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Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and the Laboratory for Clinical and Experimental Immunology, and
Department of Internal Medicine, Renal Transplant Unit, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Department of Research, Evanston Hospital, Northwestern University, Evanston, IL 60201;
§
Department of Pathology, Free University Hospital, Amsterdam, The Netherlands;
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Department of Rheumatology, Daniel Den Hoed Clinic, Rotterdam, The Netherlands; and
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Organon Technika, Boxtel, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since granzymes and PFN constitute the primary effectors of the granule exocytosis pathway, identification of these proteins by immunohistochemical (7) or molecular biologic techniques (8) has been used to assess the involvement of activated CTL or NK cells in pathologic processes. These methods, however, require the availability of tissue, which severely hampers their clinical utility. Studies in vitro have shown that granzymes may be released extracellularly during cytotoxic cell degranulation (9). Therefore, we hypothesized that levels of extracellular granzymes may reflect endogenous CTL or NK cell activity in vivo. To test this hypothesis we developed ELISAs that accurately identify native human GrA or GrB. We measured the plasma levels of the granzymes in healthy volunteers, in patients with rheumatoid arthritis (RA), and in those suffering infections with EBV or HIV-1. The results show that soluble granzymes are present at low concentrations in the blood of healthy individuals and at significantly higher levels in these disease states.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GrB was isolated from the human leukemia T cell line YT-Indy by HPLC (10). GrA was purified to homogeneity from IL-2-activated lymphocytes (11). Human neutrophil elastase was obtained from Elastin Products Co., Inc. (Owensville, MO); proteinase 3 was a gift from Dr. K. Koth (Chiron Corp., Emeryville, CA); human mast cell tryptase was a gift from Dr. R. Lutter (Academic Medical Center, Amsterdam, The Netherlands). Biotinylated goat anti-mouse polyclonal Abs were obtained from the Department of Immune Reagents of the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands).
Production and purification of mAbs against native GrA and GrB
BALB/c mice were immunized s.c. with 25 µg of native GrA or
GrB suspended in CFA, followed by three to six subsequent injections of
25 µg of the protein suspended in IFA at 2-wk intervals. At the time
the animals produced high Ab titers, fusion of the spleen and lymph
node cells with mouse myeloma Sp2/0-Ag 14 cells was performed according
to standard procedures (12). Conditioned medium from the hybridomas was
screened for the presence of anti-GrA or anti-GrB mAb using rat
anti-mouse Ig-Sepharose suspensions together with
125I-radiolabeled native GrA or GrB as described
previously for anti-C3 mAb (12). The supernatant of Ab-producing
hybridomas was concentrated by ammonium sulfate precipitation (50%,
w/v) followed by dialysis against PBS, pH 7.4. Thereafter, mAb were
purified by protein G affinity chromatography (Pharmacia Fine
Chemicals, Uppsala, Sweden) according to the instructions of the
manufacturer. Subclasses of mAb were determined by an isotyping
dipstick method (Innogenetics, Antwerp, Belgium). All mAb were of the
IgG1 
subclass.
Competition RIA for the initial characterization of the mAb
Limiting amounts of Sepharose-coupled mAb were added to 125I-radiolabeled GrA or GrB (2–4 ng) that had been preincubated for 1 h at room temperature with 2 µg of a second mAb. The mixture was then incubated by head-over-head rotation for at least 4 h at room temperature. Next, the Sepharose beads were washed with PBS/0.05% (w/v) Tween-20, and the amount of bound 125I-labeled GrA or GrB was measured. An mAb was considered to bind to similar or overlapping epitopes when interaction of the second Ab with 125I-labeled GrA or B was inhibited by >50%.
Biotinylation of Abs
Purified Abs were biotinylated using long chain biotinyl-N-hydroxysuccinimide ester sulfonic acid (Pierce Chemical Co., Rockford, IL) according to the instructions of the manufacturer.
