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Hashimoto’s Thyroiditis with Heterogeneous Antithyrotropin Receptor Antibodies: Unique Epitopes May Contribute to the Regulation of Thyroid Function by the Antibodies¹

Department of Medicine and Clinical Science (T.A., H.H., M.S., K.N.), Kyoto University School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan; and Cell Regulation Section (L.D.K., K.T.), Metabolic Diseases Branch, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address correspondence and requests for reprints to: Takashi Akamizu, M.D., Department of Medicine and Clinical Science, Kyoto University School of Medicine, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: akataka{at}kuhp.kyoto-u.ac.jp

	Abstract

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

 
Blocking-type TSH-binding inhibitor Igs (TBIIs) are known to cause hypothyroidism and an atrophic thyroid gland in patients with primary myxedema. They can block the activity of thyroid-stimulating antibodies (TSAbs) in Graves’ patients as well as the activity of TSH. The majority of the epitopes for these blocking-type TBIIs have been, and are shown herein, to be present on the C-terminal region of the extracellular domain of the human TSH receptor (TSHR), whereas those for Graves’ TSAbs are on the N-terminus. We report on a patient with Hashimoto’s thyroiditis who suffered from mild hypothyroidism and a moderately sized goiter. Her serum had a potent blocking-type TBII and a weak TSAb in human and porcine TSHR systems. Using human TSHR/lutropin-CG receptor chimeras, we determined that the functional epitope of her blocking-type TBII was uniquely present on the N-terminal, rather than the C-terminal, region of the extracellular domain of the TSHR, unlike the case for blocking-type TBIIs in primary myxedema patients. The epitope of her TSAb was also unusual. Although the functional epitopes of most TSAbs are known to involve the N-terminal region of the receptor, her TSAb epitope did not seem to be present solely on the N- or C-terminus of the extracellular domain of the receptor. Blocking-type TBIIs from patients with primary myxedema blocked her TSAb activity as well as stimulation by TSH; her blocking-type TBII was able to only partially block her TSAb. In contrast, her blocking-type TBII almost completely blocked TSAbs from Graves’ patients. Thus, we suggest that the unique epitopes of this patient’s heterogeneous population of TSH receptor antibodies, at least in part, contribute to regulation of her thyroid function.

	Introduction

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

 
ANTI-TSH RECEPTOR antibodies (TRAbs) are found in most patients with Graves’ disease and a portion of patients with primary myxedema (1, 2, 3). They are considered to induce hyperthyroid or hypothyroid states, depending on their activity (1, 2, 3). TRAbs are measured by a binding assay as TSH-binding inhibitor Igs (TBIIs), or by biological assays as thyroid-stimulating antibodies (TSAbs), TSH-stimulation blocking antibodies (TSH-BAbs), and TSAb-stimulation blocking antibodies (TSAb-BAbs). TSH-BAb and TSAb-BAb are often associated with high TBII levels and are, therefore, sometimes called blocking-type TBIIs rather than thyroid-stimulation blocking antibodies (TSBAb). TBII and TSAb assays are commercially available and are considered by some to be useful to assess therapeutic responses in Graves’ patients after antithyroid drug treatment and in their diagnostic evaluation (1, 4).

Patients with primary myxedema who have hypothyroidism with blocking-type TBIIs usually exhibit severe hypothyroidism with an atrophic thyroid gland and have a very potent TBII and TSH-BAb (5, 6). In patients with goitrous Hashimoto’s thyroiditis, TBIIs and TSH-BAbs are rarely found and are usually not strongly bioactive, even if present (1, 6). In the present case of goitrous Hashimoto’s thyroiditis with mild hypothyroidism, the patient possessed a potent blocking-type TBII and a weak TSAb. To explore the contributions of her TRAb to her thyroid function, we tried to identify their functional epitopes on the TSH receptor (TSHR). We found unique functional epitopes of her TSH-BAb and TSAb by comparison with idiopathic myxedema or Graves’ patients, respectively. The action on these heterogeneous TRAbs with unique functional epitopes seemed to contribute to the symptoms of her intriguing clinical state.

