Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes.

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein significantly reversed the TBII as well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of these rabbit antisera, we identified three discrete regions of the TSH R, represented by amino acids 202-221, 292-311 and 367-386, as TBII epitopes and four regions represented by amino acids 352-371, 367-386, 382-401 and 392-415 as TSBAb epitopes. These data demonstrate that rabbit antibodies that bind to amino acids 367-386 mediate their TSBAb activity by inhibiting the binding of TSH to TSHR; whereas, antibodies to regions 352-415, excluding aa 367-386, exert their TSBAb activity by affecting a step subsequent to TSH binding. Coincident with the elevation of TBII and TSBAb activity, serum total T4 levels declined and thus suggested that the antibodies exerted functional effects on thyroid in vivo. Together, these data demonstrate that glycosylated hET-gp protein is a more potent immunogen and it can induce a broader antibody response directed against multiple TBII and TSBAb epitopes.

Soluble ecto-domain mutant of thyrotropin (TSH) receptor incapable of binding TSH neutralizes the action of thyroid-stimulating antibodies from Graves' patients.

A soluble form of the amino-terminal extracellular (ecto-) domain of the human TSH receptor was generated. This protein was capable of binding TSH and autoimmune antibodies found in Graves' patients. A deletion mutant of the ectodomain lacking nine amino acids in the C-terminal region lost its ability to interact with TSH but retained binding to Graves' IgGs. In cells expressing recombinant TSH receptors, cotreatment with the mutant protein blocked the cAMP production induced by stimulating antibodies from all Graves' patients tested but was without effect on TSH action. The ability to dissociate the actions of TSH and Graves' IgGs provides a tool with which to study the mechanisms underlying Graves' disease and the possibility of neutralizing the undesirable effects of thyroid-stimulating antibodies without altering the normal responses to TSH.

Flow cytometric analyses of antibody binding to Chinese hamster ovary cells expressing human thyrotropin receptor.

To develop a method that can be used to directly detect binding of antibodies to TSH receptor (TSHr), we employed Chinese hamster ovary (CHO) cells permanently transfected with a human TSHr complementary DNA (CHOR). These cells showed increased cAMP production when treated with either human TSH or thyroid-stimulating antibodies and decreased TSH-mediated cAMP production when treated with stimulation-blocking antibodies. We employed flow cytometry and rabbit antibodies against the extracellular domain of the TSHr (ETSHr) to test whether these cells can be used to directly detect and quantitate the binding of anti-TSHr antibodies. Rabbit anti-ETSHr bound specifically to CHOR cells, and the binding could be blocked with purified ETSHr. To test the feasibility of using these cells for epitope mapping, we tested the binding of rabbit antibodies raised against several synthetic TSHr peptides. Rabbit antipeptide 92 (amino acids 12-30) and 91 (amino acids 32-46) showed little or no binding to the CHOR cells. In contrast, antibodies raised against peptides 93 (amino acids 316-330), 95 (aa 325-345), 3A (aa 357-372), 367 (aa 367-386), and 1B (aa 362-376) showed significant binding to the CHOR cells. The specificity of binding of antipeptide antibodies was demonstrated by a complete inhibition of binding by corresponding peptides. When TSH-binding inhibitory Ig-positive sera from 15 patients with hyperthyroidism were tested, 8 of them showed specific binding to the CHOR cells compared to their relative binding to normal CHO cells; sera from all normal individuals tested did not exhibit specific binding to CHOR cells. These studies showed the usefulness of CHOR cells and flow cytometry in epitope mapping using sera with known specificities and the potential usefulness of the technique to detect anti-TSHr antibodies in patient sera.

Differential reactivities of recombinant glycosylated ectodomains of mouse and human thyrotropin receptors with patient autoantibodies.

