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The Journal of Immunology, 2002, 169: 1050-1057.
Copyright © 2002 by The American Association of Immunologists

Prevention and Induction of Autoimmune Exocrinopathy Is Dependent on Pathogenic Autoantigen Cleavage in Murine Sjögren’s Syndrome1

Kaoru Saegusa*, Naozumi Ishimaru*, Kumiko Yanagi*, Kenji Mishima*, Rieko Arakaki*, Takashi Suda{dagger}, Ichiro Saito* and Yoshio Hayashi2,*

* Department of Pathology, Tokushima University School of Dentistry, Tokushima, Japan; and {dagger} Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, Ishikawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The in vivo role of autoantigen cleavage during apoptosis in autoimmune diseases remains unclear. Previously, we found a cleavage product of 120-kDa {alpha}-fodrin as an important autoantigen in the pathogenesis of primary Sjögren’s syndrome (SS). In the murine primary SS model, tissue-infiltrating CD4+ T cells purified from the salivary glands bear a large proportion of Fas ligand, and the salivary gland duct cells constitutively possess Fas. Infiltrating CD4+ T cells, but not CD8+ T cells, identified significant 51Cr release against mouse salivary gland cells. In vitro studies demonstrated that apoptotic mouse salivary gland cells result in a specific {alpha}-fodrin cleavage into 120 kDa and that preincubation with caspase inhibitor peptides blocked {alpha}-fodrin cleavage. In vivo treatment with caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and N-acetyl-Asp-Glu-Val-Asp-al-CHO into the murine model results in dramatic inhibitory effects on the development of autoimmune lesions and in restoration of sicca syndrome. Furthermore, we found that immunization with recombinant {alpha}-fodrin protein identical with an autoantigen into normal recipients induced autoimmune lesions similar to SS. These data indicate that prevention and induction of autoimmune exocrinopathy is dependent on autoantigen cleavage via caspase cascade and that caspase inhibitors might provide a new therapeutic option directed at reducing tissue damage in the murine model for SS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Organ-specific autoimmune diseases are characterized by tissue destruction and functional decline due to autoreactive T cells that escape self-tolerance (1, 2). Sjögren’s syndrome (SS)3 is an autoimmune disorder characterized by lymphocytic infiltrates, destruction of the salivary and lacrimal glands, and systemic production of autoantibodies to the ribonucleoprotein particles SS-A/Ro and SS-B/La (3, 4, 5). Although the specificity of CTL function has been an important issue of organ-specific autoimmune response, the mechanisms responsible for tissue destruction in SS remain to be elucidated.

We reported previously that a cleavage product of 120-kDa {alpha}-fodrin may be an important autoantigen in the pathogenesis of primary SS in both the animal model and in humans (6). {alpha}-Fodrin is a ubiquitous, calmodulin-binding protein (7) found to be cleaved by calcium-activated protease (calpain) in apoptotic T cells and by calpain and/or caspase 3 (CPP32) (8) in anti-Fas-stimulated Jurkat cells and/or neuronal apoptosis (9, 10, 11, 12). It was demonstrated that the fodrin {alpha} subunit is cleaved in association with apoptosis and that the 120-kDa fragment is a breakdown product of the mature form of 240-kDa fodrin {alpha} subunit (11, 12). Recent reports have shown that caspase 3 is required for {alpha}-fodrin cleavage during apoptosis (12, 13, 14). In Jurkat cells, caspase 3-like proteases have been reported to cleave {alpha}-fodrin and poly(ADP-ribose) polymerase (PARP) but with differential sensitivity to the caspase 3 inhibitor N-acetyl-Asp-Glu-Val-Asp-al (DEVD)-CHO (14). In neuroblastoma cells, treatment with staurosporin induced cleavage of {alpha}-fodrin at both caspase 3 and calpain cleavage sites (15). Accumulating evidence indicates that the interaction of Fas with Fas ligand (FasL) regulates a large number of pathophysiological processes of apoptosis (16, 17). It has been reported that both Fas and FasL are present in thyrocytes and that their concomitant expression on thyrocytes, independent of infiltrating T cells, is responsible for thyrocyte destruction in Hashimoto’s thyroiditis (18). In contrast, expression of Fas by pancreatic {beta} cells has been shown to have a major influence on the susceptibility of tissue destruction in nonobese diabetic mice to diabetes (19, 20). Thus, we speculate that Fas-mediated cytotoxicity and caspase-mediated {alpha}-fodrin proteolysis are involved in the progression of tissue destruction in SS.

