Thyrotropin receptor antibodies: new insights into their actions and clinical relevance

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1 Best Practice & Research Clinical Endocrinology & Metabolism Vol. 19, No. 1, pp , 2005 doi: /j.beem available online at 3 Thyrotropin receptor antibodies: new insights into their actions and clinical relevance Takao Ando Rauf Latif Terry F. Davies* Department of Medicine, One Gustave L Levy Place, P.O. Box 1055, Mount Sinai School of Medicine, New York, NY 10029, USA The thyrotropin receptor (TSHR) is a G-protein-coupled receptor with a large ectodomain. TSH, acting via TSHR, regulates thyroid growth and thyroid hormone production and secretion. The TSHR undergoes complex post-translational processing involving dimerization, intramolecular cleavage, and shedding of its ectodomain, and each of these processes may influence the antigenicity of the TSHR. The TSHR is also the major autoantigen in Graves disease, as well as a leading candidate autoantigen in both Graves ophthalmopathy and pretibial myxedema. The naturally conformed TSHR is most effectively presented as an autoantigen to the immune system, causing the production of stimulating TSHR-Abs. There are also autoantibodies which block the TSHR from TSH action, and neutral TSHR-Abs which have no influence on TSH action. TSHR- Abs can be detected by competition assays of TSHR-Abs for labeled TSH, or monoclonal TSHR- Ab binding to solubilized TSHRs, or by bioassays using thyroid cells or mammalian cells expressing recombinant TSHRs. Keywords: autoimmune thyroid disease; autoantibody; epitope; Graves disease; post-translational processing. The thyrotropin receptor (TSHR) is a G-protein-coupled receptor with a large extracellular domain. TSH binds to the TSHR expressed on the thyroid epithelial cell surface and regulates thyroid growth and thyroid hormone production and release. 1 3 The TSHR is thought to be initially expressed on the plasma membrane surface as holoreceptor glycoproteins 4,5, but subsequently undergoes intramolecular cleavage with the formation of two subunits and the loss of a 50-amino-acid ectodomain region (Figure 1(A)). 3 * Corresponding author. Tel.: C ; Fax: C address: terry.davies@mssm.edu (T.F. Davies) X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

2 Figure 1. TSHR structure. (A) A model of full-length TSHR. TSHR has a large extracellular domain with nine leucine-rich repeats which forms a horseshoe-like structure and determines its ligand specificity. 26 Adapted from Ando Tet al. (2004, Endocrinology 145: ) with permission. (B) A model of leucine-rich repeats. Each leucinerich repeat (LRR) is made of amino acids forming a small b-strand (arrows) followed by a small a-helix (cylinder). The repeat units are arranged parallel to a common axis and organized spatially to form a horseshoe-shaped molecule, with the small b-strands and small a-helices making the concave and convex surfaces of the horseshoe, respectively. From Figure 3, Smith G et al. (2003 EMBO Journal 22: ) with permission. 34 T. Ando et al

3 Thyrotropin receptor antibodies 35 The extracellular a-(or A-) subunit is subsequently shed from the plasma membrane. 6 The heavily glycosylated and conformed ectodomain of the receptor is also the major site for TSH binding, while the seven transmembrane domains and cytoplasmic tail of the TSHR are involved in signal transduction. TSHR is a major target for autoantibodies in autoimmune thyroid disease (AITD). Stimulating TSHR autoantibodies (TSHR-Abs) activate the TSHRs, resulting in overproduction of thyroid hormone, as seen in Graves disease (GD). Less commonly, some TSHR-Abs block the binding of TSH to the TSHR, and these blocking TSHR-Abs induce thyroid atrophy and hypothyroidism, as seen in patients with atrophic thyroiditis (AT). 1,2 Immune reactivity to the TSHR antigen is also widely observed in Graves ophthalmopathy (GO), where accumulation of glycosaminoglycans in the retro-orbital space secondary to the local lymphocytic infiltration results in the swelling of the retroorbital tissues. 7 A similar phenomenon is thought to be the cause of pretibial myxedema (PTM). TSHR-Abs have been most commonly measured by a protein binding assay technique involving competition for labeled TSH binding to solubilized TSHRs. 2 This type of assay can detect TSHR-Abs which stimulate the TSHR as well as antibodies which block the TSHR. These two types of TSHR-Abs can only be distinguished by cellbased bioassays. In addition, there are some TSHR-Abs which are undetectable in these assays. These TSHR-Abs do not inhibit TSH binding to the TSHR and, therefore, they have been called neutral TSHR-Abs. The clinical significance of neutral TSHR-Abs is unclear. TSHR STRUCTURE The human TSHR size, deduced from the cdna 8 10, is 764 amino acids long, with 418 amino acids in the ectodomain. It includes a 21-amino-acid signal peptide and six putative N-linked glycosylation sites. In addition, the ectodomain has nine leucine-rich repeats (Figure 1(B)) characteristic of a subgroup of G protein-coupled receptors such as the LH (leutropin) receptor and the FSH (follitropin) receptor. 1,3 The ectodomain of the TSHR possesses two unique regions which are not present in the other closely related members of this subgroup of receptors. These regions are in the N-terminus of w10 amino acids (38 45) and in the C-terminus of the ectodomain of w50 amino acids ( ). 3 The cysteine clusters in the N-terminus have been shown to be important for the proper folding and/or intracellular trafficking of the receptor 11, whereas clusters at the C-terminus of the ectodomain serve in disulfide bond formation after intramolecular cleavage which forms the a and b (or A and B) subunits. TSHR glycosylation The molecular weight of the full-length TSHR deduced from the amino acid sequence corresponds to w84 kda. 3 However, recombinant full-length TSHR generated in mammalian cells, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), appears to be present in w100 and w120 kda forms. 3 The former is the mannose-rich precursor not expressed on the membrane. 4,5 The latter is the fully matured glycosylated receptor which is expressed on the plasma membrane and which subsequently undergoes cleavage. 4,5,12

