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The Journal of Immunology, 2006, 177: 5051-5058.
Copyright © 2006 by The American Association of Immunologists, Inc.

Identification of a Novel Human Granzyme B Inhibitor Secreted by Cultured Sertoli Cells1

Simonetta Sipione2,{dagger}, Katia C. Simmen2,{dagger}, Sarah J. Lord2,*, Bruce Motyka{dagger}, Catherine Ewen{ddagger}, Irene Shostak{dagger}, Gina R. Rayat*, Jannette M. Dufour*, Greg S. Korbutt*, Ray V. Rajotte3,* and R. Chris Bleackley3,{dagger}

* Department of Surgery, {dagger} Department of Biochemistry, and {ddagger} Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sertoli cells are found within the seminiferous tubules of the testis where they line the inner lumen of the tubule and are closely associated with one another via tight junctions that form the blood-testis barrier. Their main function is to support the growth and development of new sperm (1).

Cotransplantation of islets with Sertoli cells isolated from rodent testis has been found to successfully protect islets from allo- and autoimmune mechanisms of graft destruction (2, 3, 4, 5). However, what remains to be determined is the mechanism of how Sertoli cells achieve this feat. One potential way Sertoli cells may prevent graft rejection is through the inhibition of CTL killing.

CTLs provide essential protection against virus infection and intracellular pathogens; however, they can also cause harm in cases of autoimmune disease, graft rejection, and graft-vs-host disease. The major mechanism of CTL-mediated killing is the granzyme B pathway (6, 7, 8). When a CTL comes into contact with a target cell it delivers a "lethal hit" of cytolytic molecules, that include perforin and granzyme B, resulting in target cell death by apoptosis (9, 10, 11). Briefly, the CTL-granzyme B pathway involves the calcium-dependent release of granzyme B and perforin, stored in the CTL lytic granules, in the direction of the target cell. Granzyme B, a mannose-6 phosphorylated (M6P)4 protein, binds its receptor, the mannose-6 phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor, on the surface of the target cell and, along with perforin, is endocytosed by the target cell (12). Once inside the target cell, granzyme B remains arrested in the endocytic vesicle, unable to mediate apoptosis, until released into the cytoplasm by perforin or some other sort of lytic agent (e.g., adenovirus (Adv)) (13). In the cytoplasm, granzyme B, a serine proteinase, cleaves procaspases at aspartic acid residues, activating them and initiating the caspase cascade to DNA fragmentation and apoptotic cell death (14, 15, 16, 17, 18).

Sertoli cells have been found to express many proteins that could potentially block the CTL-granzyme B pathway to apoptotic cell death. For example, Sertoli cells have been shown to secrete M6P glycoproteins and IGF-II, which are ligands for the M6P/IGF-II death receptor for granzyme B (19, 20). Some of these M6P glycoproteins include prosaposin, procathepsin L, and TGF-beta (1, 19). TGF-beta, in particular, is a known immunosuppressant agent that has been implicated in the mechanism of how Sertoli cells protect islets from autoimmune destruction in the NOD mouse (21). Sertoli cells may block the granzyme B pathway to apoptotic cell death through the secretion of proteins that are ligands for the M6P/IGF-II receptor. These proteins could down-regulate or block the receptor, effectively preventing granzyme B uptake and subsequent target cell killing.

Human Sertoli cells have additionally been found to express human proteinase inhibitor-9 (PI-9), a serpin that is a potent inhibitor of granzyme B (22, 23, 24, 25). Serpins are serine proteinase inhibitors that inhibit proteolysis through the formation of an essentially irreversible complex with their proteinase (26). PI-9 has been found to inhibit granzyme B in vitro and in vivo and PI-9-transfected cells have been shown to be protected against granzyme B-mediated apoptosis (24, 25). This may explain why Sertoli cells are an immune-privileged tissue, but it cannot account for the protective effect exerted on bystander cells, because PI-9 is a cytosolic protein that is not secreted in the extracellular environment. No studies have been conducted so far to characterize the effect of Sertoli cell-secreted factors on granzyme B-mediated killing.

The focus of the present study was to test the affect of Sertoli cell-conditioned medium (SCCM) on granzyme B-mediated apoptosis. We found that Sertoli cells secrete a factor that inhibits granzyme B enzymatic activity through the formation of a stable complex that effectively reduces granzyme B-mediated apoptosis. This new granzyme B inhibitor is serpina3n, a protease inhibitor belonging to the superfamily of serpins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, cell lines, and reagents

Male BALB/c mice (University of Alberta, Edmonton, Alberta, Canada) were used as Sertoli cell donors. Care and maintenance of all animals was in accordance with the guidelines of the Canadian Council of Animal Care.