In this study, we demonstrate that an increased activity of apoptotic proteases is required for the {alpha}-fodrin proteolysis during development of murine SS. In addition, we present evidence that a cleavege product of autoantigen induces autoimmune exocrinopathy in normal recipients and that treatment with caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (z-VAD-fmk) and DEVD-CHO protect animals against the development of autoimmune exocrinopathy in the SS model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female NFS/N strain mice carrying the mutant gene sld (21) were reared in our specific pathogen-free mouse colony and given food and water ad libitum. Thymectomy was performed on day 3 after birth (3d-Tx) in NFS/sld mice (22). A total of 156 mice, consisting of 74 3d-Tx and 82 nonthymectomied (non-Tx) NFS/sld mice, were investigated in the present study. C57BL/6 mice (n = 24) purchased from Charles River Japan (Atsugi, Japan) were used as controls.

Histology and immunohistology

All organs were removed from the mice, fixed with 4% phosphate-buffered formaldehyde (pH 7.2), and prepared for histologic examination. The sections were stained with H&E. Histological grading of the inflammatory lesions was done according to the method proposed by White and Casarett (23). Immunohistology was performed on freshly frozen sections (4 µm in thickness) by the biotin-avidin immunoperoxidase method using avidin-biotin immunoperoxidase complex reagent (Vector Laboratories, Burlingame, CA). The mAbs used are as follows: biotinylated rat mAbs to CD3 (Life Technologies, Grand Island, NY), B220, CD4, CD8, Mac-1 (BD Biosciences, San Jose, CA), murine Fas (clone 13; Transduction Laboratories, Lexington, KY), and murine FasL (K-10; BD PharMingen, San Diego, CA).

TUNEL

Apoptotic cells were detected in sections using the in situ TUNEL kit (Wako Pure Chemical, Osaka, Japan). Briefly, sections were incubated with proteinase K (20 µg/ml) for 10 min and then presoaked in TdT buffer (0.5 mmol/L cacodylate, 1 mmol/L CoCl, 0.5 mmol/L DTT, 0.05% BSA, 0.15 mol/L NaCl) for 10 min. Sections were incubated for 2 h at 37°C in 25 µl of TdT solution containing 1x terminal transferase buffer, 0.5 nmol of biotin-dUTP, and 10 U of TdT (Wako Pure Chemical). After the TdT reaction, sections were soaked in TdT blocking buffer (300 nmol/L NaCl, 30 mmol/L tri-sodium citrate-2-hydrate), incubated with HRP-conjugated streptavidin for 30 min at room temparature, and developed for 10 min in phosphate-buffered citrate (pH 5.8) containing 0.6 mg/ml diaminobenzidine. Nuclei were counterstained with hematoxylin.

Flow cytometric analysis

Surface markers were identified by mAbs with an EPICS flow cytometer (Beckman Coulter, Miami, FL). Rat mAbs to CD3 (Life Technologies), B220, CD4, CD8 (BD Biosciences), murine Fas (Jo2, BD PharMingen), and murine FasL (BD PharMingen) were used. Double-labeled surface phenotypes such as CD3/B220, CD4/FasL, and CD8/FasL were analyzed. FasL expressions were analyzed on tissue-infiltrating lymphocytes in 3d-Tx NFS/sld mice gated on CD4 and CD8. Apoptotic cells were detected with an EPICS flow cytometer (Beckman Coulter) using the Annexin VFITC Apoptosis Detection Kit (Genzyme, Cambridge, MA). For detection of T cell activation makers, single cell suspensions were stained with Abs conjugated to PE (anti-CD3, Life Technologies; anti-CD4, Cedarlane Laboratories, Hornby, Ontario, Canada; B220, BD PharMingen) and FITC (anti-CD8, Cedarlane Laboratories; Thy1.2, anti-CD44, anti-CD45RB, anti-Mel-14, BD PharMingen) and were analyzed with an EPICS flow cytometer (Beckman Coulter).

RT-PCR

Total RNA was extracted from homogenized tissues with TRIzol reagent (Life Technologies), and cDNA was prepared from RNA with 50 pmol oligo (dT) 18 and 200 U of murine leukemia virus reverse transcriptase (Life Technologies). Two microliters of the cDNA mixture was used in a PCR with 10 pmol of forward and reverse primers and 2.5 U of Taq DNA polymerase (PerkinElmer/Cetus, Norwalk, CT). The sequences of the specific sense and anti-sense oligonucleotide primer pairs were as follows: Fas, 5'-ATCCGAGCTCTGAGGAGGCGGGGTTCATGAAAC-3' and 5'-GGAGGTTCTAGATTCAGGGTCATCCTG-3'; {beta}-actin, 5'-ATGGATGACGATATCGCT-3' and 5'-ATGAGGTAGTCTGTCAGGT-3'. Samples were amplified through 30 cycles at an annealing temperature of 58°C in a PCR Thermal Cycler (PerkinElmer/Cetus). PCR products were blotted onto nylon membrane and hybridized with 32P-labeled cDNA probes.

