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The Journal of Immunology, 2001, 166: 5801-5809.
Copyright © 2001 by The American Association of Immunologists

Possible Involvement of EBV-Mediated {alpha}-Fodrin Cleavage for Organ-Specific Autoantigen in Sjogren’s Syndrome1

Hiroko Inoue*,{dagger}, Kazuo Tsubota{dagger}, Masafumi Ono{dagger}, Yasuhiro Kizu{ddagger}, Fumio Mizuno§, Kenzo Takada, Koichi Yamada*, Kumiko Yanagi*, Yoshio Hayashi* and Ichiro Saito2,*

* Department of Pathology, Tokushima University School of Dentistry, Tokushima, Japan; Departments of {dagger} Ophthalmology and {ddagger} Oral Medicine, Tokyo Dental College, Chiba, Japan; § Department of Microbiology, Tokyo Medical University, Tokyo, Japan; and Department of Virology, Cancer Institute, Hokkaido University, School of Medicine, Sapporo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A cleavage product of {alpha}-fodrin may be an important organ-specific autoantigen in the pathogenesis of Sjogren’s syndrome (SS), but the mechanisms of {alpha}-fodrin cleavage remain unclear. Since EBV has been implicated in the pathogenesis of SS, we determined whether EBV activation could induce the SS-specific 120-kDa autoantigen {alpha}-fodrin. ZEBRA mRNA expression, a marker for activation of the lytic cycle of EBV, was found in the salivary gland tissues from SS patients, but not in those from control individuals. ZEBRA-expressing lymphoid cells were also found in the SS glands in double-stained immunohistochemistry. Furthermore, a significant link between production of Abs against 120-kDa {alpha}-fodrin and reactivated EBV Ag was found in sera from patients with SS, but not in those from control individuals. EBV-activated lymphoid cells showed specific {alpha}-fodrin cleavage to the expected 120-kDa fragments in vitro. Pretreatment with caspase inhibitors inhibited cleavage of {alpha}-fodrin. Thus, an increase in apoptotic protease activities induced by EBV reactivation may be involved in the progression of {alpha}-fodrin proteolysis in the development of SS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sjogren’s syndrome (SS)3 is an organ-specific autoimmune disease caused by the progressive loss of exocrine glands and is associated with several autoimmune phenomena. Several reports have suggested that viral infection could be involved in the induction of SS (1, 2, 3, 4). Evidence for an association between EBV infection and SS has been accumulating. EBV Ags and increased levels of EBV DNA have been found in infiltrating B lymphocytes and a few salivary gland epithelial cells of SS patients (5, 6). Infectious EBV is present in both the saliva of SS patients (7, 8, 9) and culture supernatants of B cell lines established from SS patients (10). Other defined manifestations of an active EBV infection are the presence of infected B cells that can transform into B cell lymphomas in the circulation (11). Mariette et al. (6) previously used in situ hybridization to detect EBV DNA in a substantial proportion of lymphoid cells and epithelial cells in salivary glands from patients with SS. It has also been shown that Abs against EBV Ags are elevated in sera of SS patients (12, 13). Since EBV is known to induce strong immune responses (14, 15), these reports suggest that a reactivated EBV infection may play a role in SS, contributing to the initiation or perpetuation of an autoimmune response in target organs. However, the pathological roles of this virus remain obscure.

We recently identified a 120-kDa organ-specific autoantigen from salivary gland tissue of an animal model for primary SS in NFS/sld mutant mice, which was found to be identical to that of the human cytoskeletal protein {alpha}-fodrin (16). This 120-kDa {alpha}-fodrin reacted with sera from patients with SS, and immunization with the Ag prevented the disease in mice. These results indicate that the anti-120-kDa {alpha}-fodrin immune response plays a critical role in the development of primary SS. The fodrin {alpha} subunit is cleaved in association with apoptosis, and the 120-kDa fragment is a breakdown product of the fodrin {alpha} subunit (17). However, no trigger for the proteolysis of fodrin in human SS glands has been determined. The proteolysis of fodrin during apoptosis may be a consequence of unknown protease activation. Taken together, these observations raise the possibility that an increase in the enzymatic activity of proteases through unknown mechanisms is involved in the progression of {alpha}-fodrin proteolysis during initial stages in the development of primary SS. We therefore speculate that an increase in the enzymatic activity of apoptotic proteases by EBV reactivation is involved in the progression of {alpha}-fodrin proteolysis during SS development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human sera

Ten sera of primary SS outpatients of the Ichikawa General Hospital of Tokyo Dental College who were diagnosed according to the criteria of Fox and Saito (18) and 10 normal sera from healthy volunteers were used for Ab analysis.

