TSH-binding Inhibitor Immunoglobulin Activities Research Articles

High prevalence and little change in TSH receptor blocking antibody titres with thyroxine and antithyroid drug therapy in patients with non-goitrous autoimmune thyroiditis.

We have reevaluated the prevalence and pathogenetic importance of TSH receptor blocking antibodies (TRBAb) in autoimmune hypothyroidism, and investigated the changes in TRBAb activities during thyroxine and antithyroid drug treatment. Serum TSH binding inhibitor immunoglobulin (TBII) and thyroid stimulation blocking antibody (TSBAb) were measured serially in all patients with non-goitrous autoimmune thyroiditis (AT) and measured monthly during methimazole treatment in 6 patients. Ninety patients with non-goitrous AT and 95 patients with goitrous AT were entered consecutively into this study. All patients with non-goitrous AT were treated with thyroxine and followed at intervals of 6 months for 2 years initially and then yearly intervals. The duration of follow-up was 1-8 years. Six patients from the TRBAb-positive non-goitrous AT group who were treated with thyroxine were randomly selected and given additional treatments with methimazole (40 mg per day) for 6 months. Serum TBII was measured by a radioreceptor assay, TSBAb by using FRTL-5 cells, and antithyroid peroxidase and antithyroglobulin antibodies by radioimmunoassay. The prevalences of TBII and TSBAb is non-goitrous AT were 47.8 and 58.9%, respectively, which were significantly higher than those in goitrous AT (6.3% for TBII, 10.5% for TSBAb). All but one patient showed persistent TBII and TSBAb activities during the thyroxine treatment for up to 8 years. A high dose of methimazole (40 mg per day) did not affect the titres of TBII and TSBAb in 5 out of 6 patients with non-goitrous AT tested. However, antithyroid peroxidase and antithyroglobulin antibodies activities were significantly decreased during the methimazole treatment. The high prevalence of TSH receptor blocking antibodies (TRBAb) suggests that TRBAb may play a major role in the development of hypothyroidism and thyroid atrophy in the vast majority of patients with non-goitrous autoimmune thyroiditis. Most TRBAb activities are stable for at least 8 years and are now affected by thyroxine and antithyroid drug treatment.

Preparation and characterization of monoclonal antithyrotropin receptor antibodies obtained from peripheral lymphocytes of hypothyroid patients with primary myxedema.

Anti-TSH receptor antibodies (TSH-R Ab), which have been detected in the serum of some patients with primary myxedema, are themselves considered to induce hypothyroidism. These are termed blocking-type TSH-R Ab (TSH-R BAb), because they inhibit adenylate cyclase stimulation by TSH on thyrocytes or nonthyroidal cells transfected with TSH-R complementary DNA. We prepared monoclonal TSH-R BAb and characterized them. Peripheral lymphocytes from three patients with primary hypothyroidism and potent TSH-R BAb were transformed by Epstein-Barr virus, and the culture supernatants were screened by TSH binding inhibitor immunoglobulin (TBII) assay. Twenty positive and 7 negative lymphocyte clones were obtained; their monoclonality was confirmed by Southern blot analysis, using an immunoglobulin (Ig) JH probe. These monoclonal antibodies were then tested for TSH-R BAb activity. TSH-R BAb activity ranged from 24.1-58.5% (normal range, < 24%) in all 20 TBII-positive clones and in 2 of 7 TBII-negative clones. An enzyme-linked immunosorbent assay showed that the Ig isotypes of these clones with TBII and/or TSH-R BAb activity were IgG in 8 and IgM in 14. Another enzyme-linked immunosorbent assay and Southern blot analysis of the light chains revealed that 13 clones had kappa-chains, whereas the light chains could not be determined in the other 9 clones. To summarize, 1) we obtained 22 clones that produced monoclonal TSH-R BAb, including 8 IgG-type clones. 2) The clones exhibited dominant usage of the kappa-chain. 3) Although all TBII clones had TSH-R BAb activity, their TBII and TSH-R BAb activities were not significantly correlated, and two TSH-R BAb clones did not show TBII activity.

