Dendritic cells infected with adenovirus expressing the thyrotrophin receptor induce Graves' hyperthyroidism in BALB/c mice

Abstract

Dendritic cells (DCs) are the most potent antigen-presenting cells and a prerequisite for the initiation of primary immune response. This study was performed to investigate the contribution of DCs to the initiation of Graves' hyperthyroidism, an organ-specific autoimmune disease in which the thyrotrophin receptor (TSHR) is the major autoantigen. DCs were prepared from bone marrow precursor cells of BALB/c mice by culturing with granulocyte macrophage-colony stimulating factor and interleukin-4. Subcutaneous injections of DCs infected with recombinant adenovirus expressing the TSHR (but not beta-galactosidase) in syngeneic female mice induced Graves'-like hyperthyroidism (8 and 35% of mice after two and three injections, respectively) characterized by stimulating TSHR antibodies, elevated serum thyroxine levels and diffuse hyperplasitc goiter. TSHR antibodies determined by ELISA were of both IgG1 (Th2-type) and IgG2a (Th1-type) subclasses, and splenocytes from immunized mice secreted interferon-gamma (a Th1 cytokine), not interleukin-4 (a Th2 cytokine), in response to TSHR antigen. Surprisingly, IFN-gamma secretion, and induction of antibodies and disease were almost completely suppressed by co-administration of alum/pertussis toxin, a Th2-dominant adjuvant, whereas polyriboinosinic polyribocytidylic acid, a Th1-inducer, enhanced splenocyte secretion of IFN-gamma without changing disease incidence. These observations demonstrate that DCs efficiently present the TSHR to naive T cells to induce TSHR antibodies and Graves'-like hyperthyroidism in mice. In addition, our results challenge the previous concept of Th2 dominance in Graves' hyperthyroidism and provide support for the role of Th1 immune response in disease pathogenesis.

Fig. 1

Transgene expression in DCs infected with adenovirus. (a, b) DCs were left untreated (a) or infected with AxCALacZ at a MOI of 10 000 particles per cell under centrifugation for 2 h at 2000 g at 37°C (b), and stained with x-gal. (c) DCs were left untreated or infected with AdCMVTSHR at a MOI of 10 000 particles per cell under centrifugation for 2 h at 2000 g at 37°C, followed by flow cytometric analysis of TSHR expression with sera from a control mouse and a mouse with Graves’ hyperthyroidism [15].

Fig. 2

T₄, TSI and TBII values in sera from mice immunized with DCs infected with AdCMVTSHR or AxCALacZ alone or in combination with alum or poly (I:C). The normal upper limits are shown by horizontal lines. Data are means of duplicate determinations. Closed circles, hyperthyroid mice; open circles, euthyroid mice. Lane 1, control mice; lane 2, mice immunized 2× with AdCALacZ; lane 3, mice immunized 2× with AdCMVTSHR; lane 4, mice immunized 3× with AdCALacZ; lane 5, mice immunized 3× with AdCMVTSHR; lane 6, mice immunized 3× with AdCMVTSHR and poly (I:C); lane 7, mice immunized 3× with AdCMVTSHR and alum.

Fig. 3

IgG subclass distribution of anti-TSHR antibodies in ELISA assays. Total IgG, IgG1 and IgG2a of anti-TSHR antibodies were determined in sera from control mice and those immunized thrice with (i) DCs infected with AxCALacZ, (ii) DCs infected with AdCMVTSHR (DCs/TSHR), (iii) DCs/TSHR and alum or (iv) DCs/TSHR and poly (I:C). Data are means of duplicate determinations obtained with sera from individual mice diluted 1 : 300, 1 : 100 and 1 : 30 (from left to right).

Fig. 4

IFN-γ production from splenocytes of mice immunized with DCs infected with AdCMVTSHR alone or in combination with alum or poly (I:C). Mice were immunized twice with (i) DCs infected with AxCALacZ, (ii) DCs infected with AdCMVTSHR (DCs/TSHR), (iii) DCs/TSHR and alum or (iv) DCs/TSHR and poly (I:C). Splenocytes were isolated 10 days after the second immunization. IFN-γ in supernatants of splenocytes cultured in the presence or absence of TSHR-289 (5 µg/ml) for 5 days was determined by ELISA. Data are mean ± s.e. (n = 3) and expressed as pg/ml. Open and solid bars, splenocytes cultured in absence or presence of TSHR-289, respectively. *P < 0·05.

Fig. 5

Histology (H&E-staining) of the thyroid glands from euthyroid, control (a) and hyperthyroid mice (b). Original magnification, × 200.