Quantitation of Abs in human sera

ELISA was performed according to standard procedure, and reactivity with recombinant {alpha}-fodrin expressed in Escherichia coli was measured (16). Briefly, recombinant proteins were coated on 96-well microtiter plates at 5 µg/ml. Sera were incubated at a 1:200 dilution and Ab binding was detected using peroxidase-labeled mouse monoclonal anti-human IgG-specific antiserum (MBL, Nagoya, Japan). Tetramethylbenzidine (Sigma, St. Louis, MO) was added and color development was measured at 450 nm (A450) using a microplate reader. The ELISA index was defined as follows: (patients serum A450 - normal control A450)/(positive control A450 - normal control A450) x 100. The assay result was considered positive if the ELISA indices were above the mean value of 83 immunodiffusions defined as anti {alpha}-fodrin-negative normal control sera.

The titer of IgM Ab against viral capsid Ag (VCA) was measured by the indirect immunofluorescence method (19) using doubling dilutions of sera and an initial dilution of 1:10.

Biopsy samples

Twelve patients with SS (outpatients of the Ichikawa General Hospital, Tokyo Dental College) underwent minor salivary gland biopsies for the differential diagnosis of SS (18) with their consent. Their biopsies revealed a focus score of >2, but did not show evidence of lymphoma in routine histologic and immunohistochemical analysis. Each of these patients showed keratoconjunctivitis sicca with decreased tear production, <5 mm on a Schirmer test with anesthesia and of <10 mm on another Schirmer test with nasal stimulation (20). Cornea and conjunctival epithelia showed positive Rose-Bengal and fluorescein staining. Decreased secretion of saliva was confirmed by the gum test of <5 ml. These patients had not received glucocorticoids or immunosuppressive agents for at least 6 mo before biopsy. Normal healthy individuals lacking sicca symptoms underwent minor salivary gland biopsies as part of the diagnostic workup for other oral problems (n = 3). Histologically normal salivary gland tissues were also obtained at the time of autopsy (n = 3).

Double immunofluorescence staining

Unfixed tissue blocks were placed in OCT compound (Miles, Naperville, IL), snap frozen in liquid nitrogen, and stored at -80°C. Cryostat sections (4 µm) from each block were transferred onto glass slides for immunohistochemical analysis. Consecutive sections were subsequently placed in a sterile Eppendorf tube for RNA extraction. Sections that were cut before and after the sections taken for RNA extraction were stained with hematoxylin and eosin using the standard method. Double immunofluorescence staining for confocal microscopy was performed. Briefly, acetone-fixed sections placed on gelatin-coated slides were double stained with primary Abs, murine mAbs directed against CD21 (Coulter Immunology, Hialeah, FL) and ZEBRA (Cappel, Aurora, OH) and rabbit polyclonal Ab to synthetic {alpha}-fodrin peptide (16). Secondary fluorescent Abs used were Texas Red (TR)-labeled goat anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L) and FITC-labeled goat anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L; Molecular Probes, Eugene, OR). For the double staining with anti-ZEBRA and anti-CD21 Abs, murine monoclonal anti-ZEBRA Ab followed by TR-labeled goat anti-mouse IgG (H + L) and FITC-conjugated anti-CD21 Ab were used. The stained sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) were examined with the use of a confocal laser scanning microscope (Bio-Rad Radiance2000, Bio-Rad, Hercules, CA) equipped with an argon/krypton-mixed gas laser.

Cell culture and activation

Akata cells, which are a Burkitt’s lymphoma cell line that demonstrates prompt and synchronous activation of latent EBV genomes after cross-linking of its membrane Ig with anti-IgG Ab (21, 22, 23), and EBV-negative Akata cells (24) were maintained in RPMI 1640 medium supplemented with streptomycin (100 µg/ml), penicillin (100 IU/ml), and 10% FCS. These cells were suspended to a final concentration of 1 x 106 cells/ml with fresh medium. Then the cells were cultured for the indicated time in the presence or absence of 100 µg/ml anti-human IgG (Cappel), 2.5 µg/ml anti-human CD40 (Ancell, Bayport, MN), and protease inhibitors.

