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Autoantibodies to the Amino-Terminal Fragment of -Fodrin Expressed in Glandular Epithelial Cells in Patients with Sjögren’s Syndrome¹

Masataka Kuwana^2,*, Tetsuroh Okano, Yoko Ogawa^*, Junichi Kaburaki and Yutaka Kawakami^*

^* Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan; Department of Clinical Immunology, Kitasato University School of Allied Health Science, Sagamihara, Japan; and Department of Internal Medicine, Tokyo Electric Power Company Hospital, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sjögrens’s syndrome (SS) is an autoimmune disease characterized by destruction of lacrimal and salivary glands, but the mechanisms underlying the disease process are unclear. By immunoscreening a HepG2 cDNA library with serum from an SS patient we isolated a cDNA encoding amino-terminal 616 aa of -fodrin, a membrane skeleton protein associated with ion channels and pumps. Serum Ab to the amino-terminal fragment of -fodrin was frequently detected in SS patients compared with rheumatic disease patients without SS or healthy controls (70 vs 12 or 4%; p < 0.00001). All the anti--fodrin-positive sera recognized the amino-terminal fragment with no homology to -fodrin. Anti--fodrin Abs in patients’ sera as well as mouse polyclonal sera raised against the amino-terminal -fodrin fragment did not react with intact -fodrin, but recognized the 65-kDa amino-terminal fragment generated through cleavage by caspase-3 or granzyme B. When expression of intact and fragmented -fodrin in lacrimal glands was assessed by immunohistochemistry, the antigenic amino-terminal fragment was distributed diffusely in acinar epithelial cell cytoplasm, whereas the carboxyl-terminal fragment and/or intact -fodrin were localized in peripheral cytoplasm, especially at the basal membrane, in SS patients. In contrast, intact -fodrin was detected primarily at the apical membrane of epithelia, and the amino-terminal fragment was scarcely detected in control patients with chronic graft-vs-host disease. These findings suggest that cleavage and altered distribution of -fodrin in glandular epithelial cells may induce impaired secretory function and perpetuate an autoimmune response to -fodrin, leading to autoantibody production and glandular destruction in SS.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sjögren’s syndrome (SS)³ is an autoimmune exocrinopathy characterized by keratoconjunctivitis and xerostomia sicca (1). Prominent immunologic findings in SS patients include the presence of autoantibodies to various intracellular Ags, such as SSA/Ro and SSB/La, and polyclonal hypergammaglobulinemia (1). Pathologic findings in salivary and lacrimal glands have revealed glandular attrition in acinar and ductal epithelia concomitant with infiltration of mononuclear cells consisting of T cells, B cells, and plasma cells (2). Both immune-dependent and independent pathways are proposed as mechanisms for inducing destruction of glandular epithelial cells and resulting in impaired secretory capacity of salivary and lacrimal glands (3).

Fodrin or a generalized form of erythroid spectrin (II1/II1), a heterodimer composed of and subunits with M_r of 240 and 235 kDa, respectively, is an abundant protein of eukaryotic cell membrane skeleton (4). The /-fodrin dimers self-associate head-to-head into tetramers and serve as the basic structural element of the membrane skeleton. Both - and -fodrin share a homologous internal 106-aa repeating motif called spectrin repeat, and 22 and 17 spectrin repeats are included in - and -fodrin, respectively. -Fodrin has additional amino- and carboxyl-terminal regions with no homology to -fodrin (5). Fodrin is shown to associate with membrane ion channels and pumps and supports their composition in several kinds of epithelial cells (4). Recently, it has been shown that sera from an animal model of SS as well as those from SS patients specifically recognize the 120-kDa amino-terminal fragment of -fodrin (6, 7). The 120-kDa -fodrin fragment was expressed in salivary glands, but not in other organs, of the NFS/sld mouse model of SS (6). In addition, the 120-kDa -fodrin fragment was detected in tissue homogenates of lip biopsies from patients with primary SS, but not in those from control individuals (6). Based on these findings, the 120-kDa amino-terminal -fodrin fragment is thought to be an important organ-specific autoantigen in an animal model of SS as well as in SS patients. Although anti--fodrin Abs are well characterized, autoantibodies reactive with -fodrin have not been reported to date.

