killing of the target cell by cleaving the GzmB inhibitor PI-9 (30).

The physiological roles of both GzmK and GzmM remain to be established. However, there is evidence that the presence of different granzymes with distinct substrate specificity and unique pathways to induce cell death might provide a way to circumvent evasion strategies developed by several viruses to escape GzmB-induced cell death. Poxviruses inhibit several caspases and possibly GzmB by producing the serpin CrmA (3133). GzmB knockout mice (GzmB–/–) were not as susceptible to infection by the poxvirus ectromelia as GzmA–/– mice, whereas GzmAxB–/– mice rapidly succumbed to overwhelming infection. In an infection with ectromelia, GzmA seems to provide an essential backup system against viral evasion strategies (3436). However, GzmAxB–/– are still unimpaired in their ability to fight several other viruses as well as in rejection of several experimental tumors (3539), suggesting that other granzymes might also provide some kind of backup system. Furthermore, the fact that various target cells have been shown to exhibit a different susceptibility to death induced by different granzymes (4, 40) indicates that the condition of the target cells may define the importance of the individual granzymes.

Evidence for a differential expression of the granzymes has come from studies which identified distinct regulatory elements in the promoters of some granzyme genes (4145). Using a PCR-based assay, Kelso et al. (46) also reported the differential expression of GzmA, GzmB and GzmC in murine cytotoxic lymphocytes. Similarly, a study on human lymphocytes described a discordant expression pattern between human GzmA and GzmB as assessed by flow cytometry (21), while others show the discordant regulation of GzmH and GzmB expression (12). These data, along with the findings about the constitutive granzyme expression demonstrated in our study, support the hypothesis that individual cells express different combinations of perforin and granzymes. Together with the most recent evidence suggesting that granzymes use distinct pathways to induce target cell death (3, 6, 30, 47), this implies that granzymes are not redundant but might rather exert distinct apoptotic and regulatory functions that are important for immune protection against viral infections and tumors.


    Acknowledgements
 
We thank E. Wilharm, C. Hink-Schauer and F. Kurschus for providing recombinant granzymes and D. Schillinger for technical assistance. This work was supported by grants from the ‘Studienstiftung des deutschen Volkes’ (B.B.) and the PRO INNO program of the German ‘Bundesministerium für Wirtschaft und Technologie (BMWi)’ (W.L. and J.L.).


    Abbreviations
 
Act   active protease
APC   allophycocyanin
Gzm   granzyme
RT   reverse transcription
Zy   zymogen

