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Discordant regulation of granzyme H and granzyme B expression in human
lymphocytes1,2
Karin A. Sedelies3, Thomas J. Sayers4, Kirsten M. Edwards3,9, Weisan Chen5, Daniel G.
Pellicci6,8, Dale I. Godfrey6,8 and Joseph A. Trapani3,7
3Cancer Immunology Laboratory, Peter MacCallum Cancer Centre, Locked Bag 1,
A’Beckett Street, East Melbourne, 8006, Australia
4Basic Research program, SAIC-Frederick Inc., NCI Frederick, MD, 21702
5Ludwig Institute for Cancer Research, Austin and Repatriation Medical Center,
Studley Road, Heidelberg, 3084, Australia
6Department of Pathology and Immunology, Monash University Medical School,
Melbourne, Victoria 3181, Australia.
7address for correspondence: Dr. J. Trapani, Cancer Immunology Laboratory, Peter
MacCallum Cancer Institute, Locked Bag 1, A’Beckett Street, East Melbourne, 8006,
Australia. Email address: [email protected]
Phone: +61-3-9656-3726; FAX: +61-3-9656-1411
8Present address: Department of Microbiology and Immunology, University of
Melbourne, Parkville, 3052, Australia
9Present address: Department of Pathology and Harvard Centre for Cancer Biology,
Harvard Medical School, Boston, MA 02115
1JAT and KAS are supported by project grants and a research fellowship (JAT) from the
National Health and Medical Research Council of Australia. This work was also
JBC Papers in Press. Published on April 6, 2004 as Manuscript M312481200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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supported in part by federal funds from the National Cancer Institute, National Institutes
of Health, under Contract N01-CO-56000. The content of this publication does not
necessarily reflect the views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial products or organizations imply
endorsement by the US government.
2Abbreviations used in this manuscript: gzm, granzyme; TCC, T cell clone; PBMC,
peripheral blood mononuclear cell; CTL, cytotoxic T lymphocyte; CL, cytotoxic
lymphocyte; NK, natural killer; mAb, monoclonal antibody
Running title: granzyme H regulation in blood leukocytes
Word count excluding tables, references and legends: 5,500
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Summary
We analyzed the expression of granzyme H in human blood leukocytes, using a novel
mAb raised against recombinant granzyme H. 33kDa granzyme H was easily detected
in unfractionated peripheral blood mononuclear cells, due to its high constitutive
expression in CD3-CD56+ NK cells, whereas granzyme B was less abundant. The NK
lymphoma cell lines, YT and Lopez also expressed high granzyme H levels.
Unstimulated CD4+ and particularly CD8+ T cells expressed far lower levels of
granzyme H than NK cells, and various agents that classically induce T cell activation,
proliferation and enhanced granzyme B expression failed to induce granzyme H
expression in T cells. Also, granzyme H was not detected in NKT cells, monocytes or
neutrophils. There was a good correlation between mRNA and protein expression in
cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription
is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and
gzmH genes are tightly linked, expression of the proteins is quite discordant in T and
NK cells. The finding that granzyme H is frequently more abundant than granzyme B in
NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic
function of granzyme B in human NK cells.
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Introduction
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes
(CL) that are responsible for inducing rapid apoptosis of virus-infected or transformed
cells (1,2). CL utilize two pathways for killing target cells, both of which require direct
cell contact. The first pathway involves exocytosis of potent toxins from secretory
granules stored in the effector cell cytoplasm, whereas the second is triggered by
clustering of death receptors on the target cell membrane following interaction with
their respective ligands (TNF superfamily members) expressed on the killer cell (1,2).
Cytotoxic granules contain two major types of toxin that co-operatively induce target
cell apoptosis: granzymes (gzms), a family of serine proteases, and a pore-forming
protein, perforin. The precise mechanism of gzm/perforin synergy has not been
clarified, however it is likely that gzms enter the target cell by endocytosis (3,4), while
perforin enables pro-apoptotic gzms access to their substrates in the target cell cytosol
by destabilizing endosomes (5,6). The more traditionally held notion of perforin
permitting access to the cytosol through plasma membrane pores may also apply in
some circumstances (6).
Gzms are closely related to one another structurally, and their genes are clustered in
several loci on distinct chromosomes (7). In both humans and rodents, each locus
encodes proteases with a single broad type of substrate cleavage. Gzms A and K are
trypsin-like proteases (‘tryptases’) that cleave proteins at basic residues, and their genes
are linked on human chromosome 5 (8). Gzm M preferentially cleaves at residues with
long, uncharged side-chains (Met, Leu), and its gene is closely linked to the neutrophil
elastase gene on human chromosome 19 (9). The genes for gzms B and H are located on
chromosome 14, tightly linked with the gene encoding cathepsin G, another
chymotrypsin-like protease (‘chymase’) that, unlike the gzms, is expressed in cells of
the myeloid lineage, but not in lymphocytes (7,10). These three genes all map to within
60kb of one another. Gzm B cleaves its substrates adjacent to acidic residues,
particularly Asp (11), and plays a central role in eliciting death of the target cell by
perturbing mitochondria and activating caspases (12,13). Its absence from the granules
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of lymphocytes from gene knockout mice slows the rate of target cell DNA damage
(14).