By immunoscreening of a HepG2 cDNA library using serum from a patient with SS secondary to systemic sclerosis (SSc), we have isolated a cDNA fragment encoding the amino-terminal portion of human -fodrin. In this study different portions of -fodrin were expressed as recombinant proteins and used to detect serum anti--fodrin Abs in SS patients. In addition, a possible association of the anti--fodrin autoantibody response with the pathogenic process of SS was examined.

	Materials and Methods

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Patients and controls

Serum samples from 37 patients with primary SS and 45 patients with secondary SS were examined in this study. Additional rheumatic diseases in patients with secondary SS were systemic lupus erythematosus (SLE) in 12 and SSc in 33. Sera from 32 SLE patients and 27 SSc patients who lacked clinical symptoms of SS as well as those from 100 healthy individuals were used as controls. All SS patients satisfied the San Diego criteria as definite SS (8), and SLE and SSc patients met the American College of Rheumatology classification criteria (9, 10). Serum from patient KA, a 74-yr-old male with SS and diffuse cutaneous SSc, was used for the cDNA library screening.

For immunohistochemical studies, lacrimal gland biopsy specimens were obtained from five patients with SS (four primary and one secondary). As a control, lacrimal gland tissues were obtained from five patients who had dry eye and dry mouth as a part of symptoms related to chronic graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation for hematological malignancies (11). A written informed consent approved by the institutional review board was granted by all patients before obtaining biopsy materials.

Autoantibodies

Serum anti-SSA/Ro, anti-SSB/La, and anti-U1 RNP Abs were determined by RNA immunoprecipitation assay (12). Anti--fodrin Abs were detected by immunoblots using apoptotic HeLa cell lysates as an Ag source (described below). Sera recognizing a protein with a relative M_r consistent with that of the 120-kDa fragment recognized by anti--fodrin mAb were considered positive for anti--fodrin Abs (6).

cDNA library screening

Plaques (2 x 10⁵) of a randomly primed HepG2 cell cDNA library (Clontech Laboratories, Palo Alto, CA) were screened with the KA serum as described previously (13). Positive clones were isolated, and their nucleotide sequences were determined on an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA).

Expression of recombinant -fodrin fusion proteins

Recombinant -fodrin fusion proteins, including FOD_1–60, FOD_1–103, FOD_1–272, FOD_1–463, FOD_1–616, and FOD_273–616, which encompass aa 1–60, 1–103, 1–272, 1–463, 1–616, and 273–616, respectively, of the 2365 aa of -fodrin (5), were prepared as previously described (14). Briefly, a series of -fodrin cDNA constructs was prepared from a cDNA encoding a portion of -fodrin by PCR using specific primer pairs. The cDNA constructs were subcloned in-frame into the pMAL-c2 expression vector (New England Biolabs, Beverly, MA) (15) and were transformed into a competent Escherichia coli strain DH5 (Toyobo, Osaka, Japan). Nucleotide sequences of both strands of each DNA construct were determined to verify the translational frames and insert sequences. Expression of recombinant maltose-binding protein (MalBP) fusion protein was induced by isopropyl--D-thiogalactopyranoside, and bacterial lysates containing MalBP or -fodrin fusion proteins were directly analyzed by SDS-PAGE. In some experiments FOD_1–272 was purified by amylose-resin affinity chromatography (15, 16).

HeLa cell cultures and induction of apoptosis

HeLa cells were cultured in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Apoptosis was induced by irradiation with UV-B, followed by incubation overnight (17). Adherent cells and floating populations were pooled and used as a source of Ag for immunoblotting (18). HeLa cell apoptosis was confirmed by classic apoptotic morphology and internucleosomal DNA fragmentation (17). In some experiments apoptosis was induced in HeLa cells in the presence of various protease inhibitors, including Z-VAD.fmk (100 µM; Research Biochemicals International, Natick, MA), E64d (3.5 µg/ml; Peptide Institute, Osaka, Japan), and leupeptin (210 µM). Z-VAD.fmk and E64d are membrane-permeable inhibitors of caspases and calpain, respectively (19, 20). Protease inhibitors were added to the cultures 1 h before induction of apoptosis.