Granzyme B ELISA
Purified mAb GB11 was incubated at a concentration of 2 µg/ml in 0.1 M sodium carbonate/bicarbonate buffer, pH 9.6, for 16 h at 4°C in microtiter plates (Nunc Maxisorb Immunoplate, Nunc, Copenhagen, Denmark; 100 µl/well). The plates were then washed with PBS/0.02% (w/v) Tween-20. An identical washing procedure was performed after each incubation step which consisted of 100 µl, except for the blocking step (150 µl). After coating, residual binding sites were blocked by a 45-min incubation with PBS/2% (v/v) cow milk. All samples to be tested as well as standards (purified native GrB at various concentrations) were pretreated with 40 µg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO) for 30 min at room temperature in PBS/0.02% Tween-20. Samples were then appropriately diluted in high performance ELISA buffer (Central Laboratory of The Netherlands Red Cross Blood Transfusion Service) and incubated for 1 h. Next, the plates were incubated with an excess of biotinylated GB10 mAb (0.5 µg/ml) together with 1% (v/v) normal mouse serum for 1 h, then the plates were incubated for 30 min with streptavidin-polymerized horseradish peroxidase (Department of Immune Reagents, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service), whereafter bound peroxidase was visualized by incubation with a solution of 100 µg/ml of 3,3',5,5'-tetramethylbenzidine (Merck, Darmstadt, Germany) and 0.003% (v/v) H2O2 in 0.11 M sodium acetate buffer, pH 5.5. The reaction was stopped by addition of an equal volume of 2 M H2SO4 to the wells. Finally, the absorbance at 450 nm was read on a Titer-Tek Multiscan plate reader (Labsystems, Helsinki, Finland).
Granzyme A ELISA
The GrA ELISA was essentially performed as described for that of GrB, except that GA28 (2 µg/ml) was the coating mAb, and biotinylated GA34 (0.5 µg/ml) was used to detect bound GrA.
Degranulation experiments
JS-136 cells (a gift from Dr. J. Borst, Dutch Cancer Institute, Amsterdam, The Netherlands) were harvested after 6 days of stimulation with JY cells (13). Cells were washed in Iscove’s supplemented with 0.02% (w/v) BSA and resuspended at a concentration of 1 x 106 cells/ml in Iscove’s modified Dulbecco’s medium (IMDM)/0.02% (w/v) BSA, containing three mAbs directed against CD2 (4B2, 6G4, and Hic 27; Central Laboratory of The Netherlands Red Cross Blood Transfusion Service) each at 1 µg/ml and PMA at a final concentration of 1 ng/ml. Control cells (not stimulated with anti-CD2 and PMA) were resuspended in IMDM/0.02% BSA. Cell suspensions (100 µl/well) were put into 96-well round-bottom microtiter plates (Greiner, Frickenhausen, Germany), and incubated for various times. Aliquots of the supernatant were collected and centrifuged for 8 min at 1500 rpm to remove intact cells. Medium was stored at -20°C until testing.
Clinical samples
Plasma samples of healthy volunteers were collected by venipuncture using tubes (Venoject, Terumo Europe N.V., Leuven, Belgium) containing soybean trypsin inhibitor (0.1 mg/ml), 10 mM EDTA, and 20 mM benzamidine (final concentrations).
RA plasma samples (n = 10), obtained at the Daniel den Hoed Clinic in Rotterdam, were collected in tubes containing 10 mM EDTA and 0.05% (w/v) Polybrene (Janssen Chimica, Beerse, Belgium) (14). Simultaneously, synovial fluid (SF) samples were taken from involved knees using a plastic syringe and immediately transferred into a siliconized Vacutainer tube (Becton Dickinson, Plymouth, U.K.) containing 10 mM EDTA and 0.05% Polybrene. All patients fulfilled the revised American Rheumatism Association criteria for definite RA (15).