We expressed the extracellular domain of the mouse TSH receptor (mET-gp) using the baculovirus expression system. The recombinant protein was identified as mET-gp by immunoblotting and N-terminal amino acid sequencing. Carbohydrate analysis of the recombinant protein showed that the protein is glycosylated. Experimental antibodies raised against the extracellular domain of the human TSHr (ETSHr) were assayed for reactivity against mET-gp and glycosylated human ETSHr (ETSHr-gp) in an ELISA and found to be comparable. Similarly, both mET-gp and ETSHr-gp proteins neutralized the TSH binding inhibitory immunoglobulin (TBII) activity of rabbit anti-ETSHr antibodies in a RRA. However, when these proteins were compared for their ability to neutralize TBII and blocking activities (TSBAb) of IgG from patients with thyroid autoimmune disorders, only ETSHr-gp was able to neutralize these activities. In contrast, mET-gp partially neutralized, whereas ETSHr-gp completely neutralized the stimulatory (TSAb) activities of IgG from patients. Analyses of reactivities of these two proteins against a panel of antipeptide and monoclonal antibodies and their protein sequences showed differences in some specific epitopes. These data showed that in spite of significant homology between the two proteins, they exhibit specific epitope differences that are sufficient to cause divergence in their ability to react with patient autoantibodies to TSHr. This suggests that the two proteins might differ in their three-dimensional structure.

Requirement of glycosylation of the human thyrotropin receptor ectodomain for its reactivity with autoantibodies in patients' sera.

To understand the role of glycosylation on autoantibody reactivity, we expressed cDNA encoding amino acid residues 22 to 416 of the human thyrotropin receptor (TSHR), along with the baculovirus-encoded glycoprotein 67 signal sequence (ETSHR-gp) in insect cells. N-terminal sequence analysis revealed that the signal peptide was cleaved and confirmed the identity of ETSHR-gp protein. The molecular mass of the ETSHR-gp protein was 63 kDa and was higher than the expected molecular mass of 45 kDa, suggesting that the protein was glycosylated. Carbohydrate analysis showed that the protein was glycosylated and that mannose was the major oligosaccharide. A nonglycosylated recombinant ETSHR protein expressed earlier in our laboratory neutralized TSH-binding-inhibitory Ig (TBII) activity in the sera of rabbits immunized with the protein but did not neutralize TBII activity in the sera of patients. In contrast, the glycosylated ETSHR-gp protein neutralized TBII activity in the sera of both experimental animals and patients with autoimmune thyroid disorders. Furthermore, only the ETSHR-gp protein completely neutralized the activities of stimulatory and blocking Abs in the sera of patients with hyperthyroidism and hypothyroidism, respectively. These data clearly show that glycosylated ETSHR-gp, but not the nonglycosylated ETSHR protein, can react with autoantibodies in patients' sera and that it has the epitopes required for the binding of TBII, thyroid stimulatory Abs, and thyroid stimulatory blocking Abs. Moreover, these data suggest that glycosylation might be an important determinant of autoantigenicity of human TSHR.

Androgen regulation of growth hormone binding protein.

Male puberty is associated with elevated plasma concentrations of growth hormone (GH) and insulin-like growth factor-I (IGF-I), as well as accelerated linear growth. These effects can be reproduced by administration of testosterone (T). To further elucidate the mechanisms underlying pubertal growth, we treated 14 boys with delayed puberty and short stature with either T (n = 7) or 5alpha-dihydrotestosterone (DHT) (n = 7) and compared the effect on plasma concentrations of GH, IGF-I, and GH binding protein (GHBP). Before treatment and after either three or four doses of T enanthate or DHT heptanoate, mean 12-hour GH concentration (8 AM to 8 PM) and plasma IGF-I, T, DHT, and GHBP levels were measured, and height velocity (HV) was measured over this interval. T treatment resulted in an increase of mean GH from 3.3 to 12.0 microg/L (P < .005) and of IGF-I from 22.3 to 45.4 nmol/L (P < .01). During treatment, HV was 11.0 +/- 1.1 cm/yr, consistent with normal pubertal growth, and plasma T was 22.5 +/- 5.3 nmol/L. GHBP decreased in this group from 937 to 521 pmol/L (P < .025). DHT treatment resulted in a small decrease of mean GH from 4.3 to 2.9 microg/L (P < .025) and of IGF-I from 29.4 to 27.2 nmol/L (nonsignificant [NS]). During treatment, HV was 9.3 +/- 1.1, not significantly different from the HV obtained with T treatment, and plasma DHT was 24.2 nmol/L at 1 week and 29.2 at 2 weeks postinjection. Likewise, there was a decrease in GHBP from 928 to 698 pmol/L (P < .025). The decline in GHBP with T treatment was apparently due to an androgen receptor-dependent mechanism, since the same effect was seen during treatment with the nonaromatizable androgen, DHT. This effect is opposite to the normal chronological trend upward for GHBP, which occurs from infancy into midpuberty. Factors determining the upward trend are not known, but are evidently independent of the plasma concentration of sex hormones and GH. The increase in IGF-I in response to T treatment despite a moderate decline in GHBP (and possibly GH receptor) levels is most likely due to the large increase in GH, which may override a modest decrease in GHBP/GH receptor.