Isolation of tissue-infiltrating cells from salivary glands

We isolated tissue-infiltrating mononuclear cells from affected salivary glands, as described previously (24, 25). In brief, affected submandibular glands from five mice were removed, cut into small pieces with scissors, passed through a 100-gauge stainless steel mesh, and suspended in RPMI 1640 containing 10% FCS, 10 mM HEPES buffer, penicillin (100 U/ml), and streptomycin (100 µg/ml). After washing twice with medium, infiltrating cells were isolated from parenchymal cells by Ficoll-Isopaque density-gradient centrifugation. CD4+ and CD8+ T cells were purified from infiltrating cells using magnetic beads (Dynal Biotech, Oslo, Norway).

Primary culture of mouse salivary gland (MSG) cells

Primary cultures of MSG cells were prepared as reported previously (26). Briefly, the salivary gland cells were isolated from NFS/sld mice at 3–5 wk by enzymatic digestion with 0.76 mg/ml EDTA and mixture of collagenase (type I, 750 U/ml) and hyaluronidase (type IV, 500 U/ml), plated in 24-well plates, and maintained in MEM containing 10% FCS for 5–7 days before flow cytometric analysis and cytotoxic assay. Apoptosis was induced in MSG cells by anti-Fas Ab (Jo2, 300 ng ml-1; BD PharMingen). On Western blotting, cytosolic extracts were prepared from MSG cells (1 x 107 cells), which were treated at 37°C with apoptotic stimuli for various times.

Western blot analysis

Western blot analysis with mouse mAb to {alpha}-fodrin (Affiniti, Mamhead, U.K.) and PARP (Transduction Laboratories) were performed. Briefly, the cells were homogenated in 20 mM Tris-HCl buffer (pH 7.4) containing 5 mM diisopropylfluorophosphate, 5 mM EDTA, 5 mM benzamidine, 2 mM PMSF, and 2 mM N-ethylmaleimide. After centrifugation for 20 min at 12,000 rpm at 4°C, supernatant was extracted and used for cytoplasmic protein. Pellets were homogenized in 20 mM Tris-HCl buffer containing 2% Triton X-100. Protein binding was visualized with ECL Western blotting reagent (Amersham, Arlington Heights, IL). Protease inhibitors included mixtures (Sigma-Aldrich, St. Louis, MO), leupeptin (Wako Pure Chemical), E64 (Wako Pure Chemical), and caspase inhibitors (z-VAD-fmk and Ac-DEVD-CHO; ICN Pharmaceuticals, Costa Mesa, CA). Control for protein loading was provided by actin, tubulin, and myosin. To detect serum autoantibodies against 120-kDa {alpha}-fodrin Ag (6), mouse IgG was isolated from serum samples collected from 3d-Tx NFS/sld mice. Samples were solubilized by heating and separated by 10% SDS-PAGE. The autoantigen was electrotransferred to nitrocellulose and then quenched with 1% powdered milk in borate-buffered saline. Nitrocellulose membranes were incubated with testing serum at a 1/200 dilution in borate-buffered saline and then incubated with peroxidase-conjugated horse anti-mouse IgG (Vector Laboratories) at a 1/1000 dilution.

Cytotoxic assay

MSG cells (2 x 106) in 7.5 ml of MEM, supplemented with 5% FBS, were labeled overnight at 37°C in 5% CO2 with 300 µCi of sodium 51Cr-chromate. CD4+ and CD8+ T cells isolated from salivary gland tissues and spleen (2–3 x 106) in 0.2 ml of RPMI 1640 were supplemented with 10% FBS. Each well of 96-well microtiter plates received, in a total volume of 200 µl, target cells, effector cells in the indicated ratios, and either medium. Microplates were centrifuged for 1 min at 1500 rpm and incubated for 4 h at 37°C. After another centrifugation, 100-µl aliquots of the supernatants were assayed for radioactivity. The fraction of the total radioactivity released was then calculated, and the results, averaged from triplicates, were expressed as percent specific 51Cr release (percent experimental 51Cr release minus percent 51Cr release from target cells alone). To examine the role of FasL for cytotoxic activities, anti-murine FasL inhibitory mAb (FLIM58) (27) was used. FLIM58 neutralizes mouse but not human FasL activity (27).