Cell lysis

All cell lysis procedures were performed at 4°C. At the end of an experiment, the medium was removed. Cells were washed once with ice-cold PBS, lysed with ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1% Triton X-100). Protein concentration was determined by a modified Lowry assay (Bio-Rad).

Immunoblotting

The protein obtained from EBV-positive Akata cells and EBV-negative Akata cells was boiled in sample buffer (500 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 2% mercaptoethanol) for 5 min. Equal amounts of total protein were loaded on each lane and run on SDS-PAGE, followed by electrophoretic transfer of the proteins to a polyvinylidene difluoride membrane. Membranes were first blocked in Block Ace containing 5% nonfat dry milk for 1 h and then incubated with the mouse anti-{alpha}-fodrin Ab or human sera for 1 h. After three sequential 5-min washes with washing buffer (20 mM Tris-HCl (pH 7.4), 2.5% Block ace, and 0.1% Tween 20), the membranes were incubated with anti-human or anti-mouse peroxidase-conjugated secondary Abs (Zymed Laboratories, San Francisco, CA) for 1 h at room temperature and then again washed as described above. Bound protein was developed with ECL detection reagents (Amersham, Arlington Heights, IL) and exposed to x-ray films for 3 min.

Immunoprecipitation

Cell lysates from IgG-cross-inked Akata cells were prepared as above. Immunoprecipitations were performed using anti-ZEBRA Ab (Cappel). Forty micrograms of cell lysates was incubated with 0.5 µg of anti-ZEBRA Ab overnight at 4°C. Protein was precipitated with protein G-Sepharose beads, separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with serum (1:100) from SS patients or healthy donors. The blots were developed by ECL.

RT-PCR

Total RNA was extracted from homogenized tissues with TRIzol reagent (Life Technologies, Rockville, MD), and cDNA was prepared from RNA with 50 pmol of oligo(dT) and 200 U of reverse transcriptase (Takara Shuzo, Kyoto, Japan); 2 µl of a 10-µl cDNA mixture was used for a PCR with 10 pmol each of forward and reverse primers and 2.5 U of Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT). The sequences of the specific sense and antisense oligonucleotide primer pairs were as follows: ZEBRA, 5'-TTCCACAGCCTGCACCAGTG-3' and 5'-GGCAGCAGCCACCTCACGGT-3' and GAPDH, 5'-GGTACCTCTTCCGACCCC-3' and 5'-GTTTCAACAGTACCTACTGG-3'. Samples were amplified through 30 cycles at an annealing temperature of 55°C in a PCR Thermal Cycler (Perkin-Elmer/Cetus).

Apoptosis detection

DNA from whole-cell populations was extracted with NaI solution (25). Briefly, the cells were resuspended in lysis buffer (10 mM EDTA, 200 mM NaCl, 0.1 mg/ml protease K, 1 mg/ml RNase, 0.5% SDS, 50 mM Tris (pH 8.0)) and incubated for 30 min at 37°C. This mixture was added to NaI solution (6 M NaI, 13 mM EDTA, 0.5% sodium-N-lauroylsarcosinate, 10 mg/ml glycogen, 26 mM Tris (pH 8.0)) and incubated for 15 min at 60°C. DNA was precipitated by addition of isopropanol. Samples were then centrifuged at 13,000 x g for 15 min. Pelleted DNA was washed with isopropanol, dried, and resuspended in TE buffer. DNA was then loaded on a 1.7% agarose gel containing 0.1 µg/ml ethidium bromide, and UV fluorescence was photographed. Alternatively, in situ TUNEL staining was performed with an in situ apoptosis detection kit (Apoptag; Oncor, Gaithersburg, MD) according to the manufacturer’s instruction.

Reagents for protease inhibitor

z-VAD-fmk and z-DEVD-fmk were obtained from ICN Pharmaceuticals (Costa Mesa, CA). Ac-YVAD-cmk was from Bachem (Torrence, CA). Leupeptin was obtained from Peptide Institute (Osaka, Japan). Pepstatin, calpain inhibitor, and a protease inhibitor mixture (AEBSF, pepstatin, E64, bestatin, leupeptin, and aprotinin) were purchased from Sigma.