Mouse polyclonal sera to the amino-terminal fragment of -fodrin

To raise mouse polyclonal anti--fodrin Abs, purified FOD_1–272 was used to immunize BALB/c mice following a standard protocol (21). Mouse sera were pooled and stored at -80°C. Before use in immunoblotting and immunohistochemistry, sera were diluted 1/100 and incubated with purified MalBP immobilized on nitrocellulose membranes to absorb Abs to MalBP. Depletion of anti-MalBP Abs in the treated sera was confirmed by immunoblotting. Mouse preimmune sera diluted 1/100 were used as a control.

Immunoblotting

Reactivities to recombinant -fodrin fusion proteins and endogenous -fodrin were examined by immunoblotting (14). Briefly, bacterial lysates containing recombinant -fodrin fusion proteins or whole cell lysates of intact or apoptotic HeLa cells were fractionated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were incubated with a 1/100 dilution of patients’ sera that were preincubated with bacterial lysates containing MalBP to remove Abs reactive with bacterial proteins and MalBP. Rabbit polyclonal anti-MalBP Ab (New England Biolabs), mouse anti--fodrin mAb (AFFINITI, Exeter, U.K.), mouse anti--fodrin mAb (Nippon Shinyaku, Kyoto, Japan), mouse anti-FOD_1–272 polyclonal sera, and mouse control sera were also used as the primary Ab. In some experiments, FOD_1–272-specific Abs were purified from anti--fodrin-positive sera as described previously (22) and used as the primary Ab in immunoblotting. In addition, mouse anti-FOD_1–272 polyclonal sera and anti--fodrin mAb as well as anti--fodrin-positive patients’ sera were preincubated with purified FOD_1–272 or MalBP (5 or 50 µg/ml) before they were applied to immunoblots. The membranes were subsequently incubated with alkaline phosphatase-conjugated goat anti-human, -mouse, or -rabbit IgG (Cappel, Aurora, OH), and reactivities were visualized by development with 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate. In some experiments immunoreactivities were visualized using a chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden) and subsequently analyzed using a Molecular Imager FX (Bio-Rad, Hercules, CA). The intensity of the protein band was expressed as a relative expression, which was calculated as percentage to a control preparation.

In vitro cleavage of endogenous -fodrin by caspase-3, caspase-8, or granzyme B

The abilities of caspase-3, caspase-8, and granzyme B to cleave -fodrin were examined using the method described by Casciola-Rosen et al. (23). Briefly, HeLa cells were labeled with [³⁵S]methionine/cysteine (DuPont-NEN, Boston, MA), and endogenous -fodrin was immunoprecipitated with anti--fodrin mAb coupled with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech). The beads bound to radiolabeled -fodrin were incubated with recombinant human caspase-3 or caspase-8 (Chemicon International, Temecula, CA) in the presence of 5 mM DTT or cell culture-derived human granzyme B (Calbiochem-Novabiochem, La Jolla, CA) in the presence of 2 mM iodoacetamide at 37°C for 15 min (caspase-3 and granzyme B) or 1 h (caspase-8). Samples were then electrophoresed on 7% SDS-polyacrylamide gels, and radiolabeled fragments were visualized by autoradiography.

Immunohistochemistry

Immunohistochemical analysis of a lacrimal gland specimen was performed following a standard protocol (24). Briefly, frozen sections of lacrimal gland specimens were incubated with anti--fodrin mAb, mouse anti-FOD_1–272 polyclonal sera, or control mouse sera diluted 1/100 at room temperature for 2 h in a humidified chamber. This was followed by incubation with peroxidase-conjugated rabbit anti-mouse IgG diluted 1/100 for 45 min. Bound peroxidase was detected using diaminobenzidine tetrahydrochloride and H₂O₂. Cell nuclei were counterstained with hematoxylin or methyl green.