L cells (C3H mouse fibroblast cell line) were grown in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS (HyClone), 2 mM L-glutamine, 100 U/ml penicillin, and 50 µg/ml streptomycin. Mouse lymphocytic leukemia L1210 cells were maintained in RPMI 1640 medium supplemented with 20 mM HEPES, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM sodium pyruvate (Invitrogen Life Technologies), 0.1 mM 2-ME (Sigma-Aldrich), and 10% FBS. C57 cells (B6 mouse CTL cell line) were generated from splenocytes isolated from spleen of B6 mice that were stimulated with BALB/c mouse spleen cells. C57 cells were grown in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS, 10–4 M 2-ME, 100 µg/ml penicillin/streptomycin, 20 mM HEPES, and 80 U/ml human recombinant IL-2 (RHFM). Cells were maintained at a concentration of 5 x 105 cells/ml and were stimulated once a week with irradiated BALB/c splenocytes (2500 rad) at a ratio of 1 (C57) to 14 (splenocytes).

Human granzyme B was purified from the cytolytic granules of YT INDY cells as previously described (44). Human replication-deficient Adv was prepared as previously described (27). Mouse degranulate granzyme B material was prepared from CTL stimulated with immobilized anti-mouse CD3{epsilon} Ab (clone 145-2C11; BD Biosciences/BD Pharmingen) as previously described (28).

Isolation of mouse Sertoli cells and preparation of SCCM

Testicles were isolated from 25 9- to 12-day-old male BALB/c mouse donors, and placed in HBSS containing 0.5% BSA (Sigma-Aldrich), on ice. Testicles were chopped and digested with collagenase (1 mg/ml, type V; Sigma-Aldrich) in a shaking water bath for 6 min at 37°C. The tissue was washed three times with HBSS and further digested with DNase (0.4 mg/ml; Boehringer Mannheim) and trypsin (1 mg/ml; Boehringer Mannheim) in calcium-free medium containing 1 mM/L EGTA and 0.5% BSA (Sigma-Aldrich) in a siliconized 250-ml flask in a shaking water bath for 6 min at 37°C. Following the second digestion, the cells were washed with HBSS, filtered through a 500-µm nylon mesh, and then washed three more times before plating. Cell viability was determined by trypan blue exclusion. The number of GATA-4-positive Sertoli cells and smooth muscle {alpha} actin-positive peritubular myoid cells in culture was determined by immunohistochemistry as previously described (29), using mouse monoclonal anti-GATA-4 (1:50; Santa Cruz Biotechnology) and mouse monoclonal anti-smooth muscle {alpha} actin (1:50; DakoCytomation). In each preparation, a minimum of 500 cells were counted. On average, 5 x 107 cells were obtained from each preparation (corresponding to 2 x 106 cells/mouse) 74% of which were Sertoli cells.

For the preparation of conditioned medium, Sertoli cells were plated at the concentration of 5 x 107 cells in 30 ml of serum-free HAM’s F10 culture medium supplemented with 0.5% BSA (no BSA was added when SCCM was prepared for Western blot analysis), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were cultured for 3 days at 37°C and 5% CO2 after which the supernatant was collected and centrifuged twice for 5 min at 2000 rpm to remove cellular debris. The resulting SCCM was then concentrated with an AmiconYM-10 Centricon devise (molecular mass cutoff of 10 kDa; Millipore) for 90 min at 7000 rpm (4°C) down to a volume of 5 ml (6x concentration). Therefore, the concentrated SCCM used in our experiments corresponded to ~107 secreted cell equivalents/ml. Serum-free HAM’s F10, with and without 0.5% BSA, was concentrated in a similar manner to be used as a control medium. Protein concentration was determined with Bradford protein assay (Bio-Rad). SCCM was stored at 4°C until used.

CTL killing assay

[3H]Thymidine-labeled L1210 cells were preincubated with HAM’s F-10 control medium or SCCM (50 µl or ~0.5 x 106 secreted cell equivalents) for 1 h at 37°C. C57 effector cells were then mixed with the target cells at an E:T ratio of 5:1 and incubated for 3 h at 37°C. Sample lysis buffer (1% Triton X, 200 µl) was then added to each sample and tubes were vortexed for 1 min. Tubes were subsequently spun at 1400 rpm for 10 min at 4°C. Supernatants were transferred to liquid scintillation vials, aqueous counting scintillant was added, and samples were counted in a beta counter for the determination of amount of [3H]thymidine release. The percent-specific [3H]thymidine release per sample was calculated as follows: ((sample count (target and effector) – spontaneous count (target alone))/(totals count – spontaneous count)) x 100.

