A Novel Murine Model of Graves’ Hyperthyroidism with Intramuscular Injection of Adenovirus Expressing the Thyrotropin Receptor

Abstract

In this work we report a novel method to efficiently induce a murine model of Graves’ hyperthyroidism. Inbred mice of different strains were immunized by i.m. injection with adenovirus expressing thyrotropin receptor (TSHR) or β-galactosidase (1 × 1011 particles/mouse, three times at 3-wk intervals) and followed up to 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c (H-2d) and 25% of female C57BL/6 (H-2b) mice developed Graves’-like hyperthyroidism with elevated serum thyroxine (T4) levels and positive anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and TSH-binding inhibiting Ig (TBII) activities. In contrast, none of female CBA/J (H-2k), DBA/1J (H-2q), or SJL/J (H-2s) mice developed Graves’ hyperthyroidism or anti-TSHR autoantibodies except SJL/J, which showed strong TBII activities. There was a significant positive correlation between TSI values and T4 levels, but the correlations between T4 and TBII and between TSI and TBII were very weak. TSI activities in sera from hyperthyroid mice measured with some chimeric TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared similar to those in patients with Graves’ disease. The thyroid glands from hyperthyroid mice displayed diffuse enlargement with hypertrophy and hypercellularity of follicular epithelia with occasional protrusion into the follicular lumen, characteristics of Graves’ hyperthyroidism. Decreased amounts of colloid were also observed. However, there was no inflammatory cell infiltration. Furthermore, extraocular muscles from hyperthyroid mice were normal. Thus, the highly efficient means that we now report to induce Graves’ hyperthyroidism in mice will be very useful for studying the pathogenesis of autoimmunity in Graves’ disease.

Prevention of Autoantibody-Mediated Graves’-Like Hyperthyroidism in Mice with IL-4, a Th2 Cytokine

“…On this basis, the normal range of T 4 was defined as 2.03-7.01 g/dl (mean Ϯ 3 SD). Disease incidence at this time point was 60% (12 of 20) in mice immunized with AdCMVTSHR alone (TSHR group), similar to our previous study (15,21), 53% (8 of 15) in those with AdCMVTSHR and AdRGDCMVIL-12 (TSHR plus IL-12), but only 13% (2 of 15) in those with AdCM-VTSHR and AdRGDCMVIL-4 (TSHR plus IL-4) (Figs. 3A and 4A).…”

“…TSAb activities in murine sera were measured with FRTL5 cells as previously described (15). The cells were seeded at 3 ϫ 10 4 cells/well in a 96-well culture plate and incubated in 50 l of hypotonic HBSS containing 0.5 mM isobutyl-methylxanthine, 20 mM HEPES, 0.25% BSA, and 5 l of serum for 2 h at 37°C.…”

“…Adenovirus was propagated in 293 human embryonal kidney cells and purified through two rounds of CsCl density gradient centrifugation. The viral particle concentration was determined as previously described (15). The multiplicity of infection (MOI) was defined as the ratio of total number of particles used in a particular infection divided by the number of cells.…”

“…Adenovirus expressing the human TSHR (AdCMVTSHR) has previously been described (15). Adenoviruses expressing mouse IL-12 or mouse IL-4 were constructed according to a recently described method (16,17).…”

“…Intramuscular injection of adenovirus coding the TSHR induces TSAb and hyperthyroidism in Ͼ50% of female BALB/c mice (15). In the present study, we determined the outcome of injecting adenoviruses expressing the TSHR in combination with adenovirus expressing IL-4 or IL-12.…”