4 36 T. Ando et al It has been shown in Chinese hamster ovary (CHO) cells that complete inhibition of TSHR glycosylation by mutagenesis 13,14 or pharmacological treatments 15,16 prevented the TSHR from being expressed on the cell surface. Additionally, mutagenesis has shown that six N-glycosylation sites (Asn residues at 77*, 99, 113*, 177, 198*, 302*) are effectively glycosylated 13, and four of these sites (indicated with asterisks) have been confirmed by mass spectrometry. 17 However, mutation of up to two of any of these N-glycosylation sites has been shown not to alter TSHR function. 13 Therefore, glycosylation seems to play a quantitative role in folding and surface expression of the TSHR. 13 TSHR intramolecular cleavage and shedding A fully glycosylated TSHR is expressed on the plasma membrane as a single holoreceptor. 4,5 This mature TSHR undergoes intramolecular cleavage by a currently undefined enzyme or enzymes akin to the matrix metalloproteases. 6,18 In this process a w50-amino-acid region on the C-terminus of the ectodomain (w ) is lost. This results in a two-subunit structure (a and b) connected via disulfide bonds formed between the cysteine clusters at the C-terminus of the ectodomain. 3 In primary thyrocytes, most of the TSHRs are fully glycosylated and cleaved 4,19, but b subunits on the plasma membrane are predominant, being fold more than a subunits. 19 In transfected mammalian cells such as CHO cells and other fibroblasts expressing functional recombinant TSHRs, the uncleaved holoreceptor may be the dominant form due to inefficient processing 4, and the b predominance is still seen even in such TSHRtransfected CHO cells. 5 This b predominance may be explained by a subunit shedding 6, with reduction of disulfide bonds between the two TSHR subunits catalyzed by PDI (protein disulfide-isomerase). 20 However, the phenomenon of subunit shedding has been detected only in vitro, in primary thyrocytes and mammalian cells over-expressing the TSHR. 6,21 There have been no convincing reports demonstrating shed TSHR subunits in vivo. Dimerization and multimerization of the TSHR The ability to form homomeric multimers 22 is another important post-translational process that the TSHR gains, either during its synthesis or after being expressed on the plasma membrane. This has recently been confirmed by the fluorescent resonance energy transfer (FRET) technique using CHO cells over-expressing TSHRs tagged at the C-terminus of the TSHR. 23 However, it is currently not known which region(s) of the TSHR are involved in this receptor receptor interaction. TSHR structure and hormone binding It has been shown that the TSHR ectodomain, tethered with a short hydrophilic tail, GPI (glycosylphosphatidyl-inositol), is sufficient for high-affinity binding of TSH. 24,25 As shown by a mutagenesis-based computer simulation approach 26, ligand specificity between TSH, FSH, and LH to their cognate receptors is determined by the conformation of a horseshoe structure made up of the leucine-rich repeats in the a subunit (Figure 1(B)). In addition, the N-terminus portion of the b subunit is important for TSH binding, as shown by loss of TSH binding after disruption of sulfation of tyrosine residues (Tyr 385 and Tyr 387 ). 27 Recent epitope (Zantibody binding site) studies