Granzyme B-mediated apoptosis and TUNEL assay

L cells were seeded into a 96-well plate at a concentration of 2 x 105 cells/well and preincubated with 25 µl of concentrated SCCM (corresponding to ~0.25 x 106 secreted cell equivalents) or HAM’s F10 (control) for 30 min at 37°C. Increasing concentrations of human granzyme B and 100 PFU/well of Adv, Adv alone, or granzyme B alone were added to the cells. Cells were incubated for 3 h at 37°C, washed with PBS supplemented with 2% FBS, and fixed with 2% paraformaldehyde and 1% FBS overnight at 4°C. A TUNEL assay was used to measure the amount of DNA fragmentation, a hallmark feature of apoptosis that occurs in target cells upon incubation with granzyme B. Following the overnight fixation procedure, L cells were washed three times with PBS/2% FBS and permeabilized with 0.1% saponin in PBS for 1 h at room temperature. Cells were then washed three times with PBS/2% FBS and incubated with TUNEL mix (20 µl; Roche Diagnostic) and incubation for 1.5 h at 37°C. Following two washes in PBS/2% FBS, the cells were resuspended in PBS/2% FBS and analyzed by FACS (FACScan; BD Biosciences) to derive the percentage of TUNEL-positive cells.

M6PR expression and granzyme B uptake

L cells were added to 96-well plates at a concentration of 2 x 105 cells/well and preincubated with SCCM or HAM’s F10 control medium for 1 h at 37°C. For CI-MPR and CD-MPR staining, L cells were incubated for 1 h at 4°C with PBS (0.1% BSA, control), rabbit anti-bovine CI-MPR (1/500; W. Brown, Cornell University, Ithaca, NY), or rabbit anti-human CD-MPR (1/100; W. Sly, St. Louis University, St. Louis, MO), both of which cross-react with the mouse proteins (12). After washing, cells were incubated for 20 min at 4°C with goat anti-rabbit conjugated to FITC (1/100; Jackson ImmunoResearch Laboratories). Cells were then washed with PBS supplemented with 2% FBS and fixed in PBS with 2% paraformaldehyde and 1% FBS overnight at 4°C. Acquisition and analysis were performed with a FACScan (BD Biosciences).

For the detection of granzyme B binding and uptake, L cells were added to 96-well plates at a concentration of 2 x 105 cells/well and preincubated with SCCM or HAM’s F10 control medium for 1 h at 37°C. For granzyme B binding to L cells, cells were incubated for 1 h at 4°C with PBS (0.1% BSA) and granzyme B conjugated to Alexa 488 (Molecular Probes). Cells were then washed with PBS and fixed as described above before performing FACS analysis. For granzyme B uptake into L cells, cells were incubated for 1 h at 37°C with DMEM (0.1% BSA) and granzyme B conjugated to Alexa 488. Cells were then washed with DMEM with 0.1% BSA, fixed, and analyzed by FACS.

Granzyme B enzymatic activity assay

Human purified granzyme B and mouse CTL degranulate granzyme B were preincubated with SCCM (corresponding to ~0.4 x 106 secreted cell equivalents), with serpina3n or control medium for 40 min at 37°C in 96-well plates. Granzyme B enzymatic activity was then measured as previously described (30). Briefly, a reaction mix containing 50 mM HEPES (pH 7.5), 10% (w/v) sucrose, 0.05% (w/v) CHAPS, 5 mM DTT, and 300 µM Acetyl-Ile-Glu-Pro-Asp-paranitroanilide (Ac-IEPD-pNA) (Kamiya Biomedical) was added to each sample. The plate was then incubated for 4–5 h at 37°C. Hydrolysis of Ac-IEPD-pNA was measured at 405 nm at time 0 and every hour thereafter, using a Multiskan Ascent spectrophotometer (Thermo Lab System).

Western blotting for granzyme B And serine proteinase inhibitor-6 (SPI-6)

Human granzyme B (36 ng) was incubated with 40 µl of concentrated SCCM (50 µg/ml, BSA-free, corresponding to ~0.4 x 106 secreted cell equivalents), with the same amount of concentrated HAM’s F10 medium or with PBS for 2 h at 37°C. SDS sample buffer was added to the samples which were then denatured by heating at 100°C for 5 min. Proteins were separated on a 10% SDS-polyacrylamide gel at 30 mA/gel for 1.5 h and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore).

Immunodetection of granzyme B was performed with a mouse monoclonal anti-human granzyme B Ab (clone 2C5, 1/500 dilution; Santa Cruz Biotechnology). The secondary Ab used was an anti-mouse HRP-conjugated Ab (1/3,000; Bio-Rad). SPI-6 immunodetection was performed with two different Abs, a rabbit anti-mouse SPI-6 Ab (1/5,000 dilution; provided by Dr. J. P. Medema, Leiden University Medical Center, Leiden, The Netherlands) and a mouse anti-human PI-9 Ab (P19-17, 8.5 µg/ml; Alexis Biochemicals) known to cross-react with SPI-6 (22, 31). An anti-rabbit HRP-conjugated Ab (1/20,000; Bio-Rad) or an anti-mouse HRP-conjugated Ab (1/3,000; Bio-Rad) were used as secondary Abs, respectively. Detection of immunoreactive bands was performed by ECL Plus (Amersham Biosciences). Where indicated, PVDF membranes were stripped with 62.5 mM Tris-HCl (pH 6.7) containing 2% SDS and 100 mM 2-ME for 30 min at 60°C in a shaking water bath, before reprobing with a different Ab.

