Similarity and dissimilarity between clinical and laboratory findings, especially anti-thyrotropin receptor antibody in ophthalmic Graves' disease without persistent hyperthyroidism and hyperthyroid Graves' disease

The aim of this study was to investigate thyroid states, significance of anti-TSH receptor antibodies and the clinical courses of patients with euthyroid Graves' ophthalmopathy. The clinical and laboratory finding of 30 patients with euthyroid Graves' ophthalmopathy were briefly as follows: 1) normal sized thyroid or small goiter; 2) negative or weakly positive thyrotropin binding inhibitor immunoglobulin (TBII); 3) normal thyroid [99 m-Tc] pertechnetate uptake; and 4) frequent observations of low serum TSH values. Besides TBII, thyroid stimulating antibody (TSAb) was measured under low salt and isotonic conditions using FRTL-5 rat thyroid cells. Both TBII and TSAb titers were lower in euthyroid Graves' ophthalmopathy than in hyperthyroid Graves' disease. Serum TSH levels frequently became low in patients considered as euthyroid upon the first examination as well as in Graves' patients in remission, reflecting preceding or mild hyperthyroidism. In follow-up studies, these patients with mildly elevated thyroid hormone levels and low TSH levels seldom reached a state of persistent hyperthyroidism, when TBII was negative or only weakly positive.     

Activity of thyroid stimulating antibody and thyroid stimulation blocking antibody determined by radioiodine uptake into FRTL-5 cells

To investigate the pathophysiology of patients with autoimmune thyroid diseases, we measured serum thyroid stimulating antibody (TSAb) activity and thyroid stimulation blocking antibody (TSBAb) activity by determining the radioiodine (125I) uptake into FRTL-5 cells. FRTL-5 cells were pre-incubated for seven days with 5H medium and then incubated for 48 hours with patients' crude IgG prepared by polyethylene glycol precipitation. In order to measure TSBAb, 10 microU/ml TSH was also added. 125I was added one hour before the end of the 48 hour incubation period. After the incubation, the medium was aspirated, and the radioactivity in the cells was counted. In patients with untreated hyperthyroid Graves' disease, TSAb was detectable in 18 of 20 patients, the detectability being 90%, and activity showed a statistically significant positive correlation with TSAb activity determined by c-AMP accumulation. Out of 41 patients with hypothyroidism, TSBAb determined by 125I uptake was positive in six cases, the detectability being 14.6%. The inhibition of 125I uptake by one of these six IgGs was suggested to be at the TSH receptor level because it inhibited TSH induced c-AMP accumulation and showed positive thyrotropin binding inhibitor immunoglobulin (TBI I) activity, but did not inhibit the forskolin- and (Bu)2cAMP-induced 125I uptake. Inhibition of another IgG was suggested at the post-receptor level because it did not inhibit TSH induced cAMP accumulation and showed negative TBI I activity, but inhibited forskolin- and (Bu)2cAMP-induced 125I uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible binding site of thyrotropin binding inhibitor immunoglobulin (TBII) on the thyrotropin (TSH) receptor, which is different from TSH binding site

A synthetic decapeptide, P-194, which has the sequence No. 103 to 111 of hTSH receptor structure with an additional N-terminal tyrosine, did not bind TSH nor affected its receptor binding and thyroid stimulating activity. Preincubation of P-194 with sera from thyroid patients caused a significant decrease in TBII activity in almost all 12 TBII positive sera and an increase of thyroid stimulating activity in 3 of 7 Graves' IgG studied. In addition, [125I] P-194 bound to serum IgG fraction from thyroid patients with a positive correlation with TBII (N = 35, r = 0.509, p less than 0.01). The P-194 portion may be, at least a part of, TBII binding site distinct from the TSH binding site on the TSH receptor.     

Relationship among thyrotropin (TSH), thyroid stimulating immunoglobulins, and results of triiodothyronine (T3) suppression test in patients with Graves' disease

To investigate the relationship between TSH and abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease in whom normal thyroid hormone levels in the serum were maintained by antithyroid drug therapy and in patients with euthyroid Graves' disease, determinations were made of the TSH concentration, action of thyroid stimulating immunoglobulins (TSAb and TBII), and T3 suppression. Out of thirty-three patients with hyperthyroid Graves' disease, twelve patients with subnormal TSH levels were all non-suppressible according to the T3 suppression test results and the detectability of TSAb and/or TBII was as high as 75%. In three out of five patients with euthyroid Graves' disease, the serum TSH level was subnormal. All three showed non-suppressibility in the T3 suppression test and positive action of either TSAb or TBII. One of them became clinically thyrotoxic when the TSAb activity was further increased and TBII became positive, and was therefore diagnosed as having hyperthyroid Graves' disease. The present findings suggest that there are still abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease who have low TSH, even if their thyroid hormone concentrations remain normal. Moreover, it is likely that some of the patients with euthyroid Graves' disease are actually in a state of subclinical hyperthyroidism because of the presence of abnormal thyroid stimulator(s).     