Rabbit antibodies toward extracellular loops of the membrane spanning region of human thyrotropin receptor possess thyroid stimulating activities

We have synthesized three different peptides, E1 (amino acid residues 478–497), E2 (amino acid residues 561–580) and E3 (amino acid residues 649–652), corresponding to the first, the second and the third extracellular loops of the membrane spanning region of human thyrotropin receptor (TSH-R), respectively. We have produced rabbit antibodies toward these peptides and evaluated their thyroid stimulating antibody (TSAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. Although only slight TSAb activity was observed in E1 antibodies, E2 and E3 antibodies possessed strong TSAb activities, the values of which were 1118% and 910%, respectively. None of these antibody had TBII activities. These results suggest that antibodies against the extracellular loops of the TSH-R can stimulate cAMP formation in thyroid cells and that these regions may be one of the candidates for the epitope against autoantibodies from patients with Graves' disease.

Natural Course of Graves’ Disease After Subtotal Thyroidectomy and Management of Patients with Postoperative Thyroid Dysfunction

The Natural course of Graves' disease after subtotal thyroidectomy was studied in 67 patients who had subtotal thyroidectomy for Graves' disease and did not receive any medical treatment for 8 to 12 years after surgery. Postoperative thyroid status was determined by serum free thyroxine (T4), free triiodothyronine (T3), and thyrotropin (TSH) levels in the first period (1 year after surgery), second period (3.9 +/- 1.0 years), and third period (8.8 +/- 1.5 years). Serum thyroid stimulating antibody (TSAb) and TSH binding inhibitor immunoglobulin (TBII) activities were also measured. A total of 53 patients (79%) changed thyroid status during the observation period. One year after surgery, 50 percent of euthyroid subjects developed abnormal thyroid function in the next period. Reversible latent hypothyroidism was the most common type of thyroid dysfunction seen in up to 46% of patients. Interestingly, hyperthyroidism and hypothyroidism that developed in the postoperative period were temporary disorders in most cases. Serum TSAb and TBII activities did not help predict the postoperative changes of thyroid status. Our study indicates that the instability of the thyroid function is common after subtotal thyroidectomy.

Development of chicken antibodies toward the human thyrotropin receptor peptides and their bioactivities

We have synthesized four peptides (P2, P4, E3 and P1) corresponding to different segments of human thyrotropin (TSH) receptor. We have obtained antibodies by immunizing them to chickens, and antibodies are evaluated for their thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. None of the antibodies had TSAb activity. Antibodies against human TSH receptor specific region such as P2 and P4 (P2: No. 372–397, P4: No. 341–358) had TSBAb and TBII activities. Anti-E3 antibody (E3: the third putative extracellular loop, No. 649–661) had only TSBAb activity. Anti-P1 antibody (P1: high homology with pig LH CG receptor, No. 398–417), however, had none. These results suggest that anti-TSH receptor antibodies to different antigenic epitopes show heterogeneity in their biological activities.

Comparison of atrophic and goitrous auto-immune thyroiditis in children: Clinical, laboratory and TSH-receptor antibody studies

We studied the clinical features, laboratory and thyroid functions and thyrotropin (TSH)-receptor and thyroid-stimulation antibodies in 21 patients with atrophic auto-immune thyroiditis (AAT) and 48 patients with goitrous auto-immune thyroiditis (GAT) of childhood onset. The clinical features of patient with AAT were cessation of growth and obesity, while asymptomatic enlargement of the thyroid gland was the sole symptom in most patients with GAT. Although the ages at diagnosis were comparable in both groups, the estimated ages at onset were much lower in patients with AAT than in those with GAT. Patients with AAT exhibited more severe hypothyroidism when evaluated by serum thyroxine (T4), tri-iodothyronine (T3), TSH, cholesterol levels and basal metabolic rates. The 24 h 123I-thyroidal uptake was significantly lower in patients with AAT than in those with GAT. None of the 19 patients with AAT possessed TSH-binding inhibitor immunoglobulins (TBII). On the other hand, 3 of the 32 GAT patients tested, possessed weak to potent TBII activities. Three TBII-positive patients with GAT also possessed thyroid-stimulation blocking antibodies. These findings suggest that: 1. Pathogenesis of AAT in children whose onset of hypothyroidism was before puberty is not due to TSH-receptor blocking antibodies, which are often found in patients with AAT of postpubertal onset. 2. AAT in children is considered not to be due to the later stage of GAT. 3. Some patients with GAT possessed TSH-receptor blocking antibodies. The aetiology and pathogenesis of AAT in children have yet to be elucidated.