Granzyme B immunoprecipitation and characterization of the serpin-granzyme B complex

Human granzyme B (1 µg) was incubated for 2 h at 37°C with 1 ml of SCCM previously concentrated as indicated above. Preclearing of the sample was performed by adding 1 ml of PBS containing 1% Nonidet P-40 and 0.5% sodium deoxycholate (binding buffer) and 100 µl of protein G-Sepharose (2 mg of protein G/ml drained medium; Amersham Biosciences) for 1 h at 4°C. Immunoprecipitation of granzyme B was conducted overnight at 4°C with a monoclonal anti-human granzyme B Ab (clone 2C5; Santa Cruz Biotechnology) followed by incubation with protein G-Sepharose for 3 h at 4°C. The immunoprecipitate was washed three times with binding buffer and four times with PBS, resuspended in SDS sample buffer, and denaturated at 100°C for 10 min. The immunoprecipitated proteins were resolved by SDS-PAGE and protein bands in the gel were revealed by Coomassie blue R staining. Small aliquots of the sample, collected before and after immunoprecipitation, were run on the same gel and transferred onto a PVDF membrane. Western blot for granzyme B was performed as indicated above and compared with the pattern of bands revealed by Coomassie blue-staining of the gel. The band in the gel that matched the high molecular mass-immunoreactive band in the western blot was cut and analyzed by MALDI-TOF mass spectrometry at the Institute for Biomolecular Design (IBD, University of Alberta). Briefly, an automated in-gel tryptic digestion was performed on a Mass Prep Station (Micromass). The gel pieces were destained, reduced (DTT), alkylated (iodoacetamide), digested with trypsin (sequencing grade; Promega) and the resulting peptides extracted from the gel and analyzed via liquid chromatography/mass spectrometry/mass spectrometry. liquid chromatography/mass spectrometry/mass spectrometry was performed on a CapLC HPLC (Waters) coupled with a Q-ToF-2 mass spectrometer (Waters). Tryptic peptides were separated using a linear water/acetonitrile gradient (0.2% formic acid) on a Picofrit reversed-phase capillary column, (5 µm, BioBasic C18, 300 Angstrom pore size, 75 µm ID x 10 cm, 15 µm tip; New Objectives), with an inline PepMap column (C18, 300 µm ID x 5 mm; LC Packings) used as a loading/desalting column.

Protein identification from the generated MS/MS data was done searching the National Center for Biotechnology Information nonredundant database using the Mascot search engine (Mascot Daemon; Matrix Science) at <www.matrixscience.com>, with stringency of 0.6 Da. Search parameters included carbamidomethylation of cysteine, possible oxidation of methionine, and one missed cleavage per peptide.

Cloning and expression of serpina3n

Hemagglutinin (HA)-tagged serpina3n (serpina3n-HA) was cloned by RT-PCR from mouse liver total RNA, using Superscript II and Platinum TaqDNA polymerase (Invitrogen Life Technologies), according to the manufacturer’s instruction.

The serpina3n cDNA was amplified with the following specific primers: 5'-CGCGGATCCATGGCTTTCATTGCAGCTCTGG-3' (forward) and 5'-CGCCTCGAGTCAGGCGTAGTCGGGGACGTCGTAGGGGTAGAATTTGGGGTTGGCTATCTTGGC-3' (reverse). The forward primer included a BamHI restriction site and the reverse primer included a XhoI restriction site for subsequent cloning. The reverse primer also included a short sequence coding for HA tag at the C-terminal of the serpin. The cDNA was digested with BamHI and XhoI restriction enzymes and cloned into pcDNA3 vector (Invitrogen Life Technologies).

Jurkat cells were electroporated with serpina3n-HA-pcDNA3 and single neomycin-resistant cells were sorted by FACS for clonal expansion. Expression of serpina3n-HA in the transfected clones was verified by immunoblotting with anti-HA Ab (clone HA.11, 1/1000; Covance Research Products) followed by anti-mouse HRP-conjugated Ab (1/3000; Bio-Rad).

In vitro binding of serpina3n-HA to human granzyme B

Radiolabeled ([35S]methionine) serpina3n-HA protein was produced in vitro using TNT Coupled Reticulocyte Lysate Systems (Promega) according to the manufacturer’s instruction. One microgram of DNA was used for each reaction (50 µl). Two microliters of the reaction volume were incubated with purified human granzyme B in PBS for 30 min at room temperature. Samples were then resolved by SDS-PAGE and visualized by autoradiography and by immunoblotting for granzyme B as indicated above.

