Stimulating TSH Receptor Autoantibodies Research Articles

Functional Water Channels Within the TSH Receptor - a new paradigm for TSH action with disease implications.

The TSH receptor (TSHR) transmembrane domain (TMD) lives in the plasma membrane consisting of lipids and water molecules. To understand the role of TSHR-associated water molecules we used molecular dynamic simulations of the TMD and identified a network of putative receptor associated transmembrane water channels. This result was confirmed with extended simulations of the full length TSHR with and without TSH ligand binding. While the transport time observed in the simulations via the TSHR protein was slower than via the lipid bilayer itself we found that significantly more waters traversed via the TSHR than via the lipid bilayer which more than doubled with the binding of TSH. Using rat thyroid cells (FRTL-5) and a calcein fluorescence technique we measured cell volumes after blockade of aquaporins 1 and 4, the major thyroid cell water transporters. TSH showed a dose-dependent ability to influence water transport and similar effects were observed with stimulating TSHR autoantibodies. Small molecule TSHR agonists, which are allosteric activators of the TMD, also enhanced water transport illustrating the role of the TMD in this phenomenon. Furthermore, the water channel pathway was also mapped across two activation motifs within the TSHR TMD suggesting how water movement may influence activation of the receptor. In pathophysiological conditions such as hypothyroidism and hyperthyroidism where TSH concentrations are highly variable this action of TSH may greatly influence water movement in thyroid cells and many other extrathyroidal sites where the TSHR is expressed thus affecting normal cellular function.

Fingolimod Improves the Outcome of Experimental Graves' Disease and Associated Orbitopathy by Modulating the Autoimmune Response to the Thyroid-Stimulating Hormone Receptor.

Graves' disease (GD) and Graves' orbitopathy are associated with stimulating thyrotropin receptor (TSHR) autoantibodies and autoreactive T cells. Recent in vitro studies suggested that sphingosine-1-phosphate (S1P) signaling is involved in the pathogenesis of orbitopathy. In this study, we explored the immune modulatory potential of S1P receptor antagonist fingolimod in a murine model for GD. Fingolimod was orally administered preventively during disease onset or therapeutically after disease onset. Administration of fingolimod during disease onset completely prevented the formation of TSHR-stimulating autoantibodies. Intervention after disease onset rarely reduced TSHR-stimulating autoantibodies and blocking autoantibodies were induced in some animals. Consequently, autoimmune hyperthyroidism characterized by elevated serum thyroxin levels, hyperplastic thyroid morphology accompanied by T cell infiltration, weight gain, enhanced body temperature, and tachycardia did not manifest preventively and showed milder manifestation in therapeutically treated animals. Importantly, examination of orbital tissue showed significant amelioration of orbitopathy manifestations through reduction of T cell infiltration, adipogenesis, and hyaluronan deposition. Autoimmune hyperthyroidism and orbitopathy were accompanied by changes in peripheral and splenic T cell proportions with high CD3+, CD4+, and CD8+ T cells. Activated T cells CD4+CD25+ were elevated whereas regulatory T cells CD4+Foxp3+ cells remained unchanged in spleens. Fingolimod decreased elevated T cell levels and increased CD4+CD25+Foxp3+ regulatory T cell populations. Analysis of total disease outcome revealed that treatment during disease onset protected animals against autoimmune hyperthyroidism and orbitopathy. Of note, therapeutic intervention after disease onset suppressed disease in half of the animals and in the other half disease remained at mild stages. The results of this study support a clinical trial to investigate the immunologic and clinical benefits of early treatment with S1P-based drugs in GD.

Graves' disease in clinical perspective.

Graves' disease (GD) is the most common cause for hyperthyroidism in iodine-replete areas. The disease is caused by the appearance of stimulating TSH receptor autoantibodies (TRAb) leading to hyperthyroidism. Blocking and neutral TRAb have, however, also been described. TRAb can be measured either by competition assays, assays using a bridge technology or bioassays (for discriminating stimulating vs. blocking antibodies). Therapy of GD with antithyroid drugs belonging to the group of thionamides is the first-line treatment to be continued for 12 up to 18 months. In case of relapse, thyroid ablative therapy including radioiodine therapy or thyroidectomy, respectively, should be performed. Risk factors for relapse are a large thyroid volume, persistence of high TRAb serum titer, smoking, and others. Within this review, we will give insights into the pathogenesis of GD including the pathogenesis of Graves' ophthalmopathy. We also describe recent developments of TRAb measurement, which is used for the diagnosis of GD as well as for outcome prediction. Finally, we discuss therapy aspects as well as the important issue of GD and pregnancy.