PRESENCE OF BOTH STIMULATING AND BLOCKING TYPES OF TSH‐RECEPTOR ANTIBODIES IN SERA FROM THREE PATIENTS WITH PRIMARY HYPOTHYROIDISM

A case report of three patients with primary hypothyroidism who had potent TSH-binding inhibitor immunoglobulins (TBII) and both thyroid stimulating (TSAb) and thyroid stimulation-blocking antibodies (TSBAb) has been described. Two patients displayed symptoms and signs indistinguishable from those in primary myxoedema (cases 1 and 2), and another patient had a history of Graves' disease (case 3). TBII, TSAb and TSBAb activities were 90.0, 1084.2 and 94.5% in case 1, 91.5, 826.6 and 95.8% in case 2, 76.0, 230.0 and 95.0% in case 3, respectively (normal range, less than 11.0%, less than 145.0 and less than 22.0%, respectively. The results indicate that both stimulating and blocking types of TSH-receptor antibodies exist in these patients. The possible mechanism whereby hypothyroidism developed has been discussed.

Role of blocking TSH receptor antibodies on the development of hypothyroidism and thyroid atrophy in primary myxedema.

We studied blocking type TSH receptor antibodies in 28 patients with primary myxedema and 21 patients with goitrous Hashimoto’s thyroiditis by measuring the ability of their IgG to inhibit TSH binding to its receptor, and to inhibit TSH-stimulated cAMP increases and 3H-thymidine incorporation in a rat thyroid cell line, FRTL-5. The incidences of TSH binding inhibitor immunoglobulin (TBII), thyroid stimulation blocking antibody (TSBAb) and thyroid growth blocking antibody (TGBAb) in patients with primary myxedema were 53.6%, 75% and 65.2%, respectively. However, in goitrous Hashimoto’s thyroiditis, these were 14.3%, 0% and 17.7%, respectively. These antibodies inhibited the receptor binding of 125I-bTSH dose-dependently, and also inhibited dose-dependently not only TSH-stimulated but also Graves’ IgG-stimulated cAMP increase and 3H-thymidine incorporation. TBII activities of patients with primary myxedema were significantly correlated with both their TSBAb (r=0.665; p<0.01) and TGBAb (r=0.618; p<0.01) activities. Thirteen patients whose TBII activities were more than 50% had both strong TSBAb (75.1–100%) and TGBAb (57.4–100%) activities. Transient neonatal hypothyroidism was found in an infant born to a mother having potent TBII activities. Serum of the baby also had potent TBII activities and the baby’s IgG inhibited TSH-stimulated cAMP increase and 3H-thymidine incorporation.These data suggest that a significant proportion of patients with primary myxedema have potent blocking type TSH receptor antibodies. These might play a role in primary myxedema causing hypothyroidism and thyroid atrophy through inhibition of TSH-stimulated cAMP generation.

Role of Thyrotropin Receptor Antibodies in the Development of Hyperthyroidism: Follow-Up Studies on Nine Patients with Graves’ Disease

TSH binding inhibitor immunoglobulin (TBII) and thyroid-stimulating antibody (TSAb) activities were measured serially for 4-32 months in nine patients before and during development of hyperthyroidism due to Graves' disease. Initially, all were euthyroid, seven had thyroid enlargement, one had proptosis, and seven had high serum titers of antithyroid microsomal antibodies. The occurrence of hyperthyroidism was preceded by detection of both TBII and TSAb in four patients and detection of TSAb alone in four patients. One patient had neither TBII nor TSAb when euthyroid. The mean initial TBII and TSAb activities were 10.2 +/- 15.2% (+/- SD) and 2677 +/- 4620%, respectively, when these patients were euthyroid. When they became hyperthyroid, both TBII and TSAb activities increased in all patients. At that time, TBII was detected in all but one (eight of nine subjects; 88.9%), with a mean activity of 58.8 +/- 23.4% (+/- SD), and TSAb was detected in all nine patients, with a mean value of 4508 +/- 4429%. These findings not only indicate the crucial role of TSH receptor antibodies in the development of hyperthyroidism due to Graves' disease, but also suggest that a certain period of subclinical Graves' disease exists before the onset of overt hyperthyroidism in most patients, in the sense that they have TSH receptor antibodies, especially TSAb, in their serum even though they are euthyroid.