	Case Report

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

 
A 62-yr-old woman had been suffering from primary hypothyroidism for 2 yr. She was diagnosed as having Hashimoto’s thyroiditis, based on the presence of a diffuse goiter, mild hypothyroidism [TSH, 35.3 µU/mL (normal range, 0.3–3.9 µU/mL); with free T₄ (FT₄), 0.68ng/100 mL], the absence of exophthalmos, and positive antithyroglobulin (anti-Tg) and antimicrosomal antibody tests, measured in a hemagglutination assay. She was treated with 25 µg levothyroxine sodium (T₄). She was also being given a daily dose of 2.5 mg predonisolone, 100 mg bucillamine, and 400 mg indometacin farnesil to control rheumatoid arthritis. In addition, she took 2.5 mg amlodipine, 6 mg bunazosin HCl, and 40 mg isosorbide mononitrate because of hypertension, old myocardial infarcts, and angina pectoris. On September 28, 1998, she was admitted to Kyoto University Hospital for coronary artery catheterization.

On admission, she had a moderately sized, diffuse goiter with an elastic consistency, yet no tenderness. She had no ophthalmologic abnormalities. Thyroid function tests revealed subclinical hypothyroidism despite replacement therapy with 25 µg T₄ administration [TSH, 21.3 µU/mL (normal range, 0.3–3.9 µU/mL); with FT_4, 1.03 ng/100 mL (normal range, 0.98–1.77 ng/100 mL)]. Both anti-Tg and antimicrosome antibodies, by the hemagglutination assay, were very potent, 102,400- and >1,638,400-fold higher than controls, respectively. TSH-binding inhibitor immunoglobulins (TBII) and TSAb activities, measured by commercial kits using porcine thyrocyte systems, were 89.7% (normal range, approximately -10 to +10%; RSR Ltd., Cardiff, UK) and 264% (normal range, 80–180%; Yamasa Corp., Choshi, Japan), respectively.

Ultrasonographs of the thyroid gland showed only a moderately sized diffuse goiter with markedly heterogeneous and coarse echogenicity. There were no apparent space-occupying lesions. A fine-needle biopsy specimen of the thyroid gland revealed regeneration of thyroid follicular cells, compatible with a diagnosis of Hashimoto’s thyroiditis. ^99mTc pertechnetate thyroid scintigraphy revealed a goiter with uneven trapping and a lower normal ratio of iodide uptake (1.0%; normal, 0.4–3.0%). A triiodothyronine suppression test could not be performed because of her accompanying ischemic heart disease.

A TRH test was performed to examine her thyroid response to endogenous TSH. Serum TSH increased significantly (from 35.7 to 128 µU/mL), 30 min after TRH injection; but T3 changed only slightly (from 71 to 81 ng/mL), at 180 min. The exaggerated TSH but impaired T3 response were compatible with primary hypothyroidism caused by Hashimoto’s thyroiditis and presence of a potent TSH-BAb.

	Subjects and Methods

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

 
Measurement of TBII, TSAb, TSH-BAb, and TSAb-BAb

TBII assays in a porcine thyroid membrane system were performed using the manufacturer’s protocol (RSR Ltd.). TBII assays in human and rat thyroid cell systems were performed, as described, using human or rat TSHR-transfected Chinese hamster ovary (CHO) cells, respectively (7, 8). Briefly, 100 µL ¹²⁵I-TSH (10⁴ cpm) and 100 µL of a patient or control IgG sample were mixed and incubated with the cells for 2 h. Washing and incubation buffer was modified HBSS (NaCl replaced by 222 mmol/L sucrose) containing 0.5% BSA and 20 mmol/L HEPES at pH 7.4. Specific binding was calculated by subtracting values obtained in the presence of a 1000-fold excess of unlabeled bovine TSH (100 mU/mL; Sigma, St. Louis, MO). All assays were performed in duplicate. TBII activity was expressed as: TBII% = 100 x (1 - ¹²⁵I-TSH-specific binding to the cells in the presence of sample Ig/¹²⁵I-TSH-specific binding to the cells in the presence of control Ig). The normal range for the porcine system is -10% to 10%.

