NOTE a Case of Graves' Disease with Anti-Triiodothyronine Antibodies The

Endocrinol. Japon. 1985, 32 (1), 181-188

NOTE

A Case of Graves' Disease with Anti-triiodothyronine Antibodies

SHIGEKI SAKATA, TAKASHI KOMAKI, SHIGENORI NAKAMURA, KEITA KAMIKUBO, KAORU TAKAKUWA*, MASAAKI KAMETANI*, NAOKI TOKIMITSU*, AND KIYOSHI MIURA

The Third Department of Internal Medicine, Gifu University School of Medicine, Gifu 500 *Department of Internal Medicine, Takayama Red Cross Hospital, Takayama 504

Abstract

A case of Graves' disease with high serum thyroxine (T4) and low triiodothyronine (T3) levels which was therefore initially diagnosed as a T4-thyrotoxi- cosis is reported. Examination of the serum from the patient showed the presence of unusual protein which bound T3. It was later confirmed as IgG class anti-T3 antibodies. In addition to treatment with methylmercaptoimidazole (MMI), the patient was treated with prednisolone for 30 days (total amount 500mg). Titers of and-T3 antibodies in the sera were unchanged before and after prednisolone treatment. Our present case indicates that it is clinically important to bear the presence of autoantibodies in mind to account for a possible error in measuring T3 and T4 by radioimmunoassay (RIA). In the case that RIA determination gives an unexpectedly high or low T3 and/or T4 value, the presence of autoantibodies to them should be considered and a test for them is recommended.

Circulating autoantibodies to thyroid mone in Hashimoto's thyroiditis. The ac- hormone have been reported in various cumulation of reports up to the present sug- thyroidal (Robbins et al., 1956; Ikekubo et gest that the spontaneous occurrence of al., 1978; Ginsberg et al., 1878; Jcbrgensen antibodies to T4 and/or T3 in man may not et al., 1979; Inada et al., 1980; Pearce et be a rare phenomenon, especially in patients al., 19 81) and nonthyroidal illnesses (Tri- with Hashimoto's thyroiditis. In Graves' marchi et al., 1982; Ordonez-Llanos et al., disease, however, only several cases with 1984). This was originally reported by autoantibodies to thyroid hormone have Robbins et al. (1956) in a patient with been so far reported (Staeheli et al., 1975; papillary carcinoma of the thyroid treated JOrgensen et al., 1979; Inada et al., 1980). with 131I. Later Premachandra et al. (1967) We wish to report here a patient with demonstrated autoantibodies to thyroid hor- Graves' disease associated with circulating anti-T3 IgG class antibodies. Received July 27, 1984 Enclocrinol. Japon. 182 SAKATA et al. February 1985

Table 1

Materials and Methods (A)

Serum T4, T3, 3, 3', 5'-triiodothyronine (rT3), thyroid stimulating hormone (TSH) and thyroxine binding globulin (TBG) in this patient were measured with commercially available RIA kits by single antibody (T4, T3, rT3: Dainabot RIA kit, TBG: RIA-gnost TBG, Hoechst) and by the double antibody method (T4, T3: Eiken RIA kit, TSH: Daiichi TSH kit). The normal range of T4, T3, rT3 and TBG are 4.5-13.0ƒÊg/dl, 90-210ng/dl, 177-368pg/ml and 17.0-23.0ƒÊg/ml, respectively. The normal range of TSH is below 8.0ƒÊU/ml. Anti-thyroglobulin antibodies were detected either with a commercially available hemagglutination kit (Thyroid test, Fuji Zoki) or by solid phase RIA using 125I-protein A developed and evaluated (B) in our laboratory (Sakata et al., 1983a). Anti- microsomal antibodies were examined with a commercially available kit (Microsome test, Fuji Zoki). In order to examine the presence of anti- thyroid hormone antibodies, 20ƒÊl of the patient's serum obtained 31 months after the beginning of the MMI treatment and a control serum were incubated with 380ƒÊl of 0.06 M barbital buffer, pH 8.6 with or without 120ƒÊg of 8-anilino-1- naphthalene sulfonic acid (ANS) and 100ƒÊl of 125I-T3(20pg)or 125I-T4(100pg)fbr 24 hours

at 40℃, followed by precipitation with 12.5%

polyethylene glycol(PEG, MW 7,500). Radio- activities of the precipitates were counted. Immunoglobulin fraction from the sera of the Table 1. Bir ding of 125I-T3 (A) and 125I-T4 (B) to the ƒÁ-globulin fraction of the presented patient. patient and healthy subjects was obtained by 125I-T3 (100pg precipitating 4 times weth 50% ammonium sul- , 31,000cpm) and 125I-T4 (500pg, fate, followed by extensive dialysis against phos- 45,000cpm) were added and co-precipitated 125I- T3 and 125I-T4 with 12.5%PEG were expressed phate-buffered saline (PBS, pH 7.4). The volume of the dialyzed fraction was diluted twice as the by both cpm bound and by percentage of bound original volume with PBS and kept frozen at radioactivities. Ni, N2, N3 represent three normal controles. -20℃ until use . Ten microliters of immuno.

