Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves' disease.

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.

Development and characterization of monoclonal antibodies specific for the murine thyrotropin receptor.

We have characterized 10 monoclonal antibodies (Mabs) to recombinant murine thyrotropin receptor extracellular domain (mTSHR-ecd). Affinity purified mTSHR-ecd (amino acids 22-415), expressed in a baculovirus-insect cell system, was refolded in vitro and used to hyperimmunize female Balb/c mice. Spleens were removed 10 days after a final boost of 25 microg mTSHR-ecd intraperitoneally and intravenously, and the cells were fused to SP-2 cells and cloned. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) with folded mTSHR-ecd antigen. Ten of 18 higher affinity hybridomas were selected at random and ascites fluids prepared. Nine of the monoclonals were of IgG 1 isotype, and one was IgM. Five Mabs (M3, M4, M5, M6, and M9) inhibited the binding of 125I-TSH to functional hTSHR expressed on Chinese hamster ovary (CHO) cells, and four (M1, M3, M5, and M9) blocked the TSH-stimulated generation of cyclic adenosine monophosphate (cAMP), using the same cells. The remaining Mabs appeared to be neutral in their interaction with native TSHR. The Mabs were also compared for their reactivity to mTSHR-ecd under folding (ELISA) and unfolding (reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) conditions. Most Mabs demonstrated reactivity to both conformational (folded) and linear (unfolded) forms of mTSHR-ecd, suggesting that they were generated primarily against linear epitopes although one Mab (M4) showed affinity for only folded antigen indicating a preference for a conformational epitope. Mapping the Mab epitopes using 26 overlapping peptides spanning the human (h)TSHR-ecd showed that 6 bound peptide 397-415, 1 bound peptide 352-371, and 1 peptide 22-41. These epitope mapped Mabs to the mTSHR-ecd, both receptor blocking and receptor neutral, will provide further insight into the structure-function of the TSHR ectodomain.

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 Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25-50 microg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein 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 ectodomain were antigenic. The earliest epitope included aa 22-41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function 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), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.

Folding-dependent binding of thyrotropin (TSH) and TSH receptor autoantibodies to the murine TSH receptor ectodomain.

The mouse TSH receptor ectodomain (mTSHR-ecd) was amplified from murine thyroid complementary DNA and ligated into the pAcGP67B insect cell vector, and the nucleotide sequence was confirmed. Employing a baculovirus-insect cell system, the mTSHR-ecd (amino acids 22-415) was expressed as a fusion protein with the gp67 insect cell signal sequence at the NH2-terminus and a C-terminal six-histidine tag. Protein expression was assessed by Western blot using a murine monoclonal antibody (recognizing amino acids 22-35) and a rabbit antipeptide antibody (recognizing amino acids 397-415). These antibodies detected two principal species of mTSHR-ecd, one glycosylated (66 kDa) and one nonglycosylated (52 kDa), in cell lysates of infected insect cells. More than 10% of these species were present in a water-soluble (cytosolic) fraction. This fraction was then used to purify, under native conditions, 100-microg amounts of mTSHR-ecd using nickel-nitrilo-triacetic (Ni-NTA) resin chromatography. The purified cytosolic mTSHR-ecd migrated as a homogeneous 66-kDa band visible on Coomassie blue-stained gels and was confirmed by Western blotting. We also purified the mTSHR-ecd from total cell lysates under denaturing conditions, followed by "in vitro" refolding on the Ni-NTA column. Under these conditions, milligram amounts of soluble mTSHR-ecd were obtained. This material consisted primarily of the 66-kDa glycosylated form, but in addition contained four or five lower molecular mass, partially glycosylated intermediates and the 52-kDa nonglycosylated form. Deglycosylation with either endoglycosidase F or H, reduced all mTSHR-ecd glycosylated species to a 52-kDa nonglycosylated form. Both the cytosolic and refolded mTSHR-ecd preparations inhibited the binding of [125I]TSH to the full-length human TSHR expressed in Chinese hamster ovary cells in a dose-dependent manner, with similar affinities. The affinity of such interactions was 3 orders of magnitude less than observed with native porcine TSHR and was further reduced by unfolding the mTSHR-ecd preparations. The cytosolic and refolded mTSHR-ecd were also recognized by hTSHR autoantibodies in the serum of patients with hyperthyroid Graves' disease. Such autoantibody binding to mTSHR-ecd was also markedly reduced by unfolding the antigen. These results demonstrated the successful production of large quantities of well characterized, biologically active, mTSHR-ecd antigen. In addition, the data showed that although the ectodomain of the mTSHR bound TSH, intact holoreceptor may be required for high affinity ligand binding. Whether the transmembrane region is required for direct ligand binding, as seen for other G protein-linked receptors, or whether it is needed to stabilize the ligand binding to the ectodomain and maintain a correctly folded state, remains unclear.