Statistical analysis

Comparisons between the two patient groups were performed using ² tests. The relative expression levels of the protein bands were compared using Student’s t test. p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of cDNA encoding the amino-terminal portion of -fodrin

A 1861-bp cDNA was isolated by immunoscreening of a HepG2 cDNA library. The nucleotide sequence was highly homologous to nt 298-2158 of the 7561 nt of the full-length human -fodrin cDNA (5). Two nucleotides were different from the reported -fodrin cDNA sequence. The adenine at positions 1183 and 1237 in the reported sequence was substituted by guanine in our cDNA, but these nucleotide substitutions did not result in amino acid changes. As a result, the isolated cDNA encoded the amino-terminal portion of -fodrin encompassing amino acid residues 1–616 of the entire 2365 aa and corresponding to the -fodrin-specific region and three complete spectrin repeats (Fig. 1A).

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Recombinant -fodrin fragments used in this study. A, Schematic representation of -fodrin and a series of recombinant -fodrin fragments. Numbers denote the amino acid residue numbers. -Fodrin consists of 17 spectrin repeats and amino- and carboxyl-terminal -fodrin-specific regions. FOD1–616 contains the amino-terminal -fodrin-specific region and three complete spectrin repeats. B, Bacterial lysates containing MalBP or MalBP--fodrin fusion proteins were fractionated on a SDS-10% polyacrylamide gel and stained with Coomassie blue. M, molecular mass markers.

Screening for anti--fodrin Abs in SS patients

Anti--fodrin Abs were examined in the sera from 37 patients with primary SS, 45 with secondary SS, 32 with SLE without SS, 27 with SSc without SS, and 100 healthy controls by immunoblots using FOD_1–616 as Ag. The frequencies of serum IgG anti--fodrin Abs in patients with or without SS and in healthy controls are summarized in Table I. Anti--fodrin Abs were detected in 51 and 84% of patients with primary and secondary SS, respectively, and these frequencies were significantly greater than those in healthy controls. In SLE and SSc patients, the frequencies of anti--fodrin Abs were significantly higher in patients with SS than in those without. In addition, anti--fodrin Abs were detected more frequently in patients with secondary SS than in those with primary SS, and this difference was statistically significant (p = 0.003).

View this table: [in this window] [in a new window]   Table I. Frequency of serum IgG Abs to the amino-terminal portion of -fodrin in rheumatic disease patients with or without SS

Autoantibodies coexisting with anti--fodrin Abs

To examine SS-related autoantibodies preferentially coexisting with anti--fodrin Abs, the frequencies of anti-SSA/Ro, anti-SSB/La, anti-U1 RNP, and anti--fodrin Abs were compared between SS patients (including primary and secondary SS) positive and negative for anti--fodrin Abs (Table II). Anti--fodrin Abs more frequently occurred together with anti--fodrin Abs. In contrast, anti--fodrin Abs were detected preferentially in SS patients without anti-SSA/Ro or anti-SSB/La Ab, and the frequency of anti-SSB/La Ab in anti--fodrin-positive SS patients was significantly lower than that in anti--fodrin-negative SS patients.

View this table: [in this window] [in a new window]   Table II. Frequencies of anti-SSA/Ro, anti-SSB/La, anti-U1RNP, and anti--fodrin Abs in sera from 82 SS patients with or without anti--fodrin Abs