    Notes
 
Transmitting editor: A. Kelso

Received 13 April 2005, accepted 15 August 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Barry, M. and Bleackley, R. C. 2002. Cytotoxic T lymphocytes: all roads lead to death. Nat. Rev. Immunol. 2:401.[ISI][Medline]
  2. Trapani, J. A. and Smyth, M. J. 2002. Functional significance of the perforin/granzyme cell death pathway. Nat. Rev. Immunol. 2:735.[CrossRef][ISI][Medline]
  3. Lieberman, J. 2003. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat. Rev. Immunol. 3:361.[CrossRef][ISI][Medline]
  4. Shi, L., Kam, C. M., Powers, J. C., Aebersold, R. and Greenberg, A. H. 1992. Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J. Exp. Med. 176:1521.[Abstract/Free Full Text]
  5. MacDonald, G., Shi, L., Vande Velde, C., Lieberman, J. and Greenberg, A. H. 1999. Mitochondria-dependent and -independent regulation of granzyme B-induced apoptosis. J. Exp. Med. 189:131.[Abstract/Free Full Text]
  6. Kelly, J. M., Waterhouse, N. J., Cretney, E. et al. 2004. Granzyme M mediates a novel form of perforin-dependent cell death. J. Biol. Chem. 279:22236.[Abstract/Free Full Text]
  7. Wagner, C., Iking-Konert, C., Denefleh, B., Stegmaier, S., Hug, F. and Hansch, G. M. 2004. Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils. Blood 103:1099.[Abstract/Free Full Text]
  8. Hochegger, K., Eller, P. and Rosenkranz, A. R. 2004. Granzyme A: an additional weapon of human polymorphonuclear neutrophils (PMNs) in innate immunity? Blood 103:1176.[Free Full Text]
  9. Metkar, S. S. and Froelich, C. J. 2004. Human neutrophils lack granzyme A, granzyme B, and perforin. Blood 104:905.[Free Full Text]
  10. Grossman, W. J. and Ley, T. J. 2004. Granzymes A and B are not expressed in human neutrophils. Blood 104:906.[Free Full Text]
  11. Grossman, W. J., Revell, P. A., Lu, Z. H., Johnson, H., Bredemeyer, A. J. and Ley, T. J. 2003. The orphan granzymes of humans and mice. Curr. Opin. Immunol. 15:544.[CrossRef][ISI][Medline]
  12. Sedelies, K. A., Sayers, T. J., Edwards, K. M. et al. 2004. Discordant regulation of granzyme H and granzyme B expression in human lymphocytes. J. Biol. Chem. 279:26581.[Abstract/Free Full Text]
  13. Smyth, M. J., O'Connor, M. D., Kelly, J. M., Ganesvaran, P., Thia, K. Y. and Trapani, J. A. 1995. Expression of recombinant human Met-ase-1: a NK cell-specific granzyme. Biochem. Biophys. Res. Commun. 217:675.[CrossRef][ISI][Medline]
  14. Sayers, T. J., Brooks, A. D., Ward, J. M. et al. 2001. The restricted expression of granzyme M in human lymphocytes. J. Immunol. 166:765.[Abstract/Free Full Text]
  15. Wilharm, E., Parry, M. A., Friebel, R. et al. 1999. Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma. J. Biol. Chem. 274:27331.[Abstract/Free Full Text]
  16. Luttmann, W., Herzog, V., Virchow, J. C., Jr, Matthys, H., Thierauch, K. H. and Kroegel, C. 1996. Prostacyclin modulates granulocyte/macrophage colony-stimulating factor release by human blood mononuclear cells. Pulm. Pharmacol. 9:43.[CrossRef][ISI][Medline]
  17. Lohrmann, J., Sweere, U., Zabaleta, E. et al. 2001. The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis. Mol. Genet. Genomics 265:2.[CrossRef][ISI][Medline]
  18. Peters, P. J., Borst, J., Oorschot, V. et al. 1991. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J. Exp. Med. 173:1099.[Abstract/Free Full Text]
  19. Andersson, J., Kinloch, S., Sonnerborg, A. et al. 2002. Low levels of perforin expression in CD8+ T lymphocyte granules in lymphoid tissue during acute human immunodeficiency virus type 1 infection. J. Infect. Dis. 185:1355.[CrossRef][ISI][Medline]
  20. Trimble, L. A. and Lieberman, J. 1998. Circulating CD8 T lymphocytes in human immunodeficiency virus-infected individuals have impaired function and downmodulate CD3 zeta, the signaling chain of the T-cell receptor complex. Blood 91:585.[Abstract/Free Full Text]
  21. Grossman, W. J., Verbsky, J. W., Tollefsen, B. L., Kemper, C., Atkinson, J. P. and Ley, T. J. 2004. Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood 104:2840.[Abstract/Free Full Text]
  22. Jacobs, R., Hintzen, G., Kemper, A. et al. 2001. CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells. Eur. J. Immunol. 31:3121.[CrossRef][ISI][Medline]
  23. Chen, G., Shankar, P., Lange C. et al. CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection. Blood 98:1564.
  24. Krenacs, L., Smyth, M. J., Bagdi, E. et al. 2003. The serine protease granzyme M is preferentially expressed in NK-cell, gamma delta T-cell, and intestinal T-cell lymphomas: evidence of origin from lymphocytes involved in innate immunity. Blood 101:3590.[Abstract/Free Full Text]
  25. Smyth, M. J., Sayers, T. J., Wiltrout, T., Powers, J. C. and Trapani, J. A. 1993. Met-ase: cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells. J. Immunol. 151:6195.[Abstract/Free Full Text]
