Title: Autoimmunity against a Tissue Kallikrein in IQI/Jic Mice
Abstract: We have recently characterized IQI/Jic mice as a model for Sjögren's syndrome (SS), a chronic autoimmune disease in humans. In SS, local lymphocytic infiltrations into salivary and lacrimal glands frequently develop to the involvement of systemic exocrine and nonexocrine organs, and the mechanism for progression of this disease remains obscure. Herein, we report identification of an autoantigen shared by various target organs in IQI/Jic mice. Polypeptides identified based on immunorecognition by autoantibodies in sera from IQI/Jic mice affected with autoimmune disease (>12 weeks of age) were tissue kallikrein (Klk)-1 and -13 and were cross-reactive to the autoantibodies. Interestingly, Klk-13, but not Klk-1, caused a proliferative response of splenic T cells from IQI/Jic mice from the age of 4 weeks onward. In addition, remarkably enhanced expression of Klk-13 was observed in the salivary glands of the mice in accordance with the development of inflammatory lesions. These results indicate that Klk-13 acts as an autoantigen and may increase T cells responsive to organs commonly expressing Klk-13, playing a pivotal role in the etiology of progression of disease in IQI/Jic mice. Our findings provide insights into the contributions of autoantigens shared by multiple organs in the progress of SS from an organ-specific to a systemic disorder. We have recently characterized IQI/Jic mice as a model for Sjögren's syndrome (SS), a chronic autoimmune disease in humans. In SS, local lymphocytic infiltrations into salivary and lacrimal glands frequently develop to the involvement of systemic exocrine and nonexocrine organs, and the mechanism for progression of this disease remains obscure. Herein, we report identification of an autoantigen shared by various target organs in IQI/Jic mice. Polypeptides identified based on immunorecognition by autoantibodies in sera from IQI/Jic mice affected with autoimmune disease (>12 weeks of age) were tissue kallikrein (Klk)-1 and -13 and were cross-reactive to the autoantibodies. Interestingly, Klk-13, but not Klk-1, caused a proliferative response of splenic T cells from IQI/Jic mice from the age of 4 weeks onward. In addition, remarkably enhanced expression of Klk-13 was observed in the salivary glands of the mice in accordance with the development of inflammatory lesions. These results indicate that Klk-13 acts as an autoantigen and may increase T cells responsive to organs commonly expressing Klk-13, playing a pivotal role in the etiology of progression of disease in IQI/Jic mice. Our findings provide insights into the contributions of autoantigens shared by multiple organs in the progress of SS from an organ-specific to a systemic disorder. Sjögren's syndrome (SS) 1The abbreviations used are: SS, Sjögren's syndrome; Klk, kallikrein; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride. 1The abbreviations used are: SS, Sjögren's syndrome; Klk, kallikrein; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride. is a chronic autoimmune disease characterized by lymphocytic infiltration and tissue damage to the salivary and lacrimal glands, which results in dryness of the mouth (xerostomia) and eyes (keratoconjunctivitis sicca) (1Talal N. Arthritis and Allied Conditions. 12th Ed. Lea & Febiger, Philadelphia, PA1993: 1343-1356Google Scholar, 2Fox R.I. Stern M. Michelson P. Curr. Opin. Rheumatol. 2000; 12: 391-398Crossref PubMed Scopus (230) Google Scholar). Patients with SS often show a wide spectrum of autoimmune-related disorders, including pancreatitis, sclerosing cholangitis, interstitial nephritis, and interstitial pneumonitis. In these target organs, different epithelial cells in exocrine and nonexocrine tissues are primarily affected, and SS has been generally referred to as "autoimmune exocrinopathy," "dry gland syndrome," or, more recently as "autoimmune epithelitis" (3Strand V. Talal N. Bull. Rheum. Dis. 1979; 30: 1046-1052PubMed Google Scholar, 4Epstein O. Chapman R.W. Lake-Bakaar G. Foo A.Y. Rosalki S.B. Sherlock S. Gastroenterology. 1982; 83: 1177-1182Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 5Moutsopoulos H.M. Clin. Immunol. Immunopathol. 