Sequential activation of caspase 1-like and caspase 3-like proteases

The caspase 1 (IL-1-converting enzyme)- and caspase 3 (CPP32)-like activity in anti-Fas Ab treated with MSG cell extracts was determined using fluorescent substrate (28). Cell lysates were diluted with 0.5 ml of IL-1-converting enzyme standard buffer and incubated at 30°C for 30 min with 1 µM fluorescent substrate. The caspase inhibitors z-VAD-fmk or Ac-DEVD-CHO were added to the reaction mixture at a concentration of 1 µM. Specific caspase 1- and caspase 3-like activities were determined by subtracting the values obtained in the presence of inhibitors. The fluorescent substrates, MOCAc-YVAD(dnp)-NH2 and MOCAc-DEVD(dnp)-NH2 were custom-synthesized at the Peptide Institute (Osaka, Japan). The fluorescence of the cleaved substrates was determined using a spectrofluorometer set at an excitation wavelength of 328 nm and an emission wavelength of 393 nm.

In vivo administration of caspase inhibitors

To examine the therapeutic effects of i.v. injection of caspase inhibitors, a 1 mg/ml (50 µg/head) z-VAD-fmk (n = 11) and Ac-DEVD-CHO (n = 9) in DMSO were injected i.v. three times per week into 3d-Tx NFS/sld mice from 4 to 7 wk, because autoimmune lesions in the salivary and lacrimal glands start to develop at 3 wk of age. Dose of caspase inhibitor was determined according to the previous report (29). Mice were examined histopathologically at 8 wk and compared with 3d-Tx NFS/sld mice injected with DMSO alone (n = 6). In addition, autoantibody production against the 120-kDa {alpha}-fodrin in serum was tested in both treated and nontreated mice. Detection of tear and saliva volume of the treated mice and control SS animal model of 3d-Tx NFS/sld mice was done according to a modified method as described (26).

In vivo immunization with autoantigen

Recombinant {alpha}-fodrin protein identical with a 120-kDa cleavage product (JS-1, 5 µg/ml) (6) emulsified with CFA (Difco, Detroit, MI) was administerred twice s.c. into syngeneic normal NFS/sld mice at 4 and 6 wk (n = 12). At 4 and 8 wk after the s.c. injection, mice were examined on histopathological and immunological analysis. For controls, mice injected with rat brain {alpha}-fodrin (5 µl/ml; n = 10), recombinant {alpha}-fodrin protein encoding full-length {alpha}-fodrin (JS-1, 1–1784 bp; 2.7A, 2258–4884 bp; 3'DA, 3963–7083 bp; a mixture of 5 µg/ml) (n = 10) (6), lysozyme (5 µg/ml; n = 10), GST emulsified with CFA (50 µl/head; n = 10), and CFA alone (50 µl/head; n = 10) were examined. Rat brain {alpha}-fodrin was purified according to the method described previously (30).

Proliferative T cell responses

Single cell suspensions of spleen cells from treated and control mice were cultured in 96-well flat-bottom microtiter plates (5 x 105 cells/well) in RPMI 1640 containing 10% FCS, penicillin/streptomycin, and 2-ME. Cells were cultured with recombinant {alpha}-fodrin (JS-1, 5 µg/ml), rat brain {alpha}-fodrin (5 µg/ml), lysozyme (5 µg/ml, Sigma-Aldrich), and OVA (5 µg/ml, Sigma-Aldrich). During the last 8 h of the 72-h culture period, 1 µCi of [3H]thymidine was added per well, and the incorporated radioactivity was determined using an automated beta liquid scintillation counter.

ELISA

Serum autoantibodies were detected using recombinant {alpha}-fodrin (JS-1). After coating with the recombinant {alpha}-fodrin in 96-well ELISA plates, biotinylated anti-mouse IgG (Vector Laboratories) was added as a second Ab. Measurements of specific autoantibodies were read by automatic ELISA reader (Flow Laboratories, McLean, VA) at 492 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Involvement of apoptotic cascade in tissue destruction

