Two NOD Idd-associated intervals contribute synergistically to the development of autoimmune exocrinopathy Sjgren's syndrome on a healthy murine background.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 5, May 2002, pp. 1390–1398 DOI 10.1002/art.10258 © 2002, American College of Rheumatology Two NOD Idd–Associated Intervals Contribute Synergistically to the Development of Autoimmune Exocrinopathy (Sjögren’s Syndrome) on a Healthy Murine Background Seunghee Cha, Hiroyuki Nagashima, Vinette B. Brown, Ammon B. Peck, and Michael G. Humphreys-Beher† Objective. The NOD mouse is genetically predisposed to the development of at least 2 autoimmune diseases, autoimmune diabetes and autoimmune exocrinopathy (AEC). More than 19 chromosomal intervals (referred to as Idd regions) that contribute to diabetes susceptibility in the NOD mouse model have been identified, but only 2 chromosomal intervals (associated with Idd3 and Idd5) have been shown to control sialadenitis. In the present study, we bred the Idd3 and Idd5 chromosomal intervals from NOD mice into nonautoimmune C57BL/6 mice to determine if these intervals recreate a Sjögren’s syndrome (SS)–like phenotype. Methods. C57BL/6.NODc3 mice carrying Idd3 and C57BL/6.NODc1t mice carrying Idd5 were crossed and intercrossed to generate a C57BL/6.NODc3.NODc1t mouse line homozygous for the Idd3 and Idd5 chromosomal intervals on an otherwise disease-resistant genetic background. C57BL/6.NODc3.NODc1t mice were evaluated for biochemical, pathophysiologic, and immunologic markers characteristic of the SS-like phenotype present in the NOD mouse. Results. C57BL/6.NODc3.NODc1t mice fully manifested the SS-like phenotype of the NOD mouse, including decreased salivary and lacrimal gland secretory flow rates, increased salivary protein content due in part to less fluid, aberrant proteolytic enzyme activity, decline in amylase activity, appearance of autoantibodies to exocrine gland proteins, and glandular lymphocytic focal infiltrates. Loss of secretory function occurred more rapidly in C57BL/6.NODc3.NODc1t mice (by 12 weeks of age) than in NOD mice (by 16 weeks of age). No signs of insulitis or autoimmune (type 1) diabetes were observed in the C57BL/6.NODc3.NODc1t mice. Conclusion. Genes located within the 2 chromosomal intervals Idd3 and Idd5 appear necessary and sufficient for manifestation of AEC. We propose that this murine model of SS-like disease be designated C57BL/6.NOD-Aec1Aec2. Identification of specific genes within the Aec1 and Aec2 genetic regions should help elucidate the mechanism(s) underlying SS-like disease. Sjögren’s syndrome (SS) (also referred to as “autoimmune exocrinopathy” [AEC]) is an autoimmune disease characterized by exocrine gland dysfunction resulting from an immunologic attack primarily against the salivary and lacrimal glands. Primary SS defines a chronic autoimmune attack restricted to the exocrine glands, while secondary SS is accompanied by other autoimmune diseases, often involving the connective tissues (1–4). The loss of protective exocrine secretions results in increased bacterial and fungal infections of the ocular and oral mucosa, tissue injury, and periodontal disease (2–4). In addition to the salivary and lacrimal glands, other exocrine tissues, including skin, lungs, the gastrointestinal tract, and the vaginal region, may be involved, with concomitant irritation due to excessive dryness of the mucosal surface (5). Supported by grants from the NIH (DE-13769 and DE13290) and by funds from the College of Dentistry, University of Florida, Gainesville. Dr. Cha’s work was supported by a graduate student fellowship from the Department of Oral Biology, University of Florida, Gainesville. Seunghee Cha, DDS, PhD, Hiroyuki Nagashima, DDS, PhD, Vinette B. Brown, BS, Ammon B. Peck, PhD: University of Florida, Gainesville. † Dr. Humphreys-Beher is deceased. Drs. Cha and Nagashima contributed equally to this work. Address correspondence and reprint requests to Ammon B. Peck, PhD, Department of Oral Biology, PO Box 100424, University of Florida, Gainesville, FL 32610. E-mail: email@example.com. Submitted for publication August 31, 2001; accepted in revised form January 11, 2002. 1390 SS IN A NEW C57BL/6 MOUSE MODEL Diagnosis of SS often involves the detection of infiltrating lymphocytic populations in the minor salivary glands, by histopathologic analyses of a labial gland biopsy specimen (2,3). Foci of lymphocytes infiltrating the lacrimal and salivary glands are predominantly CD4⫹, with smaller CD8⫹ and B cell components, and are defined as foci if there are ⬎50 lymphocytes per 4 mm2 on histologic sections. A preferential T cell receptor repertoire has been reported, along with up-regulation of numerous cytokines, including interleukin-1␤ (IL-1␤), IL-6, IL-10, tumor necrosis factor ␣, and interferon-␥ (1–3). The significance of such factors in the development of the autoimmune pathology has yet to be determined. Serologic evaluations