TSAb assays in human and rat systems were performed using FRTL-5 or hTSHR-transfected CHO cells, as described previously (9, 10, 11). TSAb was measured in the porcine system, according to the manufacturer’s instructions (Yamasa TSAb kit , Yamasa Corp.). In all cases, 45 µL of a patient’s IgG was incubated with the cells for 2 h. The cAMP levels of supernatants were measured with RIA kits (Yamasa Corp.). TSAb activity was expressed as the percentage of generated cAMP, relative to control IgG. All assays were performed in duplicate. The normal ranges for porcine, rat, and human are less than 150% (12), 150% (10), and 130% (9), respectively. The coefficient of variation of the Yamasa kit is less than 20% (n = 5).

TSH-BAb activities for TSH stimulation were determined using CHO cells transfected with wild-type or chimera TSHR complementary DNAs and used a slight modification of previously described methods (9, 11). Briefly, after 40 µL of patient Ig, precipitated with PEG, was preincubated for 15 min with 10⁵ CHO cells, 10 µL bovine TSH (final concentration, 100 µU/mL; Sigma) was added; and the cells were incubated for 1 h. cAMP levels in the culture medium were measured with an RIA kit. In the case of porcine assays, porcine frozen cells provided in the TSAb kit (Yamasa) were used according to the manufacturer’s instructions. All assays were performed in duplicate. TSH-BAb activity was expressed as: TSH-BAb% = 100 x {1 - [(TSH + patient’s Ig) - patient’s Ig]/[(TSH + control Ig) - control Ig]}. In TSAb-BAb measurements, 20 µL TSBAb or control Ig was preincubated for 15 min with cells before 20 µL TSAb or control Ig was added; and the cells were incubated for 2 h. TSAb-BAb% was expressed similarly as TSH-BAb%: TSAb-BAb % = 100 x{1 - [(TSAb + patient’s Ig) - (control Ig + patient’s Ig)]/[(TSAb + control Ig) - control Ig]}. The normal range was less than 25% (11). The conversion assay was performed by the method described previously (13). After incubation of 0.2 mL porcine thyroid cells (Yamasa Corp.) with 50 µL of patient’s IgG, the cells were incubated with 100 µL IgG fraction of goat antihuman IgG (Fab fragment specific, 0.2 mg/mL, Organon Teknika Corp., Durham, NC). cAMP concentrations in the supernatant were measured as in the TSAb assay.

Throughout the study, because of the limited amount of patient sera, all the assays were performed in duplicate and twice, and the representative data of similar results were shown.

	Results

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

 
Species specificity of TSAb, TSH-BAb, and TBII

The patient was diagnosed as having goitrous Hashimoto’s disease and exhibited only mild hypothyroidism. Despite this, the patient’s serum had potent TBII and mild TSAb activity in our routine assays, which use porcine thyroid systems (Table 1). We initially used a rat bioassay system to see whether her serum had TSH-BAb activity. Surprisingly, her Ig exhibited a potent TSH-BAb but a negative TSAb activity in the rat system (Table 1). The discrepancy in TSAb activity between porcine and rat systems led us to examine all three activities in systems from three different species, including human thyrocyte systems (Table 1). Although the values are variable among species, TBII and TSH-BAb activities were potent in all three systems. TSAb activity was positive in porcine and negative in the rat system. In the conversion assay, the patient’s IgG exhibited great elevation of TSAb activity (from 210% to 29,210%) as other TSH-BAbs.