Histological and immunohistological characteristics of autoimmune exocrinopathy in 3d-Tx NFS/sld SS model mice were described in detail (22, 31, 32). To determine the possible involvement of the apoptotic cascade in tissue destruction, we examined apoptotic cells in the salivary gland specimens from 3d-Tx and non-Tx NFS/sld mice. Immunohistology revealed that the majority of tissue-infiltrating lymphoid cells in the salivary glands bear FasL in the SS model (Fig. 1GoA) and that epithelial duct cells express Fas Ag on their cell surface (Fig. 1GoB). We found that tissue-infiltrating CD4+ T cells isolated from the affected glands bear a large proportion of FasL (>85%), compared with CD8+ T cells bearing FasL on flow cytometry (<23%; p < 0.01; Fig. 1GoE). A minor proportion of infiltrating CD4+ T cells express Fas (<31%), and CD8+ T cells bearing Fas were negligible (<5%). Primarily cultured MSG cells isolated from 3d-Tx, non-Tx NFS/sld and C57BL/6 mice constitutively express Fas with high proportion (51–60%) on flow cytometry (Fig. 2GoA). Immunohistochemically, epithelial duct cells in non-Tx NFS/sld and C57BL/6 salivary glands are positive for Fas (Fig. 2GoB). RT-PCR analysis demonstrated that Fas mRNA was constitutively present in the salivary glands of the SS model, non-Tx NFS/sld, and normal C57BL/6 mice (Fig. 2GoC). MSG cells isolated from these mice did not express FasL on flow cytometric analysis (Fig. 2GoD). A significant increase of TUNEL+-apoptotic epithelial duct cells in the salivary glands was observed in SS model mice, compared with those in non-Tx NFS/sld and C57BL/6 mice at all ages (Fig. 3Go, A–C). We next investigated whether tissue-infiltrating T cells are responsible for tissue destruction as judged by in vitro 51Cr release cytotoxic assay against MSG cells. Infiltrating CD4+ T cells, but not CD8+ T cells, identified significant 51Cr release against MSG cells (Fig. 3GoD). These cytotoxic activities were almost entirely inhibited by incubation with anti-murine neutralizing FasL mAb (FLIM58, 1 µg/ml) (Fig. 3GoD). No significant cytotoxicities were found in splenic CD4+ and CD8+ T cells toward MSG cells.



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  		FIGURE 1. FasL and Fas expression on tissue-infiltrating cells in the salivary gland tissues from 3d-Tx NFS/sld mice. A, Immunohistochemical detection of infiltrating lymphocytes strongly positive for FasL in 3d-Tx NFS/sld mice. B, Epithelial duct cells were stained positively with Fas in 3d-Tx NFS/sld mice. Isotype-matched control for FasL (C) and Fas (D) were stained negatively. E, Flow cytometric analysis of FasL and Fas expression on tissue-infiltrating lymphocytes isolated from salivary glands of 3d-Tx NFS/sld mice gated on CD4 and CD8. FasL expression on tissue-infiltrating CD4+ T cells was prominent compared with that on CD8+ T cells. A minor proportion of infiltrating CD4+ T cells express Fas, and CD8+ T cells bearing Fas were negligible. Five mice in each group were analyzed at 8 and 12 wk of age, and the mean percentages were statistically significant at p < 0.01 (Student’s t test).

 


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  		FIGURE 2. Fas expression on primary cultured MSG cells. A, Flow cytometric analysis of Fas expression on MSG cells from 3d-Tx, non-Tx NFS/sld, and normal C57BL/6 mice. Fas expression was constitutively observed on MSG cells from each group of mice. B, Epithelial duct cells in the salivary glands were constitutively stained with Fas in normal C57BL/6 mice. C, RT-PCR analysis in salivary gland tissues. Fas mRNA expression was present in C57BL/6, non-Tx NFS/sld mice, and 3d-Tx NFS/sld from two different mice. D, Flow cytometric analysis of FasL expression on MSG cells from 3d-Tx, non-Tx NFS/sld, and normal C57BL/6 mice. No significant positive cells were observed. Five mice in each group were analyzed.

 


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  		FIGURE 3. In situ TUNEL detection was frequently positive for epithelial duct cells in 3d-Tx NFS/sld mice (12 wk old) (A), but not in non-Tx NFS/sld mice (12 wk old) (B). C, A significant increase of apoptotic duct cells was observed in the salivary gland tissues from 3d-Tx NFS/sld mice at all ages. The percentage of duct cells staining positively with TUNEL was enumurated using a 10 x 20 grid net micrometer disc covering an objective of area 0.16 mm2. Data were analyzed in 10 fields per section and were expressed as mean percentage ± SD in five mice examined per group. (*, p < 0.01; **, p < 0.001; Student’s t test). Five mice in each group were analyzed at 8, 12, and 16 wk of age. D, Detection of cytotoxic activity in tissue-infiltrating CD4+ T cells toward MSG cells. These activities were almost entirely inhibited by anti-murine neutralizing FasL mAb (FLIM48, 1 µg/ml). No significant cytotoxic activities were found in tissue-infiltrating CD8+ T cells or in splenic CD4+ and CD8+ T cells toward MSG cells. The results, averaged from triplicates, were expressed as a percentage of specific 51Cr release.