Preparation of serpina3n-containing medium

Jurkat cell clones expressing serpina3n-HA and control cells transfected with pcDNA3 vector were incubated overnight in Opti-MEM I (Invitrogen Life Technologies) at the concentration of 5 x 106 cells/ml. For inhibition of granzyme B activity, cell-conditioned medium was concentrated to one-fifth of its original volume using AmiconYM-10 Centricon filters as described above and immediately used for the experiments.

Statistics

Statistical significance of differences between two independent groups was calculated with a paired Student’s t test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization Of Sertoli cell preparation

Immediately after isolation the viability of the cell preparation was 100%, as assessed by trypan blue exclusion assay. Sertoli cells accounted for 74% of the cells in the preparation, as assessed by immunostaining for the Sertoli cell marker GATA-4. Other cell types found in the preparation included myeloid cells (6%) and germ cells (whole morphology, 20%). Germ cells do not survive at 37°C in the culture conditions used (32) and after 3 days the cell culture was constituted mainly by Sertoli cells. Protein concentration in the conditioned medium, collected and concentrated as described in Materials and Methods, was 50 µg/ml.

SCCM affects granzyme B-mediated killing

We first tested whether SCCM was able to protect target cells from CTL-mediated killing. L1210 cells undergo apoptotic cell death when treated with C57 CTL. Our laboratory has shown previously that killing by this CTL line is primarily a result of granzyme B and not Fas ligand (data not published). Treatment of L1210 cells with SCCM significantly reduced CTL killing (Fig. 1A).


Figure 1
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  		FIGURE 1. SCCM reduces granzyme B-mediated killing. A, [3H]Thymidine release from L1210 cells after 3-h incubation with CTL in the presence of HAM’s control medium or SCCM. B, TUNEL labeling of L cells after 3-h incubation with 24, 120, or 600 ng/ml granzyme B and Adv in the presence of HAM’s control medium or SCCM. Data shown as the mean ± SEM of at least three different experiments conducted on different preparations of SCCM. *, p < 0.05

 
To assess whether SCCM affected the granzyme B-mediated pathway of killing, we performed a killing assay using purified human granzyme B. Granzyme B treatment of target cells resulted in dose-dependent DNA fragmentation and cell death, as assessed by TUNEL analysis. However, in the presence of SCCM granzyme B-mediated DNA fragmentation in target cells was dramatically reduced (Fig. 1B). The reduction in DNA fragmentation was found to be significant at doses ≥120 ng/ml granzyme B (p < 0.05).

SCCM inhibits granzyme B enzymatic activity

Because no significant effect of SCCM on M6P/IGF-II receptor expression or granzyme B uptake was found (Fig. 2), we examined whether SCCM could affect granzyme B enzymatic activity. Indeed, preincubation of human granzyme B with SCCM resulted in up to 83% decrease of its activity, with respect to incubation with control HAM’s F10 medium (Fig. 3A). Similar results were observed with mouse granzyme B that was obtained from CTL degranulate material (Fig. 3B).


Figure 2
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  		FIGURE 2. SCCM has no effect on mannose-6 phosphate receptor (MPR) expression or granzyme B (grB) uptake. A and B, Cation independent (CI)- and cation dependent (CD)- forms of the MPR expression in L cells after 1-h incubation in the presence of HAM’s control medium or SCCM. MPR expression was determined using Abs specific for CI- and CD-MPR followed by incubation with a FITC conjugated secondary Ab and flow cytometric analysis. C and D, Binding and uptake of granzyme B in L cells after 1-h incubation in the presence of HAM’s control medium or SCCM. Granzyme B was conjugated to Alexa 488 for the determination of binding and uptake in L cells through flow cytometric analysis. Data are presented as the relative mean fluorescence intensity (MFI) (B and D), and percent-positive cells (A and C). Data are shown as the mean ± SEM of at least three independent experiments conducted on different preparations of SCCM.

 

Figure 3
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  		FIGURE 3. SCCM reduces granzyme B enzymatic activity. A, Cleavage of Ac-IEPD-pNA by human purified granzyme B at three different concentrations of granzyme B (24, 120, or 600 ng/ml) in the presence of HAM’s control medium or SCCM. B, Cleavage of Ac-IEPD-pNA by mouse CTL degranulate granzyme B in the presence of HAM’s control medium or SCCM. Data shown as the mean ± SEM of at least three different experiments conducted on different preparations of SCCM. *, p < 0.05