5 using multiple mabs with blocking activity 28,29, confirmed these multiple and discontinuous TSH-TSHR interacting sites, with at least two on the TSHR a subunit and at least one on the b subunit. Therefore, interaction between TSH and the TSHR is highly dependent on the complex structure of the receptor. INFLUENCE OF POST-TRANSLATIONAL PROCESSING ON TSHR FUNCTION Glycosylation N-glycosylation is seen only in the TSHR ectodomain, the major TSH binding site. However, the role of glycosylation in TSH binding has been controversial. As mentioned above, glycosylation of the TSHR is not involved in TSH binding 13, therefore studies showing lowered TSH binding affinity with TSHR produced in Escherichia coli 30 and insect cells 31,32 should be due to altered protein folding and expression due to imperfect glycosylation. Intramolecular cleavage The TSHR loses a w50-amino-acid polypeptide region by a cleavage phenomenon, causing the formation of a two-subunit structure. This cleaved form of the TSHR is the major form expressed on the thyroid cell membrane. 4,19 It has been shown that this region can be deleted from the TSHR without affecting TSH binding and TSHR activation induced by TSH. 33 In agreement with these observations, this region appears to be outside of the modeled TSH binding pocket. 28 Therefore, this w50-amino-acid region is not involved in TSH binding. It has been shown that TSH enhances this intramolecular cleavage. 21,34 a subunit shedding Following intramolecular cleavage, TSHR a subunits may shed, thus explaining the excess TSHR b subunits on the cell membrane. 5,19 Furthermore, the shed TSHRs have been shown to bind TSH 6 even without the sulfated tyrosine residues on the b subunit. 27 It has also been shown that this shedding is up-regulated by TSH. 6,21 TSHR multimerization While it has been shown that TSHRs expressed on the plasma membrane are constitutively oligomeric 22,23, the effect of dimerization and multimer formation on TSHR function is unknown. However, oligomeric TSHRs have been shown to dissociate into monomeric forms in response to TSH 35, suggesting that the dimeric or multimeric receptor forms are functional and can bind TSH with high affinity. Conclusion Thyrotropin receptor antibodies 37 Taken together, of these four post-translational processes, TSH regulates TSHR monomerization, cleavage, and a subunit shedding. This suggests a physiological relation between these phenomena during and/or following TSHR activation.

6 38 T. Ando et al THE TSHR IN AUTOIMMUNITY Autoimmune thyroid disease (AITD) and the immune response to the TSHR The TSHR is one of the major immune targets, along with Tg and TPO, of autoreactive T cells and autoantibodies in human AITD, including autoimmune hyperthyroidism (GD) and autoimmune hypothyroidism (AT). The reasons for the failure of tolerance to these antigens are uncertain but include a complex mixture of genetic susceptibility and environmental influences (as reviewed 36 ). The major characteristic of TSHR-Abs is their influence on thyroid function via the TSHR by either stimulating or blocking the receptor from activation. This occurs despite the low serum concentrations of TSHR- Abs (!10 mg/ml). 17,37,38 The estimated serum concentration of TSHR-Ab is significantly less than that of thyroid antibodies to TPO and Tg. In addition, TSHR- Abs are commonly oligoclonal and of the IgG 1 subclass as shown by isotyping of heavy and light chains 39,40, although other isotypes have been reported. 41 Stimulating TSHR-Abs Stimulating TSHR-Abs are the cause of hyperthyroidism seen in GD and, therefore, a hallmark of this disease. This was first studied in rodents by demonstrating a prolonged release of radio-iodine from the thyroid gland in response to serum and later IgG from patients with GD compared to TSH (LATS, long-acting thyroid stimulator). 42 This has recently been reproduced by measuring thyroid hormone increases in response to a stimulating TSHR-mAb (Figure 2). 43 Stimulating TSHR-Abs bind to and activate the TSHR and induce excessive thyroid hormone production and secretion from the thyroid gland which, therefore, loses the regulation normally applied by TSH. The resulting thyrotoxicosis explains the constitutional signs and symptoms of GD. Stimulating antibodies also cause transient neonatal hyperthyroidism by transition of IgG through the placenta from mother to fetus. 44 Epitopes of stimulating TSHR-Abs The precise epitope(s) to which stimulating TSHR-Abs bind are not known. This is due to their conformational nature. There is accumulating evidence to indicate that the major binding region resides on the TSHR a subunit where TSH also binds: (1) Stimulating TSHR-Abs from patients with GD can be absorbed by a subunit protein with an intact conformation 37,38 (2) Stimulating TSHR-Abs retain their activity on the chimeric TSHR in which the b subunit has been replaced with corresponding regions from the LHR 45,46 (3) Animal models of GD have been induced by immunization with TSHR a subunits 47,48 (4) The conformational binding site of stimulating mabs to the TSHR generated from rodents 34,49 is on the a subunit (w316 amino acids) 29 (5) Binding of stimulating mabs cannot be seen with short (w20 amino acids) peptide fragments or denatured TSHR antigen, but only with native TSHRs 34,49,50 (6) Stimulating TSHR-Abs from patients with GD stimulate TSHRs which lack tyrosineresidue sulfation on the b subunit known to be critical for TSH binding 27