Blocking of 120-kDa {alpha}-fodrin cleavage by protease inhibitors and quantitation of {alpha}-fodrin

Cross-linking of Akata cells or EBV-negative Akata cells with 100 µg/ml anti-IgG Ab was conducted at the end of a 1-h treatment with protease inhibitors. The intensity of 120-, 150-, and 240-kDa bands was measured by densitometric analysis using a color scanner and NIH Image 1.5. The amount of 120-kDa {alpha}-fodrin induced in the presence of protease inhibitors was expressed as percentage of the relative amount to that induced in the absence of protease inhibitor: (120-kDa/120-kDa + 150-kDa + 240-kDa)i/(120-kDa/120-kDa + 150-kDa + 240-kDa)c x 100. "i" and "c" represent in the presence and absence of protease inhibitor, respectively.

Detection of IL-1-converting enzyme (ICE)-like and CPP32-like protease activation

The ICE- and CPP32-like activities in anti-IgG Ab-treated Akata cell extracts were determined as reported previously (26). Cell lysates were diluted with 0.5 ml of ICE standard buffer and incubated at 30°C for 30 min with 1 µM fluorescent substrate. The specific inhibitor for ICE (Ac-YVAD-cmk) or CPP32 (z-DEVD-fmk) was added to the reaction mixture at a concentration of 20 µM. Specific ICE- and CPP32-like activities were determined by subtracting the values obtained in the presence of inhibitors. The fluorescent substrates, MOCA-YVADPK (dnp)-NH2 and MOCA-DEVAPK (dnp)-NH2 were custom synthesized at the Peptide Institute. 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.

Statistical analysis

Statistical analysis was performed using the Mann-Whitney U test for the value of anti-{alpha}-fodrin Ab (results shown in Table IGo), and the {chi}2 test for the expression of ZEBRA mRNA and anti-ZEBRA Ab (results shown in Figs. 1Go and 2Go).


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  		Table I. Comparison of Abs to {alpha}-fodrin, ZEBRA, and VCA in SS and normal sera1

 


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  		FIGURE 1. ZEBRA expression in sera of SS patients and normal individuals. Immunoprecipitation analysis of ZEBRA was performed on sera from SS patients (n = 10) and normal individuals (n = 10). ZEBRA protein from IgG-cross-linked Akata cells was precipitated with anti-ZEBRA mAb and separated by SDS-PAGE as described in Materials and Methods. The 20 serum samples (at 1:100) were tested by immunoblotting (arrowheads indicate ZEBRA-specific bands), classified based on their immunoreactivity (summarized in Table IGo). Ig-cross-linked or -uncross-linked Akata cells were used as positive and negative controls, respectively. A representative result of three independent experiments is shown.

 


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  		FIGURE 2. A, RT-PCR analysis of ZEBRA mRNA expression in salivary gland tissues from six patients and three normal controls. Ig-cross-linked Akata cells were used as a positive control and Raji cells as a negative control. GAPDH was used for normalization of mRNA level under the experimental condition. The results shown are a representative of three independent experiments. A high frequency of ZEBRA expression in SS salivary glands was detected when compared with that of normal controls (p < 0.05). B, Double immunofluorescent staining for ZRBRA, CD21, and 120-kDa {alpha}-fodrin in SS and normal salivary glands. Cryostat sections were double stained with TR-conjugated anti-mouse IgG to anti-ZEBRA mAb and FITC-conjugated anti-rabbit IgG to anti-{alpha}-fodrin polyclonal Ab (left panel), TR-conjugated anti-rabbit IgG to anti-{alpha}-fodrin polyclonal Ab and FITC-conjugated anti-mouse IgG to anti-CD21 mAb (middle panel), and TR-conjugated anti-mouse IgG to anti- ZEBRA mAb and FITC-conjugated anti-CD21 mAb (right panel). These stained sections were observed by a laser scanning confocal microscope. Overlap of red and green fluorescence generates yellow (indicated by arrowheads).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increase in 120-kDa {alpha}-fodrin and EBV activation in SS patients