are used to identify the presence of rheumatoid factor, hypergammaglobulinemia, and specific antinuclear antibodies (ANAs) in SS, especially antibodies to SSA/Ro and SSB/La (1–4). Additional autoantibodies which are reactive with the cellular components of the exocrine glands, including ␣-fodrin and the type 3 muscarinic acetylcholine receptor (M3R), are present (6–8). Xerophthalmia and xerostomia are assessed by specific tests for changes in exocrine gland flow rates and biochemical changes in protein composition. While the mechanisms responsible for the loss of secretory function in SS are not yet known, the disruption of normal neurosecretory processes is a critical factor contributing to the development of dry mouth and dry eye complications (9). As with other autoimmune diseases, SS both shows a genetic predisposition with familial clustering and has an unknown environmental trigger (10,11). The most revealing hereditary markers of autoimmune disease susceptibilities have been those encoded by genes of the major histocompatibility complex (MHC). However, studies of populations of various ethnicities have yielded inconsistent results, suggesting a weak class II MHC association for SS, an observation also reported for the NOD (nonobese diabetic) mouse model of SS (12–15). High levels of circulating autoantibodies further suggest that there may be immunoglobulin genes relevant to the disease itself, while family studies indicate that autosomal genes not linked to either HLA or immunoglobulin genes are involved in the underlying genetic susceptibility to AEC (11). Environmental triggers, ranging from viral infections to hormonal and/or X-linked factors, have all been advanced as potential inducers of disease. While certain disease characteristics have been elucidated in studies using human tissues, the underlying mechanisms of the disease have been difficult to assess, 1391 since patients commonly present with end-stage disease. To circumvent this, much research has turned to the study of animal models of SS, especially the NOD mouse model (12,15–19). Similar to findings in human disease, the broad range of immunologic defects in NOD mice are under polygenic control, and isolation of the genetic intervals involved in the autoimmune pathology in the NOD mouse has been an area of intense investigation that has led to the development of the “threshold” model of autoimmunity (13,14). Generation of numerous congenic partner strains of the NOD mouse has been helpful in understanding both the genetic and the immunologic components involved not only in diabetes, but also in sicca syndrome, exhibited by these mice (13,14,20). Studies using NOD-scid and NOD.Ignull mice have played a role in showing the dependence of secretory dysfunction on the presence of autoantibodies in SS (7,12,15). Functional change in the differentiated exocrine glands appears to be dependent on genetically determined nonimmunity factors that lead to tissue targeting and immune activation against the exocrine tissue (21– 23). The exocrine gland changes include morphologic abnormalities (e.g., delayed organogenesis), aberrant tissue-specific gene expression of various proteins (e.g., parotid secretory protein expression in adult submandibular gland tissues), increased apoptotic protease activity, and novel matrix metalloproteinase (MMP) (e.g., MMP-2 and MMP-9) activity (21–24). In the present study, we showed that these pathophysiologic abnormalities, together with the appearance of lymphocytic infiltrates within the exocrine glands, are under the additive and hierarchic control of genes associated with a 43-cM region of chromosome 3 (Idd3) and a 47-cM region of chromosome 1 (Idd5) (18). We propose the designation of Aec1 for the genetic interval corresponding to Idd3 and Aec2 for the genetic interval corresponding to Idd5. MATERIALS AND METHODS Mice. C57BL/6J, NOD/Lt, NOD.B6-Idd3.B10-Idd5, C57BL/6.NODc3 (Idd3, Idd10, Idd17), and C57BL/6.NODc1t (Idd5) mice were bred and maintained at the animal facility of the Department of Pathology, University of Florida. C57BL/ 6.NODc3 and C57BL/6.NODc1t mice were crossed and intercrossed, and then appropriate breeding pairs were selected for sibling matings to produce C57BL/6.NOD-Idd3Idd5 congenic mice. C57BL/6.NOD-Idd3Idd5 mice (to be designated C57BL/ 6.NOD-Aec1Aec2) were generally healthy, showed no aberrant weight gains or losses, bred as expected of C57BL/6J parental mice, and showed no signs of autoimmune diabetes. Both male and female mice of each strain were analyzed at 12 weeks and 16 weeks of age for pathophysiologic and immuno- 1392 logic markers of autoimmunity. All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Histologic evaluation. Freshly excised lacrimal and salivary glands from control and experimental mice were placed into 10% neutral buffered formalin (pH 7.2) (no. HT50-1; Sigma, St. Louis, MO). The fixed tissue was embedded in paraffin, sectioned, stained with hematoxylin