7 Thyrotropin receptor antibodies µg 5 µg 2.5 µg Ctl (a) 30 * T4 (µg/dl) (h) Figure 2. Acute thyroid stimulation in vivo by MS-1, stimulating TSHR-mAb. (a) CBA/J mice were injected with the indicated doses of MS-1 or control antibody and T4 levels were studied every 24 h. A broken line indicates the upper level for control serum T4; * indicates P!0.01 in T4 levels between 48 h and 72 h with a 10 mg injection. (b) Example of thyroid histology at 96 h in control (left) and MS-1 (10 mg) treated animals (right). Note epithelial hypertrophy. Magnification X 400. Adapted from Ando Tet al. (2004, Journal of Clinical Investigation 113: ) with permission. Therefore, stimulating TSHR-Abs either from GD patients or animal models of GD recognize conformational binding site(s) on the a subunit. It has been shown that TSH competing activity can be detected in most patients with untreated GD (see below). Sera from patients with GD were recently studied in binding inhibition assays using a mouse stimulating TSHR-mAb. 49 There was a linear correlation between TSHR-mAb binding inhibition and TSH competing activity. Therefore, TSH competing activity detected in serum from GD patients reflected mostly TSHR-Abs to the stimulating TSHR-Ab binding sites. In addition, stimulating antibodies either from patients with GD or from animal models of GD recognized the same or closely related conformational binding site(s) as for TSH on the TSHR a subunit. 29,49,50 These data support the concept of a single epitope for stimulating TSHR-Abs. Blocking TSHR-Abs In AT, the predominant pathology is T-cell-mediated apoptosis of thyroid epithelial cells. 51 However, patients with the AT develop blocking TSHR-Abs which bind to

8 40 T. Ando et al the TSHR but fail to activate and block TSH binding to the TSHR, contributing to the resulting hypothyroidism. Blocking TSHR-Abs can also be found in GD patients coincident with the stimulating variety and may contribute to fluctuations in thyroid function, explaining the poor correlation between thyroid function and serum titers of TSHR-Abs. 52,53 However, we have recently shown that such poor correlation can be also explained by TSHR inactivation, desensitization and down-regulation induced by TSHR-Abs. 43 Very rarely, blocking TSHR-Abs may cause neonatal transient hypothyroidism due to the passage of blocking antibodies into the fetal circulation via the placenta. 44 Epitopes for blocking TSHR-Abs Epitopes for blocking TSHR-Abs are widely distributed. Experimentally produced TSHR-mAbs with potent blocking activity have been shown to bind to at least two independent linear (amino acids and amino acids ) 28 and two independent conformational epitopes 29 on the a subunit. There is also at least one linear epitope on the b subunit (amino acids or its vicinity) which has been identified. 27,54,55 We have recently shown that one of these conformational epitopes is also recognized by stimulating TSHR-mAbs by binding competition assay. 29 Autoantibodies from GD patients (w90% of cases examined) and AT (15 out of 16 cases) have also recently been shown to compete with a blocking mab to the N-terminus of the TSHR b subunit, amino acids The same group has demonstrated that purified TSHR-Abs from patients with GD and AT showed similar competition profiles. 57 Independently, Sanders et al. have recently shown that a human stimulating mab competed with autoantibodies seen in AT. 50 Taken together, at least in human AITD, there are at least two binding sites for blocking antibodies, one is on the a subunit and conformational, and the other is on the b and linear. These studies also indicate that binding competition assays using stimulating mabs will still measure both stimulating and blocking antibodies. Neutral TSHR-Abs The neutral TSHR-Abs by definition do not affect TSH binding to the TSHR or TSHR function. The presence of neutral antibodies in AITD was first reported in a patient with GD and monoclonal gammopathy. 58 There have also been reports that many sera from patients with GD bound to short peptide fragments of the TSHR which could represent neutral TSHR-Abs. 3 Using affinity-purified TSHR, it has also been suggested that normal control sera may contain neutral TSHR-Abs. 38 However, TSHR-Abs in healthy individuals have not been detected 59,60 or, when detected, are very few. 41,56,57 In animal models of GD 61 64, the major linear epitopes recognized are mainly N- terminus peptides (w20 amino acids) and/or the TSHR cleaved region. 65 Neither of these epitopes is involved in the TSH binding pocket. 28 Neutral mabs have been also generated from animal models. 28,29,34,61 However, there has not been an extensive study of this class of TSHR-Abs, and therefore their role in immunopathology is poorly understood.

