TSH Binding Inhibitory Immunoglobulin Activity Research Articles

Inhibitory Effect of Thyroid Blocking Antibody (TBAb) on the Thyroid Stimulatory Effect of Human Chorionic Gonadotropin (HCG ) and Equine Chorionic Gonadotropin (ECG)

We examined the inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulating activity of human chorionic gonadotropin (HCG) and equine CG (ECG). Five TBAb positive sera obtained from patients who had been hypothyroid but were currently on T4 treatment. The TSH binding inhibitory immunoglobulin (TBII) activities of the sera were 60-160 IU/L. Inhibition of TSH binding to the TSH receptor (TSHR) [TSH binding inhibition (TBI) activity] of HCG or ECG, and inhibition of TBAb on HCG or ECG-stimulated cAMP production were examined. Both HCG and ECG preparations showed weak TBI activity in the presence of small amounts of protein [bovine serum albumin (BSA)] but were negative in the presence of large amounts of protein [normal human serum (NHS) or BSA]. Four thousand IU/mL of HCG and ECG preparation caused cAMP production similar to 100 microU/mL of bovine (b) TSH. The inhibitory effect of TBAb on cAMP production by this amount of HCG or ECG was then examined. The inhibitory effect of TBAb on cAMP production by HCG and ECG was similar to bTSH, and TBAb positive sera with more than 40 IU/L TBII activity completely blocked cAMP production by HCG, ECG and bTSH. This suggests that common alpha -subunit of both HCG and TSH are involved in the inhibitory effect of TBAb. Previous reports demonstrated that the thyroid stimulating activity of thyroid stimulating antibody (TSAb) was blocked by deglycosylated HCG (competitive antagonist of TSH binding to TSHR). The fact and our present study suggest that TSH, HCG ECG, TSAb and TBAb have a similar binding site (alpha-subunit-mimicking binding site) on the TSH receptor.

In vitro production of interferon-gamma by peripheral blood from patients with Graves' disease, Hashimoto's thyroiditis and rheumatoid arthritis

The production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC), CD4 cells, or CD8 cells in response to interleukin-2 (IL-2) stimulation has been studied; the samples were obtained from 12 healthy control subjects, 19 patients with Graves' disease (10 hyperthyroid and nine euthyroid), 13 patients with Hashimoto's thyroiditis (four hypothyroid and nine euthyroid), and 15 patients with rheumatoid arthritis (11 active and four inactive). A dose of IL-2 (25 U/ml) was utilized to induce IFN-gamma by PBMC from all four groups. The incremental increase in IFN-gamma values (with IL-2 stimulation minus without stimulation) was significantly less in PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis than that in PBMC from control subjects. The values from PBMC in patients with Graves' disease in a euthyroid state were below normal but greater than those from patients with Graves' disease in a hyperthyroid state. The incremental increase in IFN-gamma values from Graves' disease PBMC correlated with the serum TSH values (r = 0.622, P less than 0.01), but not with thyroid autoantibodies (anti-thyroid microsomal antibodies, anti-thyroid microsomal antibodies, nor TSH-binding inhibitory immunoglobulin activities). The incremental increase in IFN-gamma from PBMC from both control subjects and Graves' disease was correlated with that from CD4 cells (r = 0.711, P less than 0.01), but not with that from CD8 cells. The production of IFN-gamma in response to IL-2 from PBMC in Graves' disease correlated inversely with thyroid function, appearing to reflect the very effect of hyperthyroidism in this process. The precise explanation of these phenomena remains unclear. The decreased response of IFN-gamma to IL-2 stimulation by PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis seems to be a non-specific phenomenon occurring in both organ specific autoimmune disease and systemic autoimmune disease. It may be due to a down-regulation in autoimmune disease of CD4 cells in response to IL-2, a decreased level of IL-2 cellular receptors or a decreased receptor affinity, associated increased soluble IL-2 receptors, or a defect of the intra-CD4 cellular IL-2 signal to produce or release IFN-gamma in the conditions studied.

Regulation of thyrotropin receptor protein expression in insect cells.