Adipocyte-TSH-receptor-related antibodies in Graves' disease detected by immunoprecipitation

Fat cell TSH receptor-related antibodies were detected by immunoprecipitation of 125I-TSH-receptor complexes and the nature of the antibodies was analyzed. To 125I-TSH prebound to Triton-solubilized receptors from guinea pig fat tissues, 50 micrograms of immunoglobulin G (IgG) was added and precipitation was effected by the addition of antihuman IgG. Immunoprecipitation values in 13 patients with Graves' disease were significantly (p less than 0.05) higher than those in 11 normal subjects. No significant increase in the values was seen in 8 patients with Hashimoto's disease. No correlation was observed between immunoprecipitation values and titers of antimicrosomal and antithyroglobulin antibodies. Neither was there any correlation between the values and TSH-binding inhibitor immunoglobulins (TBII) detected by the radioreceptor assay. The IgG fractions positive for the immunoprecipitation antibody were found to be poor human thyroid stimulators (HTS) relative to their TBII activities. And a highly significant correlation was observed between TBII and HTS activities among IgGs without detectable antibody by immunoprecipitation (r=0.907; p less than 0.005; n=7). These findings 1) demonstrate that immunoprecipitation assay using fat cell TSH receptor may detect TSH receptor-related antibodies different from TBII in patients with Graves' disease and 2) suggest the antibodies may recognize determinants on the receptor or its vicinity that do not participate in the binding of TSH or thyroid stimulating antibody, and may interfere with thyroidal response to these stimulators.     

Transient neonatal hypothyroidism due to transplacental transfer of maternal immunoglobulins that inhibit TSH binding, TSH-induced cAMP increase and cell growth

Transient neonatal hypothyroidism due to transplacental transfer of maternal blocking type TSH receptor antibodies (TRAb) was found in a baby born to a 27-yr-old mother, who had been receiving thyroxine medication for primary myxedema. Maternal IgG inhibited radiolabelled TSH binding to its receptor (TBII), TSH-stimulated thyroid adenylate cyclase (AC) activation (TSII) and TSH-stimulated 3H-thymidine uptake (TGII) in cultured rat thyroid cells (FRTL-5). At birth, the baby's IgG showed similar activities to maternal IgG but all these activities decreased gradually, and disappeared from her serum within 12 weeks of age. In the baby, initially nonvisualized thyroid was clearly visualized on 99 m-Tc thyroid scintigraphy when all these blocking activities disappeared, TSII and TGII being decreased more slowly than TBII, and the baby remained euthyroid after discontinuation of thyroxine. This study suggests that such IgGs induced hypothyroidism and thyroid atrophy in the mother and were responsible for transient neonatal hypothyroidism in the baby.     

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.     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

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.     

A synthetic oligopeptide derived from human thyrotropin receptor sequence binds to Graves' immunoglobulin and inhibits thyroid stimulating antibody activity but lacks interactions with TSH.

An 11-residue oligopeptide, P-195, was synthesized to match human thyrotropin (TSH) receptor structure from No. 333 to 343 of amino acid sequence. Preincubation of 5 Graves' IgGs with P-195 up to 10 micrograms resulted in dose-dependent reductions of thyroid stimulating antibody (TSAb) activity. [125I] labeled P-195 was found to bind Graves' IgG. The bound radioactivity correlated significantly with their TSAb activity (N = 25, r = 0.587, p less than 0.01). A peptide having a completely reverse sequence as P-195 did not show such biological activity. The peptide did not affect TSH and thyrotropin binding inhibitor immunoglobulin (TBII) on their receptor binding nor biological activities. P-195 was concluded to have a part of TSAb binding sites.     

Molecular cloning of a human thyrotropin receptor cDNA fragment. Use of highly degenerate, inosine containing primers derived from aligned amino acid sequences of a homologous family of glycoprotein hormone receptors

Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH-R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used polymerase chain reaction and highly degenerate, inosine containing oligonucleotides derived from sequence alignments of the transmembrane regions 2 and 7 of a number of G-binding protein receptors including the lutropin/choriogonadotropin (LH/CG) receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH-R revealed that one of the groups (GT1) containing seven clones which had been sequenced belong to the human TSH-receptor. The sequence of all 7 GT1 clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH-R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G-binding proteins where the primers are biased in their sequence to the LH/CG receptors it is possible to amplify the TSH-R receptor sequence.     