9 Thyrotropin receptor antibodies 41 GRAVES OPHTHALMOPATHY (GO), PRETIBIAL MYXEDEMA (PTM) AND THE ROLE OF THE TSHR GD is associated with retro-orbital inflammation producing the characteristic eye manifestations which are clinically apparent in 25 50% of patients 7 or more if detected radiographically. 66 Much less common are the skin manifestations of PTM. 7 The pathology of both these conditions is thought to arise from a local inflammatory response providing a cytokine milieu causing fibroblasts to secrete and cause the accumulation of glycosaminoglycans, including hyaluronic acid and chondroitin sulfate. 7 This is seen in the interstitial spaces of the retro-orbital fibroblasts, adipose and muscle tissues alongside the diffuse lymphocytic infiltration. The end result is tissue swelling leading to clinical proptosis and muscle dysfunction. In the same way, accumulation of these glycosaminoglycans is seen in the skin pathology of PTM. 7 Humeral immunity to the TSHR antigen is widely observed in GO and PTM. Patients with the highest titers of TSHR-Abs have the most severe form of GD, with marked eye and skin involvement 67, and the titers of TSHR-Abs correlate with the activity of GO. 68 In support of these observations, the expression of the TSHR in retro-orbital fibroblasts of patients with GO has been shown to increase in response to local immune regulators, such as cytokines (e.g. IL-1b 69, IL-6 70 ). The resulting up-regulation of TSHR expression may further promote the infiltration of T cells and the local accumulation of glycosaminoglycans. Hence, TSHR-Abs appear to have an additive role in GO in maintaining the T-cell infiltrate. WHAT MAKES THE TSHR ANTIGENIC? Post-translational processing The major functional TSHR species on the thyroid gland is the cleaved form. 4,19 This TSHR contains a conformational epitope on the a subunit recognized by stimulating and some blocking antibodies as well as the linear binding sites for blocking antibodies on the b subunit. The intervening w50-amino-acid region is not present in the cleaved form of the TSHR. However, this region appears to be one of the major linear epitopes of the TSHR in the animal model of GD. 29,65 These paradoxical findings may be explained by evidence suggesting that stimulating and blocking TSHR-Abs are able to prevent the TSHR from TSH-induced TSHR cleavage and thus may increase the uncleaved holoreceptor species (our unpublished observation). Therefore, the generation of stimulating and/or blocking TSHR-Abs may alter the antigenicity of the TSHR by inhibiting intramolecular cleavage. It has been suggested that shed TSHR a subunits, when shed in vivo, may trigger autoimmune responses to the TSHR, since the a subunit contains the conformational binding site for stimulating TSHR-Abs. Indeed, the shed TSHR from in vitro cell culture medium showed a similar binding of TSHR-Abs from patients with GD as observed with the full-length TSHR. 38 It has also been shown that TSHR-Abs from patients with GD can recognize TSHR multimers. 71,72 However, we have recently found that TSHR-mAb stimulation, in contrast to TSH 35, does not induce monomer formation (Figure 3). 21 These findings may explain the mechanism for the prolonged TSHR stimulation with stimulating TSHR-Abs. However, the role(s) of oligomerization in antigenicity to the TSHR is uncertain.