globulin fraction thus obtained had approximately 0.04mg of ƒÁ-globulin as measured by paper electrophoresis. Twenty microliters of immunoglobulin fraction thus obtained from the patient examined in the following manner. Each 10 pl and two healthy subjects was incubated with 125I- of the serum from the patient was incubated with T3 and titrated with increasing amounts of rabbit 950 ƒÊl of 0.06 M barbital buffer, pH 8.6, which anti-human IgG which was raised in a rabbit contains 250 ƒÊg of ANS, 50ƒÊl of 125I-T3 and 50 (GI-1). After centrifugation, the precipitates were μl of cold T3 of various concentration.s. After washed twice with 0.15M NaCl and radioac- incubation for 24 hours at 25•Ž, equal amounts tivities were counted. of 25% PEG were added to each tube, followed The binding constants (Ka) and binding capa- by centrifugation. Then the radioactivities of cities of and-T3 antibodies in this patient were the precipitaes were counted and analyzed with Vol.32, No.1 GRAVES' DISEASE WITH ANTI-T3 ANTIBODIES 183

Scatchard's plots. 125I-T3 and 125I-T4 with specific activities of 1160ƒÊCi/ƒÊg and 1250ƒÊCi/ƒÊg, respectively, were obtained from New Engl. Nucl. Protein A was purchased from Sigma Chemi- cals and radioiodinated with 125INa (Amersham, A UK) by the chloramine T method (Hunter and Greenwood., 1962). The specific activity of 125I protein A was 4-6ƒÊCi/ƒÊg. T4 and T3 were obtained from Sigma Chemicals and further purified with HPLC. The other chemicals em- ployed were all reagent grade. B

Case

A 45-year-old woman consulted the Takayama Red Cross Hospital in January 1979 complaining of swelling of the neck, finger tremor, palpitation, excessive sweating, weight loss and irregular menstrual cycles. She had been in good health until July 1978 when she began to notice symptoms. Her

past history was non-contributory and the family history was negative for thyroidal C disorders. On physical examination, she was found to be 152cm tall and her weight was 55kg. Her pulse was 100/min and regular. Blood

pressure was 138/70mmHg. Her skin was warm, moist and exophthalmos was present. Her eye changes were classified as class 2 according to the classification of the Ameri- can Thyroid Associations (Werner, 1969). Fig. 1. Thyroid function tests prior to (A), and The thyroid was symmetrically enlarged with after MMI treatment (C) and change in the a soft, smooth surface. Auscultation over concentration of T3, T4 and TSH (B) are the thyroid gland revealed a systolic mur- shown. mur. The 24-hour 131I-uptake of the thyroid in the patient was markedly increased nation technique (Thyroid test) was negative

(96.2%). T4 and T3 measured by single while the solid phase RIA using 125I-protein antibody RIA were 24.1ƒÊg/d1 and 124ng/dl, A proved positive. respectively (Fig. 1). Her basal TSH was She was started on treatment with MMI 0.8ƒÊU/ml and did not respond for two 30mg daily and her T4 as well as T3 levels hours to the intravenous injection of 500ƒÊg dropped rapidly. The clinical course of the of TRH (TRH test). The concentration of patient is summarized in Fig. 1. After be- thyroxine binding globulin was in the normal ginning treatment with MMI, she occasional- range. The test for circulating anti-thyroglo ly showed T4 levels above the normal range bulin antibodies detected by the hemaggluti- and a constantly low T3 level. Serum TSH Endocrinol. Japon. 184 SAKATA et al. February 1985 levels were suppressed throughout the in- In Oct. 1981, because she was in slightly vestigation period. hyperthyroid, an needle biopsy of the thyroid

Fig. 2. Histology of the thyroid g'and. of this patient obtained by neae biopsy.