Graves\u2019 orbitopathy occurs sex-independently in an autoimmune hyperthyroid mouse model

Graves’ orbitopathy (GO) is the most common extra thyroidal complication of Graves’ disease (GD) and occurs predominantly in women but more severe in men. The reason for this effect of gender on GO is unknown. Herein we studied the manifestation of GO in both sexes of an induced mouse model in absence of additional risk factors present in patients like advanced age, genetic variabilities or smoking. Male and female mice were immunized with human TSHR A-subunit encoding plasmid. Both sexes comparably developed autoimmune hyperthyroidism characterized by TSHR stimulating autoantibodies, elevated T4 values, hyperplastic thyroids and hearts. Autoimmune mice developed inflammatory eye symptoms and proptosis, although males earlier than females. Serial in vivo1H/19F-magnetic resonance imaging revealed elevated inflammatory infiltration, increased fat volume and glycosaminoglycan deposition in orbits of both sexes but most significantly in female mice. Histologically, infiltration of T-cells, extension of brown fat and overall collagen deposition were characteristics of GO in male mice. In contrast, female mice developed predominately macrophage infiltration in muscle and connective tissue, and muscle hypertrophy. Apart from sex-dependent variabilities in pathogenesis, disease classification revealed minor sex-differences in incidence and total outcome. In conclusion, sex does not predispose for autoimmune hyperthyroidism and associated GO.

Clinical Evaluation of the First Automated Assay for the Detection of Stimulating TSH Receptor Autoantibodies.

Until recently, stimulating TSH receptor autoantibodies (sTRAbs) could only be measured by bioassays. A new assay system, which directly detects sTRAb in sera by applying bridge technology, has been established and is now available as automated chemiluminescence (bridge) immunoassay. We evaluated the automated bridge assay in clinical routine and compared it with a conventional automated TRAb assay (competition assay). Altogether, 226 Graves' disease (GD), 57 autoimmune thyroiditis, 74 non-autoimmune nodular goiter and 49 thyroid cancer patients, as well as 41 healthy controls were retrospectively evaluated. ROC plot analysis based on sera of newly diagnosed GD patients revealed an area under curve of 0.99 (95% CI: 0.99-1.0) indicating a high assay sensitivity and specificity. The highest sensitivity (100%) and specificity (99%) were seen at a cut-off level of 0.55 IU/l. The calculated positive predictive value was 94%, whereas the negative was 100%. Applying a ROC plot-derived cut-off of≥0.30 IU/l, derived from sera of GD patients already receiving antithyroid drug therapy for≤6 months, the sensitivity was 99% whereas the specificity was 98%. Detailed comparison of both assay systems used revealed a slightly different distribution of sTRAb and TRAb. Measurement of sTRAb during follow-up revealed a steady decline over one year of follow-up. In summary, our results demonstrate that the new automated bridge assay system for detecting sTRAb has a high sensitivity and specificity for diagnosing GD and to discriminate from other thyroid diseases, respectively. Our study, however, does not provide full evidence that the bridge assay is specific for sTRAb only.

Structure and activation of the TSH receptor transmembrane domain

PurposeThe thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves’ disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD–M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown.MethodsWe have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available.ResultsMost conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs.ConclusionsInsight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves’ disease.

A Drug-Like Antagonist Inhibits Thyrotropin Receptor–Mediated Stimulation of cAMP Production in Graves' Orbital Fibroblasts

Fibroblasts (FIBs) within the retro-orbital space of patients with Graves' disease (GOFs) express thyrotropin receptors (TSHRs) and are thought to be an orbital target of TSHR-stimulating autoantibodies in Graves' ophthalmopathy (GO). Recently, we developed a low molecular weight, drug-like TSHR antagonist (NCGC00229600) that inhibited TSHR activation in a model cell system overexpressing TSHRs and in normal human thyrocytes expressing endogenous TSHRs. Herein, we test the hypothesis that NCGC00229600 will inhibit activation of TSHRs endogenously expressed in GOFs. Three strains of GOFs, previously obtained from patients with GO, were studied as undifferentiated FIBs and after differentiation into adipocytes (ADIPs), and another seven strains were studied only as FIBs. ADIP differentiation was monitored by morphology and measurement of adiponectin mRNA. FIBs and ADIPs were treated with the TSH- or TSHR-stimulating antibody M22 in the absence or presence of NCGC00229600 and TSHR activation was monitored by cAMP production. FIBs contained few if any lipid vesicles and undetectable levels of adiponectin mRNA, whereas ADIPs exhibited abundant lipid vesicles and levels of adiponectin mRNA more than 250,000 times greater than FIBs; TSHR mRNA levels were 10-fold higher in ADIPs than FIBs. FIBs exhibited higher absolute levels of basal and forskolin-stimulated cAMP production than ADIPs. Consistent with previous findings, TSH stimulated cAMP production in the majority of ADIP strains and less consistently in FIBs. Most importantly, NCGC00229600 reduced both TSH- and M22-stimulated cAMP production in GOFs. These data confirm previous findings that TSHR activation may cause increased cAMP production in GOFs and show that NCGC00229600 can inhibit TSHR activation in GOFs. These findings suggest that drug-like TSHR antagonists may have a role in treatment of GO.