Thyrotropin (TSH) receptor antibodies (TSHrAb) can inhibit TSH-mediated cyclic adenosine 3',5'- monophosphate production in thyroid cells by either blocking TSH binding or affecting a step subsequent to TSH binding.

In the present study, rabbit antibodies that possess thyroid stimulation-blocking activity were used to investigate potential mechanisms by which TSH receptor antibodies can inhibit thyroid cell function. The antibodies were produced against two synthetic peptides corresponding to amino acids 357-372 (p357) and 367-386 (p367) of the human TSHr (hTSHr). By enzyme-linked immunosorbent assay, both antisera (alpha 357 and alpha 367) had high titers ( > 1:100,000) of IgG against their respective peptides and recombinant extracellular TSHr protein (ETSHr); alpha 357 had a low IgG titer to p367 (1:800), and alpha 367 had a low IgG titer to p357 ( < 1:200). Based on competitive inhibition studies, alpha 357 and alpha 367 displayed similar relative binding affinities for their respective peptides and for recombinant ETSHr. When tested by commercial RRA, alpha 357 did not block (TSH binding inhibition index, -3.7%), whereas alpha 367 blocked TSH binding to TSHr (TSH binding inhibition index, 53.9%). The blocking effect of alpha 367 could be reversed by incubating the antiserum with p367 before assay. When applied alone to FRTL-5 cells, IgG from alpha 357 inhibited [compared to normal rabbit IgG (NRI); P < 0.01] based cAMP production by the cells, whereas IgG from alpha 367 did not. IgG from both alpha 357 and alpha 367, however, were able to inhibit (P < 0.001) TSH-mediated cAMP production by FRTL-5 cells [bovine (b) TSH, 2.5 x 10(-10) M; cAMP (mean +/- SD; picomoles per ml): NRI, 62.5 +/- 6.1; alpha 357, 12.2 +/- 2.4; alpha 367, 36.2 +/- 3.5]. Alpha 357 continued to inhibit (P < 0.05) cAMP production by FRTL-5 cells in 10(-8) M bTSH, whereas alpha 367 no longer inhibited cAMP production at bTSH concentrations above 5 x 10(-10) M. Compared to NRI, both alpha 357 and alpha 367 were also able to inhibit (P < 0.001) Graves' IgG-mediated cAMP production by FRTL-5 cells. When IgG were tested on FRTL-5 cells in the presence of 10(-7) M forskolin, only alpha 357 inhibited (P < 0.001) cAMP production (NRI, 75.1 +/- 4.8; alpha 357, 52.3 +/- 4.5; alpha 367, 77.2 +/- 1.4). To determine whether the inhibitory effect of alpha 357 on forskolin-mediated stimulation was thyroid cell dependent, IgG were tested on Chinese hamster ovary (CHO) cells transfected with the complementary DNA of the hTSHr (CHO-R). Again, alpha 357 inhibited (P < 0.005) cAMP production mediated by forskolin (at 10(-7) M; NRI, 68.7 +/- 4.4; alpha 357, 36.8 +/- 5.7; alpha 367, 64.6 +/- 8.5). alpha 357 did not inhibit forskolin-mediated cAMP production by untransfected CHO cells (CHO-N), indicating that the inhibitory effect of alpha 357 on forskolin stimulation was TSHr dependent. In addition, alpha 357 inhibited (P < 0.01) basal cAMP production by CHO-R cells, but not by CHO-N cells. alpha 367 had no effect on the basal cAMP production in either CHO-R or CHO-N cells. Neither alpha 357 nor alpha 367 inhibited cholera toxin-mediated cAMP production in FRTL-5 cells. In all relevant bioassays, the inhibitory effects of alpha 357 and alpha 367 could be reversed by preincubating the IgG with the respective peptides. From these data, we conclude that 1) alpha 367 binds to the ETSHr and blocks TSH-mediated cAMP production by inhibiting TSH from binding to its receptor; 2) alpha 357 binds to the TSHr and, without blocking TSH binding, inhibits TSH-mediated cAMP production at a step(s) subsequent to ligand binding that affects adenylate cyclase activity; and 3) forskolin-mediated cAMP production by thyroid cells can be inhibited by IgG that bind directly to the TSHr.