Blood from HIV-1-infected patients was collected in tubes (Venoject) containing 15 U of USP heparin. Samples were prepared from specimens sent to the Department of Clinical Viroimmunology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, to measure various immune parameters of HIV-1 infection. Diagnoses were based on the presence of Abs against HIV-1 p24 protein in plasma from the patients.
All plasma samples were centrifuged for 10 min at 1300 x g, whereafter supernatants were centrifuged again for 5 min. The final supernatants were stored in small aliquots at -70°C until use.
In addition, serum samples were obtained from 14 patients in various stages of acute EBV infection as determined by immunoblotting for EBV early AgD, viral capsid Ag (VCA), or Epstein-Barr nuclear Ag (16). EBV-IgG and IgM levels were further defined by peptide ELISA (17).
Statistical analysis
Data are presented as the median and range unless indicated otherwise. For statistical analysis, values below the detection limit were set at 1 pg/ml. The significance of the differences between subject groups was assessed using the nonparametric Wilcoxon-Mann-Whitney test. A double-sided p < 0.05 was considered to indicate a significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were immunized with native human GrB isolated from the
human leukemia cell line YT-Indy. From the fusion experiments three mAb
of the IgG1 subclass were obtained that bound
125I-labeled GrB in solution: GB10, GB11, and GB12.
Purified mAb were either coupled to Sepharose beads or biotinylated. As
determined by RIA competition experiments and sandwich ELISA
experiments, mAb GB11 and GB12 recognized the same or overlapping
epitopes, whereas mAb GB10 appeared to bind to a different epitope.
Pilot experiments revealed that the optimal combination was GB11 as the
coating Ab and biotinylated GB10 as the detecting Ab. Using this
combination, a highly reproducible ELISA was developed in which soluble
GrB was detected at a range of 3 ng/ml to 4 pg/ml (Fig. 1
). Addition of soybean trypsin inhibitor
(100 µg/ml, final concentration), which blocks the active site of
GrB, did not affect the detection of GrB in the ELISA (result not
shown). Finally, GrB mAb did not cross-react with purified GrA in the
ELISA (Fig. 1).
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A procedure similar to that described for GrB mAb was used to
raise mAb against purified human GrA. Twenty-six Abs (GA10–GA35), all
of the IgG1 subclass, were obtained. After purification, coupling
to Sepharose beads, and biotinylation, mAb were characterized by RIA
competition experiments and sandwich ELISAs. Five groups of mAb were
identified, each group recognizing one or overlapping epitopes (results
not shown). Testing combinations of mAb from the different groups
revealed that mAb GA28 as a catching and GA34 as a detecting Ab
provided the most sensitive assay for soluble GrA. With this highly
reproducible ELISA, GrA was detected at a range from 16 pg to 18
ng/ml (Fig. 2). Addition of
benzamidine (up to 100 mM) to the protein, which inhibits the
proteolytic activity of GrA, did not affect detection of the protein in
the ELISA (result not shown). As can be seen in Figure 2, GrB did not
cross-react in this system.
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To ascertain that the mAb used in the ELISAs were specific for
either GrA or GrB and did not react with other serine proteases
homologous to granzymes, we performed additional ELISA experiments.
Wells were coated with human neutrophil elastase, mast cell tryptase,
proteinase 3, and cathepsin G and incubated with mAb GA28, GA34, GB10,
and GB11. GrA mAbs only bound to GrA and not to GrB, neutrophil
elastase, tryptase, proteinase 3, or cathepsin G (Fig. 3). Similarly, mAb GB10 and GB11 only
bound to GrB and not to neutrophil elastase, mast cell tryptase,
proteinase 3, or cathepsin G (not shown).