Antigenic -fodrin fragments

Reactivities to amino- or carboxyl-terminal deletion fragments of FOD_1–616 were examined by immunoblotting using randomly selected 40 sera positive for anti--fodrin Abs. These sera included 10 from patients with primary SS, 22 from patients with secondary SS, five from patients with SSc without SS, and three from healthy controls. As shown in Fig. 2, representative SS sera (panels 3–5) reacted with FOD_1–272, FOD_1–463, and FOD_1–616, but not with FOD_1–60 or FOD_273–616. One SS serum showed an additional reactivity to FOD_1–103 (panel 4). Mouse anti-FOD_1–272 polyclonal sera recognized all -fodrin fusion proteins except FOD_273–616 (Fig. 2, panel 2). It is interesting to note that patients’ sera as well as mouse anti-FOD_1–272 polyclonal sera preferentially bound to the degradation products of FOD_1–616, especially to the fragment with a molecular size similar to that of FOD_1–272, rather than the intact FOD_1–616.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Reactivities to a series of recombinant -fodrin fusion proteins in SS sera positive for anti--fodrin Abs. Bacterial lysates containing MalBP or MalBP--fodrin fusion proteins were fractionated on SDS-10% polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with anti-MalBP polyclonal Ab (panel 1), mouse anti-FOD1–272 polyclonal sera (panel 2), and SS sera positive for anti--fodrin Abs (panels 3–5).

Autoantibody reactivities to endogenous -fodrin

To examine endogenous proteins recognized by anti--fodrin Abs in SS sera, whole cell lysates from nontreated HeLa cells were used as Ag in immunoblotting. When 10 anti--fodrin-positive SS sera were examined, there was no apparent protein commonly recognized by these sera (data not shown). Since it has been shown that anti--fodrin Abs in SS sera react with the 120-kDa amino-terminal fragment of -fodrin generated during apoptosis, but not with an intact -fodrin (6), apoptotic HeLa cells were used as Ag in immunoblots. As shown in Fig. 3A, representative SS sera positive for anti--fodrin Abs (OY, NK, and YN) and mouse anti-FOD_1–272 polyclonal sera commonly reacted with the 65-kDa protein, which was not recognized by anti--fodrin-negative SS serum YS. Anti--fodrin mAb recognized the 235-kDa intact -fodrin and the 170-kDa protein, but did not recognize the 65-kDa protein. Anti--fodrin mAb recognized the 240-kDa intact -fodrin and several smaller fragments, including the antigenic 120-kDa fragment. The 65-kDa protein in apoptotic HeLa cells was recognized by 10 additional anti--fodrin-positive SS sera, but not by 10 anti--fodrin-negative SS sera. Based on these findings, it is plausible that the 65-kDa protein is an amino-terminal -fodrin fragment generated by cleavage during apoptosis and contains the determinants commonly recognized by anti--fodrin Abs in SS sera. In contrast, the 170-kDa protein appears to be a carboxyl-terminal -fodrin fragment containing the epitope recognized by anti--fodrin mAb.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. The endogenous -fodrin fragment in apoptotic HeLa cell lysates is recognized by anti--fodrin Abs. Apoptotic HeLa cell lysates were fractionated on SDS-7.5% polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with anti--fodrin-positive SS sera (A), anti-FOD1–272-specific Abs purified from SS sera (B), or mouse anti-FOD1–272 polyclonal sera preincubated with purified FOD1–272 as a competitor (C). A: Lane 1, Mouse anti-FOD1–272 polyclonal sera; lanes 2–4, SS sera positive for anti--fodrin Abs; lane 5, SS serum negative for anti--fodrin Abs; lane 6, anti--fodrin mAb; lane 7, anti--fodrin mAb. B: Lane 1, Anti--fodrin mAb; lane 2, anti--fodrin mAb; lane 3, mouse anti-FOD1–272 polyclonal sera; lane 4, mouse control sera; lanes 5–7, FOD1–272-specific Abs purified from anti--fodrin-positive SS sera; lanes 8–10, mock eluted Abs. C: Lanes 1–3, Mouse anti-FOD1–272 polyclonal sera preincubated with or without MalBP; lanes 4–6, mouse anti-FOD1–272 polyclonal sera preincubated with or without FOD1–272; lanes 7–9, anti--fodrin mAb preincubated with or without FOD1–272.

