We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.
To evaluate the B cell response to the extracellular domain of the human TSH receptor (hTSHR-ecd), we used recombinant hTSHR-ecd to immunize BALB/c mice (group A) and CBA/J mice (groups B and C). Mice from groups A and B were boosted once, and mice from group C received three antigen boosts. All individual mice developed highly specific hTSHR-ecd antibodies (hTSHR-ecd-Ab), confirmed by Western blot analyses. The B cell epitopes recognized by these murine hTSHR-ecd-Ab were mapped by enzyme-linked immunoassays using 26 synthetic overlapping peptides spanning the entire mature hTSHR-ecd [amino acids (aa) 22-415], i.e. without the signal sequence. Although all BALB/c and CBA/J mice antisera recognized peptide 1 (aa 22-41), the hyperimmunized CBA/J mice (group C) demonstrated recognition of additional peptides (numbers 21-26) clustered toward the carboxyl-terminus of the hTSHR-ecd (aa 322-415). Furthermore, group C serum blocked the binding of [125I]bTSH to native porcine TSHR, whereas sera from groups A and B were inactive. We were also able to map the B cell epitopes of antisera from rabbits immunized repeatedly with hTSHR-ecd and found the same recognition pattern of peptide 1 and additional peptides clustered near the carboxyl-terminus of the hTSHR-ecd (aa 322-341 and 367-415). These rabbit antisera also inhibited the binding of [125I]bTSH to native porcine TSHR. These data provide a comprehensive B cell epitope-mapping study of induced hTSHR-ecd-Ab and demonstrate intramolecular spreading of the epitopes recognized. Although the N-terminal region was highly antigenic, repeated immunization induced hTSHR-ecd-Ab targeted to a region critical for TSH binding.
In order to replicate a recently described murine model of Graves' disease, we immunized AKR/N (H-2k) mice i.p., every 2 weeks, with either a clone of fibroblasts expressing both the human TSH receptor (hTSHR) and murine major histocompatibility complex (MHC) class II molecules or with fibroblasts expressing the MHC class II molecules alone. Mice were bled, and their thyroid hormone levels measured, at 6, 12, and up to 18 weeks after the first immunization. Between 11-12 weeks after immunization, a significant number of mice began to die spontaneously and were found to have developed large goiters. Thirty to 40% of mice immunized with hTSHR transfected fibroblasts showed markedly increased serum T3 and T4 hormone levels by 12 weeks compared with controls, with the highest thyroid hormone levels being T3: 420 ng/dl (normal < 70) and T4: 16.5 microg/dl (normal < 5). The murine serum demonstrated the presence of antibodies to the TSHR, as evidenced by inhibition of labeled TSH binding to the hTSHR, and these sera had in vitro thyroid stimulating activity. Many of the hyperthyroid mouse exhibited weight loss and hyperactivity and, on examination, their thyroids had the histological features of thyroid hyperactivity including thyroid enlargement, thyroid cell hypertrophy, and colloid droplet formation--all consistent with Graves' disease. In contrast, a small number of mice (< 5%) developed hypothyroidism with low serum T4 levels and markedly increased TSH concentrations and evidence of thyroid hypoplasia. Both hyperthyroidism and hypothyroidism were successfully transferred to naive mice using ip cells of immunized mice. Surprisingly, hypothyroidism occurred in many recipient mice even after transfer from hyperthyroid donors. These results confirmed that immunization with naturally expressed hTSHR in mammalian cells was able to induce functional TSHR autoantibodies that either stimulated or blocked the mouse thyroid gland and induced hyperthyroidism or thyroid failure. Furthermore, both blocking and stimulating antibodies coexisted in the same mice as evidenced so clearly by the transfer of hypothyroidism from hyperthyroid mice. The addition of a Th2 adjuvant (pertussis toxin) caused approximately 50% of the animals to become hyperthyroid beginning early at 9 weeks, whereas a Th1 adjuvant (CFA) delayed the disease onset such that only 10% were hyperthyroid by 12 weeks. As with human autoimmune thyroid disease, the T cell control of this murine model may be critical and requires more extensive investigation.
The TSH receptor (TSHR) has a large glycosylated ectodomain comprising the amino-terminal half of the molecule (394 of 743 residues) implicated in TSH binding, as well as autoantibody recognition in Graves' disease. In this study we employed antibodies specific for the amino-terminus (Ab1), midportion (Ab2), and carboxyl-terminus (Ab3) of the TSHR-ectodomain, previously mapped using recombinant receptor proteins, to detect the natural receptor present in detergent-solubilized porcine thyroid cell membranes via immunoblotting. Several forms of the receptor were detected. In reduced samples Ab1 detected full-length holoreceptors present in both nonglycosylated and glycoslylated forms of apparent molecular masses 80 and 90 kDa, respectively, as well as apparent dimeric nonglycosylated and dimeric glycosylated holoreceptor forms resistant to reduction. Also detected by Ab1 were a glycosylated amino-terminal 47- to 52-kDa fragment of the holoreceptor (gly alpha-subunit), reduced to 42 kDa (alpha-subunit) by Endo F deglycosylation. Ab2 detected all of the same forms. Ab3 detected primarily a carboxy-terminal, nonglycosylated fragment of 35 kDa (beta-subunit). In unreduced samples, the recognition pattern was unchanged with Ab1. Ab2 detected monomeric and dimeric beta-subunits, as well as higher order complexes. The different TSHR forms present in unreduced preparations were resolved by ammonium sulfate precipitation, confirming their autonomy. The data demonstrate the presence of multiple forms of the natural TSHR. Their roles in TSH action and TSHR autoimmunity require further exploration.
To examine the heterogeneity of autoantibodies to the human TSH receptor (hTSHR), we evaluated 20 sera from patients with Graves' disease for their recognition of prokaryotic (unglycosylated) and eukaryotic (insect cell glycosylated) recombinant hTSHR extracellular domain (ecd) in an unfolded (linear) and a folded (nonlinear) state. With the prokaryotic antigen, 12 (60%) bound folded hTSHR ecd monomer, 8 (40%) bound to the unfolded monomer, and 3 (15%) bound to a tetrameric species. Such binding to different hTSHR antigens was not mutually exclusive. In addition, 7 (35%) sera showed an apparently higher reactivity for the folded than the unfolded monomer. When reacted against the glycosylated insect cell hTSHR ecd, 9 (45%) sera recognized both the unfolded and folded monomer, and 5 (25%) recognized the tetrameric form. In all of our testing, 17 of the 20 sera (85%) bound to 1 or more of the recombinant hTSHR ecd antigens, and the recognition pattern appeared to be heterogeneous in at least 4 (20%) of the serum samples, with hTSHR antibodies recognizing linear, folded, and glycosylated hTSHR ecd monomers. We conclude, therefore, that patients with Graves' disease have autoantibodies that recognize multiple epitopes on the hTSHR ecd and that it is possible to classify them according to their recognition of linear, folded, and glycosylated products.
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