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.

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 TSHR, 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.

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.

Subtypes of Anti-TSH Receptor Antibodies Classified by Various Assays Using CHO Cells Expressing Wild-Type or Chimeric Human TSH Receptor

To analyze the heterogeneity of anti-TSH receptor antibodies (TSHRAb), we measured serum TSH-binding inhibitory immunoglobulin (TBII), thyroid-stimulating antibody (TSAb), and thyroid stimulation blocking antibody (TSBAb) activities in 31 patients with positive TSHRAb, using CHO cells expressing wild-type TSHR (WT) or TSHR chimera (Mc2) wherein residues 90-165 were substituted by the LH/CG receptor. Using membranes from WT cells, we detected TBII activity in all 31 patients; 10 (32%), all with TSAb activity only, completely lost TBII activity using Mc2 membranes. TSAb activity was found in 26 sera using WT cells; 20 (77%) completely lost TSAb activity in Mc2 cells. Comparisons of TBII and TSAb activity in WT cells did not exhibit a strong positive correlation (r = 0.52). Of the 20 sera that completely lost TSAb activity in Mc2 cells, 10 retained some TBII activity in Mc2 cells. In each of the sera with retained TBII activity, TSAb activity was recovered in Mc2 cells using the conversion assay, which measures the conversion of a nonstimulating TSHRAb to a TSAb by the action of an anti-human IgG. Additionally, the TBII and conversion assay values in Mc2 cells exhibited a strong positive correlation (r = 0.86). Of the 31 sera, TSBAb was found in 7 samples, with no difference in WT and Mc2 cells. TBII activity was detected in all 7 sera with WT cells; TSAb activity in only 2. In the 5 sera with TSBAb but no TSAb activity, and with only a minimal or no decrease in TBII activity in Mc2 cell membranes, the in vitro conversion assay uncovered TSAb activity. Analyzing these data, we classify the sera into 5 groups containing multiple, different TSHR autoantibodies, including two different TSAbs, three different TBIIs, and one nonfunctional antibody. The heterogeneity of TBIIs as well as TSAbs provides a basis to explain the lack of correlation between TBII and TSAb activities in some past studies of Graves' sera.

Characterisation of the Antibody Response to the Extracellular Region of Recombinant Thyrotropin Receptor

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

Studies on the Action of Thyroid Stimulation Blocking Antibody (TSBAb) on Thyroid Cell Membrane

The effect of thyroid stimulation blocking antibody (TSBAb) on stimulated cyclic AMP (cAMP) production induced by adenylate cyclase stimulators in porcine thyroid membrane (PTM) and porcine thyroid cells (PTC) has been studied. Ten TSBAbs with high TSH binding inhibitory immunoglobulin (TBII) activities significantly blocked TSH-stimulated cAMP production in PTC. The blocking effect of TSBAb on the cAMP increase induced by forskolin or GTP-gamma S stimulation in PTC was found in a few cases. However, there was no blocking action of TSBAb on the cAMP increase stimulated by forskolin, GTP gamma S, or NaF in isolated PTM. When TSBAb-globulin was absorbed with PTM or guinea pig epididymal fat membrane (GPFM), the TBII activity in TSBAb-globulin was significantly absorbed by these membranes. A decrease of TSBAb activity (blocking activity for TSH-stimulated cAMP production in PTC) by PTM absorption, but no decrease by GPFM absorption, was found in six cases. This suggests that the potent TSBAb-neutralizing component may be associated with a non-TSH receptor site in the thyroid membrane. The other four cases showed a decrease of TSBAb activity by absorption with both PTM and GPFM. This suggests that the TSBAb-neutralizing activity may be associated with the TSH receptor site of both PTM and GPFM. The results of the present study suggest that TSBAb may block TSH action either via the TSH receptor itself or via a non-TSH receptor component of the thyroid membrane and not at a postreceptor level.

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.

The role of thyroid stimulating antibody (TSAb) in the thyroid function of patients with post-partum hypothyroidism.