10 42 T. Ando et al Prebleach % recovery Postbleach Time (sec) TSH MS 1 Fab Control MS 1 lgg Figure 3. Lateral movement of TSHR by using fluorescence recovery after photobleaching. The GFP (green fluorescent protein) molecules in TSHR GFP were subjected to spot photobleaching using a 488 nm argon laser. The fluorescence intensity of the spot before and after photobleaching was measured. The % intensity indicates the percentage fluorescent intensity of the bleached points divided by that of unbleached reference point. Cells were pretreated for 20 min before the experiment as indicated. TSH caused a significant increase (P!0.001) in % recovery, as did MS-1 Fab (P!0.05). Intact MS-1 decreased the % recovery (P!0.05). Evidence from animal models of GD The Shimojo mouse model of GD 64 was the first breakthrough in the field of experimental GD modeling. Using fibroblasts expressing both the TSHR and MHC class II antigens, it was found that immunization of syngeneic mice caused the development of GD-like features including goiter, thyroid hypertrophy, and stimulating TSHR-Abs in w20% of mice immunized. 64 MHC class II expression on the fibroblasts was shown to be required to induce stimulating, but not blocking, TSHR-Abs. 73 These studies pointed out the importance of the intact conformation of the TSHR to induce stimulating TSHR-Abs, which was confirmed in other models of GD, utilizing TSHR-expressing plasmid vectors 61,62 and an adenovirus vector. 63 Insights into the induction of stimulating TSHR-Abs In the Shimojo mouse model it has been shown that the first 260 amino acids of the TSHR ectodomain, chimeric with the C-terminus ectodomain of LHR, was essential to induce TSHR-Abs. 48 However, TSHR-Abs were not induced when this region was replaced with the corresponding LHR sequence. 48 Similarly, it has been shown that TSHR ectodomain w289 amino acids expressed by adenovirus vector induced Graves hyperthyroidism more frequently than the wild type. 47 These studies again suggest the importance of this region as a conformational epitope for stimulating TSHR-Abs. Insights into the repertoire of TSHR-Abs Recent evidence has suggested that expressing truncated and secreted TSHR ectodomains in mice induced Graves hyperthyroidism in w60 80%, but only blocking antibodies were induced in animals immunized with a vector expressing a non-cleavable TSHR. 47 As reviewed above, the a subunit is recognized by both stimulating and

11 Thyrotropin receptor antibodies 43 blocking TSHR-Abs and their epitopes appear to be the same or closely related. 29,50 Thus, these results indicate the importance of the form of TSHR antigen exposed in an animal and not just the epitopes presented. These studies also indicate the possible antigenic role of shed TSHR, similar to the truncated a subunit, in the induction and maintenance of GD. 47 A NOTE ON TSHR-SPECIFIC T CELLS Autoimmune disease is initiated by the development of antigen-specific T cells which initiate the immune response. T cells specifically reactive to the TSHR have been characterized in AITD and in particular in patients with GD. 74 The T cells in the immune infiltrate of AITD are markedly restricted in their T-cell receptor V gene use, indicating their prime role in the immune response. 75 The T-cell reaction results in the development of a dominant Th2 response in GD, as evidenced by the secretion of TSHR-Abs, or a dominant Th1 response in Hashimoto s thyroiditis, as evidenced by thyroid cell apoptosis. 51 DETECTION OF TSHR-ABS Competition assays Once it was learned how to label TSH without destroying its biological activity, it was possible to use a protein binding assay for TSH competing activity. Such assays have been widely used for detecting TSHR-Abs since their introduction 30 years ago and have been mostly based on competition for TSH binding to different TSHR preparations. 2 The initial assays used crude homogenized thyroid membranes as a source of TSHRs, but they were troubled by high backgrounds and non-specific effects. 76 The use of detergent-solubilized TSHRs markedly improved such assays which were then introduced commercially. 77 Serum TSHR-Abs inhibit labeled TSH binding to the TSHRs in a dose- and time-dependent manner. These assays are only able to detect TSHR-Abs which bind to the TSHR, both stimulating and blocking TSHR-Abs, and do not detect their biological activity and do not detect neutral TSHR-Abs. The new assays for TSHR-Abs Commercial protein binding assays for TSHR-Abs have been improving in sensitivity with optimization of the contributing components. The original techniques used polyethylene glycol (PEG) precipitation of serum antibodies which then competed for radiolabelled TSH binding to solubilized thyroid membranes (the first-generation assays). 77 Once TSHR-mAbs were available it was possible to immobilize the TSHR and develop solid-phase assays which have demonstrated superior sensitivity (the secondgeneration assays). For example, in one system 78 solubilized recombinant human TSHR is bound to a solid phase by TSHR-mAb recognizing normally conformed TSHR ectodomains without inhibiting TSH binding. 61 In other systems, an ELISA format has been generated in which solubilized porcine TSHR is bound to a solid phase via mab to the cytoplasmic tail of the TSHR. 79 This second generation of assays detect TSHR-Abs in w95% of untreated patients with GD without any loss of specificity. 78,80 84