1994; 72: 162-165Crossref PubMed Scopus (372) Google Scholar). These clinical observations have led to the assumption that autoimmune reactions against antigens commonly expressed in epithelial cells play a pathogenic role in this disease (6Nishimori I. Bratanova T. Toshkov I. Caffrey T. Mogaki M. Shibata Y. Hollingsworth M.A. J. Immunol. 1995; 154: 4865-4873PubMed Google Scholar). Characterization of novel autoantigens associated with the systemic involvement in SS would provide useful information to better understand its pathogenesis and to develop new diagnostic and therapeutic strategies. Previous efforts have detected circulating antibodies and/or T cells reactive with various autoantigens in SS, including several nuclear complexes (SS-A/Ro, SS-B/La, SS-56, etc.) (7Billaut-Mulot O. Cocude C. Kolesnitchenko V. Truong M.J. Chan E.K. de la Hachula E. Tribonniere X. Capron A. Bahr G.M. J. Clin. Invest. 2001; 108: 861-869Crossref PubMed Scopus (35) Google Scholar), the type 3 muscarinic acetylcholine receptor (8Waterman S.A. Gordon T.P. Rischmueller M. Arthritis Rheum. 2000; 43: 1647-1654Crossref PubMed Scopus (212) Google Scholar), a cleavage fragment of α-fodrin produced during the apoptosis of salivary epithelial cells (9Haneji N. Nakamura T. Takio K. Yanagi K. Higashiyama H. Saito I. Noji S. Sugino H. Hayashi Y. Science. 1997; 276: 604-607Crossref PubMed Scopus (387) Google Scholar, 10Saegusa K. Ishimaru N. Yanagi K. Mishima K. Arakaki R. Suda T. Saito I. Hayashi Y. J. Immunol. 2002; 169: 1050-1057Crossref PubMed Scopus (55) Google Scholar), and islet cell autoantigen 69 (11Winer S. Astsaturov I. Cheung R. Tsui H. Song A. Gaedigk R. Winer D. Sampson A. McKerlie C. Bookman A. Dosch H.M. Lancet. 2002; 360: 1063-1069Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, the molecular nature of autoantigens causing the autoimmunity against the different epithelial cells in multiple organs in SS has remained obscure. Several animal models have been used to investigate the pathogenesis of SS. They include autoimmune-prone mice that develop SS-like pathology associated with other autoimmune conditions such as systemic lupus erythematosus, rheumatic arthritis, and insulitis (12Kessler H.S. Am. J. Pathol. 1968; 52: 671-685PubMed Google Scholar, 13Hoffman R.W. Alspaugh M.A. Waggie K.S. Durham J.B. Walker S. Arthritis Rheum. 1984; 27: 157-165Crossref PubMed Scopus (135) Google Scholar, 14Carlsten H. Tarkowski A. Jonsson R. Nilsson L.A. Scand. J. Immunol. 1990; 32: 21-28Crossref PubMed Scopus (18) Google Scholar, 15Miyagawa J. Hanafusa T. Miyazaki A. Yamada K. Fujino-Kurihara H. Nakajima H. Kono N. Nonaka K. Tochino Y. Tarui S. Virchows Arch B Cell Pathol. Incl. Mol. Pathol. 1986; 51: 215-225Crossref PubMed Scopus (59) Google Scholar) and other rodent strains requiring experimental manipulations such as antigen sensitization and neonatal thymectomy to develop inflammatory lesions (6Nishimori I. Bratanova T. Toshkov I. Caffrey T. Mogaki M. Shibata Y. Hollingsworth M.A. J. Immunol. 1995; 154: 4865-4873PubMed Google Scholar, 16Uchida K. Okazaki K. Nishi T. Uose S. Nakase H. Ohana M. Matsushima Y. Omori K. Chiba T. Lab. Invest. 2002; 82: 411-424Crossref PubMed Scopus (106) Google Scholar, 17Haneji N. Hamano H. Yanagi K. Hayashi Y. J. Immunol. 1994; 153: 2769-2777PubMed Google Scholar). Of these, IQI/Jic mice are the most recently identified (18Saegusa J. Kubota H. J. Vet. Med. Sci. 1997; 59: 897-903Crossref PubMed Scopus (40) Google Scholar, 19van Blokland S.C. Versnel M.A. Clin. Immunol. 2002; 103: 111-124Crossref PubMed Scopus (89) Google Scholar). They spontaneously develop autoimmune infiltration of lymphocytes in the salivary gland at the age of 8 weeks, thereafter progressing to systemic disorder with the involvement of the lacrimal gland, pancreas, kidney, and lung (20Konno A. Takada K. Saegusa J. Takiguchi M. Autoimmunity. 2003; 36: 247-254Crossref PubMed Scopus (23) Google Scholar, 21Takada K. Takiguchi M. Konno A. Inaba M. Rheumatology. 