Characterization of soluble, disulfide bond-stabilized, prokaryotically expressed human thyrotropin receptor ectodomain.

To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein; it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21-415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21-35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain alpha F'), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

Characterisation of the antibody response to the extracellular region of recombinant thyrotropin receptor.

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.

Multimeric complex formation by the thyrotropin receptor in solubilized thyroid membranes.

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.

Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells.

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.

Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading.

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.

Folding of the recombinant human thyrotropin (TSH) receptor extracellular domain: identification of folded monomeric and tetrameric complexes that bind TSH receptor autoantibodies.

We have analyzed protein folding and disulfide bond formation in the extracellular domain of the human TSH receptor (hTSHR-ecd) expressed in Escherichia coli. This domain, which begins at the amino-terminus and ends at residue 415, is a major autoantigen in human autoimmune thyroid disease. Refolding of reduced and denatured hTSHR-ecd occurred in polyacrylamide gels treated with 0.25 M KCl for visualization of protein bands. Under conditions of partial renaturation, at least three forms of the hTSHR-ecd were resolved by reelectrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 1) unfolded monomers, 2) folded monomers, and 3) tetramers. Disulfide bond formation was implicated in both folding and tetramerization, as reduction of these forms produced only unfolded monomers. A natural variant of the hTSHR (v1.3), sharing 231 N-terminal amino acids with hTSHR-ecd, formed folded monomers and dimers on renaturation, but not tetramers, implicating one or more of the five cysteine residues residing between positions 231-415 in the association of dimers into tetramers. Binding of three different sources of hTSHR antibodies to these various forms of the hTSHR-ecd was assessed by immunoblotting using: 1) murine monoclonal antibodies (MAbs) generated against hTSHR-ecd, 2) rabbit polyclonal antisera generated against overlapping synthetic peptides spanning residues 37-71 of the hTSHR-ecd, and 3) human immunoglobulin G from patients with Graves' disease and detectable hTSHR-Ab. One of the MAbs, shown to recognize residues 21-35, and the rabbit polyclonal antibodies bound to all three forms of the hTSHR-ecd. Some of the hTSHR autoantibodies bound predominantly to the monomeric forms of the hTSHR, but autoantibodies were also identified that recognized tetrameric hTSHR-ecd. These data demonstrate that hTSHR-Abs recognize differing nonlinear and linear epitopes in the hTSHR-ecd and provide a methodology that should be useful for further defining the structural requirements for folding of this functionally and immunologically important domain of the hTSHR.

Human autoantibodies to the thyrotropin receptor: recognition of linear, folded, and glycosylated recombinant extracellular domain.

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.

Congenital adrenal hyperplasia with female pseudohermaphroditism in a 26-year-old patient.

The paper presents the case of a 26-yr-old patient admitted for intersexuality. Clinical examination shows statural deficit, female phenotype, melanoderma, glabrous tegmina except for the pubic area presenting horizontally inserted pilosity, labioscrotum devoid of contents, pseudomicropenis with hypospadias. The Barr cytogenetic test is positive (56%) and hormone assay shows plasma cortisol at the lower limit and adrenal androgenic hormones and their metabolites in excess, suppressible by dexamethasone. The patient had a history of repeated admissions to intensive care units for severe dehydration, vomiting, diarrhea and collapse.

Cardiac disturbances in thyrotoxicosis: diagnosis, incidence, clinical features and management.

In the present paper we studied 403 patients with different etiopathogenic and clinical forms of thyrotoxicosis: toxic multinodular goiter (36.7%), toxic adenoma (4.9%), Graves' disease (27.04%), transient thyrotoxicosis (subacute thyroiditis, painless thyroiditis, Hashitoxicosis) (21.09%), T3-thyrotoxicosis (9.42%), thyrotoxicosis factitia (0.74%). Eighty-seven patients (21.5%) had cardiac disturbances. The following arrhythmias were most common: atrial fibrillation (4.00%), ventricular premature beats (2.77%), paroxysmal supraventricular tachycardia (2.23%), atrial flutter (1.00%). Congestive heart failure occurred in 10.42% of the cases. Paroxysmal tachyarrhythmias were converted to sinus rhythm in 90% of the subjects, by a selected and sustained treatment: drug therapy (carbimazole 30-40 mg/day, Lugol solution 1/2/20, 10-15 drops/day, beta-adrenergic blockers (propranolol--60-120 mg/24 hrs), calcium channel blockers (verapamil--40-60 mg/24 hrs), cardiac glycosides (deslanosid) or DC cardioversion. In order to prevent recurrences and/or complications, drug therapy was subsequently completed with subtotal thyroidectomy or radioactive iodine (131I) therapy. Thus, we succeeded in maintaining the patients in a euthyroid state, in sinus rhythm and with an adequate cardiovascular function in 95.4% of the cases.