The thyroid "microsomal" antigen is an epitope on the thyrotropin receptor

Antimicrosomal antibodies are present in the sera of most patients with autoimmune thyroiditis, and Graves' disease. It has, in general, been difficult to separate antimicrosomal activity from that directed against the thyrotropin (TSH) receptor in Graves' IgG preparations. The "microsomal" antigen has been localized to the endoplasmic reticulum and microfollicular aspect of thyrocytes; its structure is however unknown. In an attempt to identify the thyroid microsomal antigen, we studied the interaction of Hashimoto's IgG with high microsomal antibody titre and negative for thyroglobulin with purified thyroid plasma and light microsomal membranes. We allowed Hashimoto's, Graves', and control IgGs to bind to protein blots of thyroid plasma membranes resolved on SDS-PAGE under non-reducing conditions. All seven Hashimoto's IgG at a concentration of 2 mg/ml interacted with an M approximately 197,000 polypeptide corresponding to the TSH holoreceptor. By contrast to Graves' IgG (which were positive at 1 mg/ml), however, this binding was not blocked by pretreatment of the protein blots with TSH. Normal IgGs showed no binding at concentrations of up to 2 mg/ml. Both Hashimoto's and Graves' IgG interacted with TSH-affinity column-purified receptor preparations. Two of the Hashimoto's IgGs induced adenylate cyclase activation in thyroid plasma membranes, three inhibited TSH-stimulated enzyme activation, and two were without effect. Two classes of autoantibodies, other than TSH receptor directed, were encountered; one class raised to antigens common to all seven patients and another class unique to individual patients, eg, Mr 210,000 and Mr 20,000 polypeptides. We propose that the TSH receptor has multiple epitopes (functional domains), and the one to which antimicrosomal antibody bind is likely to be spatially separated from that with which Graves' IgG and TSH interact. Differences in affinity or number of sites allows for the demonstration of Graves' IgG against a background of antimicrosomal antibody.     

Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients

To examine the identity of binding sites for thyrotropin (TSH) and thyroid stimulating antibodies (TSAbs) associated with Graves' disease, we constructed eight human TSH receptor/rat LH-CG receptor chimeras. Substitution of amino acid residues 8-165 of the TSH receptor with the corresponding LH-CG receptor segment (Mc1 + 2) results in a chimera which retains high affinity TSH binding and the cAMP response to TSH but loses both the cAMP response to Graves' IgG and Graves' IgG inhibition of TSH binding. Two of three IgGs from idiopathic myxedema patients which contain thyroid stimulation blocking antibodies (TSBAbs) still, however, react with this chimera. Chimeras which substitute residues 90-165 (Mc2) and 261-370 (Mc4) retain the ability to interact with TSH, Graves' IgG, and idiopathic myxedema IgG. The data thus suggest that residues 8-165 contain an epitope specific for TSAbs and that TSH receptor determinants important for the activities of TSAbs and TSH are not identical. Further, binding sites for TSBAbs in idiopathic myxedema may be different from receptor binding sites for both Graves' IgG TSAb as well as TSH and may be different in individual patients.     

Highly sensitive immunoradiometric assay for TSH and thyroid radioiodine uptake as predictors of thyroid suppression test

Suppression of TSH and thyroid radioiodine uptake by doses of either T4 or T3 were compared in 33 patients in whom Graves' thyrotoxicosis had been treated with thioamide drugs and the medication was discontinued for at least 4 months. Thyroidal radiodine uptake was suppressed in 19 patients and was not suppressed in the remaining 14 patients. Basal TSH levels before suppression were 2.07 microU/ml in the former, significantly exceeding those of the latter (0.91 microU/ml). A TSH level of at least 1.2 microU/ml before suppression is a good predictor of positive thyroid radioiodine suppression with a predictive value of 76%. A level lower than 0.7 microU/ml before suppression is a good predictor of negative thyroid radioiodine uptake suppression with a predictive value of 89%. The determination of TSH levels before the thyroid suppression test was helpful in predicting the result, but there were limitations. In the thyroid suppression test positive group, circulating T4 was depressed by doses of T3. In them, the magnitude of T4 depression correlated with the levels of thyroid radioiodine uptake before suppression. The levels of TSH correlated neither to changes in T4 nor to those in thyroid radioiodine uptake. This indicates that the thyroid glands which show high radioiodine uptake are sensitive to TSH and are also sensitive to suppression. The elevated sensitivity to TSH probably warrants the disappearance of abnormal thyroid stimulation more precisely.     

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro

Summary FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, >1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro. This work was supported by Grant CA-11198 and CA-20329 awarded by the National Institutes of Health, and a Biomedical Research Support Grant awarded to R. T. Mulcahy.     

Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor

For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2-L bench-scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared. After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.