2004; 43: 858-862Crossref PubMed Scopus (27) Google Scholar). Based on this characteristic unique to IQI/Jic mice, we previously suggested them to be favorable for studies on the pathogenesis of autoimmune epithelitis in SS and hypothesized that there are autoimmune reactions against antigens shared by target organs in IQI/Jic mice. Although the inflammatory lesions contain a large number of B cells suggestive of a progressive humoral immune response, serum autoantibodies against salivary tissues, one of the prominent pathophysiological features in patients with SS and animal models (7Billaut-Mulot O. Cocude C. Kolesnitchenko V. Truong M.J. Chan E.K. de la Hachula E. Tribonniere X. Capron A. Bahr G.M. J. Clin. Invest. 2001; 108: 861-869Crossref PubMed Scopus (35) Google Scholar, 22MacSween R.N. Goudie R.B. Anderson J.R. Armstrong E. Murray M.A. Mason D.K. Jasani M.K. Boyle J.A. Buchanan W.W. Williamson J. Ann. Rheum. Dis. 1967; 26: 402-411Crossref PubMed Scopus (57) Google Scholar, 23Feltkamp T.E. van Rossum A.L. Clin. Exp. Immunol. 1968; 3: 1-16PubMed Google Scholar, 24Atkinson J.C. Royce L.S. Wellner R. Pillemer S.R. Bermudez D. Fox P.C. J. Oral Pathol. Med. 1995; 24: 206-212Crossref PubMed Scopus (15) Google Scholar, 25Sakamoto M. Miyazawa M. Mori S. Fujisawa R. J. Oral Pathol. Med. 1999; 28: 20-25Crossref PubMed Scopus (9) Google Scholar), have not been detected by immunohistochemistry in IQI/Jic mice (18Saegusa J. Kubota H. J. Vet. Med. Sci. 1997; 59: 897-903Crossref PubMed Scopus (40) Google Scholar). In this study, we first demonstrated the presence of IgG autoantibodies characteristic to IQI/Jic mice by immunoblotting and identified the targets of these autoantibodies as kallikrein (Klk)-1 and -13, which are known to be localized mainly in glandular tissues (26Olsson A.Y. Lundwall A. Biochem. Biophys. Res. Commun. 2002; 299: 305-311Crossref PubMed Scopus (58) Google Scholar). Then we examined expression of and T cell proliferative responses to Klk-1 and -13 to investigate roles of these molecules in autoimmune pathogenesis in IQI/Jic mice. Animals—IQI/Jic mice were bred from breeding pairs purchased from Crea Japan (Tokyo, Japan) and maintained in our own animal facility. Female IQI/Jic mice of various ages were utilized for this study. BALB/c and C57BL/6 mice purchased from Japan SLC (Shizuoka, Japan) were used as sex- and age-matched controls. The animals were maintained under specific pathogen-free conditions. All of the experimental procedures met with the approval of Laboratory Animal Experimentation Committee, Graduate School of Veterinary Medicine, Hokkaido University. Immunoblotting—Various tissues from 16-week-old IQI/Jic mice were homogenized with a Polytron mixer (Kinematica, Lucerne, Switzerland) in 20 mm Tris-HCl (pH 7.2), 100 mm NaCl, and 1% Triton X-100. After centrifugation at 105,000 × g for 30 min, the proteins in the supernatant were treated with 3% β-mercaptoethanol (Wako, Osaka, Japan) at 95 °C for 3 min, separated by SDS-PAGE, and transferred onto polyvinylidene difluoride filters (Millipore, Bedford, MA). The filters were incubated with sera (diluted 1:100) from different strains, followed by the reaction with a horseradish peroxidase-conjugated anti-mouse IgG antibody (Bio-Rad) and visualization with the ECL system (Amersham Biosciences). Salivary extracts of IQI/Jic mice of various ages and BALB/c and C57BL/6 mice were also prepared and subjected to immunoblot analysis with sera from IQI/Jic mice. Purification and Identification of the Salivary Antigens—Detergent-free extracts of the salivary glands from IQI/Jic mice at the ages of 16–32 weeks were separately prepared in 20 mm phosphate-NaOH (pH 5.0) and 100 mm Tris-HCl (pH 9.0), for the purification of 18- and 16-kDa antigens, respectively. Extracted proteins in phosphate-NaOH buffer were separated on a Q Sepharose FF column (Amersham Biosciences) with a linear gradient of 0–0.5 m NaCl in the same buffer. The extract in Tris-HCl buffer was similarly fractionated using an S Sepharose FF (Amersham Biosciences) column and then a Q Sepharose FF column. Antigen-containing fractions from each sample were identified by immunoblotting with sera of IQI/Jic mice and were separately fractionated on a Superdex 75HR column (Amersham Biosciences). Antigen-containing fractions were subjected to further purifications by reversed phase high performance liquid