We investigated the association between thyroid function and the biological activities of thyroid stimulating antibodies (TSAb) and thyroid stimulation blocking antibodies (TSBAb) in patients with post-partum hypothyroidism. A prospective study. We studied 25 patients with post-partum hypothyroidism who visited our thyroid clinic during the period from 1985 to 1990. We measured TSH binding inhibitory immunoglobulin (TBII) and TSAb activity at the initial presentation of each of the 25 patients. Women found to have elevated TSAb activity were followed up. Upon finding negative TSAb activity along with positive TBII activity in the serum at the initial presentation, we measured TSBAb activity. Women found to have elevated levels of TSBAb at the initial presentation were also followed up. Elevated TBII activity was found in six of the 25 patients, as was high TSAb activity (205-2651%, normal 55.0-145.0%) in five of these six and in one other patient at the initial presentation. Markedly elevated TSBAb activity (89%) was found in one TBII positive patient. We were able to follow up serially five TSAb positive patients and the TSBAb positive patient over periods ranging from 11.5 to 26.5 months post-partum. The maximal value of TSAb activity was observed at the initial presentation in all TSAb positive patients, following which the activities gradually decreased. One of these patients developed Graves' hyperthyroidism associated with high TSAb activity (1223%) at 10.5 months post-partum. One of the other patients was restored to euthyroid with elevated TSAb activity (279%), but thereafter developed hypothyroidism in conjunction with the disappearance of TSAb activity at 26.5 months post-partum. In the other two patients, normalization of thyroid function was observed with elevated TSAb activity. Thereafter, thyroid function remained within the normal range even with the disappearance of TSAb activity. In the other patient, normalization of thyroid function was observed at 11.5 months post-partum, 3 months after the disappearance of TSAb activity. In the TSBAb positive patient, TSBAb activity decreased to 21% by 17.5 months post-partum associated with normalization of thyroid function. The present study demonstrates the presence of elevated levels of TSAb activity in some patients with post-partum hypothyroidism. In these patients, Graves' hyperthyroidism may be induced by TSAb activity, and hypothyroidism may reoccur with the disappearance of the TSAb activity. Furthermore, post-partum hypothyroidism may be due to increased TSBAb activity in some patients.

Monoclonal antibodies to the thyrotropin receptor bind to a 56-kDa subunit of the thyrotropin receptor and show heterogeneous bioactivities.

Four monoclonal antibodies to the thyrotropin (TSH) receptor were established by fusing human peripheral lymphocytes of patients from Graves' disease with a human myeloma cell line. Of two antibodies with TSH-binding inhibitory immunoglobulin activity (TBII), one inhibited TSH stimulation of adenylate cyclase and another stimulated adenylate cyclase. These antibodies showed competitive and noncompetitive modes of binding inhibition, respectively. Of the other two antibodies without TBII activity, one stimulated adenylate cyclase and the other inhibited TSH stimulation of adenylate cyclase. Of the two antibodies, which inhibited TSH stimulation of adenylate cyclase, one with TBII activity inhibited stimulation of adenylate cyclase by stimulating antibody with TBII activity, but another without TBII activity inhibited stimulation by both stimulating antibodies with or without TBII activity. These inhibitory antibodies did not influence the stimulation of adenylate cyclase by Forskolin and guanosine 5'-(beta,gamma-imido)triphosphate compounds which are known to affect other parts of the receptor-adenylate cyclase system than the receptor unit. Four antibodies with heterogeneous potencies to the TSH receptor reacted with glycoproteins extracted from thyroid membranes. One stimulating antibody without TBII activity also interacted with the glycolipid fraction of the membrane preparation, and the binding decreased after desialylation or deglycosylation of the membrane components. In order to identify the binding sites of these monoclonal antibodies, receptor proteins interacting with antibodies were visualized by Western blot analysis and by the label transfer cross-linking method. All of these antibodies with different characteristics reacted with a 56-kDa molecule.