PDGF Enhances Orbital Fibroblast Responses to TSHR Stimulating Autoantibodies in Graves' Ophthalmopathy Patients

Thyroid-stimulating hormone receptor (TSHR) stimulating autoantibodies are associated with Graves' ophthalmopathy (GO), the orbital manifestation of Graves' disease (GD). TSHR autoantibody levels and orbital TSHR expression levels correlate positively with GO disease activity. Platelet-derived growth factors (PDGF) are increased in GO and potently activate orbital fibroblast effector functions. We investigated the possible relationship between PDGF and TSHR expression on orbital fibroblasts and how that influences the immunopathological effects of TSHR autoantibodies on orbital fibroblast activity. Orbital fibroblasts were stimulated with PDGF-AA, PDGF-AB, and PDGF-BB, and TSHR expression was determined by flow cytometry. Stimulatory effects of bovine TSH and GD immunoglobulins on orbital fibroblasts (with or without PDGF-BB preincubation) were determined by IL-6, IL-8, chemokine (C-C motif) ligand (CCL)-2, CCL5, CCL7, and hyaluronan ELISA. The TSHR blocking antibody K1-70 and the cAMP inhibitor H89 were used to determine involvement of TSHR signaling. PDGF-AB and PDGF-BB stimulation increased TSHR expression on orbital fibroblasts, whereas PDGF-AA did not. Furthermore, stimulation with bovine TSH and immunoglobulins from GD patients induced IL-6, IL-8, CCL2, and hyaluronan production by orbital fibroblasts, and PDGF-BB preincubation enhanced this response of orbital fibroblasts. Blocking studies with a TSHR blocking antibody and a cAMP inhibitor inhibited these effects, indicating the involvement of TSHR signaling and thus of TSHR stimulating autoantibodies herein. These findings indicate that PDGF-B containing PDGF isoforms amplify the immunopathological effects of TSHR-stimulating autoantibodies in GO patients by stimulating TSHR expression on orbital fibroblasts.

TSH Receptor – Autoantibody Interactions

TSH receptor (TSHR) autoantibodies (TRAbs)activate the TSHR cyclic AMP cascade (stimulating TRAbs) or act as TSHR antagonist (blocking TRAbs), and both types inhibit TSH binding to the TSHR. Isolation of human monoclonal TSHR autoantibodies (stimulating M22 and blocking 5C9) has been a key milestone in studies of the TSHR and TSHR autoimmunity. Comparison of M22 and TSH interactions with the TSHR at the atomic level reveal that M22 heavy and light chains mimic TSH alpha and beta chains, respectively, in the way they bind to the receptor, but the evolutionary forces which have caused this close molecular mimicry are as yet completely unknown. More recently two more human monoclonal antibodies to the TSHR (K1-18 with stimulating and K1-70 with blocking activities) have been isolated from a single blood sample collected from a patient with hypothyroidism who previously presented with hyperthyroidism. K1-18 and K1-70 were derived from different lymphocytes as shown by V region genes analysis. This provides, for the first time, clear proof that a patient can produce both blocking and stimulating TRAbs at the same time. Although it has been postulated that stimulating and blocking TRAbs bind to different regions on the TSHR, our studies showed that antibodies of both types bind well to the TSHR containing only N-terminal amino acids 22-260. Whether TRAbs make contact with other parts of the TSHR in order to produce their biological effects (stimulation or blocking) remains to be elucidated.

Direct quantification of stimulating TSH receptor autoantibodies in Graves' disease by the first in vitro assay suitable for routine clinical diagnosis

Direct quantification of stimulating TSH receptor autoantibodies in Graves' disease by the first in vitro assay suitable for routine clinical diagnosis

The effect of thyrotropin-receptor blocking antibodies on stimulating autoantibodies from patients with Graves' disease.