In the mouse, the gene for gzm B is tightly linked to at least four other granzyme genes
predicted to code for functional chymases (gzms C-F), however only one such chymase,
gzm H performs this function in humans (15). Gzm C is now known to possess an
unusual type of pro-apoptotic activity characterised by mitochondrial swelling and
disruption that does not require prior caspase activity (16). However, no such evidence
has yet come forward for human gzm H. We recently showed that recombinant gzm H
expressed in baculovirus-infected insect cells preferentially cleaves substrates with Phe
or Tyr at the P1 position (15). Despite sharing this substrate specificity with mouse
gzms C-F, gzm H has no direct structural equivalent in the mouse, and appears to be a
uniquely human protein. Gzm H has very high amino acid identity (>90%) with many
portions of the gzm B sequence, particularly near the amino-terminus of the molecule
(10), despite performing a distinct enzymic function. It has been inferred from the high
degree of sequence similarity that the gzmH gene arose by duplication of part of the 5’
end of the gzmB gene (particularly exons 3 and 4), and fusion with the 3’ end of an
another primordial serine protease gene (10).
The function of gzm H is unknown, and its study has been greatly hindered by a lack of
reagents able to distinguish it from gzm B at the protein or mRNA levels. The cellular
expression of gzm H has been reported in just one study that assessed gzm H mRNA
expression in human T cells (10). This study concluded that like gzm B, gzm H is
expressed at high levels in activated T cells. However, in another study, a portion of the
gzmH promoter was used to preferentially direct the transgenic expression of SV-40 T
antigen to the mouse lymphoid compartment. The transgene was not activated in allo-
stimulated CTL that expressed high levels of gzm B, but was highly expressed in
activated NK cells and in NK and NK/T cell tumors that arose in these animals (17).
Although the genes encoding cathepsin G and gzms B and H are very tightly linked,
cathepsin G and gzm B have markedly different cellular expression profiles, as
cathepsin G is expressed exclusively by cells of the myeloid lineage, particularly
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neutrophils and monocytes (18). It is therefore quite feasible that gzms B and H may be
differentially expressed and regulated among leukocyte subsets. The availability of
correctly folded, proteolytically active gzm H enabled us to attempt raising
monospecific reagents for this protease. Having produced a novel gzm H-specific mAb,
we now demonstrate that gzm H is expressed solely in lymphocytes, but its constitutive
expression pattern is quite distinct from that of gzm B, as is its expression in response to
a variety of stimuli that strongly induce gzm B expression.
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Experimental procedures
Antibodies. Antibodies used in western blotting and immunohistochemistry included
several mAbs made by our laboratory: 2C5 (anti-gzm B) (19), 4H10 (anti-gzm M) (20),
PB2 (anti-perforin) (21) and 7D8 (anti-PI-9) (22). In addition, mAb GB7 (anti-gzm B)
was purchased from Serotec.
Cell lines, tissue samples and immunohistochemistry. The human tumor cell lines
used in this study were all cultured in RPMI medium supplemented with 10% fetal
bovine serum, 2mM L-glutamine, penicillin and streptomycin in a CO2 incubator at
37oC. These included YT (human NK), HL-60 and U937 (human myeloid), Daudi and
Raji (Human B cell), Jurkat and CEM (human T cell), HeLa (cervical carcinoma), Lovo
and COLO205 (colon adenocarcinoma) and MDA-MB-435 (human breast
adenocarcinoma) tumor cell lines. Lysates of homogeneous populations of human T cell
lymphoma samples of two unrelated individuals (derived from lymph node biopsies)
were also analysed. Immunohistochemical studies were performed as described
previously (23).
Recombinant gzm expression. Recombinant human gzm B and gzm H were purified
from the culture supernatants of baculovirus-infected Sf9 cells, 3-5 days after infection,
as described (15). The poly-histidine tagged gzm proteins were purified by nickel
affinity chromatography, and dialysed against PBS. The gzms were tested for their
proteolytic activity by assaying the cleavage of tripeptide substrates Ala-Ala-Asp-S-
benzyl (for gzm B, a kind gift from Drs. Chih-Min Kam and Jim Powers, Georgia
Institute of Technology, Atlanta, GA.) or Phe-Leu-Phe-S-benzyl (for gzm H, purchased
from Sigma).
mAb production and screening. Five to seven week old female BALB/c mice were
immunised into the peritoneal cavity with purified, recombinant gzm H (50ug) mixed
with Freunds’ complete adjuvant. Two to four weeks later, the mice were boosted with
the same antigen mixed with incomplete adjuvant. Three days following a further
similar immunisation, a mouse was sacrificed and its spleen cells were fused with NS-1
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mouse myeloma cells, using polyethylene glycol. Ten days later, hybridomas were
screened for secretion of anti-gzm H mAbs, using a solid phase ELISA assay in which
gzm H (or gzm B as a control) was coated onto the wells of a 96-well PVC plate.
Positive hybridomas were cloned by limiting dilution, and the clones expanded and
cryopreserved in liquid nitrogen.
Production of a rabbit polyclonal antiserum. Polyclonal antiserum was raised in
NZW rabbits by immunisation with purified recombinant human gzm B (200ug) mixed
with Freund’s complete adjuvant and injected into multiple sites subcutaneously in
volumes of <0.1 mL. Three booster immunisations were given intramuscularly with
incomplete adjuvant, at 3-4 week intervals.
Isolation of leukocyte subsets. For isolation of neutrophils, cell pellets collected after
Ficoll-Hypaque centrifugation were dispersed in PBS containing 3% dextran and left to
stand at ambient temperature for 30 min to rouleau the red cells. Neutrophils were then
pelleted from the supernatants and resuspended in hypotonic buffer containing 0.2%
NaCl for 1.5 to 2.0 min to lyse the remaining red cells. The tonicity was then adjusted to
0.9% NaCl and the cells were again pelleted and washed. Cells were >95% neutrophils,
by Giemsa staining. Monocytes were sorted from PBMC with anti-CD14 FITC, and
were at least 82% pure. CD3+CD56- T cells and CD3-CD56+ NK cells were purified
from PBMC by FACS sorting, and were reproducibly >96% pure. For isolation of NKT
cells, PBMC were cultured in medium containing IL-2, IL-7 and α-galactosylceramide
(generously provided by Kirin Breweries, Gunma, Japan) for 7 days. Cells that stained
positive with PE-conjugated CD1d/a-galactosylceramide tetramer (generously provided
by Drs Stephané Sidobre and Mitchell Kronenberg, La Jolla Institute for Allergy and
Immunology, CA) and CD3-FITC were collected by FACS sorting, and purity was
found to be 95%.