and eosin, and viewed by light microscopy. Lymphocyte infiltration was quantified by determining the number of lymphocytic foci present in sections of exocrine gland tissue. Lymphocyte aggregations containing ⬎50 cells were considered foci, and the number of foci in areas of 4 mm2 (referred to as focus score) was determined. Focus scores were expressed as the mean of the values obtained in 4 female mice per strain (18). Analyses of salivary protein. Whole saliva samples were collected for exactly 10 minutes from individual mice following intraperitoneal injection of isoproterenol and pilocarpine dissolved in saline, as detailed elsewhere (21). Salivary protein concentrations were determined using the Bradford assay kit (no. 500-0002; Bio-Rad, Emeryville, CA). Specific amylase activity in each saliva sample was measured by starch hydrolysis using the ␣-Amylase kit (no. 577; Sigma). Protease assays. Gelatinase activity in submandibular and lacrimal glands was determined using the MMP assay kit (no. ECM700; Chemicon, Temecula, CA). Lysates of exocrine glands were prepared as described elsewhere (22). A unit of activity was defined as 1.0 g fluorescein isothiocyanate (FITC)–conjugated type IV collagen substrate degraded/ minute/mg gland lysate, based on fluorescence intensity using 520 nm (emission) and 495 nm (excitation). Caspase 3 activity was determined by enzyme activity assay using the Caspase-3 Substrate 1 kit (no. 235400; Calbiochem, San Diego, CA). Organs from 4 mice were pooled for each group and assayed 4 times to ensure reproducibility. Parotid secretory protein analyses. Aberrant parotid secretory protein (PSP) expression and its proteolysis in the submandibular gland were determined by Western blot analysis of whole gland lysates as detailed elsewhere (17). Briefly, PSP between the normal 27-kd and NOD 24-kd protein isoforms was used as an indicator of proteolytic activity recognizing the NLNL cleavage site (23). Autoantibody determinations. Purified rat M3R isolated from transfected COS-7 cells was separated on a 12% sodium dodecyl sulfate–polyacrylamide gel (15 g/lane) as described elsewhere (6). A 1:500 dilution of sera from control and experimental groups of mice was used as a source of primary antibody, followed by a 1:1,000 dilution of an alkaline phosphatase–conjugated goat anti-mouse second antibody for the Western blot protocol. The reaction with receptor was visualized colorimetrically using the chromogenic reagent nitroblue tetrazolium (18). ANA determinations. ANAs in the sera of mice were detected using the ANA screening test (#1000-F; Sigma). HEp-2 substrate slides were overlaid with the appropriate mouse serum samples (diluted 1:40) and then incubated for 30 minutes. After 3 washes with phosphate buffered saline, the substrate slides were overlaid with FITC-conjugated goat anti-mouse IgG (diluted 1:250) (F5897; Sigma) for 3 hours. Nuclear fluorescence was detected by fluorescence microscopy. CHA ET AL Figure 1. Location of the Idd5 (Aec2) and Idd3 (Aec1) genetic intervals on mouse chromosomes 1 and 3, respectively, as determined by microsatellite mapping. Production of the C57BL/6.NOD-Aec1Aec2 mouse line was achieved by breeding the C57BL/6.c1t to the C57BL/ 6.c3 congenic strains, and intercrossing to obtain the F2 generation. Microsatellite mapping was used to follow the inheritance of the Idd3 and Idd5 chromosomal intervals. F2-generation mice homozygous for both intervals were inbred for additional generations. Statistical analysis. Values are presented as the mean ⫾ SEM. One-way analysis of variance, Student t-test, and paired comparison tests for significant differences in saliva and tear volume, protein concentration, amylase, and protease activities were performed with SAS computer software programs. P values less than 0.05 were considered significant. RESULTS Generation of the C57BL/6.NOD-Aec1Aec2 mouse. To determine if the chromosomal intervals defined by Idd3 and Idd5 are sufficient to permit full development of an SS-like disease, C56BL/6.NODc3 mice were mated with C56BL/6.NODc1t mice to generate F1 mice. The F1 mice were then intercrossed to breed an F2 generation, from which breeding pairs that were homozygous for both the Idd3 and Idd5 chromosomal segments were selected (Figure 1). To confirm that the resulting mouse line contained the full genetic regions and to reduce the probability that an internal crossover event had occurred, each breeding mouse was screened using the microsatellite markers D1Mit5, D1Mit15, D3Mit132, and Tshb, as well as internal probes to these markers (20). Since it is not possible to determine if the SS IN A NEW C57BL/6 MOUSE MODEL 1393 Secretory dysfunction in the C57BL/6.NODAec1Aec2 mouse. AEC-susceptible NOD/Lt mice exhibit marked loss of secretory function beginning at 16 weeks of age. As shown in Figure 2, C57BL/6.NODAec1Aec2 mice, when stimulated with an injection of isoproterenol/pilocarpine, exhibited a reduced secretory