Inhibition of thyrotropin-stimulated adenylate cyclase activation and growth of rat thyroid cells, FRTL-5, by immunoglobulin G from patients with primary myxedema: comparison with activities of thyrotropin-binding inhibitor immunoglobulins

We studied the blocking type TSH receptor antibodies in 28 patients with primary myxedema and 21 patients with goitrous Hashimoto's thyroiditis by measuring the ability of their IgGs to inhibit TSH binding to its receptor, and to inhibit TSH-stimulated cAMP increase and [3H]thymidine incorporation in a rat thyroid cell line, FRTL-5. The incidences of TSH binding inhibitor immunoglobulin, thyroid stimulation inhibiting immunoglobulin and thyroid growth inhibiting immunoglobulin in patients with primary myxedema were 54.6, 75 and 65.2%, respectively, against 14.3, 0 and 17.7%, respectively, in goitrous Hashimoto's thyroiditis. The antibodies inhibited dose-dependently not only TSH stimulated but also Graves' IgG-stimulated cAMP increase and [3H]thymidine incorporation. The TSH binding inhibitor immunoglobulin activities in patients with primary myxedema were significantly correlated with both the thyroid stimulation inhibiting immunoglobulin (r = 0.665; P less than 0.01) and the thyroid growth inhibiting immunoglobulin (r = 0.618; P less than 0.01) activity. Thirteen patients whose TSH binding inhibitor immunoglobulin activities were more than 50% had both strong thyroid stimulation inhibiting immunoglobulin (75.1-100%) and thyroid growth inhibiting immunoglobulin (57.4-100%) activities. These data suggest that the vast majority of patients with primary myxedema have potent blocking type TSH receptor antibodies. These might play a role in primary myxedema causing hypothyroidism and thyroid atrophy through inhibiting TSH-stimulated cAMP generation.

Studies on thyrotrophin receptor antibodies in patients with euthyroid Graves' disease.

Thyroid stimulating antibodies (TSAb) and TSH-binding inhibitor immunoglobulins (TBII) were assessed in 30 patients with euthyroid Graves' disease. TSAb were detected in 24 cases (80.0%), the incidence being not significantly different from that in hyperthyroid Graves' disease (29/30, 97.6%). On the other hand, the incidence of TBII in patients with euthyroid Graves' disease (12/30, 40.0%) was significantly lower than that in patients with hyperthyroid Graves' disease (30/30, 100.0%). The mean TSAb and TBII activities in the euthyroid patients were significantly lower than in the hyperthyroid patients (P less than 0.005 and P less than 0.001, respectively). Both TBII and, more closely, TSAb activities correlated with T3-nonsuppressibility and inhibition of serum TSH response to TRH stimulation. The findings supported the stimulation in vivo of the thyroid by these antibodies. Both antithyroglobulin and antimicrosomal antibody titres in euthyroid Graves' disease were significantly lower than in hyperthyroid Graves' disease (P less than 0.05, P less than 0.01, respectively). Goitre size was significantly smaller (P less than 0.001), and 99mTc thyroid uptake was significantly lower (P less than 0.001) in the euthyroid than in the hyperthyroid group. Thus, the reduced mass of thyroid tissues responding to the stimulators was considered to be one of the factors responsible for the euthyroidism despite the presence of TSAb. The high incidence of TSAb and relatively low incidence of TBII in euthyroid Graves' disease indicate that the presence of TSAb does not necessarily lead to hyperthyroidism and that the development of overt thyrotoxicosis may require augmentation of both TSAb and TBII.