12 44 T. Ando et al Inhibition of binding to TSHR (%) rd generation 2nd generation 1st generation U/L (International Standard 90/672) Figure 4. A comparison of TSHR-Ab assay. Effect of 90/672 (international standard for TSHR-Abs from the National Institute for Biological Standardization and Control) on inhibition of binding to the TSHR in different assays for TSHR-Abs. Modified and adopted from Rees Smith B et al. (2004, Thyroid 14: in press) with permission. There appears to be no advantage of human over porcine TSHRs, and both sources of TSHR give similar results. Any apparent differences in sensitivity may be due to larger test sample volumes and longer incubation periods. 85 Comparison of such assays needs to be performed by objective investigators under similar assay conditions, as done previously. 84,86 Most recently, a third generation of assay has been developed based on stimulating TSHR-mAb 50 to the TSHR with still more sensitivity for detecting TSHR-Abs. These solid-phase assays use competition for biotinylated TSHR-mAb binding to immobilized TSHRs and are much more stable than assays employing labeled TSH. 87 Using these assays O95% of untreated patients with GD have detectable TSHR-Ab (Figure 4). Bioassays TSHR-Abs were discovered by an in vivo bioassay based on the release of radio-iodine from pre-loaded guinea pigs. 42 Now stimulating TSHR-Abs can be measured by bioassays most commonly depending on the detection of camp generation in thyroid cells or TSHR-expressing CHO cells. 3 The presence of stimulating TSHR-Abs in the serum causes stimulation of the TSHR and enhanced camp accumulation. Blocking TSHR-Abs can be measured by detecting a reduction in TSH-mediated camp generation in the presence of serum. Commercial kits are available even for this technique. Clinical application of assays for TSHR-Abs Many physicians would pay dearly to obtain a marker for the disease which interests them most. Those of us interested in caring for patients with GD have such a marker. The putative clinical indications for the measurement of TSHR-Abs are, therefore, summarized in Table 1.

13 Thyrotropin receptor antibodies 45 Table 1. Indications for measurement of TSHR-Abs. Graves disease Diagnosis Prediction of remission after anti-thyroid drugs 100 Hypothyroidism Diagnosis for the atrophic thyroiditis 101 Graves ophthalmopathy Detection of underlying autoimmunity (with TPO antibody) a102,103 Assessment of disease activity 68 Multinodular toxic goiter Inclusion of co-existing GD b104,105 Neonatal transient hyper- and hypothyroidism c44 a TPO antibodies have been suggested protective in GO. 102 b High prevalence (w20 50%) of co-existence of TBI activity among multinodular toxic goiter has been reported Such cases may not be treated with radio-iodine for frequent complications. 106 c To predict neonatal thyroid function during pregnancy, the function of persistent TSHR-Abs should be determined by using bioassay. Detection of neutral TSHR-Abs There are no commercial clinical assays to measure this class of TSHR-Abs. Neutral antibodies can be detected experimentally by using CHO cells expressing the TSHR and subjecting them to flow cytometry 41,59,60, by ELISA systems using recombinant native TSHR 38,88, by immunoblotting 72,89, or by immunoprecipitation CHANGES IN TSHR-AB LEVELS WITH TREATMENT OF AITD Antithyroid drugs (ATDs) Over 50% of patients with hyperthyroid GD relapse after a 12-month course of ATDs, the actual percentage varying among populations and with their iodine intake. 92 The level of TSHR-Abs falls on treatment with ATDs secondary to their immunosuppressive action and the induced decrease in thyroid antigen expression. 93 The measurement of TSHR-Abs in patients with GD has proven to be a useful predictor of relapse and remission after ATD treatment since first used in ,94 The presence of significant titers of biologically active TSHR-Abs accurately predicted recurrence in up to 90% of GD patients. Similar data had been published previously using indirect measurements of TSHR-Abs: for example, the pioneering work of Alexander, who showed clearly that the failure of T3 to suppress radio-iodine uptake increased the chances of recurrence since a non-tsh thyroid-stimulator of radio-iodine uptake was present. 95 In an often-quoted metaanalysis different reports at that time were available which investigated the usefulness of TSHR-Abs in the prediction of relapse and remission, but the methods for TSHR-Ab measurement varied widely and prevented any valid firm conclusions. The overall assumption of the meta-analysis, therefore, was that all the different assays used in the 18 different studies were of equal sensitivity, equal precision and, most importantly, equal specificity. However, many of the studies used