T and NK cell stimulation. Human PBMC and purified populations of T and NK cells
were incubated in medium supplemented with recombinant human interleukin-2 (IL-2,
100-500 U/mL; Chiron Corporation) and / or PHA (2 ug/mL) or anti-CD3 mAb HIT3A
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(10ng/mL; BD-Pharmingen) for up to ten days prior to harvest and analysis for gzm
expression.
Protein immunoblotting. Cell lysates were fractionated on 12.5% SDS-polyacrylamide
gels, and the proteins electroblotted onto PVDF membranes. Non-specific protein
binding was blocked by pre-incubation with 5% non-fat skim milk in PBS at 4oC for
two hours. Membranes were incubated with mAb (hybridoma culture supernatant,
typically diluted 1/5 to 1/20 in PBS) at 4oC overnight, washed, then bound Ig was
detected with goat anti-mouse Ig conjugated to horse radish peroxidase.
Generation of gzm-specific cDNA probes Northern blotting, and real-time PCR
analysis. cDNA probes specific for the 3’ untranslated regions of gzm B and H
mRNA/cDNA were produced by PCR amplification of each respective cDNA clone,
using synthetic oligonucleotide primers. The gzm B probe was amplified using the
oligonucleotides 5’-GAAACGCTACTAACTACAGG-3’ and 5’-
CCACTCAGCTAAGAGGT-3’ and was 129 nucleotides in length. The gzm H probe
was similarly amplified using the oligonucleotides 5’-AGAACAATGAAGCGCCTC-3’
and 5 '-ACACGCGGCCGCGATCCATTTATTACAGTCC-3’, and was 138 nucleotides
in length. Each probe was purified from 2% agarose gels and labelled with α32P-dATP
by nick translation or random priming with synthetic hexanucleotides. The radiolabelled
probes were used to identify gzm-specific mRNA in Northern blot experiments. Total
cellular RNA was isolated from various populations of human cells and cell lines (10
ug/lane) and separated by electrophoresis on 1% formaldehyde/agarose gels, then
transferred to nylon membranes by capillary transfer. The membrane was allowed to dry
at RT, then baked at 80oC for two hours. Prehybridisation was in buffer containing 50%
formamide, 1% SDS and 5 x SSC (pH 6.8) solution at 42oC. Following hybridisation
with pre-boiled radiolabelled cDNA probe for 16 hours, the filters were washed at low
stringency, then under high stringency conditions (0.1 x SSC, 1% SDS, 60oC for 2 x 20
min), air dried and exposed to X-Ray film (Kodak) for 1-3 days at –80oC. In some
experiments, plasmid DNA was spotted onto nylon membranes (50 ng/spot), then
denatured in NaOH. The membranes were baked at 80oC for two hours, then
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prehybridised and hybridised as described above. For RT-PCR, 2ug of DNAse-treated
RNA was reverse transcribed using M-MLV Reverse Transcriptase Rnase H minus,
Point Mutant (Promega). PCR primers for each gene were designed using Primer
Express Software (Applied Biosystems, Foster City, CA, USA), with a melting
temperature at 58-60°C and a resulting product of 75-150bp. Triplicate 20ul PCR
reactions were carried out using SYBR Green Master Mix (Applied Biosystem),
initially for 15 min at 95°C, followed by 25 cycles of 95°C for 30 seconds and 60°C for
30 seconds in the ABI Prism 7700 sequence detection system. The levels of gzm H and
B mRNA were normalised to that of ribosomal protein L32. Primer pairs were as
follows: L32 forward 5′- TTCCTGGTCCACAACGTCAAG-3′, reverse 5′-
TGTGAGCGATCTCGGCAC- 3′; gzm B forward 5′-
GCGGTGGCTTCCTGATACAAG- 3′, reverse 5′- CCCCCAAGGTGACATTTATGG-
3′; gzm H forward 5′- TGGCGGCATCCTAGTGAGAA- 3′, reverse 5′-
GCCCCCAAGGTGACATTTATG- 3′. Primer efficiency for gzms B and H was
demonstrated by the overlapping amplification profiles of equal quantities of gzm B and
H cDNA.
Granule fractionation. For cell fractionation experiments, YT human NK tumor cells
(3 x 10e8) were harvested, washed several times in PBS and lysed by nitrogen
cavitation, as described (24). The cell lysate was fractionated on a continuous Percoll
density gradient as described, and 1mL fractions were collected, starting with the dense
(bottom) end of the gradient. Following the removal of Percoll by ultracentrifugation,
aliquots of each fraction were analysed by SDS-PAGE and western blot.