 
In vitro cleavage of {alpha}-fodrin by apoptotic proteases

We examined the in vitro cleavage of {alpha}-fodrin in MSG cells induced by anti-Fas mAb (Jo2, 300 ng ml-1). Anti-Fas mAb-stimulated apoptosis in MSG cells was confirmed by flow cytometry of DNA content of nuclei with propidium iodide and annexin V (Fig. 4GoA). Western blot analysis demonstrated that the 240-kDa {alpha}-fodrin in apoptotic MSG cells was cleaved to smaller fragments into 120 kDa in a time-dependent manner, and the cleavage was entirely blocked by preincubation with caspase inhibitors (z-VAD-fmk and DEVD-CHO) (Fig. 4GoB). Protease inhibitor mixtures, cysteine protease inhibitors (E64), and serine protease inhibitor (leupeptin) had no significant effect on 120-kDa {alpha}-fodrin cleavage in apoptotic MSG cells (Fig. 4GoB). The 113-kDa PARP in apoptotic MSG cells was not cleaved to smaller fragments. We next investigated whether cysteine proteases are involved in {alpha}-fodrin cleavage on apoptotic MSG cells. The caspase 1- and caspase 3-like activities in anti-Fas mAb-stimulated MSG cell extracts were determined using fluorescent substrates, and caspase inhibitors (z-VAD-fmk and DEVD-CHO) inhibited these activities at different doses (0.2, 2, and 20 µM) (Fig. 4GoC).



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  		FIGURE 4. A, The MSG cell apoptosis induced by anti-Fas mAb (Jo2) stimulation determined by flow cytometry of DNA content of nuclei with propidium iodide and annexin V. B, Western blot analysis demonstrated that the 240-kDa {alpha}-fodrin in apoptotic MSG cells was cleaved into smaller fragments of 120 kDa in a time-dependent manner, and the cleavage product was entirely blocked by preincubation with caspase inhibitors (z-VAD-fmk and DEVD-CHO) measured at 24 h. The PARP cleavage in apoptotic MSG cells was not detected. Cleavage products of PARP (89 or 24 kDa) were detected in apoptotic Jurkat cells. Protease inhibitor mixtures, cysteine protease inhibitors (E64), and serine protease inhibitor (leupeptin) had no significant effect on {alpha}-fodrin cleavage. C, Activation of caspase 1-like and caspase 3-like proteases was detected during anti-Fas-induced apoptosis, and caspase inhibitors inhibited these activities at different doses (0.2, 2, and 20 µM). Cytosolic extracts were prepared from MSG cells (1 x 107 cells), which were treated at 37°C with 300 ng ml-1 of Jo-2. The 100% activity as control was calculated using the values 300 U/ml recombinant caspase 1 or caspase 3 added to each substrate (200 µM MOCAc-YVAD(dnp)-NH2 and MOCAc-DEVD(dnp)-NH2).

 
Preventive effect of caspase inhibitors in vivo

We investigated whether the i.v. injection of caspase inhibitors protects animals against the development of autoimmune lesions. Treatment with i.v. injection of both z-VAD-fmk and DEVD-CHO (three times per week) (p < 0.005) prevented the development of autoimmune lesions in the salivary and lacrimal glands (Fig. 5Go, A and B). The average saliva and tear volume of the treated SS animal model was significantly higher than that of the control group (Fig. 5GoC). A significant decrease of autoantigen-specific T cell proliferation was observed in spleen cells from treated mice (Fig. 5GoD). In addition, serum autoantibody production against 120-kDa {alpha}-fodrin was clearly inhibited by the treatment with caspase inhibitors (Fig. 5GoE).



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  		FIGURE 5. Preventive effect of i.v. injection of caspase inhibitors. One milligram per milliliter (50 µg/head) z-VAD-fmk (n = 11) and DEVD-CHO (n = 9) in DMSO was injected i.v. three times per week into 3d-Tx NFS/sld mice for 4–7 wk. A, Mice were examined histopathologically at 8 wk and compared with 3d-Tx NFS/sld mice injected with DMSO alone (n = 7) (*, p < 0.01; **, p < 0.005; Student’s t test). B, Representative histologic features in the salivary and lacrimal glands showing preventive effects in z-VAD-fmk-treated mice at 8 wk of age. C, The average saliva and tear volume of the SS model mice treated with caspase inhibitors was significantly higher than that of control mice. Results are expressed as mean ± SEM in five mice examined per group (*, p < 0.05; **, p < 0.005; Student’s t test). D, Mice treated with caspase inhibitors showed a significant decrease of autoantigen-specific T cell proliferation in spleen cells, compared with those in controls (*, p < 0.01; Student’s t test). Data are expressed as cpm per culture in triplicate. E, Serum autoantibody production to 120-kDa {alpha}-fodrin was inhibited in sera from two different mice treated with both caspase inhibitors.