chromatography using an RPC resource column (Amersham Biosciences). Proteins in the positive fractions indicated by arrows in Fig. 1B were separated on SDS gels and blotted onto polyvinylidene difluoride filters. After staining with Coomassie Brilliant Blue, bands corresponding to the antigens were excised and analyzed for their N-terminal sequences with the Procise 492 protein sequencing system (Applied Biosystems, Warrington, UK). Reverse Transcription-PCR—Total RNA was extracted from various organs of 16-week-old IQI/Jic mice with an RNA extraction reagent (Invitrogen). The cDNA synthesis was performed using a reverse transcription kit (Invitrogen), according to the manufacturer's directions. The cDNA was amplified by PCR with the following primers: 5′-CCGCTTCACCAAATATCAATGTG-3′ and 5′-GCTCATCTGGGTATTCATATTTGACG-3′ for Klk-1 (GenBank™ accession number NM_010639); 5′-GTACTACCAAAAGGAACACATT-3′ and 5′-CAGGAGGTATTGTTTGAAGCA-3′ for Klk-13 (GenBank™ accession number NM_010115); and 5′-GAAGGTCGGTGTGAACGGATT-3′ and 5′-GAAGACACCAGTAGACTCCACGACATA-3′ for glyceraldehyde-3-phosphate dehydrogenase (GenBank™ accession number BC_083065). PCR was conducted with 35, 40, and 30 cycles for the amplification of Klk-1, Klk-13, and glyceraldehyde-3-phosphate dehydrogenase, respectively, and each cycle included denaturation at 94 °C for 40 s, annealing at 66 °C for 20 s, and extension at 72 °C for 30 s. The PCR products were subjected to electrophoresis on 2.0% agarose gels. All of the primers generated only one amplification band (see Fig. 2B). The specific amplification of each Klk transcript was confirmed by subcloning of the PCR products into pCRII plasmid (Invitrogen) and sequencing on an 8800 CEQ DNA sequencer (Beckman Coulter, Fullerton, CA). Klk-1 and -13 mRNA levels were quantified by real time PCR on a GeneAmp 5700 sequence detection system (Applied Biosystems) using SYBR green (Applied Biosystems) for detection of the amplified products. The reactions were conducted following the conditions for the conventional PCR described above and were normalized with the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA. Analysis of Autoantibody Cross-reactivity—Autoantibodies bound to purified Klk-1 and -13 on polyvinylidene difluoride filters were extracted in 0.1 m glycine (pH 2.8) and neutralized with 2.0 m Tris-HCl (pH 8.0). Reactivity of purified autoantibodies were then separately examined against Klk-1 and -13 by immunoblotting. Generation of Klk-1 Recombinant Proteins—The open reading frame encoding the full-length each subunit of heterodimeric Klk-1 was amplified by PCR using kidney cDNA of IQI/Jic mice as a template with the following primers: heavy chain, 5′-GGATCCATTGTTGGAGGATTTAACTGTGAG-3′ and 5′-GAATTCTCATTTGACGGGTGTAATGCTGC-3′; light chain, 5′-GGATCCTATGAATACCCAGATGAGCTCC-3′ and 5′-GAATTCTCAGTCATTTTCAGCCATAGTTTC-3′. The PCR products were digested with BamHI and EcoRI and ligated into BamHI/EcoRI sites of pGEX-6P-1 (Amersham Biosciences). Expression of recombinant proteins fused with glutathione S-transferase was induced in Escherichia coli BL21 by incubation with 0.1 mm isopropyl β-d-thiogalactoside (Wako). Inclusion bodies were extracted in a bacterial protein extraction reagent (Pierce) and solubilized in 6.0 m guanidine HCl (pH 8.5). The products were purified by gel permeation chromatography on a Superdex 75HR column equilibrated with 6.0 m guanidine HCl (pH 8.5). Proliferation Assay—Splenocytes (1 × 106 cells/well) were cultured in 96-well flat-bottomed plates (Corning, New York, NY) in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum, penicillin/streptomycin in the presence or the absence of Klks (0.05–50 μg/ml) or bovine serum albumin (Sigma) (10 μg/ml). During the last 20 h of the 72-hour culture period, the cells were pulsed with [3H]thymidine (1 μCi/well; ICN Biomedicals, Irvine, CA), and the incorporation was counted by liquid scintillation. To normalize pooled results, the data were presented as cpm of cultures with antigens divided by cpm of cultures without antigens (stimulation index). Serine Protease Inhibitor—To block proteolytic activity of tissue Klks, AEBSF (Sigma) was used according to a previous report (27Murakami