Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor

Abstract

Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.

Fig. 1

Proliferation of IL-2-dependent (a) or IL-4-dependent (b) cell lines in the presence of culture supernatants from CHO cells transiently transfected with the plasmids shown. Results depict incorporation of ³[H]-thymidine and are expressed as cpm means of triplicate wells ± SD. (c) IL-12-induced IFN-γ production in mouse splenocytes cultured with supernatants of CHO cells transiently transfected with pGCV-IL-12. Quantification of IFN-γ was done by sandwich ELISA. (d) Assay for functional expression of TSHR in CHO cells transiently transfected with the indicated plasmid vectors. JPO9 cells stably expressing TSHR were used as controls. Cells were cultured with or without bTSH for 2 h, lysed, and total cellular cAMP was assayed in cell lysates using a commercial kit. Results are expressed as means of duplicate values ± SD.

Fig. 2

Goitrous thyroids from thyrotoxic mice no.2 (a) or no.7 (c), i.d. challenged 3× with 50 µg of pcDNA3-TSHR, as described in Table 1. A normal thyroid is shown in (b). (d) Follicular heterogeneity in the thyroid of mouse no.2. A follicle with activated epithelial cells (a) is juxtaposed against a follicle with flat (inactive) epithelium (b). (e) Histological appearance of normal follicles. (f) Section of thyroid from mouse no.7, showing homogeneous distribution of hyperplastic follicles lined with activated thyrocytes (a) that frequently protrude into the follicular lumen (b). (d,e,f, × 200).

Fig. 3

Assays for TSHR-specific antibodies (a), TSAbs (b) and TT4 (c) in sera of BALB/c mice i.d. challenged at 0, 3, and 6 weeks with the plasmids shown. The assays were performed 8 weeks after the initial priming. Bars depict individual mice. Responses are considered significant if they exceed the mean + 3 SD of the control values (pcDNA3 challenge only), indicated by the vertical lines.