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Results
In order to determine the cellular and sub-cellular expression of gzm H, it was first
necessary to produce an antiserum that could distinguish it from other granzymes,
particularly its closest structural relative gzm B, with which it shares approximately
75% amino acid identity (25). We have previously expressed and purified catalytically
active gzm H secreted by baculovirus-infected insect cells (15), so this protein was used
as an immunogen for mAb production in BALB/c mice. Of 800 hybridoma culture
supernatants screened for mAb secretion in a solid phase ELISA assay, 48 were found
to secrete an Ig that bound to gzm H. All but one of these mAbs produced an equivalent
signal when tested on purified gzm B, indicating they detected an epitope common to
both granzymes (data not shown). However, the mAb produced by hybridoma 4G5
specifically recognised gzm H, and failed to react with gzm B in the ELISA assay
(Figure 1A). Conversely, mAb 2C5, which we have used extensively in studies on gzm
B (12,13,19,26,27), was specific for gzm B and failed to recognise gzm H. MAb 4G5
detected as little as 3 ng gzm H, but did not react at all with gzm B in western blots
(Figure 1B). In the reciprocal experiment, mAb 2C5 reacted strongly with gzm B in
western blots, but did not react with gzm H (data not shown). By contrast with both
mAbs, a polyclonal rabbit antiserum raised against gzm B showed strong cross-
reactivity with gzm H, once again demonstrating the close structural similarity of the
two granzymes (Figure 1B). Interestingly, mAb GB7, a commercially available reagent
purported to specifically detect gzmB, produced an equivalent signal with gzms B and
H (Figure 1C). Similar analyses using other recombinant granzymes also demonstrated
that both 4G5 and 2C5 did not react with recombinant human gzm M, a more distant
relative of gzms B and H (approximately 35% amino acid identity; data not shown) (7).
Having shown that 4G5 specifically detects gzm H, we next used the mAb in western
blotting on whole cell lysates made from a series of hemopoietic and non-hemopoietic
cells lines. The human NK leukemia cell lines YT (Figure 2) and Lopez (data not
shown) both demonstrated a 33kDa protein that co-migrated with recombinant gzmH.
The predicted molecular mass on reduced SDS-PAGE was consistent with a gzm H
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polypeptide backbone of 26kDa (deduced from the cDNA sequence) and approximately
7kDa of added carbohydrate. Gzm H was not expressed by the acute T cell leukemia
lines Jurkat and CEM, the B lymphoma cell lines Daudi and Raji, the myeloid cell lines
HL-60 and U937, or cells from two lymph node biopsies excised from patients with
disseminated T cell lymphoma. Similarly, no signal was detectable in the human
carcinoma cell lines 293 (embryonal kidney), HeLa (cervical), or the adenocarcinomas
Lovo, COLO 205 and MDA-MB435. As Lopez and YT cells both contain large
numbers of cytolytic granules and also express perforin and gzms B and M (24), the co-
expression of gzm H by these cells lines was consistent with a possible role for gzm H
in cytotoxic granule-mediated cell death.
To determine the subcellular localisation of gzm H, we initially performed
immunohistochemistry on pelleted YT cells, using mAbs 2C5 (anti-gzmB) and 4H10
(anti-gzmM) as controls (Figure 3A). As was shown previously, gzms B and M were
localised to granules in the cytoplasm of YT cells (20,26). Cytoplasmic staining in YT
cells was also observed with the gzmH mAb, however the strength of the staining varied
considerably from cell to cell: some YT cells (about 50%) stained strongly, a minority
stained weakly, and the remainder (about 25%) showed no staining. The heterogeneity
of staining was not due to limiting Ig, as similar numbers of strongly and weakly
staining cells were observed over a range of antiserum concentrations (data not shown).
None of the granzyme mAbs bound to Jurkat cells, which were used as a negative
control in this experiment (see Figure 2). To definitively demonstrate gzmH localization
to cytotoxic granules, a lysate of mechanically disrupted YT cells was fractionated on a
Percoll density gradient (Figure 3B). The fractions were analysed by western blotting
with antibodies specific for gzms B and H, perforin and the serpin, PI-9 which is
localised to the cytosol of CL rather than granules (26). Gzm H was co-localized with
the granule proteins perforin and gzmB in fractions 6-8 at the dense end of the gradient
(specific gravity 1.10-1.13 g/mL, data not shown). Fractions 6-8 were free of PI-9,
which is abundant in the cytosol of YT cells (represented by the least dense fractions,
14-18), but is not a significant constituent of the granules (26).
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Almost all of the human and rodent granzymes described to date are expressed
constitutively by NK cells and in an inducible manner by CD8+ (and some CD4+) T
cells, when they become activated and adopt an effector phenotype (28). The two
granzymes known to play a pivotal role in target cell apoptosis, gzm B, which is
encoded by a gene that is very tightly linked to that of gzm H, and gzm A, encoded on a
different chromosome, Chr 5 (8) are both regulated in this way. One exception to this
general “rule” is gzm M, which is constitutively expressed by NK cells but not by
human or rodent T cells when they become activated (20,23). We therefore wished to
determine which leukocyte subsets (if any) in normal human peripheral blood express
gzmH, both in their quiescent state, or following exposure to activating stimuli.
Initially, we subjected unfractionated peripheral blood mononuclear cells (PBMC) from
normal individuals to western blotting, either prior to, or after exposure to agents that
cause T cell activation and proliferation in vitro (Figure 4A). Surprisingly, freshly
isolated, unstimulated PBMC from unrelated donors showed strong constitutive gzm H
expression, whereas the expression of gzm B by the same cells was very low or absent
(Figure 4 A). From past studies, gzmB is known to be expressed at low levels in
unstimulated NK cells (23) and is induced from very low levels in stimulated CD4+ and
CD8+ T cells (10,29-31). Consistent with this pattern of expression, exposure of PBMC
to the T cell stimuli IL-2 and anti-CD3 mAb (Figure 4A) over 4 day time course
experiments caused a strong induction of gzmB protein. The response to IL-2/anti-CD3
had peaked within 24 h, then declined slightly from days 2 to 4. Next, we sorted fresh
PBMC into CD3+CD56- T cells and CD3-CD56+ NK cells (Figure 4B). The T cells
constitutively expressed no gzm B and low levels of gzm H, while the NK cells gave a
strong signal for gzm H, and a substantially weaker signal for gzm B. As NK cells make
up only 2-5% of PBMC, it is likely that gzm B would be undetectable in western
analysis of unfractionated PBMC in most instances (Figure 4A). Exposure of the T cells
to IL-2/PHA caused a strong and continual up-regulation of gzm B levels up till day
four. By stark contrast, the level of gzm H expression did not increase above
constitutive levels but remained constant (Figure 4B). In purified NK cells exposed to
IL-2, the levels of gzm H remained unchanged, whereas gzm B levels increased
somewhat (Figure 4B).
