Immunization with thyroglobulin induces Graves'-like disease in mice

We immunized AKR/N mice with bovine thyroglobulin (Tg) once every 2 weeks and monitored their time-dependent changes in 125I uptake activity in the thyroid glands. After 3 months, anti-Tg antibody was positive in all sera from the immunized mice. Serum free tri-iodothyronine (T3) and free thyroxine (T4) levels in the immunized mice (n=6) were significantly higher than those in the saline injected (control) mice (n=6). Neck counts as well as scintigraphy of the thyroid glands revealed that iodide uptake activity of the immunized mice was not suppressed, but was instead higher than that of the control mice. Two of the six immunized mice showed extremely high iodide uptake activity. The thyroid glands of these two mice were diffusely enlarged and the height of the epithelial cells was also increased. In addition, two mice with high iodide uptake activity produced a high titer of thyroid-stimulating antibody. Additional experiments showed that 4 out of 11 AKR/N mice and 3 out of 10 C57BL6 mice immunized with Tg had high serum free T3/free T4 levels, high 125I uptake activity of the thyroid, and positive thyroid-stimulating antibody activity. Diffuse goiter, thyrotoxicosis, high iodide uptake activity, and positive thyroid-stimulating antibody are the characteristics of Graves' disease. Thus, these mice exhibit the symptoms of Graves' disease. These results suggest that immunization with Tg induces Graves'-like disease in mice and that our methods will provide a new animal model of Graves' disease

Dominant negative effect of mutated thyroid stimulating hormone receptor (P556L) causes hypothyroidism in C.RF-Tshr(hyt/wild) mice.

C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice

Thyroid glands from C.RF-Tshr^wild/wild and Tshr^wild/hyt mice.

<p>Macroscopic views of thyroid glands from 12-week-old C.RF-Tshr^wild/wild (a) and Tshr^wild/hyt (b) mice observed under a stereomicroscope (Olympus SZX7). Black arrows indicate the upper poles, and red arrows indicate the lower poles of the left lobe of the thyroid gland. Histology on high magnification in C.RF-Tshr^wild/wild (d) and Tshr^wild/hyt (e) mice. Tissues were fixed with 4% formaldehyde and embedded in paraffin. Sections (5 µm) were stained with hematoxylin-eosin. Bars indicate 200 µm.</p

cAMP response to TSH and ¹²⁵I-binding activities of HEK 293 cells expressing wild-type TSHR and/or mutated TSHR.

<p>(a) Cyclic AMP levels in HEK 293 cells expressing TSHR(W) or TSHR(M) following TSH stimulation. TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W) (○-○) or the same amount of pcDNA-TSHR(M) (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (b) Cyclic AMP levels in HEK 293 cells expressing TSHR(W) and TSHR(M) following TSH stimulation. TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA (□-□), 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA-TSHR(M) (▪-▪) or 0.5 µg of pcDNA-TSHR(M)+the same amount of pcDNA (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (c) ¹²⁵I-binding activities of HEK 293 cells expressing wild-type TSHR and/or mutated TSHR. HEK 293 cells were transfected with 0.5 µg of pcDNA3-TSHR(W)+same amount of pcDNA (○-○), 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA-TSHR(M) (□-□) or 0.5 µg of pcDNA-TSHR(M)+the same amount of pcDNA (•-•). After 72 h of culture, TSH binding activities were assayed using ¹²⁵I-bovine TSH. Data are means ± S.E. of triplicate assays.</p