Because of limited amounts of patient serum and the absence of TSAb activity in the rat system, we used CHO cells stably transfected with wild-type human TSHR or with the TSHR/LH-CGR (TSHR/LH CG receptor) chimera complementary DNAs, Mc1 + 2 and Mc4 (7), instead of a transient transfection system using mutant rat TSHRs (3). In Mc1 + 2 and Mc4 TSH/LH-CGR chimeras, residues of the N-terminus (amino acid residues 9–165) and C-terminus (residues 261–370) of the extracellular domain of TSHR are replaced by homologous LH-CGR residues, respectively (7). The majority of functional epitopes of Graves’ TSAbs and TSH-BAb in patients with idiopathic myxedema have been shown to exist on the regions substituted by LH-CGR residues in Mc1 + 2 and Mc4, respectively (Fig. 1, Refs. 7, 9).

View larger version (44K): [in this window] [in a new window]   Figure 1. Schematic representation of the wild-type TSHR localizing the TRAb epitopes of the present patient and ordinary patients (A). In B and C, interactions between chimeric TSHR-LH/CGRs (Mc1 + 2 and Mc4) and TRAbs are shown. The shaded area shows a deleted or mutated region.

	Discussion

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

The patient certainly was suffering from advanced Hashimoto’s thyroiditis, as evidenced by extremely high titers of anti-Tg and antimicrosome antibodies and a markedly heterogeneous and coarse pattern in the thyroid ultrasonography. Because this patient has a potent TSH-BAb, it should have prevented TSH, elevated by her hypothyroidism, from stimulating her thyroid gland. Therefore, she should have had severe hypothyroidism unless she had a TSAb. She does have a TSAb; moreover, her TSAb could stimulate because the epitope is different from that of TSBAb. Her thyroid function could not, however, reach a normal level, probably because of destruction of thyrocytes caused by Hashimoto’s thyroiditis. Supporting the existence of her functional TSAb, she has regenerating thyroid cells and thyroid scintigraphy showing uneven trapping, albeit with lower normal ^{99 m}Tc pertechnetate uptake in the thyroid scintigraphy. It is often observed that patients with euthyroid or hypothyroid Graves’ disease with ophthalmopathy have positive TSAbs but show normal or decreased thyroid function (14, 15, 16). Although the precise mechanism is not known, it is speculated that the unresponsiveness of thyroid gland may cause these thyroid states (15, 16).

The majority of functional epitopes of TSH-BAbs from patients with primary hypothyroidism are supposed to be present in the C-terminus of the extracellular domain of TSHR, whereas the minority of them are present in the N-terminus (2, 3, 7, 9). In contrast, most of TSAb epitopes are located in the N-terminus (2, 3, 7, 9). The functional epitope of the patient’s TSH-BAb was different from those in most patients with primary hypothyroidism (Fig. 1A). Thus, it was present in the N-terminus because TSH-BAb activity was lost in Mc1 + 2 TSH/LH-CGR chimera but not the Mc4 chimera (Table 2). Not surprisingly, therefore, her TSBAb blocked the activity of all four Graves’ TSAbs, the epitopes of which are located in the N-terminus (Table 3).

The patient’s TSBAb seemed to block her own TSAb only partially. This is probably because of the unique functional epitopes of her TSAb, as well as her TSBAb. The precise functional epitope of her TSAb has not been identified, but it is clear that it was present neither solely on the N-terminus, as is the case for most Graves’ TSAbs, nor solely on the C-terminus (Fig. 1A). Thus, its activity was not lost in either Mc1 + 2 or Mc4 cells (Fig. 1, B and C). In a previous study of Graves’ TSAbs (9), a rare set of TSAbs was noted with similar properties. The functional epitopes of these TSAbs were determined to involve a combined epitope present on both the Mc1 + 2 and Mc4 region, not either alone. Alternatively, they were regions other than Mc1 + 2 and Mc4 epitopes. We hypothesize that this might be the case for this patient and are making stable TSHR/LH-CGR clones with the appropriate substitutions to test this possibility. Were this the case, her TSBAb, whose epitope is solely within the Mc1 + 2 substitution region, would not be expected to fully block her TSAb activity, which has a neither Mc1 + 2 nor Mc4 epitope alone.