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To further define which cell types were responsible for gzm H expression, we next
examined more highly purified PBMC subsets (Figure 5). As shown previously, gzm H
was expressed at very high levels in unstimulated CD3-CD56+ NK cells, and at far
lower levels in unstimulated CD4+/CD8+ T cells (Figure 5A). Consistent with previous
observations, the unstimulated NK cells also expressed small amounts of gzm B and far
larger quantities of gzm M (23). Purified CD4+ and CD8+ T cells were also examined
separately for granzyme expression upon in vitro stimulation with PHA and IL-2. Again
consistent with previous findings, both T cell subsets expressed large amounts of gzm
B, whereas gzm M was undetectable. Gzm H was expressed in activated CD4+ cells,
but contrary to previous reports (10) the level of expression was not increased in
comparison with unstimulated CD4+ cells. Despite expressing large quantities of gzm
B, the stimulated CD8+ T cells expressed no detectable gzmH (Figure 5A). As NK cells
expressed large quantities of gzm H, we were also interested in whether NKT cells also
expressed gzm H. As the number of NKT cells in PBMC is typically only 0.1%,
unfractionated PBMC from a healthy donor were stimulated in vitro with IL-2, IL-7 and
α-galactosylceramide for 7 days. This resulted in the proportion of cells staining with
PE-labelled CD1d/α-galactosylceramide tetramer increasing to approximately 1.5%
(data not shown). When these NKT cells were sorted to homogeneity and analysed by
protein immunoblotting (Figure 5B), they were found to lack expression of gzm H,
whereas small amounts of gzm B were expressed. Neither polymorphonuclear
leukocytes nor monocytes expressed detectable gzmH (Figure 5B). It was therefore
concluded that gzm H is expressed predominantly by NK cells and weakly by CD4+ T
cells, but not by CD8+ T cells in unstimulated human peripheral blood. A number of
stimuli that strongly induce gzm B expression had no effect on gzm H protein levels.
Absence of 4G5 reactivity with myeloid cells indicated that this mAb does not cross-
react with cathepsin G, a closely related serine protease expressed at high levels in
myeloid cells, but not at all in lymphocytes (18).
Our results were not in agreement with a previous report that indicated gzm H is
inducible upon T cell activation (10). This study was performed without the benefit of a
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specific gzm H antiserum, and relied on mRNA expression studies performed with
cDNA probes. To determine whether the previous findings may have resulted from
cross-reactivity of the gzm H cDNA probe with gzm B mRNA, we generated probes
specific for gzm B and gzm H mRNAs, based on unique, gzm-specific sequences
located in the 3’ untranslated regions of both genes. Unlike the respective full-length
gzmB and gzm H cDNAs, which cross-reacted strongly, even under highly stringent
hybridisation and washing conditions (Figure 6), the 3’ untranslated region probes were
highly specific for gzms B and H (Figure 6A, bottom panel). Neither set of probes
reacted with gzm A, which has a far lower degree of sequence similarity with both gzm
H and gzm B. This result shows the importance of checking for possible cross-reactivity
of all reagents purported to specifically detect either gzm B or H.
The gzmH and gzmB genes are tightly linked to that encoding cathepsin G, and all three
proteases share considerable structural and sequence similarity. Our demonstration that
gzmH protein is not expressed in myeloid cells clearly indicates that gene sequences
regulating cell-specific expression of gzm H and Cathepsin G have not been conserved.
It is also known that myeloid cells store large quantities of cathepsin G in their
cytoplasmic granules, but produce mRNA only during the promyelocyte stage of
development, switching off gene transcription during cell maturation (32). To determine
whether mature NK cells store gzm H protein in the absence of its mRNA, we used gzm
B- and gzm H-specific primers to perform quantitative real-time PCR analysis of sorted
CD3-CD56+ peripheral blood NK cells. We were able to detect mRNA encoding both
gzms B and H (Figure 7). While gzm H protein was considerably more abundant than
gzm B in NK cells (see above), gzm B and gzm H mRNAs were about equally abundant
in freshly isolated NK cells, when compared to a control mRNA, L32. Therefore, gzmH
gene transcripts are still present in mature NK cells, unlike the case with CatG
transcripts in myeloid cells (34).
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Discussion
We have produced a new mAb, 4G5 that specifically detects gzm H and used it to
compare the expression of gzms B and H in a variety of cell lines and human primary
cells. The two gzms have very similar amino acid sequences (approximately 75%
sequence identity) and are predicted to have similar three-dimensional structures, as
both are chymotrypsin-like serine proteases. It was therefore essential to firstly show
that 4G5 and the anti-gzm B mAb used in this study, 2C5, react specifically with their
respective target antigens. This was demonstrated in several ways. Firstly, the mAb
produced by hybridoma 4G5 was unique among 48 mAbs produced that recognized
recombinant gzm H in that it failed to recognise gzm B in a solid phase ELISA assay.