K. Ikigai H. Nagumo T. Tomita M. Shimamura T. FEBS Lett. 1989; 257: 400-402Crossref PubMed Scopus (6) Google Scholar). Purified Klks from the salivary extract at 37 nm were incubated with 3.7 mm AEBSF at 37 °C for 60 min. Klks treated with AEBSF were separated from free AEBSF by gel permeation chromatography on a Superdex 75HR column equilibrated with phosphate-buffered saline (pH 7.4) and then tested for the proliferative response of splenocytes. Statistical Analysis—The data are presented as the means ± S.E. Statistical significance was assessed by Student's t test. A value of p < 0.05 was considered statistically significant. Anti-salivary Autoantibodies in IQI/Jic Mice—To examine the presence of circulating autoantibodies in IQI/Jic mice, we first performed immunoblot analysis of various tissue homogenates from IQI/Jic mice with sera from IQI/Jic mice and nonautoimmune control mice, including BALB/c and C57BL/6. Sera from all strains generated similar patterns of signals around 25 and 50 kDa in size that were bound in extracts of almost all organs. These signals appeared to be derived from direct binding of the second antibody (anti-mouse IgG) with light and heavy chains of IgG in tissue extracts, because they were recognized even in immunoblotting without serum (Fig. 1A). Notably, sera from IQI/Jic mice reacted with 18- and 16-kDa polypeptides in salivary gland extracts with high intensities, whereas sera from age-matched control mice did not show such signals in immunoblotting (Fig. 1A), indicating that IQI/Jic mice had autoantibodies to these polypeptides relatively characteristic of the salivary gland, a primary target organ in their autoimmune disease. These autoantibodies appeared in sera of IQI/Jic mice from 12 weeks of age onward; autoantibodies were detected in three of six mice examined at the age of 12 weeks and in all mice at the ages of 16, 32, and 48 weeks (six of six for each age group). In addition, signal intensities in immunoblotting increased as the mice aged when tissue extracts from 16-week-old IQI/Jic mice were used for antigens (data not shown). Although sera from several IQI/Jic mice showed faint bands of some other polypeptides in nontarget organs, including the brain, liver, and/or stomach (Fig. 1A), the pathologic significance of these signals was obscure, and they were excluded from further examination in this study. Identification of the 18- and 16-kDa Salivary Antigens—To identify the autoantigens, the 18- and 16-kDa polypeptides in the salivary glands of IQI/Jic mice were purified by a series of chromatographies based on recognition with autoantibodies in immunoblotting. After a sequential procedure employing anion and cation exchange chromatographies followed by gel permeation, the 18- and 16-kDa polypeptides were separated to homogeneity on a reversed phase chromatography column (Fig. 1, B and C). Sequencing analysis gave a single N-terminal amino acid sequence for each 18- and 16-kDa polypeptide purified. The N-terminal sequence of the 18-kDa polypeptide consisting of 18 amino acid residues was identical to that of the heavy chain of murine kallikrein (Klk)-1/glandular Klk-6 (28Hosoi K. Tsunasawa S. Kurihara K. Aoyama H. Ueha T. Murai T. Sakiyama F. J. Biochem. (Tokyo). 1994; 115: 137-143Crossref PubMed Scopus (37) Google Scholar) except for two unidentified residues, the seventh and fourteenth residues, as shown in Fig. 1D. Likewise, the 16-kDa polypeptide was identified as Klk-13/glandular Klk-13 (28Hosoi K. Tsunasawa S. Kurihara K. Aoyama H. Ueha T. Murai T. Sakiyama F. J. Biochem. (Tokyo). 1994; 115: 137-143Crossref PubMed Scopus (37) Google Scholar) heavy chain (Fig. 1D). Here the first 20 amino acid residues were identical to those of Klk-13, again except for two unidentified residues. These Klks are known to be heterodimeric molecules consisting of heavy and light chains, and the primary structures of heavy chains are highly homologous (28Hosoi K. Tsunasawa S. Kurihara K. Aoyama H. Ueha T. Murai T. Sakiyama F. J. Biochem. (Tokyo). 