(H & E,•~100)

Fig. 3. Binding of 125I-T3

(10,000cpm) and 125I- T4 (20,000cpm) to the serum from the patient

presented and a normal control with_??_or

without_??_addition of 120ƒÊg of ANS. Left

panel indicates the binding of 125I-T3 and right

panel indicates the binding of 125I-T4. Vol.32, No.1 GRAVES' DISEASE WITH ANTI-T3 ANTIBODIES 185

gland was done. Histologically, diffuse were slightly higher than those obtained hyperplasia of the follicular epithelial cells from a healthy subject. The addition of without lymphocytic infiltration was com- 120ƒÊg of ANS enhanced the coprecipitated patible with Graves' disease (Fig. 2). radioactivities of 125I-T3 and 125I-T4 to In Nov. 1981, since she still had high 28.8% and 8.9%, respectively. titers of anti-T3 antibodies, she was started Accordingly, 20ƒÊl of immunoglobulin on treatment with oral prednisolone, (30mg fraction from the patient and two healthy per day for 10 days, 15 mg for 10 days, subjects (N1, N2) were incubated with 125I- and 5mg for 10 days) and the titers of T3 and titrated with increasing amounts of anti-T3 antibodies before and after treatment rabbit anti-human IgG. Fig. 4 shows the were compared. titration data. The results clearly indicate that the patient had IgG class immuno-

globulin with antibody activity predominant- Results ly to T3. The association constant and binding capacity of anti-T3 antibodies in The binding of 125I-T3 and 1251-T4 to this patient were analyzed with Scatchard's

the serum from the patient and the effect plots. As shown in Fig. 5 the results of of ANS on the binding are shown in Fig. 3. Scatchard's analysis indicate that the anti- In the case where 0.06 M barbital buffer body activities to T3 can be attributed to without ANS was used, only 6.5% of the two components. One has a stronger bind- 125I -T3 and 3.8% of the 125I-T4 were co- ing constant (Ka=5.9•~108M-1) and smaller precipitated with 12.5% PEG. These values binding capacity (2.1•~10-8mol/ml serum)

Fig. 4. Twenty microliters of immunoglobulin fraction from the patient and two normal

controls was incubated with 125I-T3 (53,000 Ffg. 5. Scatchard's analysis of T3 and anti-T3 cpm) followed by precipitation with rabbit antibodies in the patient presented. Binding

anti-human IgG. Radioactivities of coprecip- of T3 with anti-T3 antibodies was analyzed itated 125I-T3 with immunoglobulin from the in two components: showing a higher binding

patient (•›) and mean values for two normal constant (Ka=5.9•~108M-1), smaller binding controls (•¢) are shown. Radioactivities of capacity (2.1•~10-8mol/ml); and a lower bind- coprecipitated 125I-T3 from two normal cont- ing constant (Ka=2.0•~107 M-1), larger bind- rols were almost identical. ing capacity (10.0•~10-8 mol/ml). Endocrinol. Japon. 186 SAKATA et at. February 1985

Fig. 6. Titers of anti-T3 and anti-T4 antibodies (as expressed by % bound radioactivites of the sera) before and after prednisolone treatment.

and the other has a weaker binding con- other than thyroxine binding globulin (TBG) stant (Ka=2.0•~107 M-1) and larger binding in her serum. capacity (10.0•~10-8mol/ml serum). Titration of 20ƒÊl of immunoglobulin On Nov. 2, 1981, since the patient had fraction which had been incubated with 125IT3 still high titers of anti-T3 antibodies, she and precipitated with increasing was started on treatment with prednisolone amounts of rabbit anti-human IgG revealed

(30mg/day). Titers of anti-T3 antibodies that the T3 binding protein in her serum before and after prednisolone treatment were was a IgG class immunoglobulin. Although 21.7% and 20.8%, respectively. we have not examined further, from the data shown in Fig. 3 it is likely that, in addition to anti-T3 antibodies, she had anti- Discussion T4 antibodies in her serum. Since Robbins et al. (1956) first reported The patient presented here showed the the presence of anti-T4 antibodies in the typical clinical picture of hyperthyroidism, serum of a papillary carcinoma patient who which was later confirmed as Graves' disease had been treated with 131I, the presence of and not Hashitoxicosis from the thyroid anti-thyroid hormone antibodies has been tissue obtained by needle biopsy. She was reported in various thyroidal and non- started on treatment with 30mg/day of MMI thyroidal disorders. These include Hashi- and the levels of serum T4 and T3 declined moto's thyroiditis (Premachandra et al., 1967; rapidly. In contrast to the high T4 value, Ochi et al., 1972; Staeheli et al., 1975; her serum T3 value prior to beginning the Herrmann et al., 1977; Ikekubo et al., 1978; MMI treatment was within the normal range. Ginsberg et al., 1978; Wiersinga et al., 1982; After beginning MMI, her serum T3 levels Nakamura et al., 1982., Suzuki et al., 1982), as measured by single antibody RIA, were hypothyroidism during desiccated thyroid constantly low even though she occasionally hormone treatment (Staeheli et al., 1975), had high serum T4 levels. Graves' disease (Staeheli et al., 1975; These laboratory data suggested to us gensen et al., 1979; Inada et al., 1980) and the presence of unusual serum protein(s) non-thyroidal illness such as WaldenstrOm's which bound predominantly with T3. In- macroglobulinemia (Trimarchi et al., 1982) cubation of the serum from the patient with and hepatocarcinoma (Ordonez-Llanos et al., 125I-T3 with or without the addition of 120 1984). Thus, most of the cases so far re-