As we were specifically attempting to produce a gzm H-specific mAb, screening for
cross-reactivity with gzm B was included in the initial screen of hybridoma
supernatants, enabling the other 47 mAbs to be eliminated. Secondly, the specificity of
4G5 for gzm H was shown in western blot analysis using purified proteins as targets. As
recombinant proteins were used, there was no possibility that either mAb was actually
detecting small amounts of an alternative gzm present as a contaminant. Thirdly,
immunohistochemical staining of the human NK tumor cell line YT demonstrated
markedly different patterns of staining for gzms B and H. Whereas gzm B staining was
uniform and strong across all cells, gzm H staining was identified only in a subset of
cells, and the strength of staining varied considerably from cell to cell. We are currently
attempting to ascertain the factors that govern gzmH expression in individual YT cells.
This study has raised a number of unexpected results. The major finding is that the
expression of gzms B and H is quite discordant in human lymphocyte subsets, both in
unstimulated T and NK cells, and when the cells were stimulated with a variety of
agents that induce activation and proliferation. For example, gzm H is constitutively
expressed at high levels in peripheral blood NK cells, whereas gzm B is expressed at
much lower levels by the same cells. Stimuli that typically induce strong gzm B
expression also had little effect on gzm H levels in either T or NK cells exposed to
various cytokines and mitogens. Previously, Heusel and colleagues also used sensitive
nuclease protection assays to show a likely discrepancy in gzm B and H regulation in
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human lymphocytes, with highest levels of gzm H mRNA identified in stimulated NK
cells, but virtually none in T cells (33). Human NK cell (large granular lymphocyte)
tumors have also been shown to express mRNA for both gzms B and H in microarray
studies that are said to have used nucleic acid probes that can distinguish between the
two gzms (34). Thus, contrary to previous belief, the principal gzms expressed by
resting NK cells are gzms H (this report), A and M (23), not gzm B. By contrast, the
expression of all gzms is elevated in both of the NK tumor cell lines (YT and Lopez)
that have been examined to date (23,31). The findings in primary NK cells raise the
important question of how NK cells achieve target cell apoptosis when they express low
levels of gzm B, which is thought to have the strongest pro-apoptotic activity of any of
the gzms. In the absence of gzm B, gzms A, M and H would all be candidates to fulfil a
pro-apoptotic function, individually or jointly. While CD3-CD56+ circulating NK cells
expressed large amounts of gzmH, the very low numbers of circulating NKT cells in
peripheral blood meant that it was not possible to determine whether unstimulated NKT
cells also express gzm H. In order to recover enough cells for analysis, we expanded the
NKT cells in IL-2 and IL-7, raising the possibility that these stimuli may have down-
regulated gzmH expression during in vitro culture.
The pattern of expression of gzm H protein was again quite distinct from that of gzm B
in peripheral blood T lymphocytes that were stimulated in various ways in relatively
short term cultures in vitro. All of the stimuli examined in this study led to very marked
induction of gzmB protein, both in CD4+ and CD8+ PBMC. However, gzm H protein
was expressed at low levels in CD4+ T cells, but was not detected in CD8+ T cells and
was not induced by mitogens and mediators of T cell activation including IL-2, PHA,
concanavalin A and anti-CD3 used alone or in various combinations. Collectively our
data clearly indicate that gzms B and H, the products of very tightly linked genes, are
nevertheless regulated in very distinct ways. Despite this, the real-time PCR data
presented in this study also indicate that regulation of the gzmH gene is dissimilar to
CatG as CatG mRNA is not found once myeloid cell maturation occurs. That is, the
regulation of all three closely-linked genes is unique.
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By contrast with the monoclonal reagents used in this study, polyclonal antisera raised
against gzm B (Figure 1B) or gzm H (data not shown) were strongly reactive with both
gzms, as were 47/48 of the mAbs raised against recombinant gzmH. This indicates that
at least one epitope shared between the two closely related gzms is commonly
recognised by mice and other species. Given the close structural similarities between
human granzymes, we anticipated it might be problematic to produce monospecific
mouse antisera when we started to produce anti-gzm mAbs some years ago. Initially,
our strategy was to identify stretches of the gzm amino acid sequence that were
hydrophilic (suggesting orientation to the exterior of the protease) and polymorphic
among the gzms of that species. When we used synthetic polypeptides corresponding to
such portions of gzm sequences to immunise mice, we identified an apparent
immunodominant epitope mapping between residues 139 and 158 (using the N terminal
isoleucine residue of processed gzm B as residue 1) (35). These peptides proved useful
for producing monospecific mAbs for gzmB (2C5, used in the present study) (19) and
gzmM (20). However, peptides from the corresponding region of gzm H largely
produced mAbs (in many independent fusions) that were able to detect the immunising
peptide, but not native gzmH (data not shown). The availability of recombinant,
correctly folded gzmH produced in baculovirus-infected cells has allowed us to
successfully revisit this difficult endeavour.
As with serological reagents, the current study also shows that full-length cDNA probes
used in Northern blot analysis do not specifically detect either gzm B or H. Rather, we
propose that shorter cDNAs spanning the unique 3’ untranslated regions of both genes
are far more suitable for Northern blotting and related assays such as in situ
hybridisation. For other purposes, the use of gzm-specific primers for PCR
amplification may be a suitable alternative. As the reagents purporting to detect gzm B
are increasingly coming into use for diagnostic purposes both in inflammatory and
neoplastic diseases (36-38), we urge extreme caution when interpreting results obtained
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with mAbs or DNA probes whose reactivity with other gzm family members and
cathepsin G has not been adequately excluded.