response by the salivary glands at 16 weeks of age, equal to that observed in NOD/Lt mice. In contrast, secretory responses by salivary glands of control C57BL/6J mice exhibited an increased response to stimulation with isoproterenol/pilocarpine over time, reflecting normal increase in size and weight. Interestingly, in the C57BL/ 6.NOD-Aec1Aec2 mice the temporal loss of secretory responses actually occurred sooner (beginning at 12 weeks of age) than in NOD/Lt mice (Figure 2). It should be noted that NOD/Lt mice whose Idd3 and Idd5 regions have been replaced by those chromosomal regions derived from C57BL/6J mice exhibit secretory function similar to that observed in healthy C57BL/6J mice. Thus, these findings reveal the importance of genes within these 2 chromosomal intervals for the expression of secretory function. Expression of biochemical markers associated with disease pathogenesis. NOD/Lt mice ectopically express a number of differentiation marker proteins suggestive of a loss of homeostasis in exocrine gland epithelial cells (15,17,18,21–24), similar to patients with SS. An analysis of these markers in C57BL/6.NODAec1Aec2 congenic mice revealed alterations similar to those observed in autoimmune disease–expressing NOD/Lt parental strains (Tables 1 and 2). The congenic mice had reduced saliva flow rates leading to a concomitant increase in protein concentrations, similar to that Figure 2. Temporal changes in saliva volume in mice following intraperitoneal injection of an isoproterenol/pilocarpine mixture. Whole saliva was collected by micropipette from the oral cavities of 6 mice per group. Salivary flow rates were measured at 8, 12, 16, and 20 weeks. Values are the mean and SEM. chromosomal regions in this new line are exactly those of the Idd3 and Idd5 intervals defined in the NOD mouse, we have designated this new line C57BL/6.NODAec1Aec2, where Aec1 corresponds to Idd3 and Aec2 corresponds to Idd5. Characterization of the disease phenotype was carried out on offspring after 3 additional brother-sister intercrosses. Table 1. Sjögren’s syndrome–like pathophysiology in the C57BL/6.NOD-Aec1Aec2 mouse strain compared with other strains* Whole saliva Strain, age C57BL/6J, 16 weeks C57BL/6.NOD-Aec1Aec2 12 weeks 16 weeks NOD/Lt, 16 weeks NOD.B6-Idd3.B10-Idd5, 16 weeks No. of mice Protein concentration, g/l Amylase activity, units/mg protein Submandibular gland lysate caspase 3 activity, pmoles/ minute/g protein 10 3.1 ⫾ 0.1 210 ⫾ 6.9 26.1 ⫾ 5.3 10 10 8 11 4.8 ⫾ 0.8† 5.7 ⫾ 0.3‡ 5.0 ⫾ 0.2 3.4 ⫾ 0.1§ 101 ⫾ 6.5‡ 137 ⫾ 9.9† 117 ⫾ 4.6 210 ⫾ 2.1§ ND 29.5 ⫾ 7.3 31.8 ⫾ 11.3 ND * Values are the mean ⫾ SEM from 3 experiments. ND ⫽ not determined. † P ⬍ 0.05 versus parental C57BL/6J mice, by Student’s t-test (analysis of variance). ‡ P ⬍ 0.01 versus parental C57BL/6J mice, by Student’s t-test (analysis of variance). § P ⬍ 0.05 versus parental NOD/Lt mice, by Student’s t-test (analysis of variance). 1394 CHA ET AL Table 2. Analysis of MMP-9 activity in the C57BL/6.NOD-Aec1Aec2 mouse strain compared with other strains* Gland lysate tested Strain Submandibular Lacrimal C57BL/6J C57BL/6.NOD-Aec1Aec2 NOD/Lt NOD.B6-Idd3.B10-Idd5 0.413 ⫾ 0.06 0.900 ⫾ 0.32† 0.890 ⫾ 0.03 0.495 ⫾ 0.01‡ 0.020 ⫾ 0.01 0.030 ⫾ 0.01 0.050 ⫾ 0.01 0.023 ⫾ 0.01 * Matrix metalloproteinase 9 (MMP-9) activity was determined when mice were 16 weeks old. Values are the mean ⫾ SEM from 3 experiments. † P ⬍ 0.01 versus parental C57BL/6J mice, by Student’s t-test (analysis of variance). ‡ P ⬍ 0.01 versus parental NOD/Lt mice, by Student’s t-test (analysis of variance). observed in the NOD/Lt strain expressing SS-like pathophysiology (P ⬍ 0.01 versus C57BL/6 parental strain). Amylase activity in the saliva of C57BL/6.NODAec1Aec2 mice at 12 weeks was ⬃50% of the enzyme activity in the parental healthy strain (P ⬍ 0.01) (Table 1). PSP, a salivary protein not normally synthesized by acinar cells of the submandibular gland, is aberrantly expressed in the submandibular gland of adult C57BL/ 6.NOD-Aec1Aec2 congenic mice, similar to findings in NOD/Lt mice (results not shown). The PSP that is secreted into whole saliva, primarily by acinar cells of the parotid gland, has been shown to undergo enzymatic cleavage at an NLNL site located in the N-terminus region in mice that develop AEC, but not in healthy mice (25). This difference is shown in Figure 3, comparing PSP profiles from whole saliva of 20-week-old C57BL/6J and NOD/Lt mice and 8–16-week-old C57BL/ 6.NOD-Aec1Aec2 mice. As expected, with development of AEC, PSP in whole saliva of C57BL/6.NODAec1Aec2 mice exhibited a temporal change from a 27-kd to a 24-kd isoform associated with the expression of a proteolytic enzyme. However, the fact that the alteration in PSP occurred between 8 weeks and 12 weeks of age indicates that the disease phenotype probably appears earlier in C57BL/6.NOD-Aec1Aec2 mice than in NOD/Lt mice. Markers indicating onset of AEC. Salivary and lacrimal tissues targeted in AEC in