1994; 115: 137-143Crossref PubMed Scopus (37) Google Scholar, 29Kikkawa Y. Yamanaka N. Tada J. Kanamori N. Tsumura K. Hosoi K. Biochim. Biophys. Acta. 1998; 1382: 55-64Crossref PubMed Scopus (30) Google Scholar) as represented by the N-terminal sequences shown in Fig. 1D. Expression Profiling of Klk-1 and -13—Immunoblotting analysis also showed that the 18- and 16-kDa antigens in the salivary glands of IQI/Jic mice became abundant as the mice aged, that is, these polypeptides were clearly observed in salivary extracts from mice older than 16 weeks but not in those from young animals at the age of 4 weeks (Fig. 2A). These observations coincided with our previous findings that inflammatory infiltrations in the salivary glands were apparent in old mice (>16 weeks) but not in young mice (21Takada K. Takiguchi M. Konno A. Inaba M. Rheumatology. 2004; 43: 858-862Crossref PubMed Scopus (27) Google Scholar), suggesting that the production of autoantibodies and the apparent increase of the antigens were associated with the development of the disease in IQI/Jic mice. The absence or the very low intensities of 18- and 16-kDa polypeptides in tissues from BALB/c and C57BL/6 mice (Fig. 2A) raised the possibility that the contents of these antigens were extremely low or that antigenicities of these polypeptides in control mice were different from those in IQI/Jic mice. Moreover, if Klk-1 and/or Klk-13 were associated with the disease development as autoantigens, their expression in various tissues could be expected, because IQI/Jic mice spontaneously develop inflammatory lesions in the lacrimal gland, lung, pancreas, and kidney in addition to the salivary gland (21Takada K. Takiguchi M. Konno A. Inaba M. Rheumatology. 2004; 43: 858-862Crossref PubMed Scopus (27) Google Scholar), although no bands were visualized on immunoblots of extracts from these tissues (Fig. 1A). Therefore, we then examined the expression of Klk-1 and -13 in these target organs from IQI/Jic mice by reverse transcription-PCR and found that the transcripts of these Klks were present in all of these tissues (Fig. 2B). Furthermore, quantitative PCR analyses demonstrated the abundance of Klk-1 and -13 transcripts in the salivary gland at levels clearly distinguishable from those in other organs (Fig. 2C). In addition, cloning and sequencing of Klk-1 and -13 cDNAs of various organs from IQI/Jic mice demonstrated that the deduced amino acid sequences of these Klks were identical to those reported for ICR (28Hosoi K. Tsunasawa S. Kurihara K. Aoyama H. Ueha T. Murai T. Sakiyama F. J. Biochem. (Tokyo). 1994; 115: 137-143Crossref PubMed Scopus (37) Google Scholar) and BALB/c (30Evans B.A. Drinkwater C.C. Richards R.I. J. Biol. Chem. 1987; 262: 8027-8034Abstract Full Text PDF PubMed Google Scholar) mice, 2K. Takada, M. Takiguchi, and M. Inaba, unpublished data. negating the possibility of structural differences for Klks among strains as the cause of the absence or low intensities of 18- and 16-kDa bands in tissues from control mice on the immunoblot (Fig. 2A). Cross-reactivity of Serum Autoantibodies with Reduced Klks—SDS-PAGE under reduced and nonreduced conditions showed that purified Klk-1 and -13 were heterodimeric molecules in which heavy and light chains were linked via disulfide bonds (Fig. 3A). Immunoblot analysis revealed that autoantibodies found in IQI/Jic mice were clearly reactive with the 18-kDa (Klk-1) and 16-kDa (Klk-13) heavy chains of reduced forms but not with native Klks or the light chains of the reduced forms (Fig. 3A). This was verified by the observation that the recombinant glutathione S-transferase-fused protein of Klk-1 heavy chain specifically reacted with sera from IQI/Jic mice, whereas the light chain recombinant protein showed no positive reactions (Fig. 3B). Because heavy chains of murine Klk-1 and -13 are known to be highly homologous in their primary structures (28Hosoi K. Tsunasawa S. Kurihara K. Aoyama H. Ueha T. Murai T. Sakiyama F. J. Biochem. (Tokyo). 