The effects of testosterone and dihydrotestosterone on hypothalamic regulation of growth hormone secretion.

Testosterone (T) administration to pubertal boys increases spontaneous GH secretion. It is not known whether this occurs via pituitary or hypothalamic mechanisms. We evaluated the GH secretion of 12 boys, aged 13.67 +/- 0.37 yr (mean +/- SE), diagnosed with constitutional delay in growth and adolescence. The evaluation was made both before and after 3 months of treatment with T or the nonaromatizable androgen, 5 alpha-dihydrotetosterone. Serum for determination of spontaneous GH secretion was sampled every 20 min for 24 h. Pituitary responsiveness was assessed by the administration of GHRH with sampling of GH at intervals for the next 2 h. This was also done with pyridostigmine (PDS) pretreatment to assess the effects of somatostatin. The dose of androgen used was 80 mg/m2 month. All tests were then repeated during treatment. Spontaneous GH secretion was analyzed by the Cluster method. The response to GHRH was measured as the area under the curve. Somatostatin effects were quantified as the difference in responsiveness between the two GHRH tests performed at each admission: one without prior PDS administration and one in which somatostatin was blocked by PDS. Treatment with T increased mean spontaneous GH secretion from 2.25 +/- 0.34 micrograms/L before treatment to 6.77 +/- 0.69 micrograms/L (mean +/- SE; P < 0.001) and mean spontaneous peak height from 5.62 +/- 1.05 to 17.21 +/- 1.52 micrograms/L (mean +/- SE; P < 0.001). No significant differences between pretreatment and treatment evaluations for any spontaneous GH secretory parameters were seen in 5 alpha-dihydrotestosterone-treated patients, except that maximum peak height was decreased after treatment (P < 0.02). In T treated patients, the GHRH stimulation tests without prior PDS administration changed from 84.14 +/- 34.54 total micrograms/L before to 102.3 +/- 35.82 total micrograms/L (mean +/- SE; P = NS) after androgen treatment. PDS pretreatment produced an increase in responsiveness to GHRH over the test without PDS pretreatment. This increase was 127.03 +/- 35.68 total micrograms/L before T treatment; after T treatment, this increase was 78.38 +/- 57.6 total micrograms/L (mean +/- SE; P = NS). T treatment, via an estrogen-dependent mechanism, caused increased GH pulse amplitude, thereby increasing the mean serum GH concentration. This increase was not the result of increased pituitary responsiveness or decreased somatostatin tone. This indicates that T exerted its effect on GH via increased GHRH pulse amplitude.

Experimental autoimmunity to thyrotropin receptor.

Since the cloning of a full length cDNA encoding the thyrotropin receptor (TSHr), several laboratories have been actively trying to develop an optimal animal model to understand the pathogenesis of TSHr mediated autoimmune diseases and have made considerable progress. To date, results from our laboratory have indicated that the nature of the antigen, and the adjuvant used for immunization, immunogenetic background of the animal and fine specificities of antibodies elicited might play an important role in determining the qualitative nature of the antibody response. Although an ideal animal model for either Graves' disease or primary myxedema is not yet available, ongoing studies in our laboratory and elsewhere hold promise for establishing animal models for various TSHr mediated autoimmune diseases in the near future.

Thyrotropin receptor-specific antibodies in BALB/cJ mice with experimental hyperthyroxinemia show a restricted binding specificity and belong to the immunoglobulin G1 subclass.