humans and in the NOD/Lt mouse demonstrate up-regulated activities of MMPs (MMP-9) (24,26,27) and caspases (17). We found that MMP-9 activity in the submandibular gland of mice from a congenic C57BL/6J background was similar to the activity detected in NOD/Lt mice (Table 2). Although elevated over the level in the parental C57BL/6J background, the level of MMP activity in the lacrimal gland of C57BL/6.NOD-Aec1Aec2 mice did not reach that observed in the NOD/Lt mice (P ⬍ 0.05). Reciprocally, the replacement of the Idd3 and Idd5 genetic intervals in the NOD/Lt background with those intervals derived from C57BL/6J mice resulted in NOD/Lt mice with MMP activity similar to that observed in C57BL/6J mice (Table 2). Caspase 3 activity in 16-week-old C57BL/6.NOD-Aec1Aec2 mice was elevated over that in C57BL/6J control mice but did not achieve the level in NOD/Lt mice. It may be possible that, as a consequence of the early onset of disease in these mice, maximum caspase 3 expression occurs at an earlier age. The autoimmune response in C57BL/6.NODAec1Aec2 mice. NOD/Lt mice develop focal lymphocytic infiltrates in the salivary and lacrimal glands, which are accompanied by increased cytokine production and production of autoantibodies (28–30). Histologic evaluation of the submandibular and lacrimal glands indicated the presence of focal immune cell infiltrates detected at 12 weeks in the submandibular gland and at 16 weeks in the lacrimal gland of these mice (Figure 4). According to the standard grading system for determining the number of foci of lymphocytes infiltrating the exocrine tissues, the Figure 3. Temporal changes in the sodium dodecyl sulfate– polyacrylamide gel electrophoresis profiles for parotid secretory protein (PSP) in the saliva of C57BL/6.NOD-Aec1Aec2 mice at 8, 12, and 16 weeks of age. Two-microgram specimens of whole saliva from normal C57BL/6J, autoimmune exocrinopathy–susceptible NOD/Lt, and C57BL/6.NOD-Aec1Aec2 mice were studied. PSP was detected by Western blotting using a polyclonal rabbit anti-mouse PSP antibody (17). Saliva samples from 5 individual mice were examined on at least 2 separate occasions. SS IN A NEW C57BL/6 MOUSE MODEL 1395 Figure 4. Photomicrographs showing focal leukocyte infiltrates (arrows) in the submandibular (SMX) and lacrimal (LAC) glands of NOD/Lt, C57BL/6.NOD-Aec1Aec2, and NOD.B6-Idd3.B10-Idd5 mice. Exocrine tissues were freshly explanted, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned (0.4). Sections were visualized by light microscopy (hematoxylin and eosin stained; original magnification ⫻ 160). C57BL/6.NOD-Aec1Aec2 congenic mice had focus scores reflective of those in NOD/Lt mice (Table 3). C57BL/6J mice do not typically have focal infiltration of exocrine tissues at either 12 weeks or 16 weeks of age (31). Flow cytometric analyses indicated that the immune cell aggregates consisted of CD4⫹,CD8⫹ T cells, and B lymphocytes. The B cells accounted for ⬃25–30% of the lymphocytes infiltrating the lacrimal glands, simTable 3. Lymphocyte infiltration in the exocrine glands of C57BL/ 6.NOD-Aec1Aec2 mice compared with other strains as measured by focus score* Focus score Strain, age C57BL/6J, 16 weeks C57BL/6.NOD-Aec1Aec2 12 weeks 16 weeks NOD/Lt, 16 weeks NOD.B6-Idd3.B10-Idd5, 16 weeks Submandibular Lacrimal 0 0 ⬎2 ⬎2 ⬎2 ⬍1 0 1 ⬍1 ⬎1 * Foci contained ⬎50 lymphocytes/4-mm2 area of tissue. Numbers of foci were determined by 2 individuals, under blinded conditions. Values are the mean ⫾ SEM. ilar to the findings previously reported for NOD/Lt mice (30). The ratio of CD4⫹:CD8⫹ T cells was lower in the Idd3Idd5 double-congenic mice compared with the NOD parental strain (4:1 versus 2:1; P ⬎ 0.05), in both salivary and lacrimal glands. Steady-state concentrations of messenger RNA for various inflammatory cytokines in the double-congenic mice were similar to those in the parental NOD mice (data not shown), with the exception of elevated levels of transforming growth factor ␤ (TGF␤) in the double-congenic mice. In contrast to our expectation, TGF␤ levels were 88% and 56% higher in the submandibular and lacrimal glands, respectively, in the C57BL/6.NOD-Aec1Aec2 mice. The presence of ANAs was determined by immunofluorescence staining of HEp-2 cells. HEp-2 cells were first incubated with sera obtained from parental C57BL/6J and NOD/Lt mice as well as C57BL/6.NODAec1Aec2 mice, and then with FITC-conjugated goat anti-mouse IgG antiserum. Distinct staining was evident within the nucleus and slight staining evident in the cytoplasm when the HEp-2 cells were incubated with either NOD/Lt or C57BL/6.NOD-Aec1Aec2 sera, but not with C57BL/6J sera (results not shown). These sera were positive at a 1:40 dilution, a concentration known 1396 CHA ET AL to enable detection of ANAs in parental NOD/Lt mice. Western blotting with purified rat M3R protein isolated from transfected COS-7 cells (6) was used to determine the presence of autoantibodies to M3R. Again, the sera from both