1994; 115: 137-143Crossref PubMed Scopus (37) Google Scholar, 29Kikkawa Y. Yamanaka N. Tada J. Kanamori N. Tsumura K. Hosoi K. Biochim. Biophys. Acta. 1998; 1382: 55-64Crossref PubMed Scopus (30) Google Scholar), we then examined the cross-reactivity of autoantibodies in sera of IQI/Jic mice with these antigens. Immunoblot analysis showed that autoantibodies once bound to Klk-1 and -13 reacted with Klk-13 and Klk-1, respectively (Fig. 3C). Cellular Autoreactivity with Klk-13 in IQI/Jic Mice— Splenocytes from IQI/Jic mice were tested for proliferation in response to purified Klks to investigate the reactivity of T cells with these molecules. As shown in Fig. 4A, spleen cells from IQI/Jic mice of various ages showed significant proliferative responses to native Klk-1 and -13 but not to the control antigen, bovine serum albumin. The proliferative response to Klk-13 was specifically observed in IQI/Jic mice. In contrast, the response to Klk-1 was detected in normal BALB/c and C57BL/6 as well. It has been demonstrated that Klk-1 enhances the spontaneous proliferation of lymphocytes through its enzymatic activity of serine protease (27Murakami K. Ikigai H. Nagumo T. Tomita M. Shimamura T. FEBS Lett. 1989; 257: 400-402Crossref PubMed Scopus (6) Google Scholar). Treatment of antigens with a serine protease inhibitor, AEBSF, completely abolished the proliferative response to Klk-1 of splenocytes not only from control mice but also from IQI/Jic mice (Fig. 4B). However, the significant response to Klk-13 in IQI/Jic mice was evident even after the treatment with AEBSF (Fig. 4B). Fig. 4B also shows that T cell auotoreactivity with Klk-13 appeared spontaneously in IQI/Jic mice at the age of 4 weeks, much earlier than the occurrence of inflammatory lesions and production of serum autoantibodies and was maintained at a constant level afterward. Moreover, proliferation of T cells from IQI/Jic mice was elicited in response to Klk-13 in a dose-dependent manner but not in response to Klk-1 over a wide range of concentrations from 0.05 to 50 μg/ml (Fig. 4C). We previously reported that the IQI/Jic strain of mice is a unique model for SS, because it represents the autoimmune epithelitis process, progressing from salivary-specific disorders to the involvement of multiple organs with inflammatory cellular infiltrations (21Takada K. Takiguchi M. Konno A. Inaba M. Rheumatology. 2004; 43: 858-862Crossref PubMed Scopus (27) Google Scholar). In the present study, we demonstrated the presence of autoantibodies reactive with the extract of salivary glands, the primary target of autoimmunity, in the sera of IQI/Jic mice (Fig. 1A) and identified the antigens as kallikrein (Klk)-1 and -13 (Fig. 1, B–D). Autoantibodies in sera of IQI/Jic mice were cross-reactive with these Klks (Fig. 3C), and T cell proliferation occurred specifically in response to Klk-13, regardless of the treatment with a serine protease inhibitor (Fig. 4). Based on these results, we concluded that IQI/Jic mice bear T cell autoimmunity against Klk-13, and the resulting autoantibodies are reactive with Klk-1 as well, presumably because of high homology in the amino acid sequences of these molecules (28Hosoi K. Tsunasawa S. Kurihara K. Aoyama H. Ueha T. Murai T. Sakiyama F. J. Biochem. (Tokyo). 1994; 115: 137-143Crossref PubMed Scopus (37) Google Scholar, 29Kikkawa Y. Yamanaka N. Tada J. Kanamori N. Tsumura K. Hosoi K. Biochim. Biophys. Acta. 1998; 1382: 55-64Crossref PubMed Scopus (30) Google Scholar). Transcripts of Klk-13 were detected commonly in various target organs (Fig. 2B), and their expression levels were significantly higher in the salivary gland than in other organs in IQI/Jic mice (Fig. 2C). Klk-13, formerly designated prorenin converting enzyme B (31Kim W.S. Nakayama K. Nakagawa T. Kawamura Y. Haraguchi K. Murakami K. J. Biol. 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Accordingly, autoimmune reaction against Klk-13 could play a critical role in disease progression from periductal infiltrations within salivary glands to systemic autoimmune conditions targeting multiple organs in IQI/Jic mice. To ensure the correlation of autoimmunity against Klk-13 with the development or spread of the disease in IQI/Jic mice, future studies should include in vivo analyses of the adoptive transfer of Klk-13-reactive T cells (35Zekzer D. Wong F.S. Ayalon O. Millet I. Altieri M. Shintani S. Solimena M. Sherwin R.S. J. Clin. Invest. 