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Since in vitro studies have shown that granzymes may be released
during degranulation of cytotoxic cells (9), degranulation experiments
were performed, and the medium of the cells was tested for the presence
of GrA and GrB. After 6 days of stimulation of the human CTL clone JS
136, degranulation was induced by resuspending the cells in BSA
containing IMDM medium supplemented with anti-CD2 mAbs together
with PMA. At various times of incubation, samples from the medium were
collected and tested for the presence of soluble granzymes. After
30-min incubation, detectable levels of both GrA and GrB were found
(Fig. 4). Soluble GrA and GrB levels
continued to increase until 5 h of incubation.
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Granzymes are expressed by PBMC of healthy individuals (18, 19);
therefore, soluble granzymes may be present in normal blood. Indeed,
most plasma samples from healthy individuals contained detectable
levels of soluble granzymes (Fig. 5 and
Table I). The median level was 33.5 pg/ml
(range, 1–121) for soluble GrA and 11.5 pg/ml (range, 1–130) for GrB.
These values were measured in plasma samples prepared from blood
collected in soybean trypsin inhibitor (100 µg/ml), EDTA (10 mM), and
benzamidine (10 mM). Plasma samples prepared from blood collected in
citrate (10.5 mM), EDTA (10 mM), or heparin (15 USP U) yielded similar
results, as did serum samples obtained from the volunteers (data not
shown).
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Immunohistochemical and biochemical studies have shown local
production of GrA, GrB, and PFN in inflamed rheumatoid joints (8, 20, 21, 22). Therefore, plasma and synovial fluid samples of 10 RA patients
fulfilling the American Rheumatism Association criteria (15) were
tested for the presence of soluble granzymes. Both soluble GrA and GrB
were detected in the synovial fluid samples of the RA patients. In most
cases the values markedly exceeded plasma levels detected in normal
volunteers (Fig. 5
and Table I). In contrast, plasma levels of GrA were
similar to those in controls, while GrB levels tended to be somewhat
higher. Since rheumatoid factors present in plasma and SF could lead to
spurious results, we performed additional specificity control studies.
First, we did not observe absorption values significantly above
background for ELISAs where the GrA coating Ab was used combined with
the biotinylated GrB mAb, and vice versa. Second, rheumatoid factor
levels in the samples tested did not correlate with either GrA or B
levels. Third, addition of purified rheumatoid factors to the ELISAs
did not alter the levels of the granzymes detected. To ascertain
whether soluble GrA or GrB was produced locally in the joints, the
ratio between SF and plasma samples was calculated for each patient.
This ratio exceeded 1.0 in all patients, with a range from 5.66 to
69,554 for soluble GrA and from 4.4 to 3,775 for GrB. Thus, consistent
with the finding that RA synovium contains activated CTLs, soluble GrA
and GrB are produced locally in the joints of patients with
RA.
Circulating levels of soluble GrA and GrB in patients with EBV or HIV-1 infections
Cytotoxic T cells and NK cells are involved in the immune response
against a variety of viral infections (23, 24, 25). Therefore, we tested
blood samples from patients with symptomatic EBV or HIV-1 infection for
the presence of soluble GrA and GrB. Serum samples of 14 patients at
various stages of EBV infection (Table II) contained elevated levels of both
soluble GrA (median, 156; range, 1–5500) and soluble GrB (median, 60;
range, 1–4000). Levels of GrA were significantly higher than those in
the healthy control group, whereas those of GrB tended to be higher
(Fig. 5 and Table I).
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To establish whether levels of soluble granzymes fluctuated with
the stage of viral infection, levels were measured in plasma samples
taken during the acute and convalescent phases. Longitudinal plasma
samples from four EBV-infected and one HIV-1-infected patients were
available for analysis (Fig. 7). Although
both GrA and GrB levels were increased during the acute phase of the
infection, levels of GrA were consistently higher than those of GrB
(not shown). Moreover, increases in GrA were associated with markers of
early viral infection, namely IgM anti-VCA in EBV infection and p24
Ag in HIV-1 infection (Fig. 7). In the period following the acute
infection, the granzyme levels decreased to the (high) normal range.