14 46 T. Ando et al their own in-house methods for the measurement of TSHR-Abs, sometimes for the first time. Therefore, there was a lack of essential data on the assay performances which would have made a meta-analysis difficult to interpret. It is currently not known whether the recent improvements in sensitivity and/or specificity of TSHR- Ab assays will further improve the accuracy of relapse prediction and evaluation for treatment. 81,83 Potential factors interfering with the action of TSHR-Abs Logically, the presence of stimulating TSHR-Abs in the serum of a patient with recent GD must influence thyroid function unless there is some interference in the transduction of such a signal. However, there are a number of possible reasons for thyrotoxic recurrence after ATDs being unpredictable. These include: Antibody quantity: TSHR-Abs are found in very low concentrations in many GD patients 17,37,38 Antibody affinity: the affinity of TSHR-Abs for the TSHR has been shown with studies of mabs to be highly variable 29,49,50 Problems with thyroid function: there are two major potential disturbances in the thyroid reserve which may prevent an appropriate thyroid hormone response to stimulating TSHR-Abs iodine deficiency, which varies geographically, and autoimmune thyroiditis which commonly accompanies GD 2 Changes in TSHR-Ab levels following radiation and surgery TSHR-Ab levels eventually fall after treatment of GD with ATDs, radio-iodine and surgery. 52 However, TSHR-Ab levels show a marked, but transient, increase after RAI therapy, perhaps secondary to the differential loss of intrathyroidal regulatory T cells. 97 It is this increase which has been correlated with worsening of GO after RAI therapy and has led to caution in the use of this mode of treatment in patients with significant eye disease. 98 TSHR-Ab levels in pregnancy The thyroid autoimmune response diminishes during pregnancy, resulting in remission of GD in the vast majority of patients. 99 However, some patients with more severe disease and high levels of TSHR-Abs have persistence of these antibodies into the third trimester. The validity of predicting neonatal GD using the measurement of biologically active TSHR-Abs in the mother has been well documented for many years. 44 However, this is the only clinical situation where the bioactivity of TSHR-Abs may need to be known in order to predict neonatal thyroid function. Luckily, pregnancy is a time when thyroid autoantibodies generally decrease in titer, due the secretion of placental factors which are immunosuppressive, and bioactivity assessment is not necessary in many patients. 44 However, in those GD patients with higher titers of TSHR-Ab by competition assay in the third trimester a subsequent bioassay will allow the prediction of neonatal hyperthyroidism or blocking antibody-induced hypothyroidism. 44 This approach applies most importantly to women previously treated for GD who are euthyroid or on thyroxine replacement but have persisting titers of TSHR-Abs.

15 Thyrotropin receptor antibodies 47 SUMMARY The intact conformation of TSHR is of paramount importance for TSH and TSHR-Abs to stimulate the TSHR. The structure of the TSHR, because of complex posttranslational modifications, may influence the immune response. Extensive studies are needed to elucidate regulation of the TSHR structure by TSH and/or TSHR-Abs and any influence of such a regulation on the antigenicity of TSHR. The studies may reveal new mechanisms in AITD. The clinical role of TSHR-Abs in AITD is well established, but new and highly sensitive assays will increase the clinical utility of the assessment. Practice points the TSHR is the major antigen of Graves disease, both the thyroid and extrathyroidal manifestations the measurement of TSHR-Abs has specific clinical indications, including the prediction of recurrence after a course of antithyroid drugs the availability of stimulating TSHR monoclonal antibodies makes the development of highly sensitive assays for TSHR-Abs possible in the near future Research agenda the physiological role of TSHR cleavage and subunit shedding requires elucidation modeling of TSHR-Ab conformational epitopes on the TSHR will provide insight into the molecular mechanisms of Graves disease clarification of the role of extra-thyroidal TSHRs is needed, in particular their role in the maintenance of tolerance to the TSHR antigen in normals REFERENCES 1. Davies TF, Marians R & Latif R. The TSH receptor reveals itself. J Clin Invest 2002; 110: Rees Smith B, McLachlan SM & Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev 1988; 9: Rapoport B, Chazenbalk GD, Jaume JC et al. The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 1998; 19: Misrahi M, Ghinea N, Sar S et al. Processing of the precursors of the human thyroid-stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus-infected insect cells). Eur J Biochem 1994; 222: Tanaka K, Chazenbalk GD, McLachlan SM et al. Subunit structure of thyrotropin receptors expressed on the cell surface. J Biol Chem 1999; 274: Couet J, Sar S, Jolivet A et al. Shedding of human thyrotropin receptor ectodomain. Involvement of a matrix metalloprotease. J Biol Chem 1996; 271: Prabhakar BS, Bahn RS & Smith TJ. Current perspective on the pathogenesis of Graves disease and ophthalmopathy. Endocr Rev 2003; 24:

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