To confirm the presence of positive correlation between the 120-kDa {alpha}-fodrin Abs and EBV reactivation, we examined sera from patients with primary SS (n = 10) and from normal healthy individuals (n = 10). We used anti-ZEBRA and anti-VCA IgM Abs to estimate EBV reactivation. ZEBRA protein is induced as activation of the lytic cycle of EBV, which is the first transcribed EBV-encoded immediate-early gene product and plays an essential role in EBV reactivation (27). Seven of the 10 SS sera, all of which were positive for anti-ZEBRA (Fig. 1Go) and/or anti-VCA IgM, showed reactivity to the 120-kDa {alpha}-fodrin (Table IGo). The 10 normal sera from healthy individuals did not show either anti-ZEBRA or anti-{alpha}-fodrin Ab, which indicates that there are enhanced levels of anti-ZEBRA Ab (p < 0.01; Fig. 1Go) and anti-120-kDa {alpha}-fodrin Ab (p < 0.01; Table IGo) in SS patient sera. Moreover, all normal sera were positive for anti-VCA IgG Ab (data not shown). On primary infection with EBV, serum IgG specific for the VCA is produced within a few days. Thereafter, the Ab titer varies little and persists for life. In addition, we demonstrated by RT-PCR analysis that the level of ZEBRA mRNA was elevated in five of six tissue homogenates of lip biopsies taken from patients with primary SS, but the mRNA was not detected in those from three control individuals (Fig. 2GoA), and this difference was statistically significant (p < 0.05) by {chi}2 test. We further examined hisotologically, by using a double immunofluorescence staining technique, whether infiltrated B cells in SS patient salivary glands were positive for both anti-{alpha}-fodrin and anti-ZEBRA Abs. Tissue sections from salivary glands of SS patients and normal individuals were double stained as described in the legend to Fig. 2B and in Materials and Methods, and were observed by a confocal laser microscope. These results (Fig. 2GoB) show that overlap expression of CD21 (green) and {alpha}-fodrin or ZEBRA (red fluorescence) in the top middle and right panels generates a yellow color (spots indicated by arrowheads), but such overlapped spots were not detected in the sections from normal individual salivary glands (bottom middle and right panels), which indicates that infiltrated B cells are positive to anti-{alpha}-fodrin and anti-ZEBRA Abs. To confirm that infiltrated ZEBRA-positive B cells are also positive to anti-{alpha}-fodrin, we examined sections by double staining with TR-conjugated anti-mouse IgG to murine anti-ZEBRA Ab and FITC-conjugated anti-rabbit IgG to rabbit anti-{alpha}-fodrin Ab (left panel). Yellow-colored spots generated by overlap of green (anti-{alpha}-fodrin) and red (anti-ZEBRA) fluorescence were detected in SS patient salivary glands (top left panel, spots are indicated by arrowheads), but not in control individual salivary glands (bottom left panel). These data have convincingly demonstrated that EBV activation in infiltrated B cells may be associated with 120-kDa {alpha}-fodrin expression.

Induction of apoptosis and {alpha}-fodrin cleavage by EBV activation

Next, we examined the mechanism of {alpha}-fodrin cleavage using an EBV activation model cell line, Akata cells. As a control, we used EBV-negative Akata cells of the same origin, making it possible to examine the effects of EBV in Burkitt’s lymphoma cells (24). We found the induction of the 120-kDa {alpha}-fodrin (Fig. 3Go, A and B) and DNA laddering (Fig. 3Go, G and H) after surface IgG cross-linking of both Akata cells and EBV-negative Akata cells. The result of TUNEL analysis in IgG cross-linked vs staurosporin-treated Akata cells (Fig. 4GoA) emphasized that DNA laddering in these cells (Figs. 3GoG and 4B) was caused by apoptosis. We speculate that the small amount of 120-kDa {alpha}-fodrin detected in untreated Akata cells (Fig. 3GoA) might be caused by apoptosis induced during 24-h culture.



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  		FIGURE 3. The effects of anti-CD40 Ab on the expressions of {alpha}-fodrin, ZEBRA, and the induction of apoptosis in Akata and EBV-negative Akata cells caused by surface Ig cross-linking. Akata cells (A, C, E, and G) and EBV-negative Akata cells (B, D, F, and H) were cultured with or without anti-IgG Ab (100 µg/ml) for 24 h in the presence or absence of anti-CD40 Ab (2.5 µg/ml). Cells were then lysed and {alpha}-fodrin (A and B) and ZEBRA (C and D) expression levels were analyzed by immunoblotting as described in Materials and Methods. Tubulin was used for normalizing the amount of protein loaded (E and F). Shown are DNA fragmentation patterns of cultured Akata cells (G) and EBV-negative Akata cells (H) with or without IgG Ab in the presence or absence of anti-CD40 Ab visualized by UV fluorescence of ethidium bromide-stained agarose gels. The results shown are a representative of three similar experiments.