The hyperthyroidism of Graves' disease (GD) is caused by thyrotropin-receptor (TSHR) stimulating autoantibodies (TSAb), which lead to overproduction of thyroid hormones. In this study we tried to block the stimulatory effect of patients' TSAb to the TSHR with monoclonal antibodies (mAbs) and sera from hypothyroid patients. Two groups of blocking mAbs raised by different methods from two independent groups were tested for their ability to inhibit TSH binding to the TSHR, and also the binding of TSAb from the serum of patients with GD. Group 1 mAbs (7E3, 3H10, 4C1, 1B1, 4E9) bind to amino acids 378-387 and group 2 mAbs (23.1 and 31.7) to amino acids 382-415 of the human TSHR. These results were compared to the TSH- and TSAb-inhibiting effect of sera from hypothyroid patients containing bona fide thyroid blocking antibodies (TBAb) without agonistic activity. All studies were done in a conventional cyclic adenosine monophosphate (cAMP) or a modified luciferase reporter gene bioassay. TSH-induced cAMP/luciferase signal was reduced (> 70% inhibition) by all 7 mAbs, verifying the blocking nature. Comparable results (82.2%-96.3% inhibition) were seen when cells were preincubated with 8 TBAb sera. These TBAb sera also inhibited cAMP/luciferase induction of TSAb-positive sera from patients with GD (median of 27 experiments 62.2% inhibition; range, 26.8%-93.9%), and maintained inhibition greater than 20% even when diluted 1:150. However, when mAbs were incubated with these sera, results were heterogeneous: 17 of 30 sera (57%) incubated with mAb 31.7 caused reduced cAMP production compared to incubation with the control antibody, as did 18 of 34 sera (53%) incubated with mAb 7E3, 17 of 33 sera (52%) incubated with mAb 3H10, and 16 of 31 (52%) with mAb 23.1. Mixing all four mAbs did not enhance the cAMP-reductive effect (16/27 sera; 59% inhibited). Inhibition was less pronounced than with TBAb sera (0%-76% of a control antibody) and only present at antibody concentrations greater than 10 microg/mL. We conclude that despite the strong TBAb activity of the mAbs, their effect on TSAb-induced TSHR activation of sera from patients with GD was weaker than that of human TBAb autoantibodies. Thus, the latter are not only strong inhibitors of TSH activity, but also block the stimulatory effect of autoantibodies from patients with GD. However, this effect could not be reproduced by experimental mAbs to the same extent, because it may be the result of a broader spectrum of antibodies present in the TBAb sera, interacting with or in the vicinity of TSAb epitopes. Also of interest, when a TBAb serum was added to a TSAb serum, the TBAb effect was predominant even at high dilutions.

The cysteine-rich amino terminus of the thyrotropin receptor is the immunodominant linear antibody epitope in mice immunized using naked deoxyribonucleic acid or adenovirus vectors.

Experimental Graves' disease is more effectively produced by immunization approaches involving in vivo TSH receptor (TSHR) expression than by conventional immunization with TSHR protein and adjuvant. Unlike conformational epitopes that are extremely difficult to define, linear epitopes can be readily assessed using synthetic peptides. TSHR linear epitopes are well characterized in conventionally immunized animals, but there is no information for animals vaccinated with TSHR DNA in plasmid or adenovirus vectors. We used synthetic peptides to characterize linear epitopes in mice immunized by in vivo expression of TSHR DNA. TSHR adenovirus-injected mice had higher antibody levels than TSHR DNA-vaccinated mice. However, the dominant peptide recognized in both groups was the TSHR cysteine-rich N terminus (residues 22-41). Sera from TSHR adenovirus-immunized (but not TSHR DNA-vaccinated) mice interacted to a lesser extent with peptides encompassing residues 352-401, which include the region deleted following TSHR cleavage as well as the ectodomain juxta-membrane region. Although antibodies characterized using synthetic peptides are probably TSH blockers or nonfunctional, stimulating antibodies may recognize linear components in a conformational epitope. The cysteine-rich TSHR N terminus is functionally important in the action of stimulating TSHR autoantibodies in humans. The immunodominance of the same region in immunized mice suggests that this region may also be immunodominant in humans.

Use of thyrotropin receptor (TSHR) mutants to detect stimulating TSHR antibodies in hypothyroid patients with idiopathic myxedema, who have blocking TSHR antibodies.

Deletions of residues 295-306, 299-301, and 387-395 of the TSH receptor, as well as point mutations of cysteine 301 or 390 to serine, and tyrosine 385 to phenylalanine or alanine, markedly diminish the ability of a transfected receptor to measure the activity of blocking TSH receptor autoantibodies (TSHRAbs) in patients with idiopathic myxedema and hypothyroidism, but not stimulating TSHRAbs in Graves' patients. This has allowed us to use these mutants to detect stimulating TSHRAb activity in the sera of hypothyroid patients with idiopathic myxedema who have blocking TSHRAbs. In 7 such patients, we show that 50% or more have significant stimulatory activity in cells transfected with mutant receptors, as evidenced by the ability of the immunoglobulin G to directly increase cAMP levels or to enhance the ability of TSH or a Graves' stimulating TSHRAb to increase cAMP levels. Three of the TSH receptor mutants, deletions of residues 295-306 and 387-395 and the point mutation of cysteine 301 to serine, are shown to be particularly useful in these assays and may be useful to clarify the pathogenetic role and clinical significance of stimulating TSHRAbs in patients with autoimmune thyroid disease who also have blocking TSHRAbs.