 
Induction of autoimmune lesions by immunization with autoantigen

To examine the autoimmune nature of 120-kDa {alpha}-fodrin, recombinant {alpha}-fodrin protein identical with an autoantigen was administerred s.c. into normal NFS/sld mice at 4 wk. Organ-specific autoimmune lesions similar to SS developed at 8 wk after the injection in almost all mice immunized with autoantigen, but not in all groups of control (Fig. 6GoA; Table IGo). No inflammatory lesions were observed in other organs. A majority of infiltrating cells were CD4+ and FasL+, and the epithelial duct cells express Fas on their cell surface (Fig. 6GoA). A minor proportion of CD8+ and Fas+ infiltrating cells was observed (data not shown). A specific cleavage of {alpha}-fodrin into 120 kDa was detected in the salivary glands of immunized mice, but not in controls (Fig. 6GoB). The activation markers (CD44high, CD45RBlow, Mel-14low) were up-regulated in spleen cells gated on CD4 from immunized mice, compared with controls (Fig. 6GoC). Mice injected with recombinant autoantigen showed a significant increase of autoantigen-specific T cell proliferation in spleen cells (Fig. 6GoD). A high titer of serum autoantibodies against 120-kDa {alpha}-fodrin was detected in immunized mice, compared with control mice, by ELISA (Fig. 6GoE). These data strongly suggest that a cleavage product of 120-kDa {alpha}-fodrin is a pathogenic autoantigen on the development of murine primary SS.



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  		FIGURE 6. Autoimmune lesions in the salivary and lacrimal glands developing at 8 wk after the s.c. injection of autoantigen in almost all syngeneic normal mice. Representative histologic features showed in the salivary and lacrimal glands (A, upper panels, HE). A majority of infiltrating mononuclear cells were CD4+ and FasL+, and the epithelial duct cells express Fas on their cell surface (A, lower panels). B, A specific cleavage product of 120-kDa {alpha}-fodrin was detected in the salivary glands from immunized mice on Western blotting, but not in control. C, The activation markers (CD44high, CD45RBlow, and Mel-14low) were up-regulated in spleen cells gated on CD4 from immunized mice, compared with control mice. D, Mice injected with {alpha}-fodrin autoantigen showed a significant increase of autoantigen-specific T cell proliferation in spleen cells, whereas no responses were observed against lysozyme and OVA (*, p < 0.05; Student’s t test). Data are expressed as cpm per culture in triplicate. E, A higher titer of serum autoantibodies against 120-kDa {alpha}-fodrin was detected by ELISA in immunized mice compared with that of control mice (*, p < 0.05; Student’s t test).

 

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  		Table I. Frequency of inflammatory infiltrates in autoimmune exocrinopathy of autoantigen-administered normal NFS/sld mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cleavage of certain autoantigens during apoptosis may reveal immunocryptic epitopes that could potentially induce autoimmune responses in systemic autoimmune diseases (33, 34). Among the substrates cleaved during apoptosis are nuclear autoantigens targeted in systemic autoimmune diseases such as PARP (35, 36), U1–70-kDa (37), the nuclear lamin (38, 39), and DNA-dependent kinase (40). However, the in vivo role of autoantigen cleavage during apoptosis in autoimmune diseases remains unclear.