 


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  		FIGURE 4. Apoptosis detection in Akata cells. Akata cells were cultured for 24 h alone, with 100 µg/ml anti-IgG Ab or 10 µM staurosporin. A, Immunohistochemical analysis of in situ TUNEL detection was conducted as described in Materials and Methods. A significant increase of apototic cells was observed in the anti-IgG Ab and staurosporin-treated cells compared with untreated cells. B, DNA fragmentation was analyzed on an agarose gel as described in Materials and Methods.

 
The next experiments were conducted using anti-CD40 Ab to determine whether or not the 120-kDa {alpha}-fodrin was induced by EBV activation, because it is known that signaling through CD40 blocks surface Ig-mediated apoptosis of B cell lines (28, 29, 30, 31). The results demonstrate that anti-CD40 Ab strongly blocked 120-kDa {alpha}-fodrin expression and DNA fragmentation in EBV-negative Akata cells (Fig. 3Go, B and H), but had no effect on the expression of the 120-kDa {alpha}-fodrin and DNA laddering in Akata cells (Fig. 3Go, A and G). Neither ZEBRA expression in Akata cells was inhibited by anit-CD40 treatment (Fig. 3GoC). We concluded that the induction of apoptosis and {alpha}-fodrin degradation were caused by EBV activation. These results are consistent with a previous study that demonstrated breakdown of chromosomal DNA during EBV replication (32).

Cleavege of {alpha}-fodrin was followed by ZEBRA expression

We examined the kinetics of ZEBRA expression and 120-kDa {alpha}-fodrin formation in EBV-activated Akata cells by immunoblotting methods. The results (Fig. 5Go) show that 120-kDa {alpha}-fodrin formation becomes prominent after 12 h of treatment, whereas ZEBRA expression precedes the 120-kDa {alpha}-fodrin formation and becomes prominent after 6 h of treatment. A very faint 120-kDa band detected in 0- to 6-h homogenates appeared to be a degradation product of intact {alpha}-fodrin, because increasing levels of 120-kDa bands were observed within 9 h and increased steadily thereafter. These results indicate that {alpha}-fodrin cleavage may be caused by ZEBRA expression.



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  		FIGURE 5. One hundred twenty-kilodalton {alpha}-fodrin cleavage following ZEBRA expression in Akata cells. Cell lysates were prepared from Akata cells, which were treated for the indicated periods with 100 µg/ml anti-IgG Ab or for 24 h with 1 µM staurosporin. Expression of the 120-kDa {alpha}-fodrin and ZEBRA in the lysates was then determined with protein immunoblot analysis as described in Materials and Methods. Tubulin was used for normalizing the amount of protein loaded. The results shown are representative of one of three similar experiments.

 
Effects of protease inhibitors on the expression of 120-kDa {alpha}-fodrin

We further examined whether {alpha}-fodrin cleavage to 120-kDa fragments in apoptotic Akata cells induced by IgG cross-linking could be blocked by treatment with specific protease inhibitors. In apoptotic Akata cells (Fig. 6GoA), a caspase inhibitor (z-VAD-fmk) and calcium-activated protease (calpain)-specific inhibitor + z-VAD-fmk strongly suppressed 120-kDa {alpha}-fodrin formation (~75%). A protease inhibitor mixture (AEBSF, pepstatin A, E64, bestatin, leupeptin, and aprotinin) moderately suppressed the 120-kDa {alpha}-fodrin formation (~50%), but E64 and leupeptin also suppressed to the same extent when added individually. ICE-specific inhibitor (Ac-YVAD-cmk) and calpain inhibitor + CPP32-specific inhibitor (z-DEVD-fmk) suppressed slightly (~15%), and z-DEVD-fmk and calpain inhibitor had no effect. In EBV-negative Akata cells (Fig, 6B), z-VAD-fmk and calpain inhibitor slightly suppressed the 120-kDa {alpha}-fodrin formation (~25%), but z-DEVD-fmk and Ac-YVAD-cmk had no effect. These results suggest that the induction of the proteases by EBV activation is different from the surface IgG-cross-linked signal, and that induction of the cysteine protease families by EBV activation participates in the progression of {alpha}-fodrin cleavage.