Cleavage of endogenous -fodrin by caspase-3, caspase-8, and granzyme B

To identify apoptosis-related proteases involved in generation of the antigenic 65-kDa -fodrin fragment, we examined the effects of protease inhibitors on the generation of the 65-kDa fragment during apoptosis (Fig. 4). HeLa cell apoptosis was induced in the presence or the absence of Z-VAD.fmk, E64d, or leupeptin, and the cell lysates were fractionated and probed with anti--fodrin mAb and mouse anti-FOD_1–272 polyclonal sera. -Fodrin was cleaved into the 170- and 65-kDa fragments in apoptotic HeLa cells, while the 235-kDa intact -fodrin was the dominant form in nonirradiated HeLa cells. Apoptosis-induced cleavage of -fodrin was inhibited by Z-VAD.fmk, a specific inhibitor for caspases, but not by E64d or leupeptin.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Effects of protease inhibitors on the generation of the antigenic 65-kDa amino-terminal fragment of -fodrin during apoptosis. A, HeLa cell apoptosis was induced by irradiation, and the cells were cultured for 24 h in the presence or the absence of Z-VAD.fmk, E64d, or leupeptin. HeLa cell lysates (105 cells/lane) were then fractionated on SDS-7.5% polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with anti--fodrin mAb (upper panel) or mouse anti-FOD1–272 polyclonal sera (lower panel). B, The intensity of the 65-kDa fragment band was quantified by densitometry and expressed as a relative expression to preparation without apoptosis induction. Results are the mean ± SD of three independent experiments. *, The significant difference compared with expression in the absence of protease inhibitor.

View larger version (65K): [in this window] [in a new window]   FIGURE 5. Endogenous -fodrin is cleaved by caspase-3 and granzyme B, but not by caspase-8. [35S]methionine/cysteine-labeled endogenous -fodrin was immunoprecipitated by anti--fodrin mAb and incubated with caspase-3, granzyme B (A), or caspase-8 (B). Arrows represent intact -fodrin (235 kDa). Solid arrowheads denote caspase-3-induced fragments (170, 120, and 65 kDa), and open arrowheads denote granzyme B-induced fragments (110, 65, and 60 kDa). Similar results were obtained in four independent experiments.

Expression of the antigenic -fodrin fragment in SS lacrimal glands

Expression of the antigenic 65-kDa -fodrin fragment in lacrimal gland biopsies from five SS patients was examined by immunohistochemistry using mouse anti-FOD1–272 polyclonal sera. Lacrimal gland biopsies from five patients with chronic GVHD were used as controls. Several histopathologic differences were noted between SS and chronic GVHD, including an acinar lesion that was less affected in chronic GVHD compared with SS (25). Especially, two specimens from chronic GVHD patients had histologically normal acinar lesion. In a representative SS specimen, diffuse cytoplasmic staining was detected in acinar epithelial cells, but not in infiltrating lymphocytes (Fig. 6A). The cytoplasmic expression of the antigenic -fodrin fragment in acinar epithelial cells, especially within lesions with prominent lymphocyte infiltrates, was commonly observed in all five SS biopsies. In contrast, no or weak immunoreactivity to acinar epithelial cells was detected in lacrimal glands from chronic GVHD patients (Fig. 6B). Lacrimal gland specimens were also stained with anti--fodrin mAb that were reactive with intact -fodrin and the 170-kDa carboxyl-terminal fragment. As shown in Fig. 6C, anti--fodrin mAb stained the peripheral cytoplasm of acinar epithelial cells in SS lacrimal glands. In all SS specimens, peripheral cytoplasmic staining was prominent at the basal membrane, but not at the apical membrane. In contrast, the apical membrane of acinar epithelial cells was intensely stained by anti--fodrin mAb in lacrimal gland biopsies from chronic GVHD patients (Fig. 6D). No immunoreactivity was detected when lacrimal gland sections were stained with mouse control sera.