Immunization with the extracellular domain of TSH receptor (TSHR) led to the development of hyperthyroxinemia in BALB/cJ, but not C57BL/6J, SJL/J, and B10.BR, mice. Earlier, human studies had shown that thyroid-stimulating antibodies are predominantly of the immunoglobulin G1 (IgG1) subclass with a narrow specificity to TSHR, and antibodies that block thyroid function could be of any subclass with a broader specificity. Therefore, antibody responses in susceptible (BALB/cJ) and resistant (SJL/J) mice were characterized. There were no significant differences in the titers, relative affinities, or isotypes of antibodies against the TSHR. BALB/cJ and SJL/J sera reacted with 2 and 7 of 26 overlapping peptides from the extracellular domain of the TSHR. The ability of sera from BALB/cJ and SJL/J mice to block TSH binding to TSHR was reversed by 1 and 6 of the reactive peptides, respectively. BALB/cJ mice showed predominantly an IgG1 response against the TSHR and peptides, whereas SJL/J mice showed varying levels of all IgG subclasses. Although SJL/J sera reacted with peptides to which blocking antibodies bind, they did not show hypothyroidism, suggesting that their sera contained a mixture of blocking and stimulating antibodies that negated the effects of each other. In contrast, some TSHR-specific antibodies in BALB/cJ probably represented stimulating antibodies.

A region on the human thyrotropin receptor which can induce antibodies that inhibit thyrotropin-mediated activation of in vitro thyroid cell function also contains a highly immunogenic epitope.

Autoantibodies to the thyrotropin receptor (TSHr) bind to the extracellular domain of the TSHr (ETSHr) and either stimulate or inhibit thyroid cell function and/or growth. In order to investigate the regulation and the specificity of the immune response to the TSHr, our laboratory recently produced recombinant human ETSHr protein by using the baculovirus expression system. In the present study, we used the recombinant ETSHr protein, a panel of overlapping synthetic peptides derived from the TSHr, and polyclonal rabbit antibodies produced against recombinant ETSHr and synthetic peptides to define a highly immunogenic region (aa 352-388) of the TSHr. Moreover, we used competitive inhibition studies to identify a dominant epitope (aa 367-372) within this region to which ETSHr antibodies react. This immunodominant epitope lies within a region unique to the TSHr when compared to the other glycoprotein hormone receptors. These data, together with the earlier observation that antibodies against aa region 357-372 can inhibit thyrotropin (TSH)-mediated activation of thyroid cells in culture, show that aa 367-372 represents, an immunodominant epitope within a functionally important region which is unique to the TSHr. Therefore, this region may play an important role in the induction or modulation of the specific immune response against the TSHr.

High frequency of B cells capable of producing anti-thyrotropin receptor antibodies in patients with Graves' disease.

Hyperthyroidism in Graves' disease (GD) is mediated by antibodies to the thyrotropin receptor (TSHr). Patients that go into remission show a decline in antibody titer. However, upon cessation of treatment with anti-thyroid drugs a significant proportion of patients relapse and TSHr antibodies (TSHrAb) are present in their circulation. This suggests that B cells capable of producing TSHrAb persist despite treatment. To determine the frequency of these cells, B cells from six patients with GD and four healthy controls were infected with Epstein-Barr virus and cultured in 96-well plates at varying cell concentrations. A higher frequency of B cells capable of producing TSHrAbs was detected in patients with GD, relative to normal controls. For example, at 2 x 10(5) cells per well, 100% of wells containing cells from either patients with GD or controls were positive for immunoglobulin (Ig) production. In contrast, 27% of the wells containing cells from Graves' patients, and only 3% from controls, were positive for TSHrAb. Higher titers of TSHrAbs were produced in cultures containing lymphocytes from patients with GD and were predominantly of IgG isotype. All patients with GD who had high thyrotropin binding inhibitory immunoglobulins also had higher frequencies of TSHr-specific B cells. These findings show that TSHrAb-producing B cells are present at a higher frequency in the peripheral circulation of patients with GD.

Thyrotropin (TSH) interacts with multiple discrete regions of the TSH receptor: polyclonal rabbit antibodies to one or more of these regions can inhibit TSH binding and function.