Thus, during viral infection the levels of both GrA and GrB increased
during the acute phase and then declined during resolution.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Using these sensitive
assays we show here for the first time that free granzymes can be
detected during states of CTL and NK cells activation both in vitro and
in vivo. The mechanisms that contribute to the increase in extracellular granzymes is uncertain. It is commonly accepted that the secretion of the granzymes is induced by target cells during the cytotoxic cell response. Evidence indicates, however, that various chemokines may also elicit exocytosis of the granzymes independently of target cells (27). Furthermore, cytolytic cells have been reported to constitutively secrete the granzymes following activation (28). In addition to an enhanced secretion, an impaired clearance of granzymes during a cytotoxic response can contribute to an increase in extracellular granzymes. Unfortunately, the precise clearance mechanisms of circulating granzymes are not known. We have observed in 30 patients with varying degrees of renal dysfunction no correlation between creatine clearance and circulating granzyme levels (result not shown). Thus, apparently kidney function does not influence the clearance of circulating granzymes. However, as long as the precise clearance mechanisms are not known, it remains to be established to what extent impaired clearance may have contributed to an increase in circulating granzymes.
The release of granzymes during NK or CTL degranulation has been
documented previously using either esterolytic assays or immunoblotting
techniques. BLT ((benzyloxycarbonyl) lysine thiobenzyl ester) esterase
activity (substrate for GrA) and asp-ase activity (substrate for GrB)
have been detected in supernatants during cytotoxicity assays (29, 30, 31, 32, 33),
while GrA has been detected using an immunoblotting technique (34).
These approaches, however, fail to accurately measure enzymatically
active forms of the granzymes. We show that CTL stimulated to undergo
granule exocytosis secrete biologically active granzymes (Fig. 3).
Furthermore, the release of granzymes has been observed during standard
cytotoxicity assays and after activation of NK cells or CTL by such
cytokines as IL-2 (results not shown). Although the signals that
regulate the degranulation and constitutive secretion of the granzymes
and the contribution these processes make to increase extracellular
levels await further characterization, it is clear that granzymes are
positioned to mediate extracellular biologic effects independently
of PFN.
Detectable levels of extracellular granzymes were present in the plasma of healthy donors. It remains to be established whether these low plasma levels are due to degranulation or constitutive secretion of the serine proteases from cytotoxic cells (28). Since normal peripheral blood contains a small percentage of activated, granzyme-positive lymphocytes, the levels in normal subjects may reflect the constitutive release of the proteases from these activated cells (18, 35, 36). During testing of normal plasma, an occasional sample was identified with levels that exceeded the upper limit of normal (>200 pg/ml). Dose-response curves of such samples did not parallel those generated with purified granzymes. After pretreatment with hyaluronidase, a strategy used to enhance reliable measurement in SF, much lower levels were detected and those response curves parallel the internal standard. Since we have not observed hyaluronidase sensitivity among patient samples, we decided to treat all samples and standards routinely with hyaluronidase to prevent the detection of false positive signals and ensure that the measured samples parallel the internal standards. The basis for this hyaluronidase sensitivity is unclear. We are now evaluating whether this behavior is due to interaction of glycosylated granzyme with circulating hyaluronic acid followed by disproportionate binding of the complexes to the microwells.
The role of granule-mediated killing as a host defense against certain
viral infections is well established (2, 37, 38, 39). Although the release
of granzymes by CTL of patients suffering from viral infections has
been documented by insensitive enzymatic assays (40, 41), we show here
that blood samples of EBV- or HIV-1-infected patients contain elevated
levels of both native GrA and GrB. These levels varied dramatically
from virtually none to 100-fold more than normal. The marked
differences in the levels are probably related to the interindividual
variation in the intensity of the CTL/NK response as well as the stage
of infection when the samples were obtained (see Fig. 7). Importantly,
during the acute phase, granzyme levels increased simultaneously with
early markers of infection. Therefore, our results suggest that
elevated levels of soluble granzymes in plasma may indicate the
presence of an acute viral infection. Preliminary results suggest that
this association may also exist during Dengue fever and CMV infection
after renal allograft transplantation.
