Purpose Primary Sj?gren’s syndrome (SjS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands B-cell clonal expansions and an increased risk of lymphoma. mice also have decreased numbers of bone marrow pre-B cells and decreased frequencies of kappa light chain gene deletion. These findings suggest that B6.Aec1/2 mice have a defective early B-cell tolerance checkpoint. B6.56R.Aec1/2 mice unexpectedly had lower anti-dsDNA antibody levels than B6.56R mice and less salivary gland infiltration than B6.Aec1/2 mice. Conclusions These data suggest that the early tolerance checkpoint defect in B6.Aec1/2 mice is not sufficient to promulgate disease in mice with pre-formed autoantibodies such as B6.56R. Rather B6. Aec1/2 mice may require a diverse B-cell repertoire for efficient T-B-cell collaboration and disease propagation. These findings imply that therapies aimed at reducing B-cell Pimobendan (Vetmedin) diversity or T-B interactions may be helpful in treating SjS. 111 Sigma Chemical Co.) and detected with AP-anti-Ig total. Immunoprecipitation and immunoblotting K562 (human erythroleukemia cells ATCC CCL-243) were metabolically labeled with 35S-methionine/cysteine (DuPont New England Nuclear Boston MA) for 14 hours. Whole cell extracts were made by sonicating cells in 0.5M NaCl 50 Tris pH 7.5 2 mM EDTA and 0.3% NP40. Extracts were immunoprecipitated on protein A and protein G Sepharose beads (Pharmacia LKB Biotechnology Inc. Piscataway NJ) that were pre-incubated with 4 μL Pimobendan (Vetmedin) of mouse sera as described previously (51). Immunoprecipitates were washed and size separated by SDS-PAGE (8%) and analyzed by autoradiography as described previously (52). Statistical analysis Analyses were performed with a two-tailed Mann-Whitney U test Pimobendan (Vetmedin) as indicated. Pimobendan (Vetmedin) Results It has been shown previously that B6.Aec1/2 mice produce ANA and anti-muscarinic acetylcholine type 3 autoantibodies (53). However the full spectrum of antibody abnormalities in the B6.Aec1/2 model has not been defined. Are there global shifts in the antibody repertoire (as reflected by altered serum immunoglobulin levels) and what is the range of autoantibody specificities? Are the autoantibodies multireactive and have they undergone class switching or somatic mutation? Rabbit polyclonal to CapG. Understanding the range and molecular features of the autoantibodies produced in the B6.Aec1/2 model may provide insights into the nature and timing of the B-cell tolerance breakdown in this mouse model of SjS. Altered serum immunoglobulin levels in B6.Aec1/2 mice Reasoning that patients with SjS often exhibit hypergammaglobulinemia we began by analyzing serum immunoglobulin levels in the B6.Aec1/2 mouse model of SjS. Unexpectedly B6.Aec1/2 mice exhibited serum IgA levels than B6 mice and similar IgM and IgG levels (Fig. 1a). This analysis was restricted to female mice because of the previously described association of disease manifestations and female sex in the Aec mouse model (54). Differences in IgA levels were still significant when male mice were included in the analysis (data not shown). IgG levels also differed slightly but significantly between B6 and B6.Aec1/2 strains when male and female mice were included in the analysis (data not shown). Serum IgM levels were significantly higher in female than in male mice in both the B6 and the B6.Aec1/2 strains whereas serum IgG and IgA levels did not differ significantly between male and female mice of either strain (data not shown). Because sicca symptoms in the B6.Aec1/2 model are also age-dependent we stratified the data by age. Serum levels of IgG and IgA oscillated and did not show a consistent age-related trend whereas IgM increased with increasing age in B6 (Supplementary Fig. S1a). In B6.Aec1/2 mice immunoglobulin levels did not change appreciably with age (Supplementary Fig. S1a). Fig. 1 IgM IgA and Pimobendan (Vetmedin) IgG antibody levels and anti-dsDNA antibodies Analysis of anti-dsDNA and anti-chromatin antibodies in B6 and B6.Aec1/2 mice Sera from B6.Aec1/2 mice have previously been Pimobendan (Vetmedin) analyzed for ANA reactivity but the pattern often included cytoplasmic staining (55). To clarify the autoantibody specificities in B6.Aec1/2 mice we therefore surveyed B6 and B6. Aec1/2 sera for anti-dsDNA and anti-chromatin antibodies. Although IgM levels of anti-dsDNA were not significantly different IgA and IgG anti-dsDNA antibodies were higher in B6.Aec1/2 mice than in B6 mice (Fig. 1b). The higher IgA anti-dsDNA levels.