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References
1. Barry, M., and Bleackley, R. C. (2002) Nat Rev Immunol 2, 401-409 2. Trapani, J. A., and Smyth, M. J. (2002) Nat Rev Immunol 2, 735-747 3. Motyka, B., Korbutt, G., Pinkoski, M. J., Heibein, J. A., Caputo, A.,
Hobman, M., Barry, M., Shostak, I., Sawchuk, T., Holmes, C. F., Gauldie, J., and Bleackley, R. C. (2000) Cell 103, 491-500
4. Trapani, J. A., Sutton, V. R., Thia, K. Y., Li, Y. Q., Froelich, C. J., Jans, D. A., Sandrin, M. S., and Browne, K. A. (2003) J Cell Biol 160, 223-233
5. Froelich, C. J., Orth, K., Turbov, J., Seth, P., Gottlieb, R., Babior, B., Shah, G. M., Bleackley, R. C., Dixit, V. M., and Hanna, W. (1996) J Biol Chem 271, 29073-29079
6. Browne, K. A., Blink, E., Sutton, V. R., Froelich, C. J., Jans, D. A., and Trapani, J. A. (1999) Mol Cell Biol 19, 8604-8615
7. Smyth, M. J., O'Connor, M. D., and Trapani, J. A. (1996) J Leukoc Biol 60, 555-562
8. Baker, E., Sayers, T. J., Sutherland, G. R., and Smyth, M. J. (1994) Immunogenetics 40, 235-237
9. Smyth, M. J., Sayers, T. J., Wiltrout, T., Powers, J. C., and Trapani, J. A. (1993) J Immunol 151, 6195-6205
10. Haddad, P., Jenne, D., Tschopp, J., Clement, M. V., Mathieu-Mahul, D., and Sasportes, M. (1991) Int Immunol 3, 57-66
11. Odake, S., Kam, C. M., Narasimhan, L., Poe, M., Blake, J. T., Krahenbuhl, O., Tschopp, J., and Powers, J. C. (1991) Biochemistry 30, 2217-2227
12. Sutton, V. R., Davis, J. E., Cancilla, M., Johnstone, R. W., Ruefli, A. A., Sedelies, K., Browne, K. A., and Trapani, J. A. (2000) J Exp Med 192, 1403-1414
13. Sutton, V. R., Wowk, M. E., Cancilla, M., and Trapani, J. A. (2003) Immunity 18, 319-329
14. Heusel, J. W., Wesselschmidt, R. L., Shresta, S., Russell, J. H., and Ley, T. J. (1994) Cell 76, 977-987
15. Edwards, K. M., Kam, C. M., Powers, J. C., and Trapani, J. A. (1999) J Biol Chem 274, 30468-30473
16. Johnson, H., Scorrano, L., Korsmeyer, S. J., and Ley, T. J. (2003) Blood 101, 3093-3101
17. MacIvor, D. M., Pham, C. T., and Ley, T. J. (1999) Blood 93, 963-973
18. Hohn, P. A., Popescu, N. C., Hanson, R. D., Salvesen, G., and Ley, T. J. (1989) J Biol Chem 264, 13412-13419
19. Trapani, J. A., Browne, K. A., Dawson, M., and Smyth, M. J. (1993) Biochem Biophys Res Commun 195, 910-920
20. Smyth, M. J., O'Connor, M. D., Kelly, J. M., Ganesvaran, P., Thia, K. Y., and Trapani, J. A. (1995) Biochem Biophys Res Commun 217, 675-683
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
21
21. Geisberg, M., Trapani, J. A., and Dupont, B. (1990) Tissue Antigens 35, 229-233
22. Hirst, C. E., Buzza, M. S., Sutton, V. R., Trapani, J. A., Loveland, K. L., and Bird, P. I. (2001) Mol Hum Reprod 7, 1133-1142
23. Sayers, T. J., Brooks, A. D., Ward, J. M., Hoshino, T., Bere, W. E., Wiegand, G. W., Kelly, J. M., Smyth, M. J., and Kelley, J. M. (2001) J Immunol 166, 765-771
24. Davis JE, S. V., Browne KA, Trapani JA. (2003) Journal of Immunological Methods
25. Klein, J. L., Selvakumar, A., Trapani, J. A., and Dupont, B. (1990) Tissue Antigens 35, 220-228
26. Sun, J., Bird, C. H., Sutton, V., McDonald, L., Coughlin, P. B., De Jong, T. A., Trapani, J. A., and Bird, P. I. (1996) J Biol Chem 271, 27802-27809
27. Sutton, V. R., Vaux, D. L., and Trapani, J. A. (1997) J Immunol 158, 5783-5790
28. Trapani, J. A. (1998) Int Rev Cytol 182, 111-192 29. Liu, C. C., Rafii, S., Granelli-Piperno, A., Trapani, J. A., and Young,
J. D. (1989) J Exp Med 170, 2105-2118 30. Smyth, M. J., Ortaldo, J. R., Shinkai, Y., Yagita, H., Nakata, M.,
Okumura, K., and Young, H. A. (1990) J Exp Med 171, 1269-1281 31. Trapani, J. A., Klein, J. L., White, P. C., and Dupont, B. (1988) Proc
Natl Acad Sci U S A 85, 6924-6928 32. Hanson RD, C. N., Burnett D, Campbell EJ, Senior RM, Ley TJ.
(1990) J.Biol. Chem. 265, 1524-1530 33. Heusel, J. W., Hanson, R. D., Silverman, G. A., and Ley, T. J.
(1991) J Biol Chem 266, 6152-6158 34. Kothapalli, R., Bailey, R. D., Kusmartseva, I., Mane, S., Epling-
Burnette, P. K., and Loughran, T. P., Jr. (2003) Int J Oncol 22, 33-39 35. Apostolidis, V. A., Browne, K. A., Smyth, M. J., and Trapani, J. A.