NOD/Lt and C57BL/6.NOD-Aec1Aec2 mice reacted positively, while sera derived from the parental C57BL/6J mice failed to react. DISCUSSION Previously, the NOD Idd3 and Idd5 genetic intervals have been transferred separately into the genetic background of the disease-resistant strain C57BL/6J (18). The NOD-derived Idd5 (Aec2) interval resulted in elevated expression of biochemical markers of SS-like disease associated with the asymptomatic initiation phase in NOD/Lt mice. Idd3 (Aec1) did not appear to cause alterations in biochemical markers of salivary gland or immune system function. The transfer of either of these alleles did not produce evidence of insulitis or diabetes on a normal background. There was no impact on exocrine tissue secretory response in either congenic C57BL/6 strain. The transfer of individual resistance alleles did not prevent disease progression or onset of loss of secretory function. However, the transfer of both the C57BL/6 Idd3 and C57BL/10 Idd5 alleles to the NOD/Lt genetic background reduced the expression of autoimmunity markers and partially restored secretory response. This suggests that these 2 alleles are capable of acting in an additive and hierarchic manner to control AEC (18,19). From the results of the present studies, it would appear that the Idd3 and Idd5 susceptibility alleles in the NOD mouse are sufficient for the generation of SS-like disease on a previously autoimmune-resistant genetic background. Thus, the intervals identified originally as contributing to diabetes onset contain genetic material leading to AEC, with designations of Aec2 and Aec1 for Idd5 and Idd3, respectively. With transfer of the NOD Idd3 and Idd5 susceptibility alleles into the C57BL/6 disease-free mouse, there was a more rapid progression to onset of SS-like pathophysiology in terms of focal lymphocytic infiltration in the submandibular gland, and, more importantly, loss of saliva secretory capacity, than in NOD mice. The Idd5 interval on chromosome 1 and Idd3 interval on chromosome 3 encompass 47 cM and 43 cM of NOD genetic material, respectively (20). Sialadenitis in the NOD mouse has previously been mapped to the distal portion of chromosome 1, while diabetes segregates to a centromeric portion of chromosome 1 (20). Recently, the Idd5 interval has been subdivided into 2 smaller loci whose proximal Idd5.1 includes Casp8 as well as Cd152 (Ctla-4), and whose distal Idd5.2 contains Nramp1 and Cmkar2 as candidate genes for diabetes (32). Although murine Idd5.1 overlaps the orthologous CTLA-4/IDDM12 locus of humans and allelic variants in Ctla-4 contribute to diabetes susceptibility, development of AEC does not appear to rely on this genetic interval, since Aec2 is located distal to Idd5.1 but does encompass the Idd5.2 locus and Bcl-2 gene (32). Candidate genes in the Aec2 interval include fibronectin 1 (Fn1), insulin-like growth factor binding protein 2 and 5 (Igfbp2 and Igfbp5), type IV collagen (Col6a3), B cell translocation gene 2 (Btg2), IL-10 (Il10), and laminin 1 and 2 (Lamc1 and Lamc2). A genetic association between promoter region polymorphisms in the IL-10 gene and primary SS has been reported recently (33). The Idd3 allele in C57BL/6.NOD-Aec1Aec2 congenic mice contains additional susceptibility intervals Idd10 and Idd17 (20,32,34). Based on the results of a number of studies of autoimmune diabetes and experimental autoimmune encephalomyelitis, using a 0.35-cM Idd3 susceptibility segment from chromosome 3 (33–35), it has been proposed that the IL-2 gene is the candidate gene responsible for the influence on immune cell activation in autoimmunity in these mouse models (31,33,34,36–38). The other Idd alleles of chromosome 3 (Idd10, Idd17, Idd18) have been proposed to enhance the contribution of the Idd3 susceptibility interval, but are not necessary for its action in autoimmune disease (39,40). The rapid onset of secretory dysfunction in C57BL/6.NOD-Aec1Aec2 mice, correlating with the appearance of detectable autoantibodies to ANA and M3R, may result from epistatic interactions of additional susceptibility alleles. Alternatively, additional genetic susceptibility alleles that are too weak to produce autoimmune disease in the normally healthy genetic background of this strain may reside on the C57BL/6J genetic background. Our study using complementary DNA microarray technology has provided an interesting connection between genetically programmed nonimmune components and adult onset of AEC in the NOD mouse. Although identification of the major causative gene(s) and critical antigen(s) is still far from complete, candidate genes can be narrowed down further by the generation of probes from C57BL/6.NOD-Aec1Aec2 mice. This will allow us to discard a list of genes reflecting strain differences and to find common genes in the NOD mouse and the C57BL/6.NOD-Aec1Aec2 mouse, which are definitely associated with the manifestation of dis- SS IN A NEW C57BL/6 MOUSE MODEL ease phenotype. Having