 
Granzyme B is covalently modified by a factor secreted by Sertoli cells

To assess whether granzyme B was subjected to molecular modifications by factors secreted by Sertoli cells, we incubated granzyme B with SCCM and we then resolved the sample by SDS-PAGE and immunoblotting with an anti-granzyme B Ab. As shown in Fig. 4A, in control samples (granzyme B alone and granzyme B incubated with HAM’s F10 control medium) we detected a band with an approximate molecular mass of 32 kDa, as expected, and a second band with a molecular mass of ~54 kDa that was most likely a glycosylated form of granzyme B. When granzyme B was preincubated with SCCM, a new immunoreactive band appeared, with an approximate molecular mass of 78 kDa. These data indicate the formation of a stable complex between granzyme B and an unknown factor in SCCM. We suspected this factor to be a serine proteinase inhibitor or serpin. Serpins are known to bind essentially irreversibly to their cognate proteinase in a manner that is resistant to SDS and heat-denaturation, a property thought to be unique among this class of proteinase inhibitors (26). The serine proteinase inhibitors known to inhibit granzyme B enzymatic activity through the formation of a stable complex are murine SPI-6 and human PI-9. Sertoli cells have been shown to express SPI-6 and PI-9 in mouse and human testis, respectively (22, 23). Therefore, we tested the hypothesis that SPI-6 could be the factor secreted by the mouse Sertoli cells responsible for the binding and inhibition of granzyme B. Western blotting with an Ab recognizing SPI-6 showed that no SPI-6 was detectable in SCCM or in the complex with granzyme B. An immunoreactive band with a molecular mass of 42 kDa was present in the positive control (total cell lysate from C57 mouse CTL) (Fig. 4B). Fig. 4C shows the position of the granzyme B complex in the same gel, stripped and reprobed with the anti-granzyme B Ab. These data indicate that SPI-6 is not the serpin in the observed complex of SCCM and granzyme B.


Figure 4
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  		FIGURE 4. Granzyme B is covalently modified by a factor secreted by cultured Sertoli cells. A, Granzyme B was incubated 2 h with HAM’s control medium, SCCM, or PBS and then detected by immunoblotting with an anti-granzyme B Ab. The arrow indicates a higher molecular mass granzyme B-immunoreactive band that appears when granzyme B is preincubated with SCCM. B, Immunoblotting with anti-SPI-6 Ab. C, Same blot as in B stripped and reprobed with anti-granzyme B Ab. The arrow indicates the higher molecular mass complex that appears when granzyme B is incubated with SCCM and that is not detected by the SPI-6 Ab.

 
Identification of a new granzyme B inhibitor secreted by mouse Sertoli cells

To identify the nature of the complex formed upon incubation of purified granzyme B with the SCCM, we performed MALDI-TOF mass spectrometry analysis of the high-molecular mass complex immunoprecipitated with an anti-granzyme B Ab. Two proteins were identified in the complex based on their peptide mass fingerprints (Table I): human granzyme B and mouse serpina3n (also known as spi2.2), a serine proteinase inhibitor. The predicted molecular masses of serpina3n (47 kDa) and human granzyme B (32 kDa) are indeed compatible with the formation of a covalent heterodimeric complex with an apparent molecular mass of ~78 kDa as observed in our experiments. In all identified serpins, the part of the protein that interacts with the cognate protease is the reactive-center loop (RCL) (33). Table II shows the amino acid sequence of the RCL of serpina3n and the other two serpins, the mouse SPI-6 and the human PI-9, which bind and inactivate granzyme B in mouse and humans, respectively. Although granzyme B preferentially cleaves substrates at Asp or Glu residues (34, 35), it has also been shown to cleave after Met (36, 37). Therefore, the Met that is present in the RCL of serpina3n (Table II) may represent the P1 residue necessary for serpin cleavage by granzyme B. It is noteworthy that other residues in the reactive-center loop of serpina3n are conserved or at least compatible with the previously defined preferences of granzyme B for the interaction with PI-9 (35) (Table II).


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  		Table I. Peptides identified by MALDI-TOF mass spectrometry of the granzyme B complexa

 

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  		Table II. Sequence comparison of three serpins known to interact with granzyme Ba

 
Serpina3n forms a covalent complex with granzyme B in vitro

We cloned the serpina3n cDNA from mouse liver total RNA by RT-PCR. Because an anti-serpina3n Ab was not available, to facilitate detection we engineered an HA tag at the serpin C terminus. We then transcribed/translated the recombinant protein in vitro and we tested its ability to bind to purified granzyme B. As shown in Fig. 5, when granzyme B was added to the in vitro synthesized serpin, a high molecular mass complex of serpina3n with granzyme B was formed, similar to the complex observed when granzyme B is incubated with SCCM. The slightly lower molecular mass of the complex formed by the recombinant serpin (with respect to the complex formed by the serpin secreted by Sertoli cells) is likely due to lack of serpin glycosylation. These data confirmed that serpina3n is the protein secreted by Sertoli cells that binds to granzyme B.


Figure 5
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  		FIGURE 5. Serpina3n forms a complex with granzyme B. A, SDS-PAGE and autoradiography of in vitro translated/transcribed and 35S-radiolabeled serpina3n-HA incubated with human granzyme B (300 ng) or PBS. B, Granzyme B immunoblot after incubation of in vitro translated/transcribed serpina3n-HA with human granzyme B (85 ng) or PBS. Ser, 35S-serpina3n-HA; RTL, reticulocyte lysate. Data shown are representative of three independent experiments.