(1995) Mol Immunol 32, 909-917 36. Tak, P. P., Spaeny-Dekking, L., Kraan, M. C., Breedveld, F. C.,
Froelich, C. J., and Hack, C. E. (1999) Clin Exp Immunol 116, 366-370
37. Medema, J. P., de Jong, J., Peltenburg, L. T., Verdegaal, E. M., Gorter, A., Bres, S. A., Franken, K. L., Hahne, M., Albar, J. P., Melief, C. J., and Offringa, R. (2001) Proc Natl Acad Sci U S A 98, 11515-11520
38. Netto, M. V., Fonseca, B. A., Dantas, M., Saber, L. T., Castro, M. C., and Ferraz, A. S. (2002) Transplant Proc 34, 476-478
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Figure Legends.
Figure 1. Specificity of anti-gzm B and H mAbs. (A) Solid-phase ELISA assay
in which serially diluted mAbs raised against human gzm B peptide (2C5, Ref. 19)
and recombinant gzm H (4G5, this study) were tested on both recombinant purified
gzms. (B) Western blot showing reactivity of 4G5 (tissue culture supernatant,
diluted 1/10) and a polyclonal rabbit antiserum raised against recombinant human
gzmB (1/1000) with various quantities of each recombinant gzm protein. (C)
Western blot showing reactivity of mAbs 4G5, 2C5 and GB7, which has been
considered to react specifically with gzmB, with recombinant gzms B and H.
Figure 2. Expression of gzmH in human tumor cell lines. Western blot analysis
of whole cell lysates derived from a variety of hemopoietic and non-hemopoietic
tumor cell lines and two primary T cell lymphomas, as described in Materials and
Methods. The same panel of lysates was also probed with a mAb detecting tubulin,
as a loading control. Rc gzm H = recombinant gzm H, which was run as a positive
control. The numerals at left indicate the migration of molecular size markers in
kDa.
Figure 3. Expression of gzms B, H and M in YT NK leukemia cells. (A)
Immunohistochemical analysis of YT and Jurkat (human T cell leukemia) cells,
probed with mAbs specifically detecting gzms B, H and M (see Materials and
Methods). (B) Western blot analysis of alternate Percoll density gradient fractions
of a cytoplasmic lysate of YT cells. The numerals indicate fraction numbers,
collected from the bottom (dense) end of the gradient (see Materials and Methods).
The fractions were probed with mAbs specific for gzmH (4G5), the known granule
proteins gzmB (2C5) and perforin (PB2), and PI-9 (7D8), which is absent from
granules, but located in the cytosol.
Figure 4. Expression of gzms B and H in activated human PBMC. (A) Western
blot analysis with mAbs 4G5 (anti-gzm H) and 2C5 (anti-gzm B) of whole cell
lysates of unstimulated PBMC (day 0; 5x10e6 cells per lane) and of similar
numbers of cells cultured in medium supplemented with IL-2 and anti-CD3 mAb
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for the number of days indicated. (B) Western blot analysis of whole cell lysates of
unstimulated CD3+CD56- T and CD3-CD56+ NK cells (day 0; 5x10e6 cells per
lane) and of similar numbers of cells cultured in medium supplemented with
PHA/IL-2 (T cells) or 100 U/mL IL-2 (NK cells) for the number of days indicated.
Figure 5. Expression of gzms B, H and M in human lymphocyte subsets. (A)
Western blot analysis of whole cell lysates of unstimulated human CD3-CD56+
NK cells, unstimulated CD4+ and CD8+ T cells (CD4/CD8) and CD4+ or CD8+ T
cells cultured with PHA and IL-2 for 5 days. The expression of tubulin was used as
a loading control. (B) Western blot analysis of whole cell lysates of peripheral
blood NKT cells expanded in culture medium supplemented with IL-2 and IL-7 for
seven days, unstimulated CD3-CD56+ NK cells, and freshly isolated
polymorphonuclear leukocytes (PMN) and monocytes. Staining with an anti-serpin
antibody that detects the serpin PAI-2 was used as a loading control.
Figure 6. Expression of gzm B and H mRNA in YT cells and human PBMC.
DNA dot blot analysis in which full length cDNAs encoding human gzms A, B and
H cloned into the plasmid vector, pBLUESCRIPT KS+ were spotted onto nylon
filters (50ng/spot). cDNA encoding the human non-granzyme protein IFI 16 was
used as negative control. Replicate filters were probed and washed under high
stringency conditions with 32P-dATP-labelled, full-length, purified cDNA inserts
encoding gzm B (B) or gzmH (H), or with plasmid DNA (KS+). Lower panel:
Similar filters spotted with cDNAs encoding gzms A, B or H were probed and
washed under high stringency conditions with 32P-dATP-labelled, purified PCR-
generated DNA probes specific for the 3’ untranslated sequences of human gzmB
(3’B) or gzmH (3’H) cDNA (see Materials and Methods).
Figure 7. mRNAs encoding gzms B and H are both expressed in unstimulated
CD3-CD56+ NK cells. Real-time PCR amplification of RNA extracted from
freshly isolated, purified CD3-CD56+ peripheral blood NK cells and unfractionated
PBMC of a normal donor. The result is expressed as the mean expression level ±
standard deviation of triplicate assays, relative to L32 mRNA. The data shown are
representative of four experiments carried out with RNA from two unrelated
donors.
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Dale I. Godfrey and Joseph A. TrapaniKarin A. Sedelies, Thomas J. Sayers, Kirsten M. Edwards, Weisan Chen, Daniel G. Pellicci,
lymphocytesDiscordant regulation of granzyme H and granzyme B expression in human
published online April 6, 2004J. Biol. Chem.
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