high synteny between humans and mice, mouse candidate genes identified by the microarray system can be used to screen human libraries and examine common mutations or polymorphisms in the orthologous human genes, in order to determine how the candidate genes affect the organogenesis of salivary glands and function in disease pathogenesis in the subset of humans who are susceptible. In addition, more reliable screening tools for diagnosis and intervention strategies to cure SS may become more readily available. The C57BL/6 genome most likely harbors additional autoimmune susceptibility alleles. Besides I-Ag7, NOD mice are negative for the expression of a second MHC marker, I-E (31,39). The presence of an I-Enull genotype in various mouse strains, such as C57BL/6, has been reported to lead to the spontaneous occurrence of focal infiltrates in the exocrine tissues of older mice (typically ⬎1 year of age). The role of the I-E locus in autoimmunity can now be assessed in the new genetic model for SS by backcrossing the appropriate mouse MHC alleles. Generation of the C57BL/6.NOD-Aec1Aec2 mouse presents a new opportunity to elucidate the genetic control of SS. A number of techniques, e.g., positional cloning, expression microarrays, or traditional recombinant congenic analyses for Aec1 and Aec2 intervals maintaining the traits of SS-like disease, may permit the identification of candidate genes. Identified genes can then be used to reveal the mechanism underlying the development of autoimmune disease in susceptible individuals. This may open new targets for gene or drug therapy. These markers may further serve as a diagnostic tool for early detection of primary SS, and more importantly, for screening of patients with other autoimmune connective tissue diseases for the potential development of secondary SS. ACKNOWLEDGMENTS The authors would like to thank Joy Nanni and Janet Cornelius for technical assistance throughout these studies. REFERENCES 1. Fox RI, Tornwall J, Maruyami T, Stern M. Sjögren’s syndrome: evolving concepts of diagnosis, pathogenesis, and therapy. Curr Opin Rheumatol 1998;10:446–56. 2. Fox PC, Speight PM. Current concepts of autoimmune exocrinopathy: immunologic mechanisms in the salivary pathology of Sjögren’s syndrome. Crit Rev Oral Biol Med 1996;7:144–58. 3. Fox RI, Kang HI. Pathogenesis of Sjögren’s syndrome. Rheum Dis Clin North Am 1992;18:517–38. 1397 4. Talal N. Sjögren’s syndrome: historical overview and clinical spectrum of disease. Rheum Dis Clin North Am 1992;18:507–16. 5. Moutsopoulos HM, Kordosis T. Sjögren’s syndrome revisited: autoimmune epithelitis. Br J Rheumatol 1996;35:204–6. 6. Nguyen KH-T, Brayer J, Cha S, Diggs S, Yasunari U, Hilal G, et al. Evidence for antimuscarinic acetylcholine receptor antibody–mediated secretory dysfunction in NOD mice. Arthritis Rheum 2000;43:2297–306. 7. Robinson CP, Brayer J, Yamachika S, Esch T, Peck AB, Stewart C, et al. Transfer of human serum IgG into nonobese diabetic Ignull mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren’s syndrome. Proc Natl Acad Sci U S A 1998;95:7538–43. 8. Haneji N, Nakamura T, Takio K, Yanagi K, Higashiyama H, Saito I, et al. Identification of ␣-fodrin as a candidate autoantigen in primary Sjögren’s syndrome. Science 1997;276:604–7. 9. Waterman SA, Gordon TP, Rischmueller M. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjögren’s syndrome. Arthritis Rheum 2000;43: 1647–54. 10. Arnett F, Goldstein R, Duvic M, Reveille J. Major histocompatibility complex genes in systemic lupus erythematosus, Sjögren’s syndrome, and polymyositis. Am J Med 1988;85:38–41. 11. Grossman C. Possible underlying mechanisms of sexual dimorphism in the immune response: fact and hypothesis. J Steroid Biochem 1989;34:241–51. 12. Brayer JB, Humphreys-Beher MG, Peck AB. Sjögren’s syndrome: immunological response underlying the disease. Arch Immunol Ther Exp (Warsz) 2001;49:353–60. 13. Wicker LS, Todd JA, Peterson B. Genetic control of autoimmune diabetes in the NOD mouse. Annu Rev Immunol 1995;13: 179–200. 14. Serreze DV, Leiter EH. Genetic and pathogenic basis of autoimmune diabetes in NOD mice. Curr Opin Immunol 1994;6: 900–6. 15. Robinson CP, Yamachika S, Bounous DI, Brayer J, Jonsson R, Holmdahl R, et al. A novel NOD-derived murine model of primary Sjögren’s syndrome. Arthritis Rheum 1998;41:150–6. 16. Kong L, Robinson CP, Peck AB, Vela-Roch N, Sakata KM, Dang H, et al. Inappropriate apoptosis of salivary and lacrimal gland epithelium of immunodeficient NOD-scid mice. Clin Exp Rheumatol 1998;16:675–82. 17. Robinson CP, Alford CE, Cooper C, Picardo EL, Shah N, Peck AB, et al. Elevated levels of cysteine protease activity in saliva and salivary glands of the NOD mouse model for Sjögren’s syndrome. Proc Natl Acad Sci U S A 1997;94:5767–71. 18. Brayer J, Lowry J, Cha S, Robinson CP, Yamachika S, Peck AB, et al. Alleles from chromosomes 1 and 3 of NOD mice combine to influence Sjögren’s syndrome-like autoimmune exocrinopathy. J Rheumatol 2000;27:1896–904. 