It is known that TSBAb from patients with primary myxedema, whose functional epitopes are, in large measure, associated with the C-terminus of the extracellular domain, can block almost all Graves’ TSAbs (17). Our previous finding that monoclonal TSBAbs having a C-terminal epitope could block Graves’ TSAbs, whereas those with an N-terminal epitope could not (18), supports this. This suggests that TSBAbs with the C-terminal epitope can block a TSAb whose functional epitope is solely on the N-terminal region of the extracellular domain. Although the precise mechanism is unknown, we speculate that the blocking antibodies having a C-terminal epitope not only competitively inhibit TSH-binding but also might cause conformational change of the receptor or inhibit signal transduction of the receptor by TSAb. Interestingly, TSBAbs from patients with primary myxedema blocked the patient’s TSAb more efficiently than her own TSBAb (Table 4). This may reflect the potential interaction of her TSAb with a combined epitope involving Mc4, as well as the fact that her TSBAb has its functional epitope on the Mc1 + 2, rather than the Mc4, region. Thus, the TSBAb in patients with primary myxedema would block the interaction of her TSAb with both regions, whereas her own TSBAb would not, because it is unable to block her interaction with the Mc4 region.

In summary, we describe a Hashimoto’s patient wherein heterogeneous TRAbs seem to have influenced her thyroid function. We propose that the unique epitope heterogeneity in her TRAbs may contribute the unique thyroid functional state in this patient. Future investigations defining the combined TSAb epitope that we hypothesize exists in this patient’s case, plus the study of more cases, will be necessary to prove this possibility and will be important to determine pathophysiological significance of TRAbs in so-called TRAb diseases. The recent report by Grasso et al. (19) found that 47% Graves’ sera seemed to have TSH-BAb whose epitopes were N-terminal. Because their sera lost both TSH-BAb and TSAb activities in Mc1 + 2, it is difficult to evaluate interactions between their own TSAb and TSH-BAb. However, the blocking antibodies present in our patient may resemble those in their Graves’ sera, suggesting the overlap among the different autoimmune thyroid diseases, Hashimoto’s thyroiditis, primary myxedema, and Graves’ disease.

Finally, species specificity of TSAb among human, rat, and porcine TSHR is documented in this report (Table 1). The overall amino acid homology between human and rat TSHR is 86% (8). Although the entire amino acid sequence of the porcine receptor has not been published, a part of the extracellular domain of the porcine receptor corresponding to amino acid residues 261–343 of human or rat TSHR (8) showed 86% and 87% of homologies to human and rat TSHR, respectively (personal communication with Dr. Donald Sellitti and Ref. 20). Discrepancies in TSAb activities between species, e.g. human and rat (21), or human and porcine (22), have been reported. Because current commercial assays or kits for TBII and TSAbs usually rely on either porcine, human, or rat TSHR assays, there will always be the possibility of a negative value, as a result of species specificity. It becomes important to consider this possibility and to insure that a negative value is not a false-negative resulting from species specificity (21, 23).

	Acknowledgments

 
We thank Miss Maki Kochi for her excellent secretarial assistance.

	Footnotes

 
¹ Supported in part by a grant in aid from the Inamori Foundation (to T.A.).

Received July 28, 1999.

Revised February 14, 2000.

Accepted February 27, 2000.

	References

Top Abstract Introduction Case Report Subjects and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Akamizu, T. Articles by Nakao, K. Search for Related Content PubMed PubMed Citation Articles by Akamizu, T. Articles by Nakao, K.