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  		FIGURE 6. The effects of various protease inhibitors on fodrin proteolysis in IgG-cross-linked EBV-positive or -negative Akata cells. Twenty-four-hour cross-linking treatment of Akata cells (A) or EBV-negative Akata cells (B) with 100 µg/ml anti-IgG was initiated at the end of 1-h treatment with protease inhibitors (5 µM E64, 50 µM leupeptin, 200 µM pepstatin, 20 µM z-VAD, 20 µM DEVD, 10 µM YVAD, and 50 µM calpain inhibitor). One hundred twenty-, 150-, and 240-kDa {alpha}-fodrin bands were quantified by densitometry. The results were calculated and the amount of 120-kDa {alpha}-fodrin formed in the presence of protease inhibitor was expressed as described in Materials and Methods. Tubulin was used for normalizing the amount of protein loaded. Calp inh, calpain inhibitor. The results shown are representative of three independent experiments.

 
Caspase activity in apoptotic EBV-positive Akata cells

We next examined whether ICE/CED-3 family cysteine proteases (caspases) (33) are involved in {alpha}-fodrin cleavage in Akata cells. After cross-linking Akata cells with anti-IgG for various periods, the ICE- and CPP32-like activities in cell lysates were determined using fluorescent substrates. The induction of ICE and CPP32 activities was seen after 240 and 420 min, respectively (Fig. 7Go). This induction of ICE- or CPP32-like activity followed the expression of ZEBRA in apoptotic Akata cells (Fig. 5Go). Fas-mediated appearance of ICE- or CPP32-like activity proceeds without RNA or protein synthesis (34, 35, 36), and each activity peaked at 10 min or showed a sequential increase after 30 min (26). The appearance of these protease activities in apoptotic Akata cells detected later than in Fas-mediated apoptotic cells suggests that de novo protein synthesis accompanied by EBV reactivation may induce the enzymatic activity of these apoptotic proteases. This observation is consistent with a previous study that demonstrated inhibition of DNA fragmentation induction during EBV reactivation by cycloheximide but not by phosphonoacetic acid, which is a specific inhibitor of EBV DNA polymerase but has no effect on early protein production (32).



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  		FIGURE 7. Sequential activation of ICE-like and CPP32-like proteases during IgG-cross-link-induced apoptosis in Akata cells. A and B, Akata cells were treated for various times with 100 µg/ml of anti-IgG Ab alone (•) or in the presence of either 50 µM Ac-YVAD-cmk (A; {circ}) or z-DEVD-fmk (B; {circ}). The ICE-like (A) or CPP32-like (B) activity in the cell extracts (100 µg protein) was assayed using fluorescent substrates as described in Materials and Methods; one unit corresponds to the activity that cleaves 1 pmol of the respective fluorescent substrate at 30°C in 30 min. The results shown are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although particular alleles closely linked to the MHC class II locus increase the risk of developing SS (37, 38), studies of identical twins have implicated environmental factors in the initiation of the disease. Previous evidence has also implicated EBV reactivation in SS (5, 6, 7, 8, 9, 10).

EBV is a ubiquitous human herpes virus which replicates in the salivary glands during primary infection (39) and the virus remains latent at this site in immunologically normal adults (40). Since this virus is known to stimulate autoantibody production (41) and strong T cell-mediated immune responses (14), it may participate in the pathogenesis of SS and serve as a model for studying other human autoimmune disorders. Several previous reports (5, 6, 7, 8, 9, 10, 12, 13) and our present observations that anti-reactivated EBV Abs were present in SS sera and that ZEBRA expression was expressed in SS salivary glands suggest that SS may be a consequence of activated EBV. Thus, SS patients exhibit many signs of active, uncontrolled EBV reactivation. Although cleavage of certain autoantigens during apoptosis may expose immunocryptic epitopes that could potentially induce autoimmune responses in systemic autoimmune diseases (42, 43, 44, 45), it remains unclear whether autoantigen cleavage occurs in organ-specific autoimmune diseases.

{alpha}-Fodrin is a calmodulin-binding protein that was recently found to be cleaved by calpain in apoptotic T cells and by calpain and caspases in anti-Fas-stimulated Jurkat cells and/or neuronal apoptosis (17, 46, 47, 48). It is now believed that the interaction of Fas with Fas ligand regulates a large number of pathophysiological processes of apoptosis (49, 50). However, the mechanisms responsible for tissue destruction in SS remain to be elucidated. In this study, we provide evidence to suggest that EBV-mediated apoptosis may be involved in the initial cascade of tissue destruction. Analysis of proteolytic events associated with apoptosis may define the mechanisms leading to protease activation and identify key substrates whose cleavage might be linked to profound changes in cellular architecture. There is increasing evidence that proteases are overactivated in autoimmune conditions and subsequent tissue destruction (51, 52). Moreover, the cascade of caspases is a critical component of the cell death pathway (26, 53), and a few proteins have been found to be cleaved during apoptosis. These include poly(ADP)-ribose polymerase, a small U1 nuclear ribonucleoprotein, and {alpha}-fodrin, which were subsequently identified as substrates for caspases (43, 48, 54, 55). We provide evidence herein that {alpha}-fodrin is cleaved by a combination of caspases and one or more other proteases during EBV mediated-apoptosis in SS salivary glands.