As a means of identifying functional regions of the TSH receptor (TSHr), we immunized four rabbits with recombinant extracellular TSHr (ETSHr) protein and systematically evaluated their antibody response. The antibody response was characterized by testing serial serum samples for immunoglobulin G (IgG) against ETSHr protein and 26 synthetic peptides which span the entire ETSHr. Sera were also tested for their ability to block TSH binding to native TSHr. All four rabbits developed high serum IgG titers (>1:100,000) to ETSHr. None of the rabbits developed significant IgG titers against 11 of the peptides, but each showed persistent high titers against several of the others. After multiple inoculations of antigen, sera from 3 rabbits showed significant ability to block TSH binding. Based on the ability of peptides to reverse this blocking activity, we identified 3 regions of the TSHr (i.e. amino acids 292-311, 367-386, and 397-415) through which antibodies can block TSH binding. Moreover, antibodies purified on either peptide 292-311 or peptide 367-386 affinity columns could block both TSH binding and TSH-mediated activation of thyroid cells in culture. These studies show ETSHr protein is sufficient to induce production of functionally relevant antibodies. Furthermore, we have identified several sites on the TSHr through which antibodies can inhibit TSH binding, thus leading to identification of several potential TSH-binding regions.

A recombinant extracellular domain of the thyrotropin (TSH) receptor binds TSH in the absence of membranes.

We produced large quantities of the extracellular domain of the human TSH receptor (ETSHR) using the baculovirus expression system. Insect cells containing the ETSHR protein were sequentially extracted using lysis, nuclease, and high salt buffers to enrich for recombinant protein. The ETSHR protein was purified to homogeneity on a C4 reverse phase semipreparative column using HPLC. The recombinant protein was identified as ETSHR by immunoreactivity with antibodies prepared against TSHR-derived synthetic peptides. The identity of the ETSHR was further confirmed by amino acid compositional analyses, which agreed with the amino acid composition predicted from reported cDNA sequence analyses. Protein sequence analyses confirmed that the first 26 amino acids of the N-terminal region and the C-terminal amino acid were identical to the predicted amino acid sequence. The purified ETSHR was refolded in the presence of 1.5 M guanidine-HCl and 1 mM each of cystine and cysteine. [125I] TSH bound to the refolded ETSHR in vitro in a dose-dependent manner and was specifically blocked by unlabeled TSH, but not by LH or FSH. It was notable that a membrane requirement was not essential for TSH to bind to ETSHR.

Induction of hyperthyroxinemia in BALB/C but not in several other strains of mice.

We recently expressed the extracellular domain of the human TSHR (ETSHR) protein using a baculovirus expression system and purified it to homogeneity. The ETSHR specifically binds both TSH and antibodies to TSHR. In the present study, C57BL/6J, SJL/J, BALB/cJ and B10BR.SgSnJ mice were immunized with the recombinant ETSHR or an equivalent amount of control antigen. All strains of mice produced high titers of antibody against the TSHR protein which were capable of blocking the binding of TSH to native TSHR. However, only BALB/cJ mice showed significantly elevated levels of thyroxine in their sera compared to the control mice. Similarly, BALB/cJ mice primed with ETSHR and then challenged with thyroid membranes showed significantly elevated levels of thyroxine. In addition, histopathological examination of thyroid glands from affected mice showed morphological changes characterized by hydropic and subnuclear vacuolar changes and focal scalloping, with no apparent inflammation or glandular destruction. Moreover, mice with elevated thyroxine levels showed increased in vivo thyroidal uptake of 131Iodine. Together, these data suggest that BALB/cJ mice are susceptible to the induction of hyperthyroxinemia.

Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSH receptor peptides.

We prepared a series of overlapping peptides (29 in total, 20 amino acids each) containing the sequence of the entire extracellular domain of the human TSH receptor. Three peptides (181-200, 376-394, and EC3 (629-639)) bound IgG from patients with Graves' disease in an enzyme linked immunoassay. Peptide 181-200 bound IgG from 9 of 10, EC3 from 8 of 10, and 376-394 from 6 of 10 patients respectively, compared to 0 of 9 controls. We affinity purified TSHr auto-antibodies from four Graves' patients using the three above noted peptides bound to epoxy-activated sepharose. Thyroid stimulating activity was enriched in the bound fraction from at least two of the three peptide affinity columns in each of the four patients, although the pattern of affinity enrichment differed between patients. One patient was found to possess a combination of stimulatory and inhibitory TSHr antibodies and, after affinity purification, the anti-376-394 and anti-EC3 fractions were enriched in stimulatory activity, suggesting that those regions of the receptor were epitopes for stimulatory antibodies. However, affinity purification against peptide 181-200 produced an IgG preparation that was not stimulatory, but was a potent thyroid inhibitor. Thus, we have not only partially purified TSHr auto-antibodies, but also successfully separated stimulatory and inhibitory antibodies from a single patient using combination TSHr peptide affinity.