 
Serpina3n expressed in Jurkat cells is secreted into the medium and inhibits granzyme B activity

We next expressed serpina3n in Jurkat cells and selected stable clones with high transgene expression. Fig. 6A shows expression of the serpin in one of these clones, SerE12-HA, as well as its secretion into the culture medium. When the culture medium from SerE12-HA clone was incubated with purified human granzyme B, a high molecular mass complex of serpina3n and granzyme B was formed (Fig. 6B). Importantly, SerE12-HA-conditioned medium also inhibited granzyme B enzymatic activity in a dose-dependent manner (Fig. 6C). These data provide unequivocal evidence of the inhibitory interaction of serpina3n with granzyme B.


Figure 6
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  		FIGURE 6. Serpina3n secreted by transfected Jurkat cells binds to granzyme B and inhibits its enzymatic activity. A, Expression of serpina3n-HA in Jurkat cells. A total of 0.5 x 106 stable transfected cells (clone SerE12-HA) were incubated overnight in 200 µl of OPTI-MEM I medium. Serpina3n in the cell lysate corresponding to 0.25 x 106 cells (L) and in 10 µl of the conditioned medium (CM) was detected by immunoblotting with anti-HA Ab. B, Serpina3n-HA secreted into the culture medium forms a complex with human granzyme B. Purified human granzyme B (85 ng) was incubated for 2 h at 37°C with medium collected from Jurkat cells, pcDNA3-transfected cells or SerE12-HA cells. Formation of serpina3n-granzyme B complex was detected by SDS-PAGE and immunoblotting with an anti-granzyme B Ab. C, Serpina3n-HA inhibits granzyme B enzymatic activity. Granzyme B (212 ng) was preincubated for 1 h at 37°C with increasing volumes of concentrated conditioned medium from SerE12-HA cells or pcDNA3-transfected cells (corresponding to ~2.5 x 107 cell equivalents/ml), and then granzyme B activity was measured. Data are expressed as percentage of the activity of granzyme B preincubated with the medium of pcDNA3-transfected cells and are the mean ± SEM of three independent experiments performed in triplicate. *, p < 0.001

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Sertoli cells are known to protect islet auto (5), allo- (2, 3, 4), and xenografts (38, 39) from rejection. It has been suggested that Sertoli cells protect bystander cells from immune destruction through the secretion of immunosuppressive factors (21). Sertoli cell mediated protection of islets in the NOD mouse model, a model of autoimmune diabetes, has been attributed to TGF-beta secreted by Sertoli cells (21). TGF-beta is an anti-inflammatory cytokine capable of suppressing T cell, macrophage, NK cell, and B cell activity as well as the expression of many proinflammatory cytokines. In the present study, we show that Sertoli cells also secrete a factor that inhibits CTL killing via blocking the granzyme B pathway to apoptotic target cell death, a main immune effector mechanism in graft destruction.

Previous studies had demonstrated that Sertoli cells communicate with developing spermatogonia through the secretion of numerous M6P glycoproteins that interact with the cell surface M6P/IGF-II receptor (20). The cell surface M6P/IGF-II receptor has also been shown to bind and internalize granzyme B during CTL mediated target cell killing (12). Once inside the target cell, granzyme B has been shown to mediate rapid DNA fragmentation, a hallmark feature of apoptosis (16). We suspected that Sertoli cells may be inhibiting granzyme B-mediated apoptosis through the secretion of ligands for the M6P/IGF-II receptor. However, we found that the SCCM had no effect on M6P/IGF-II receptor cell surface expression, nor did it interfere with granzyme B binding or uptake. Therefore, we investigated whether the inhibitory action of the SCCM could be ascribed to a direct effect on granzyme B proteolytic activity. Indeed, we did find that a factor secreted by mouse Sertoli cells effectively reduced both human and mouse granzyme B enzymatic activity; however, the question that remained was what that factor might be.

Human PI-9 is a potent inhibitor of granzyme B enzymatic activity (24, 25). PI-9 was originally found to be expressed by T cells and proposed to protect CTLs against death mediated by misdirected granzyme B (24). PI-9 may play a similar protective role in Sertoli cells, recently found to also express granzyme B (22, 23). PI-9 inhibits granzyme B enzymatic activity through the formation of an SDS stable complex with granzyme B (26). When we incubated human granzyme B with mouse SCCM and performed Western blotting with an anti-granzyme B Ab, we observed the appearance of a new immunoreactive band at higher molecular mass, indicative of the formation of a stable complex containing granzyme B. Because the granzyme B complex was resistant to SDS and heat-induced denaturation we suspected that it might be a serine proteinase inhibitor (serpin). Therefore, we tested the hypothesis that the unidentified complex consisted of SPI-6 bound to granzyme B. However, we could not detect SPI-6 in the complex with granzyme B. This led us to believe that some other serpin secreted by Sertoli cells must be interacting with granzyme B to form an SDS-stable complex. Indeed, MALDI-TOF mass spectrometry analysis of the complex unequivocally identified a different serpin, serpina3n, as the factor bound to granzyme B. Cloning and expression of serpina3n in Jurkat cells confirmed that this protein binds to and inhibits granzyme B activity. This is the first time that a serpin other than PI-9 and SPI-6 has been shown to inhibit granzyme B. Furthermore, the inhibition is mediated by a secreted serpin and thus achieved before granzyme B enter into the target cells.

