CHARACTERIZATION OF THE MURINE IMMUNE-RESPONSE TO THE MURINE TSH RECEPTOR ECTODOMAIN - INDUCTION OF HYPOTHYROIDISM AND TSH RECEPTOR ANTIBODIES

The thyrotropin receptor (TSHR) is the major autoantigen of human Grav es' disease. In order to define the antigenicity of the TSHR in a defi ned model, we examined the immune response of BALB/c mice to immunizat ion with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25-50 mu g of insect cell expressed, purified and refolded, mTSHR-ecd in alu m adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. C ontrol mice received wild-type baculovirus-infected insect cell protei n lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the e ctodomain were antigenic. The earliest epitope included aa 22-41, but later two regions of reactivity were noted clustered towards the mid p ortion and carboxyl terminus of the ectodomain. The murine TSHR autoan tibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of b locking the native TSHR. We showed that these TSHR antibodies acted, i n vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with t he hTSHR. Hence, the antibody response to mTSHR-ecd was potentially an tagonistic in its influence on the TSHR. Assessment of thyroid functio n in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confi rming the onset of thyroid failure. However, thyroid histology remaine d grossly normal. These data demonstrate that mTSHR-ecd is a potent an tigen with three major immunogenic regions. The induced mTSHR-Abs bloc ked TSH action in vivo and reduced murine thyroid function.