View larger version (123K): [in this window] [in a new window]   FIGURE 6. Immunohistochemical staining for the intact and fragmented -fodrin in lacrimal gland tissue biopsies from a representative SS patient (A and C) and chronic GVHD patient (B and D). Lacrimal gland tissue sections were stained with mouse anti-FOD1–272 polyclonal sera (A and B) or anti--fodrin mAb (C and D). Positive immunoreactivity appears as brown color, and counterstaining as blue or green. Original magnification, x20 (A and B) or x40 (C and D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Anti--fodrin Abs frequently coexisted with anti--fodrin Abs. Although - and -fodrin have a significant amino acid homology, it is unlikely that the coexistence of these two autoantibodies is due to cross-reactivity on the basis of the following observations. First, all the sera positive for anti--fodrin Abs recognized the amino-terminal -fodrin fragment that has no amino acid homology to -fodrin. Second, -fodrin-specific Abs eluted from patients’ sera did not react with the 120-kDa -fodrin fragment. Finally, Ab reactivity to the 120-kDa -fodrin fragment was not absorbed by preincubation of SS sera with purified FOD_1–272. Therefore, both components of a functional unit of the fodrin heterodimer are targeted by the autoantibody response. In this regard, autoantibodies in sera from patients with rheumatic diseases often target multiple components of complex autoantigens, such as U1 RNP (28), histones (29), and RNA polymerases I/II/III (22). Fodrin should be included in the list of the complex molecules targeted by autoantibodies in the sera of patients with rheumatic diseases.

It is of note that anti--fodrin Abs in SS sera reacted with the 65-kDa amino-terminal fragment, but not with -fodrin in its intact form. This is analogous to anti--fodrin Abs that bound to the 120-kDa amino-terminal fragment, but not to the intact molecule (6). It seems likely that the antigenic epitope within the amino-terminal region of -fodrin is masked in the molecule containing the entire amino acid sequence even in its denatured state in immunoblots. Therefore, structural modification is required for expression of the antigenic epitope on -fodrin recognized by autoantibodies in SS sera. Since anti--fodrin Abs in SS sera preferentially reacted with the degradation product of FOD_1–616 lacking the spectrin repeats, rather than the intact FOD_1–616 (see Fig. 2), it is possible that that spectrin repeats, each of which folds into three helixes, interfere with autoantibody binding to the epitope located within the amino-terminal -fodrin-specific region.

Our results show that the antigenic 65-kDa amino-terminal fragment of -fodrin was released upon cleavage of -fodrin by caspase-3 during apoptosis. This is based on the inhibition of the -fodrin cleavage by the caspase inhibitor Z-VAD.fmk and the cleavage of -fodrin with caspase-3. Several proteins of the membrane skeleton, such as laminin, actin, and -fodrin, were shown to be susceptible to caspases (30). Cleavage of these molecules during apoptosis is believed to result in unstable cellular structures and subsequently in promoting processes in apoptosis. In contrast, Casciola-Rosen et al. (23) have recently reported that the majority of Ags targeted by autoantibodies in the sera of patients with rheumatic diseases are susceptible to granzyme B, but not to caspase-8, and that the generation of unique fragments by granzyme B was a universal feature of autoantigens. Because -fodrin was cleaved by granzyme B, but was resistant to caspase-8, and the majority of the -fodrin fragments generated by granzyme B were unique, -fodrin followed the rule proposed by Casciola-Rosen et al. (23). However, the antigenic 65-kDa fragment was commonly generated by cleavage by caspase-3 and granzyme B, but generation of the identical fragment by caspase-3 and granzyme B was shown in an another autoantigen DNA-dependent protein kinase catalytic subunit (31). Granzyme B shares with caspases a requirement for aspartic acid in the substrate P₁ position (32). Based on the fragment sizes and the known cleavage specificities of caspases and granzyme B (32, 33), a cleavage site generating the antigenic 65-kDa amino-terminal fragment of -fodrin is predicted to be VEAD⁵⁷⁶-I. In addition, a possible caspase-3-specific site of -fodrin is DEVD¹⁴⁵⁸-S, while possible granzyme B-specific sites included IVTD¹⁵⁵⁴-S and AEID¹⁹⁶¹-A, although additional studies using mutated -fodrin with amino acid substitutions at these sites are necessary to confirm these cleavage sites.

