Interactions between the mannose receptor and thyroid autoantigens.

Thyroid autoantigens require internalization and processing by antigen-presenting cells to induce immune responses. Besides pinocytosis, antigen uptake can be receptor-mediated. The mannose receptor (ManR) has a cysteine rich domain (CR) and eight carbohydrate recognition domains (CRD) that bind glycosylated proteins. The TSH receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) are glycoproteins. To investigate a role for the ManR in thyroid autoimmunity, we tested the interaction between these autoantigens and chimeric ManRs. Plasmids encoding the CR-domain linked to IgG-Fc (CR-Fc) and CDR domains 4-7 linked to IgG-Fc (CDR4-7-Fc) were expressed and purified with Protein A. Enzyme-linked immunosorbent assay (ELISA) plates were coated with human thyroid autoantigens and CR-Fc or CRD4-7-Fc binding detected with peroxidase-conjugated anti-IgG-Fc. CRD4-7-Fc binding was highest for the TSHR, followed by Tg and was minimal for TPO. CR-Fc bound to Tg but not to TSHR or TPO. The interaction between the TSHR and CRD-Fc was calcium-dependent; it was inhibited by mannose (not galactose), and required a glycosylated TSHR A-subunit. Moreover, precomplexing the TSHR A-subunit with CRD-Fc (but not CR-Fc), or adding mannose (but not galactose), decreased in vitro responses of splenocytes from TSHR-immunized mice. Our data indicate that the ManR may participate in autoimmune responses to Tg and the TSHR but not to TPO. Most important, ManR binding of heavily glycosylated TSHR A-subunits suggests a mechanism by which the minute amounts of A-subunit protein shed from the thyroid may be captured by antigen-presenting cells located in the gland or in draining lymph nodes.

Susceptibility rather than resistance to hyperthyroidism is dominant in a thyrotropin receptor adenovirus-induced animal model of Graves' disease as revealed by BALB/c-C57BL/6 hybrid mice.

We investigated why TSH receptor (TSHR) adenovirus immunization induces hyperthyroidism more commonly in BALB/c than in C57BL/6 mice. Recent modifications of the adenovirus model suggested that using adenovirus expressing the TSHR A subunit (A-subunit-Ad), rather than the full-length TSHR, and injecting fewer viral particles would increase the frequency of hyperthyroidism in C57BL/6 mice. This hypothesis was not fulfilled; 65% of BALB/c but only 5% of C57BL/6 mice developed hyperthyroidism. TSH binding inhibitory antibody titers were similar in each strain. Functional TSHR antibody measurements provided a better indication for this strain difference. Whereas thyroid-stimulating antibody activity was higher in C57BL/6 than BALB/c mice, TSH blocking antibody activity was more potent in hyperthyroid-resistant C57BL/6 mice. F(1) hybrids (BALB/c x C57BL/6) responded to A-subunit-Ad immunization with hyperthyroidism and TSHR antibody profiles similar to those of the hyperthyroid-susceptible parental BALB/c strain. In contrast, ELISA of TSHR antibodies revealed that the IgG subclass distribution in the F(1) mice resembled the disease-resistant C57BL/6 parental strain. Because the IgG subclass distribution is dependent on the T helper 1/T helper 2 cytokine balance, this paradigm can likely be excluded as an explanation for susceptibility to hyperthyroidism. In summary, our data for BALB/c, C57BL/6, and F(1) strains suggest that BALB/c mice carry a dominant gene(s) for susceptibility to induction of a thyroid-stimulating antibody/TSH blocking antibody balance that results in hyperthyroidism. Study of this genetic influence will provide useful information on potential candidate genes in human Graves' disease.

Evidence that factors other than particular thyrotropin receptor T cell epitopes contribute to the development of hyperthyroidism in murine Graves' disease.

Immunization with thyrotropin receptor (TSHR)-adenovirus is an effective approach for inducing thyroid stimulating antibodies and Graves' hyperthyroidism in BALB/c mice. In contrast, mice of the same strain vaccinated with TSHR-DNA have low or absent TSHR antibodies and their T cells recognize restricted epitopes on the TSHR. In the present study, we tested the hypothesis that immunization with TSHR-adenovirus induces a wider, or different, spectrum of TSHR T cell epitopes in BALB/c mice. Because TSHR antibody levels rose progressively from one to three TSHR-adenovirus injections, we compared T cell responses from mice immunized once or three times. Mice in the latter group were subdivided into animals that developed hyperthyroidism and those that remained euthyroid. Unexpectedly, splenocytes from mice immunized once, as well as splenocytes from hyperthyroid and euthyroid mice (three injections), all produced interferon-gamma in response to the same three synthetic peptides (amino acid residues 52-71, 67-86 and 157-176). These peptides were also the major epitopes recognized by TSHR-DNA plasmid vaccinated mice. We observed lesser responses to a wide range of additional peptides in mice injected three times with TSHR-adenovirus, but the pattern was more consistent with increased background 'noise' than with spreading from primary epitopes to dominant secondary epitopes. In conclusion, these data suggest that factors other than particular TSHR T cell epitopes (such as adenovirus-induced expression of conformationally intact TSHR protein), contribute to the generation of thyroid stimulating antibodies with consequent hyperthyroidism in TSHR-adenovirus immunized mice.