We reported previously that a cleavage product of 120-kDa {alpha}-fodrin may be an important autoantigen in the development of primary SS, and anti-120-kDa {alpha}-fodrin Abs have been frequently detected in sera from patients (6). Because it was shown that the fodrin {alpha} subunit is cleaved in association with apoptosis and the 120-kDa fragment is a breakdown product of the mature form of 240-kDa fodrin {alpha} subunit (11, 12), we examined the in vitro cleavage of {alpha}-fodrin using primarily cultured MSG cells. We clearly detected 120-kDa {alpha}-fodrin in anti-Fas-induced apoptotic MSG cells by Western blotting. We found that a significant increase of TUNEL+-apoptotic epithelial duct cells in the salivary glands was detected in the 3d-Tx NFS/sld SS model compared with those in non-Tx NFS/sld mice at all ages. MSG cells constitutively express Fas with high proportion, and tissue-infiltrating CD4+ T cells isolated from the salivary gland tissues of the SS model mice bear a large proportion of FasL. Moreover, we confirmed that tissue-infiltrating CD4+ T cells, but not CD8+ T cells, are responsible for tissue destruction as judged by in vitro 51Cr release cytotoxic assay against MSG cells in vitro. These cytotoxic activities were inhibited by incubation with anti-murine neutralizing FasL mAb. Although it has been reported that Fas-induced apoptosis seems to be the major killing pathway of the CD4+ cytotoxic T cells (41), our data suggest that one mechanism by which activated CD4+ T cells induce cytotoxicity toward salivary gland cells in the murine SS model is Fas based. When we investigated whether cysteine proteases are involved in {alpha}-fodrin cleavage, the caspase 1- and caspase 3-like activities in anti-Fas Ab-treated MSG cell extracts were determined using fluorescent substrates. In apoptotic MSG cells, caspase inhibitors (z-VAD-fmk and DEVD-CHO) inhibited the formation of 120-kDa {alpha}-fodrin, whereas protease inhibitor mixtures, other cysteine protease inhibitors (E64), and serine protease inhibitor (leupeptin) had no effect on 120-kDa {alpha}-fodrin cleavage in apoptotic MSG cells. Furthermore, the treatment of the murine SS model with i.v. injection of z-VAD-fmk and DEVD-CHO prevented the development of autoimmune conditions, resulting in restoration of saliva and tear secretion. These results suggest that increased activity of the caspase cascade is involved in the progression of {alpha}-fodrin proteolysis during the initial stages of the development of primary SS. Moreover, we obtained experimental evidence that immunization with recombinant {alpha}-fodrin protein identical with an autoantigen is sufficient to induce autoimmune SS lesions in normal recipients, but not with rat brain {alpha}-fodrin or with recombinant full-length {alpha}-fodrin. These results indicate that caspase-mediated {alpha}-fodrin cleavage into the 120-kDa fragment plays a critical role in the development of autoimmmune exocrinopathy in primary SS and suggest that the primary mediators of the disease are autoantigen-driven T cell responses.

When human T cell leukemia CEM cells were induced to undergo apoptosis, the 240-kDa {alpha}-fodrin was cleaved to a single detectable fragment of 120 kDa (42). It is plausible that the 120-kDa fragment is a breakdown product of the 150-kDa {alpha}-fodrin cleavage (9). There is increasing evidence that the cascade of caspases is a critical component of the cell death pathway (43, 44, 45), and a few proteins have been found to be cleaved during apoptosis. These include PARP, a small U1 nuclear ribonucleoprotein, and {alpha}-fodrin, which were subsequently identified as substrates for caspases (35, 36, 37). We provided evidence that {alpha}-fodrin is cleaved by one or more members of caspases during apoptotic cell death in SS salivary glands. Fodrin cleavage by caspases can potentially lead to cytoskeletal rearrangement, and it is of interest to point out that {alpha}-fodrin binds to ankylin, which contains a cell death domain (46). In contrast, it has been shown that cleavage products of {alpha}-fodrin inhibit ATP-dependent glutamate and {gamma}-aminobutyric acid accumulation into synaptic vesicles (47), supposing that a cleavage product of 120-kDa {alpha}-fodrin could be a novel component of an unknown immunoregulatory network such as cytolinker proteins (48).

Taken together, these results are strongly suggestive of essential roles of caspase cascade for {alpha}-fodrin cleavage leading to tissue destruction in autoimmune exocrinopathy of primary SS. Moreover, in vivo preventive effects against autoimmune lesions treated with caspase inhibitor have important implications for testing useful therapies.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scentific Research (12307040 and 12557022) from the Ministry of Education, Science and Culture of Japan. K.S. is a Research Fellow of the Japan Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Yoshio Hayashi, Department of Pathology, Tokushima University School of Dentistry, 3 Kuramotocho, Tokushima 770, Japan. E-mail address: hayashi{at}dent.tokushima-u.ac.jp Back

3 Abbreviations used in this paper: SS, Sjögren’s syndrome; PARP, poly(ADP-ribose) polymerase; FasL, Fas ligand; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; 3d-Tx, thymectomy performed on day 3 after birth; non-Tx, nonthymectomied; MSG, mouse salivary gland; FLIM, FasL inhibitory mAb; DEVD, N-acetyl-Asp-Glu-Val-Asp-al. Back

Received for publication December 6, 2001. Accepted for publication May 3, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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