Heterogeneity in cellular and antibody responses against thyrotropin receptor in patients with Graves' disease detected using synthetic peptides.

Graves' disease (GD) is characterized by the presence of autoantibodies to the thyrotropin receptor (TSHr). These antibodies bind to the TSHr and stimulate thyroid cells, thus causing hyperthyroidism. To understand the regulation of TSHr-specific immune responses in Graves' disease, it is important to evaluate the T-cell response in patients with GD against TSHr. In this study we used 11 different peptides that were derived from two regions (i.e. amino acid, AA 12-46 and 316-397) unique to the TSHr when compared to other glycoprotein hormone receptors, and which also have the highest predicted immunogenicity. We evaluated both lymphocyte proliferation as a measure of T-cell response and antibody binding to each of these peptides in nine patients with GD and eight healthy subjects. Patients with GD showed considerable lymphocyte proliferative and antibody responses against several of these peptides. There was considerable heterogeneity in immune responses amongst the patients. Moreover, our data suggested that several peptides contained both T cell and antibody reactive epitopes and might represent some of the highly immunogenic regions of the TSHr.

Dual mechanism of perturbation of thyrotropin-mediated activation of thyroid cells by antibodies to the thyrotropin receptor (TSHR) and TSHR-derived peptides.

To further define the epitopes with which anti-TSH receptor (anti-TSHR) antibodies react and mediate their biological effects, we used antibodies against the extracellular domain of TSHR (ETSHR) protein and nine peptides derived from the ETSHR. Peptides were chosen based on their predicted immunogenicity as well as their uniqueness to the TSHR. Antipeptide antibodies showed varying degrees of reactivity against ETSHR, with antipeptide-2-(352-366) and -3A-(357-372) showing relatively stronger reactivity with the receptor. Antibodies were tested for their ability to stimulate thyroid cells and were found to be ineffective in causing both cAMP release and iodide uptake. However, anti-3A and anti-ETSHR showed blocking TSHR antibody (TSHRAb) activities of 76.9% and 79.7%, respectively, which were significantly different (P < 0.005) compared to that of preimmune serum. Anti-2 and -91 (AA 32-46) also showed blocking TSHRAb activities of 37.5% and 35.6%, respectively (P < 0.05). Antisera were also tested for their ability to block TSH binding to thyroid membranes in a RRA. Anti-ETSHR, but not any of the antipeptide antibodies, displayed TSH binding inhibitory immunoglobulin activity. These findings suggest that there might be different mechanisms that mediate blocking TSHR antibody activity. One mechanism involves the inhibition of TSH binding to the receptor, and the other probably involves a step subsequent to TSH binding.

Immunization of mice with Yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies.

It is well established that autoimmune Graves' disease, which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHr). Although what initially triggers this autoantibody response is not known, a number of studies have suggested that Yersinia enterocolitica, an enterobacteria, could initiate the immune response against the TSHr. In this study, we produced antibodies against purified extracellular domain of human TSHr (ETSHr) and showed that anti-ETSHr antibodies reacted with envelope preparations from Y. enterocolitica. This reactivity was specifically blocked by preincubating sera with purified ETSHr. Moreover, antibodies reactive with ETSHr were induced by immunizing mice with Y. enterocolitica but not with Shigella flexneri SA100, Salmonella typhimurium TML, and Listeria monocytogenes. Anti-Y. enterocolitica antisera specifically reacted with the ETSHr protein and the reactivity could be blocked both by ETSHr and Y. enterocolitica envelope proteins. Our studies provide the first direct evidence that immunization with Y. enterocolitica can lead to the production of antibodies capable of reacting with TSHr and might provide the initial stimulus necessary for breakdown of self-tolerance to TSHr, eventually leading to the development of autoimmunity to TSHr.