19. Nishihara M, Terada M, Kamogawa J, Ohashi Y, Mori S, Nakatsuru S, et al. Genetic basis of autoimmune sialadenitis in MRL/lpr lupus-prone mice: additive and hierarchical properties of polygenic inheritance. Arthritis Rheum 1999;42:2616–23. 20. Yui MA, Muralidharan B, Moreno-Altamirano G, Chestnut K, Wakeland EK. Production of congenic mouse strains carrying NOD-derived diabetogenic genetic intervals: an approach for the genetic dissection of complex traits. Mamm Genome 1996;7: 331–4. 21. Hu Y, Nakagawa Y, Purushotham KR, Humphreys-Beher MG. Functional changes in the salivary glands of autoimmune diseaseprone NOD mice. Am J Physiol 1992;263:E607–14. 22. Cha S, van Blockland S, Versnel M, Homo-Delarche F, Nagashima H, Brayer J, et al. Abnormal organogenesis in salivary gland development may initiate adult onset of autoimmune exocrinopathy. Clin Exp Immunogenet 2001;18:143–60. 23. Robinson CP, Yamamoto H, Peck AB, Humphreys-Beher MG. 1398 24. 25. 26. 27. 28. 29. 30. 31. 32. Genetically programmed development of salivary gland abnormalities in the NOD (nonobese diabetic)-scid mouse in the absence of detectable lymphocytic infiltration: a potential trigger for sialoadenitis of NOD mice. Clin Immunol Immunopathol 1996;79:50–9. Yamachika S, Nanni JM, Nguyen KH-T, Garces L, Lowry JM, Robinson CP, et al. Excessive synthesis of matrix metalloproteinases in the exocrine tissues of the NOD mouse models for Sjögren’s syndrome. J Rheumatol 1998;25:2371–80. Robinson CP, Bounous DE, Alford CE, Peck AB, HumphreysBeher MG. Aberrant expression and potential function for parotid secretory protein in the NOD (non-obese diabetic) mouse. Adv Exp Med Biol 1999;438:925–30. Konttinen YT, Kangaspunta P, Lindy O, Takagi M, Sorsa T, Segerberg M, et al. Collagenase in Sjögren’s syndrome. Ann Rheum Dis 1994;53:836–9. Wu AJ, Lafrenie RM, Park C, Apinhasmit W, Chen ZJ, BirkedalHansen H, et al. Modulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by interferon-␥ in a human salivary gland cell line. J Cell Physiol 1997;171:117–24. Yamachika S, Brayer J, Oxford GE, Peck AB, Humphreys-Beher MG. Aberrant proteolytic digestion of biglycan and decorin by saliva and exocrine gland lysates from the NOD mouse model for autoimmune exocrinopathy. Clin Exp Rheumatol 2000;18:233–40. Skarstein K, Wahren M, Zaura E, Hattori M, Jonsson R. Characterization of the T cell receptor repertoire and anti-Ro/SSa autoantibodies in relation to sialoadenitis in NOD mice. Autoimmunity 1995;22:9–16. Robinson CP, Cornelius J, Bounous DE, Yamamoto H, Humphreys-Beher MG, Peck AB. Characterization of the changing lymphocyte populations and cytokine expression in the exocrine tissues of autoimmune NOD mice. Autoimmunity 1998;27:29–44. Li X, Golden J, Faustman D. Faulty MHC class II I-E expression is associated with autoimmunity in diverse strains of mice. Diabetes 1993;42:1166–72. Hill NJ, Lyons PA, Armitage N, Todd JA, Wicker LS, Peterson CHA ET AL 33. 34. 35. 36. 37. 38. 39. 40. LB. The NOD Idd5 locus controls insulitis and diabetes and overlaps the orthologous CTLA4/IDDM12 and NRAMP1 loci in humans. Diabetes 2000;49:1744–7. Hulkkonen J, Pertovaara M, Antonen J, Lahdenpohja N, Pasternack A, Hurme M. Genetic association between interleukin-10 promoter region polymorphisms and primary Sjögren’s syndrome. Arthritis Rheum 2001;44:176–9. Podolin PL, Denny P, Lord CJ, Hill NJ, Todd JA, Peterson LB, et al. Congenic mapping of the insulin-dependent diabetes (Idd) gene, Idd10, localizes two genes mediating the Idd10 effect and eliminates the candidate Fcrg1. J Immunol 1997;159:1835–43. Garchon H-J, Bedossa P, Eloy L, Bach J-F. Identification and mapping to chromosome 1 of a susceptibility locus for periinsulitis in non-obese diabetic mice. Nature 1991;353:260–2. Denny P, Lord CJ, Hill NJ, Goy JV, Levy ER, Podolin PL, et al. Mapping of the IDDM locus Idd3 to a 0.35-cM interval containing the interleukin-2 gene. Diabetes 1997;46:695–700. Sundvall M, Jirholt J, Yang HT, Jansson L, Engstrom A, Pettersson U, et al. Identification of murine loci associated with susceptibility to chronic experimental autoimmune encephalomyelitis. Nat Genet 1995;10:313–7. Encinas JA, Wicker LS, Peterson LB, Mukasa A, Teuscher C, Sobel R, et al. Colocalization of QTL influencing both autoimmune diabetes and autoimmune encephalomyelitis to a 0.15 cM region on mouse chromosome 3. Nat Genet 1999;21:158–60. Wicker LS, Todd JA, Prins JB, Podolin PL, Renjilian RJ, Peterson LB. Resistance alleles at two non-major histocompatibility complex-linked insulin-dependent diabetes loci on chromosome 3, Idd3 and Idd10, protect nonobese diabetic mice from diabetes. J Exp Med 1994;180:1705–13. Wicker LS, Appel MC, Dotta F, Pressey A, Miller BJ, DeLarato NH, et al. Autoimmune syndromes in the major histocompatibility complex (MHC) congenic strains of nonobese diabetic (NOD) mice: the NOD MHC is dominant for insulitis and cyclophosphamide-induced diabetes. J Exp Med 1992;176:67–77.