μg of ANS, followed by precipitation with ported are Hashimoto's thyroiditis and only 12.5% PEG showed the presence of un- several cases of Graves' disease have been usual thyroid hormone binding protein(s) reported hitherto. Vol.32, No.1 GRAVES' DISEASE WITH ANTI-T3 ANTIBODIES 187

The exact mechanism of production of spond to the insulin auto-immune syndrome anti-thyroid hormone antibodies in our pa- reported in thyrotoxic patients treated with tient as well as reported cases is obscure; MMI (Hirata et al., 1974). The latter was however relationships between anti-thyro- supported by the observation of decreased globulin (Tg) antibodies and anti-thyroid amounts of thyroidal microsomal antibodies hormone antibodies have been discussed by and TSH-receptor antibodies in a thyrotoxic many investigators (Hehrmann et al., 1977; patient during carbimazole treatment which Pearce et al., 1981; Sakata et al., 1983b). has a structure similar to MMI (McGregor We have recently examined the genetic con- et al., 1980). Thus the possibility that MMI trol of the production of anti-thyroid hor- treatment induced the production of anti-T3 mone antibodies in mice immunized with antibodies in this patient cannot be excluded, human Tg (HTg) and reported that the we think it is less likely because the data production of anti-Tg as well as anti-T4 obtained by RIA prior to MMI treatment antibodies were controlled by an immune showed hyperthyroxinemia with a normal response gene(s) (Ir-gene(s)) which is located T3 level. in the I-A subregion of H-2 (Sakata et al., In contrast to the previous report (Ochi 1983 b). Our data using various congenic et al., 1972), treatment with prednisolone in and recombinant strains of mice also re- addition to the MMI did not succeed in vealed that the titers of anti-Tg and anti- reducing the titer of anti-T3 antibodies. thyroid hormone antibodies do not correlate To sum up, a case of Graves' disease in some strains of mice. H-2 is a major with anti-T3 antibodies belonging to the IgG histocompatibility complex (MHC) in mice class was reported. Although the exact and is equivalent to human leucocyte anti- mechanism of production of anti-T3 anti gen (HLA) in man. Thus, as in the case bodies is still not clear, the role of Tg as originally reported by Robbins et al. (1956) an antigen in relation to the genetic con- of a patient, who had been treated for trol of the production of anti-T3 antibodies thyroid cancer with 131I, the damage to the was discussed. The elucidation of the exact thyroid and/or Tg molecule and the leak of mechanism of production of autoantibodies native and/or modified Tg from the thyroid to thyroid hormone must await the future gland must be the first step in the produc- accumulation of similar cases and investigation of anti-thyroid hormone antibodies. tions. The recognition of antigenic sites on the native and/or modified Tg by the immune Acknowledgements system is probably genetically controlled. This may explain why anti-thyroid hormone We wish to thank Ms. Keiko Hirata and antibodies cannot be detected in all of the Mr. Yuukoo Yamada for skillful technical assis- autoimmune thyroid diseases. tance. We also would Eke to thank Ms. Chiine Unfortunately we do not have serum of Watanabe for preparation of this manuscript. this patient prior to the beginning of the This work was supported by the grant from. MMI treatment, and the presence of anti- the ministry of Health and Walfare's "Disorders. of adrenal hormone" Research Committee, T3 antibodies was confirmed with serum obtained 31 months after the beginning of Japan. the MMI treatment. The accumulating data References indicate that the use of MMI in the treatment of Graves' disease may affect the im- Ginsberg, J., D. Segal, R. M. Ehrlich and P. G. mune system. Two controversial results so Walfish (1978). Inappropriate triiodothyronine far have been reported. The former_corre- (T3) and thyroxine (T4) radioimmunoassay Endocrinol. Japon. 188 SAKATA et al. February 1985

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