1998; 101: 68-73Crossref PubMed Scopus (148) Google Scholar, 36Winer S. Tsui H. Lau A. Song A. Li X. Cheung R.K. Sampson A. Afifiyan F. Elford A. Jackowski G. Becker D.J. Santamaria P. Ohashi P. Dosch H.M. Nat. Med. 2003; 9: 198-205Crossref PubMed Scopus (178) Google Scholar) and the induction of Klk-13-specific tolerance to IQI/Jic mice by neonatal (9Haneji N. Nakamura T. Takio K. Yanagi K. Higashiyama H. Saito I. Noji S. Sugino H. 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Additionally, accumulating evidence indicates that the kallikrein cascade plays a significant role in the initiation and maintenance of inflammatory responses (38Proud D. Kaplan A.P. Annu. Rev. Immunol. 1988; 6: 49-83Crossref PubMed Scopus (380) Google Scholar, 39Bhoola K. Ramsaroop R. Plendl J. Cassim B. Dlamini Z. Naicker S. Biol. Chem. 2001; 382: 77-89Crossref PubMed Scopus (79) Google Scholar). In this study, significant increases of Klk-1 and -13, both tissue Klks, were observed in salivary glands from IQI/Jic mice at the age of 16 weeks, subsequently being at an equivalent level afterward up to 48 weeks of age (Fig. 2A). We previously reported that, in IQI/Jic mice, minute sialoadenitis could be detected at the age of 8 weeks, and focal infiltration of mononuclear cells became apparent after 16 weeks of age, progressing to severe parenchymal destruction as they aged (20Konno A. Takada K. Saegusa J. Takiguchi M. Autoimmunity. 2003; 36: 247-254Crossref PubMed Scopus (23) Google Scholar, 21Takada K. Takiguchi M. Konno A. Inaba M. Rheumatology. 2004; 43: 858-862Crossref PubMed Scopus (27) Google Scholar). These observations indicate that up-regulation of Klks in salivary glands involves the early development of sialoadenitis in IQI/Jic mice. In humans, saliva from patients with SS has been reported to contain significantly increased levels of Klks considered to be derived from affected duct epithelial cells (40Friberg B. Jonsson R. Linde A. Clin. Exp. Rheumatol. 1988; 6: 135-138PubMed Google Scholar, 41Hernandez C.C. Donadi E.A. Reis M.L. J. Rheumatol. 1998; 25: 2381-2384PubMed Google Scholar). However, similar to our findings in IQI/Jic mice, no correlation has been found between the Klk levels in saliva and the severity of disease, including the amount of saliva production and the histological degree of inflammatory lesions in the affected tissues in these patients (40Friberg B. Jonsson R. Linde A. Clin. Exp. Rheumatol. 1988; 6: 135-138PubMed Google Scholar). Collectively, increased Klks could result from the early inflammation in salivary glands to form a basis for the progression or persistence of sialoadenitis in IQI/Jic mice. Furthermore, release of an increased amount of Klk-13 from the salivary glands infiltrated by antigen-presenting cells could abrogate the tolerance to this molecule and accelerate the immune reaction, acting as an autoantigen. Detailed immunoblot analysis revealed that the anti-Klk antibody in the sera of IQI/Jic mice binds to the autoantigens under the reduced condition but not to their native forms (Fig. 3A). This fully explains the negative results of a previous report (18Saegusa J. Kubota H. J. Vet. Med. Sci. 1997; 59: 897-903Crossref PubMed Scopus (40) Google Scholar) and our preliminary studies 3K. Takada, M. Takiguchi, and M. 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Sci. 1998; 842: 221-228Crossref PubMed Scopus (12) Google Scholar), glandular secretions are impaired by autoantibodies directed against autonomic nervous system receptors, including muscarinic receptors, on the cell surface of lacrimal and salivary glands. However, the autoantibodies in IQI/Jic mice are unlikely to cause the alteration of enzymatic activity of Klks to affect local inflammatory responses because of the lack of association between the autoantibody and native Klks. In conclusion, we identified Klk-13 as an autoantigen in the IQI/Jic mouse model for SS. Overexpression of Klk-13 in the early phase of sialoadenitis is believed to increase autoreactive T cells capable of generating an autoimmune response against multiple organs commonly expressing Klk-13. Our results provide insights into the role of autoantigens in the disease progress from organ-specific to systemic disorder in SS. We thank Drs. Masayuki Saito and Kazuhiro Kimura (Hokkaido University) for contributions to this study.