  26. Smyth, M. J., Browne, K. A., Kinnear, B. F., Trapani, J. A. and Warren, H. S. 1995. Distinct granzyme expression in human CD3– CD56+ large granular- and CD3– CD56+ small high density-lymphocytes displaying non-MHC-restricted cytolytic activity. J. Leukoc. Biol. 57:88.[Abstract/Free Full Text]
  27. Hameed, A., Lowrey, D. M., Lichtenheld, M. and Podack, E. R. 1988. Characterization of three serine esterases isolated from human IL-2 activated killer cells. J. Immunol. 141:3142.[Abstract/Free Full Text]
  28. Hanna, W. L., Zhang, X., Turbov, J., Winkler, U., Hudig, D. and Froelich, C. J. 1993. Rapid purification of cationic granule proteases: application to human granzymes. Protein Expr. Purif. 4:398.[CrossRef][ISI][Medline]
  29. Shresta, S., Goda, P., Wesselschmidt, R. and Ley, T. J. 1997. Residual cytotoxicity and granzyme K expression in granzyme A-deficient cytotoxic lymphocytes. J. Biol. Chem. 272:20236.[Abstract/Free Full Text]
  30. Mahrus, S., Kisiel, W. and Craik, C. S. 2004. Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B. J. Biol. Chem. 279:54275.[Abstract/Free Full Text]
  31. Quan, L. T., Caputo, A., Bleackley, R. C., Pickup, D. J. and Salvesen, G. S. 1995. Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A. J. Biol. Chem. 270:10377.[Abstract/Free Full Text]
  32. Turner, P. C., Sancho, M. C., Thoennes, S. R., Caputo, A., Bleackley, R. C. and Moyer, R. W. 1999. Myxoma virus Serp2 is a weak inhibitor of granzyme B and interleukin-1beta-converting enzyme in vitro and unlike CrmA cannot block apoptosis in cowpox virus-infected cells. J. Virol. 73:6394.[Abstract/Free Full Text]
  33. Turner, S. J., Silke, J., Kenshole, B. and Ruby, J. 2000. Characterization of the ectromelia virus serpin, SPI-2. J. Gen. Virol. 81:2425.[Abstract/Free Full Text]
  34. Müllbacher, A., Ebnet, K., Blanden, R. V. et al. 1996. Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia. Proc. Natl Acad. Sci. USA 93:5783.[Abstract/Free Full Text]
  35. Müllbacher, A., Hla, R. T., Museteanu, C. and Simon, M. M. 1999. Perforin is essential for control of ectromelia virus but not related poxviruses in mice. J. Virol. 73:1665.[Abstract/Free Full Text]
  36. Müllbacher, A., Waring, P., Hla, R. T. et al. 1999. Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes. Proc. Natl Acad. Sci. USA 96:13950.[Abstract/Free Full Text]
  37. Ebnet, K., Hausmann, M., Lehmann-Grube, F. et al. 1995. Granzyme A-deficient mice retain potent cell-mediated cytotoxicity. EMBO J. 14:4230.[Abstract]
  38. Davies, J. E., Smyth, M. J. and Trapani, J. A. 2001. Granzyme A and B-deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti-tumor capacity. Eur. J. Immunol. 31:39.[CrossRef][ISI][Medline]
  39. Smyth, M. J., Street, S. E. and Trapani, J. A. 2003. Cutting edge: granzymes A and B are not essential for perforin-mediated tumor rejection. J. Immunol. 171:515.[Abstract/Free Full Text]
  40. Pardo, J., Balkow, S., Anel, A. and Simon, M. M. 2002. The differential contribution of granzyme A and granzyme B in cytotoxic T-lymphocyte mediated apoptosis is determined by the quality of the target cell. Eur. J. Immunol. 32:1980.[CrossRef][ISI][Medline]
  41. Prendergast, J. A., Helgason, C. D. and Bleackley, R. C. 1992. A comparison of the flanking regions of the mouse cytotoxic cell proteinase genes. Biochim. Biophys. Acta 1131:192.[ISI][Medline]
  42. Ebnet, K., Kramer, M. D. and Simon, M. M. 1992. Organization of the gene encoding the mouse T-cell-specific serine proteinase ‘granzyme A’. Genomics 13:502.[CrossRef][ISI][Medline]
  43. Haddad, P., Wargnier, A., Bourge, J. F., Sasportes, M. and Paul, P. 1993. A promoter element of the human serine esterase granzyme B gene controls specific transcription in activated T cells. Eur. J. Immunol. 23:625.[ISI][Medline]
  44. Kelly, J. M., O'Connor, M. D., Hulett, M. D., Thia, K. Y. and Smyth, M. J. 1996. Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. Immunogenetics 44:340.[CrossRef][ISI][Medline]
  45. MacIvor, D. M., Pham, C. T. and Ley, T. J. 1999. The 5' flanking region of the human granzyme H gene directs expression to T/natural killer cell progenitors and lymphokine-activated killer cells in transgenic mice. Blood 93:963.[Abstract/Free Full Text]
  46. Kelso, A., Costelloe, E. O., Johnson, B. J., Groves, P., Buttigieg, K. and Fitzpatrick D. R. 2002. The genes for perforin, granzymes A–C and IFN-gamma are differentially expressed in single CD8(+) T cells during primary activation. Int. Immunol. 14:605.[Abstract/Free Full Text]
  47. Johnson, H., Scorrano, L., Korsmeyer, S. J. and Ley, T. J. 2003. Cell death induced by granzyme C. Blood 101:3093.[Abstract/Free Full Text]