By immunochemical analysis using anti-FOD_1–272 sera and anti--fodrin mAb, we were able to assess distribution of intact and fragmented -fodrin in lacrimal glands. In SS patients the antigenic amino-terminal fragment was distributed diffusely in acinar epithelial cell cytoplasm, whereas the carboxyl-terminal fragment and/or intact -fodrin were localized in peripheral cytoplasm, especially at basal membrane. In contrast, in chronic GVHD patients, intact -fodrin was primarily present at the apical membrane of acinar epithelial cells, while the expression of antigenic amino-terminal -fodrin fragment was very weak. The preferential expression of the 65-kDa -fodrin fragment in SS lacrimal glands is analogous to the antigenic 120-kDa -fodrin fragment, which is shown to be expressed in salivary gland epithelial cells exclusively in an animal model of SS and SS patients (6, 7). However, the present study provides the additional description of the altered distribution of the -fodrin fragment in lacrimal gland epithelial cells in SS patients.

Since fodrin is shown to associate with membrane ion channels and pumps and to support their composition as the basic structural element of the membrane skeleton in the renal and salivary gland epithelia (4, 34), it is likely that structural modification of both - and -fodrin and subsequent altered distribution may disturb the physiologic trafficking of ion channels and pumps, resulting in dysfunction of the secretory capacities of glandular epithelial cells. In fact, the altered distribution of the membrane channel proteins, including sodium-independent bicarbonate anion exchanger and aquaporin-5, in acinar epithelial cells was reported in SS salivary glands (35, 36). Furthermore, the antigenic - and -fodrin fragments expressed in abundance in glandular epithelial cells may perpetuate the autoimmune response to fodrin by revealing previously "cryptic" epitopes, resulting in the production of autoantibodies to - and -fodrin and T cell reactivities to epithelial cells leading to glandular destruction.

What is the mechanism for the structural modification of -fodrin in glandular epithelium in SS patients? Our results strongly suggest that cleavage of -fodrin is the most likely process to induce expression of antigenic determinants recognized by SS sera. Because the 65-kDa amino-terminal fragment of -fodrin was generated through cleavage by apoptosis-related proteases, such as caspase-3 and granzyme B, expression of the antigenic -fodrin fragment in lacrimal gland epithelial cells in SS patients may reflect the enhanced apoptotic cell death in glandular epithelia of SS patients (3, 37). The glandular epithelial cell apoptosis in SS patients is mediated through a Fas/Fas ligand pathway (38, 39) and a perforin-granzyme pathway (40). However, the frequency of the TUNEL-positive apoptotic cells in total epithelial cells was shown to be <1% (39, 41), and this is inconsistent with our finding of expression of the antigenic amino-terminal -fodrin fragment in the majority of acinar epithelial cells. In this regard, a recent report proposed a theory that the transmission of death signals into the nucleus might be blocked in SS glandular epithelia on the basis of infrequent epithelial cell apoptosis despite elevated expression of Fas and Fas ligand among these cells in SS salivary glands (41). Further studies examining mechanisms for the structural modification of -fodrin in lacrimal and salivary gland epithelial cells may provide a clue to the pathogenesis of SS.

	Acknowledgments

 
We thank Dr. Carol A. Feghali for reviewing this manuscript, Mutsuko Ishida for assisting in RNA immunoprecipitation assay, Dr. Kazuto Yamazaki for valuable comments on immunohistochemistry, and Drs. Shinichiro Okamoto and Yoshihisa Oguchi for coordinating lacrimal gland biopsies.

	Footnotes

 
¹ This work was supported by the Keio University Medical Science Fund, the Japanese Ministry of Health and Welfare, and the Ministry of Education, Science, Sports and Culture of Japan.

² Address correspondence and reprint requests to Dr. Masataka Kuwana, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: kuwanam{at}sc.itc.keio.ac.jp

³ Abbreviations used in this paper: SS, Sjögren’s syndrome; GVHD, graft-vs-host disease; MalBP, maltose-binding protein; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.

Received for publication June 1, 2001. Accepted for publication August 27, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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