As previously reported, calpain and caspases in Fas-mediated apoptosis (56) and in apoptotic cells by various cytokines such as TNF induced a cleavage product of 120-kDa {alpha}-fodrin (47, 48). Recent studies have shown that production of anti-{alpha}-fodrin Ab is accelerated by estrogen deficiency (57) or through age-related, Fas-mediated apoptosis (58) in SS model mice. If EBV is not sufficient to lead to 120-kDa {alpha}-fodrin formation, it may be one of many cofactors. It is known that SS salivary glands express increased levels of cytokines, in contrast to that of normal salivary glands (59, 60). Moreover, it has been reported that EBV can lead to induction of cytokine expression (61). Therefore, 120-kDa {alpha}-fodrin cleavage in SS may result from EBV reactivation and indirect immune stimulation.

Present studies have demonstrated that there is a significant link between EBV reactivation and production of anti-{alpha}-fodrin Ab in SS patients. However, since we could not show absence of such a link in individuals without SS but with reactivation of EBV, it is questionable whether the link observed is indeed significant to SS patients. In fact, it has been reported that the reactivation of EBV is not a SS-specific event (62) and that a relationship between some populations of autoimmune diseases and EBV reactivation is present (63, 64), but anti-{alpha}-fodrin Ab is a SS-specific autoantibody (16, 65). Furthermore, it cannot be detected in plasma of rheumatoid arthritis and systemic lupus erythematosus patients (16, 65) even in the plasma of these patients with EBV reactivation (H. Inoue and I. Saito, unpublished observations). These observations are strongly supportive for the absence of a link between EBV reactivation and production of anti-{alpha}-fodrin in non-SS individuals. Therefore, the link observed in the present study is significant to SS patients.

A number of properties that may be relevant to the initiation and perpetuation of the pathogenesis of SS are now known to be present in SS salivary glands, but not in other salivary gland conditions or in normal salivary glands. These include intense B lymphocyte and CD4-positive T cell infiltration (66, 67, 68, 69, 70), destruction of epithelial cells (71, 72), abnormally high levels of EBV and EBV Ags (6, 8), production of infectious EBV in saliva (7, 8), de novo expression of HLA-DR on the cell surface (8, 9), and induction of a 120-kDa autoantigen by EBV activation (present results). These observations are consistent with several scenarios in which EBV plays an important role in the initiation and/or continuation of an immune system attack in salivary and lacrimal glands.

Present in situ studies using a double immune-staining method provides evidence that infiltrated ZEBRA-positive B cells in SS salivary glands are also positive for 120-kDa {alpha}-fodrin (Fig. 2Go). In vitro study reveals EBV activation in B cells induces the apoptosis with expression of the 120-kDa {alpha}-fodrin (Fig. 3Go). Therefore, we propose that apoptotic proteases activated with EBV activation might be involved in the progression of {alpha}-fodrin proteolysis and tissue destruction in the development of a subpopulation in SS patients. Inhibition of protease activity in EBV-mediated apoptotic cells may be a potential therapeutic approach to the treatment of SS. It is also possible that other EBV-infected cells in salivary glands, such as acinar cells, might also contribute to the productivity of 120-kDa {alpha}-fodrin. Further investigation into these issues is needed.


   Footnotes
 
1 This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Ichiro Saito, Department of Pathology, Tokushima University School of Dentistry, 3-18-15, Kuramotocho, Tokushima 770-8504, Japan. Back

3 Abbreviations used in this paper: SS, Sjogren’s syndrome; ZEBRA, BamH1-Z-DNA fragment of Epstein-Barr replication activator; VCA, viral capsid Ag; ICE, IL-1-converting enzyme; TR, Texas Red. Back

Received for publication February 28, 2000. Accepted for publication February 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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