The Mechanism of Spontaneous Hypothyroidism in Patients with Graves’ Disease after Antithyroid Drug Treatment

The natural course of Graves' disease results in hypothyroidism in up to 20% of patients previously treated with antithyroid drugs. The precise mechanisms are not known, although autoimmune destruction of thyroid tissue has been proposed. We studied sequentially obtained serum samples from three patients with hyperthyroid Graves' disease previously treated with an antithyroid drug who became hypothyroid to determine possible causes of their hypothyroidism. Antithyroglobulin and antithyroid microsomal autoantibodies, TSH binding inhibitory immunoglobulin (TBII), thyroid-stimulating antibody (TSAb), and thyroid stimulation-blocking activity were measured. Autoantibodies were markedly elevated throughout the clinical course in all three patients. Patient 1 had no TBII and blocking activity and extremely high TSAb when she was euthyroid as well as hypothyroid. Hypothyroidism was probably the result of autoimmune thyroid destruction. In patient 2, TSAb disappeared, and TBII and blocking activity increased markedly when she developed hypothyroidism, which thus appeared to result from blocking antibodies. Patient 3 had intermittent periods of hyper- and hypothyroidism before becoming and remaining euthyroid. While initially hypothyroid, TBII was weakly positive, and TSAb was strongly positive; subsequently, when hyperthyroidism recurred, TBII and TSAb were strongly positive. Hypothyroidism appeared to result from focal autoimmune thyroiditis. Patients with hyperthyroid Graves' disease may develop hypothyroidism later by different means. Autoimmune thyroiditis, diffuse or focal, with thyroid destruction is one mechanism. The appearance of antibodies that block TSH stimulation may be another.

Transient Neonatal `Athyreosis' Resulting From Thyrotropin-Binding Inhibitory Immunoglobulins

Recognition of transient forms of neonatal hypothyroidism is difficult because of the urgency of thyroxine treatment. In the present report the first child born to a mother with Graves' disease developed transient hyperthyroidism during the newborn period. The mother underwent radioactive iodine treatment and was maintained euthyroid on l-thyroxine. Two subsequent children were detected by newborn thyroid screen to have low thyroxine and markedly elevated serum thyrotropin (TSH) levels. Technetium 99 metastable and iodine 123 scans at 22 days of age showed the second child to be athyreotic. The third child was not scanned. All three children were nongoitrous at birth. Patients 2 and 3 had continuous TSH suppression with thyroxine therapy for 3 and 4 years. Thyroid function measurements after discontinuation of therapy for 8 weeks were normal, and both children had normal 123I thyroid scans. The mother was found to have potent TSH-binding inhibitory immunoglobulin (TBII) levels in her serum (85.5%). A fourth child with low thyroxine and elevated TSH was born to a mother on a regimen of l-thyroxine for hypothyroidism. 99mTc scan at 26 days of age showed no thyroid tissue and was normal at 3 months. TBII activity was 35% in the maternal serum and absent in the infant's serum. The above laboratory and clinical data are compatible with the blocking nature of TBII, resulting in transient newborn hypothyroidism and an athyreotic appearance on scan. The TBII measurement can be a useful predictor of neonatal hypothyroidism as well as confirm the transient nature of the disease in newborns.

Thyrotropin-receptor autoantibodies in children and young adults with Graves' disease.

Thyrotropin (TSH)-receptor autoantibodies were assessed by measurement of 125I-bTSH binding inhibition in 32 patients with juvenile-onset Graves' disease (one of whom was studied twice) and 16 normal control subjects. Thirteen (76.5%) of 17 thyrotoxic patients had significantly elevated TSH binding-inhibitory immunoglobulin (TBII) activity compared with eight (50%) of 16 patients who were in clinical remission and none of the control subjects. Mean TBII activity was significantly greater in thyrotoxic patients than in individuals in remission, except in one unusual patient in whom there was a discordance between TBII activity and in vitro thyroid-stimulatory activity. In eight euthyroid patients who were followed up for at least five months while not receiving treatment, assessment of TBII activity did not predict who would or would not suffer relapse at a later date. Thus, TBIIs are secreted in excess in juvenile Graves' disease, the titer decreasing as the disease remits. The TBII assay cannot be used as the sole predictor of when antithyroid medication can be withdrawn safely, however.