The potential extracellular functions of the granzymes in antiviral immunity have not been extensively evaluated. GrA induces the production of IL-6 and IL-8 in fibroblasts and epithelial cells (42) and stimulates IL-6, IL-8, and TNF production in monocytes by a mechanism that does not involve activation of the thrombin receptor (43). GrB in the absence of PFN has been reported to induce apoptosis in targets infected with viruses entering cells via endocytosis (e.g., adenovirus) (6). This suggests that soluble GrB may mediate a novel form of antiviral cell-mediated cytotoxicity (44). Therefore, extracellular granzymes may control viral infections through both cytotoxic and noncytotoxic mechanisms (37).
Granzyme-positive cells have been identified in the synovial tissue (21, 22) as well as in the SF of patients with RA (20). Although RA plasma samples contained slightly elevated levels of GrA (4/10) and GrB (7/10), strikingly higher levels of both granzymes were detected in paired SF samples. Since the ratio of plasma to SF levels consistently exceeded 1.0, the data strongly suggest that the granzymes are produced and secreted within the affected joint. The significance of markedly increased SF levels in the pathogenesis of RA is unknown. GrA and GrB have been reported to degrade extracellular matrix proteins in vitro (45, 46). GrB, in particular, is able to cleave cartilage aggrecan with an efficiency comparable to that of stromelysin (6). Hence, our findings suggest a potential role for the granzymes in the tissue destruction of the rheumatoid joint.
In summary, we have developed sensitive ELISA assays that are suitable for the accurate detection of extracellular granzymes. High levels of both GrA and GrB were detected in the plasma of patients experiencing viral infections as well as in the joints of patients with active RA. The assays will provide invaluable tools for study of the involvement of CTL and NK cells in human disease, allograft rejection, and graft-vs-host disease and should stimulate interest in identifying additional functions for this novel family of granule-associated serine proteases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. E. H. A. Spaeny-Dekking, Central Laboratory of The Netherlands Red Cross Blood Transfusion Services. Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
3 Abbreviations used in this paper: PFN, perforin; GrB, granzyme B; GrA, granzyme A; RA, rheumatoid arthritis; IMDM, Iscove’s modified Dulbecco’s medium; SF, synovial fluid; VCA, viral capsid antigen.
Received for publication September 18, 1997. Accepted for publication December 5, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vi
, A. M. Van Leeuwen, J. Borst, C. Verweij, C. E. Hack. 1993. Production and characterization of monoclonal and polyclonal antibodies raised against recombinant human granzymes A and B and cross reacting with the natural proteins. J. Immunol. Methods 163:77.[Medline]
2-macroglobulin in arthritic joints. Arthritis Rheum. 34:1139.[Medline]
-thrombin. J. Immunol. 156:2585.[Abstract]
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G. M. Tremblay, A. M. Wolbink, Y. Cormier, and C. E. Hack Granzyme Activity in the Inflamed Lung Is Not Controlled by Endogenous Serine Proteinase Inhibitors J. Immunol., October 1, 2000; 165(7): 3966 - 3969. [Abstract] [Full Text] [PDF]
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F. N. Lauw, D. Pajkrt, C. E. Hack, M. Kurimoto, S. J. H. van Deventer, and T. van der Poll Proinflammatory Effects of IL-10 During Human Endotoxemia J. Immunol., September 1, 2000; 165(5): 2783 - 2789. [Abstract] [Full |
) or purified GrA (•), as a control, were tested. Data indicate
the mean ± SD of five experiments. The detection limit of the
ELISA (defined as the concentration of GrB showing an absorption twice
the background) was 2 to 4 pg/ml.
) or HIV-1 (n
= 25; 