Serpina3n is a member of a multigene family of serpins with high degree of homology with the human {alpha}1-antichymotrypsin (SERPINA3). Although in humans there is a single gene coding for {alpha}1-antichymotrypsin, repeated duplication events resulted in the appearance of a cluster of 14 closely related genes in mice (40). Among these genes, serpina3n is the one with the highest degree of homology with antichymotrypsin (61% at the amino acid level). Serpina3n shares substrate specificity with both human antichymotrypsin and human antitrypsin, being able to bind and inactivate chymotrypsin, trypsin, cathepsin G and elastase (41). Our data demonstrate that serpina3n is also an inhibitor of granzyme B.

The previously characterized inhibitors of granzyme B, PI-9 and SPI-6, require the presence of an acidic residue in P1 position of the reactive center loop to block granzyme B activity (35, 42). Other residues in the reactive center loop, specifically the residues P4-P4', have been shown to be important for the interaction with granzyme B (35). Although the reactive center loop of serpina3n does not contain acidic residues, it presents a Met in position P1 that could be cleaved by granzyme B (36, 37). Moreover, many of the residues P4-P4' in the RCL of serpina3n are also compatible with granzyme B specificity as defined by scanning mutagenesis of the PI-9 reactive-center loop (35).

Serpina3n is highly expressed in brain, testis, lung, thymus and spleen (43). Its role in these tissues in not known nor are the specific cell types that express it. In testis serpina3n secreted by Sertoli cells may act in concert with SPI-6 to modulate the activity of the locally produced granzyme B (23). Regardless of the physiological function of serpina3n in mouse testis, the finding that this newly identified granzyme B inhibitor is secreted by Sertoli cells may contribute to our understanding of the mechanism used by Sertoli cells to protect islet grafts from allo-, auto-, and xenoimmune mechanisms of destruction.

Although other molecules secreted by Sertoli cells may negatively modulate the immune response of the recipient, likely more than one mechanism is necessary to prevent graft destruction. Secreted serpina3n efficiently inhibits granzyme B activity and granzyme B-mediated killing, the major pathway of target cell death induced by CTL, and this mechanism may represent a powerful way to disable the host cell-mediated immune response.


   Acknowledgments
 
We thank Tracy Sawchuk and Deb Dixon for excellent technical assistance and Dr. Andy Holt (University of Alberta) for helpful discussion. We also extend a special thanks to William Brown of Cornell University and William Sly of St. Louis University for providing us with Ab for CI- and CD-MPR, respectively.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
S. Sipione, K. C. Simmen, S. J. Lord, R. V. Rajotte, and R. C. Bleackley have a provisional patent on serpina3n as a novel inhibitor of human granzyme B. The patent is owned by the University of Alberta.


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

1 This work was supported by grants from the Canadian Institute of Health Research (CIHR), the Edmonton Civic Employees’ Charitable Assistance Fund, the C. F. "Curly" and Gladys MacLachlan Fund, and Peter A. Allard. R.C.B. is a CIHR Distinguished Scientist, a Medical Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR), a Howard Hughes International Scholar, and a Canada Research Chair. S.J.L. was supported by an AHFMR studentship; S.S. was supported by an AHFMR research fellowship. Back

2 S.S., K.C.S., and S.J.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. R. Chris Bleackley, Department of Biochemistry, University of Alberta, 467A Medical Sciences Building, Edmonton, Alberta, T6G 2H7, Canada; E-mail address: Chris.Bleackley{at}ualberta.ca or Dr. Ray V. Rajotte, Department of Surgery, University of Alberta, 1074 Dentistry/Pharmacy Building, Edmonton, Alberta, T6G 2N8, Canada; E-mail address: rrajotte{at}ualberta.ca Back

4 Abbreviations used in this paper: M6P, mannose-6 phosphorylated; IGF-II, insulin-like growth factor-II; PI-9, proteinase inhibitor-9; SPI-6, serine proteinase inhibitor-6; Adv, adenovirus; SCCM, Sertoli cell-conditioned medium; PVDF, polyvinylidene difluoride; HA, hemagglutinin; RCL, reactive-center loop. Back

Received for publication January 13, 2006. Accepted for publication July 5, 2006.


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 Disclosures
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