Evaluation of TSH-receptor antibodies as prognostic markers after cessation of antithyroid drug treatment in patients with Graves' disease

Clinical usefulness of thyroid-stimulating antibodies (TSab) and TSH-binding inhibitor immunoglobulins (TBII) for predicting the prognosis in patients with Graves' disease after cessation of antithyroid drug treatment was evaluated, and compared with that of T3 suppression test and goitre size. Among 46 patients who had been euthyroid on a maintenance dose of antithyroid drugs for at least one year and had discontinued taking medicine, 16 relapsed within one year (group 1), 7 relapsed later than 1 year (group 2), and 23 patients remained in remission for more than 1 year (group 3). Incidence of TSab, TBII, T3 nonsuppressibility and large goitre (transverse diameter longer than means of the values for the 46 patients: greater than or equal to 4.36 cm in females; greater than or equal to 4.74 cm in males) determined at the time of discontinuation of treatment was 87.5% (14/16), 56.3% (9/16), 78.6% (11/14) and 81.3% (13/16) in group 1; 66.7% (4/6), 28.6% (2/7), 50.0% (3/6), and 57.1% (4/7) in group 2, and 56.5% (13/23), 24.1% (5/23), 35.7% (8/23), and 26.1% (6/23) in group 3, respectively. All relapsed patients showed remarkable increases in both TSab and TBII activities at the time of relapse. High incidence of TSab in patients remaining in remission suggests that a reduced functional reserve of the thyroid, probably owing to destructive changes and/or shrinkage of the gland, may cause impaired responses to TSab and is involved in the cause of remission. Development of blocking type of TBII was not considered to be a cause of remission. Remission was predictable in all patients with any two of the indices such as negative TSab, positive T3 suppressibility, and small goitre.

Thyroglobulin release-stimulating activity in immunoglobulin G from patients with Graves' disease studied by human thyroid cells in vitro.

The ability of TSH or immunoglobulin G (G-IgG) from untreated patients with hyperthyroidism due to Graves' disease to stimulate thyroglobulin (Tg) release from human thyroid cells was studied. Thyroid tissue obtained from antithyroid drug-treated Graves' hyperthyroid patients was dispersed enzymatically and cultured in monolayers; medium was changed every 3 days. The cultured cells initially released large but declining amounts of Tg, independent of the presence of TSH (approximately 5 micrograms/dish on day 3 and approximately 1.5 micrograms/dish on day 6). After 6 days, TSH had a dose-dependent stimulatory effect on Tg release, and the peak response occurred on day 15. G-IgG-induced Tg release was found on the 12th day of culture and was maximal on day 18. Thyroid cells cultured for 12 days in the absence of TSH responded to TSH and G-IgG in a time- and dose-dependent fashion. Using 12-day cultures, Tg release-stimulating activity (Tg-RSA) was tested using 5 mg/ml (7.5 mg/dish) G-IgGs from 20 patients and 72-h incubation. The Tg-RSA of individual patients varied. However, significant correlations were found between Tg-RSA values and serum Tg concentrations or Tg-RSA and thyroid-stimulating immunoglobulin activities. No correlation was found between Tg-RSA and TSH binding inhibitor immunoglobulin activities. These results suggest that Tg-RSA can be an indicator of abnormal IgG of hyperthyroid Graves' patients. Whether the activity is identical with thyroid-stimulating activity remains to be clarified.

Paired determination of thyroid-stimulating and TSH-binding inhibitory activities in patients with Graves' disease during antithyroid drug treatment

Sequential changes in thyroid-stimulating antibodies (TSAb) and TSH-binding inhibitor immunoglobulins (TBII) during antithyroid drug treatment were studied in 17 patients with Graves' disease. Before treatment, TSAb and TBII were detected in 17 (100%) and 13 (76.5%) patients, respectively, with a significant correlation between the two activities (r = 0.600, n = 17, P less than 0.02). In 9 patients who became euthyroid as early as after 1-4 months of treatment, the TSAb and TBII activities both gradually decreased, and there was a good correlation between the changes of these activities during treatment. Among the 7 patients in whom small changes in TSAb and TBII activities were observed, 4 showed poor control of the thyrotoxicosis during the whole observation period (7 months-2 years). One patient who showed a marked dissociation between the changes in TSAb and TBII activities developed hypothyroidism, when his TBII became remarkably high. These potent TBII inhibited cAMP production induced by bTSH. These findings indicate that 1) changes in TSAb and TBII activities reflect the clinical course of hyperthyroidism in most patients with Graves' disease, and 2) development of a blocking-type of TBII may induce hypothyroidism in some patients after the treatment.