J. Endocrinol. Invest. 14: 123-130,1991
Anti-bovine thyrotropin autoantibodies in patients with Hashimoto's thyroiditis, subacute thyroiditis, and systemic lupus erythematosus S. Sakata*, H. Takuno*, K. Nagai**, Y. Kimata*, H. Maekawa*, M. Yamamoto*, N. Takeda*, Y. Ochi***, and K. Miura* *Third Department of Internal Medicine, Gifu University School of Medicine, Gifu 500, **Department of Internal Medicine, Takayama Red Cross Hospital, Takayama 506, and ***Central Clinical Laboratory, Shiga University of Medical Science, Ohtsu 520-21 Japan.
ABSTRACT. We report four cases found to have anti-bovine thyrotropin (bTSH) antibodies, two with Hashimoto's thyroiditis and the other two, each with subacute thyroiditis and systemic lupus erythematosus (SLE). The unusually high negative titers of anti-TSH receptor antibodies (Case no. 1, -43.1 %; Case no. 2, -34.9%; Case no. 3, -55.2%; Case no. 4, -59.9%) led to the incidental finding of the presence of anti-bovine (bTSH) antibodies in each patient. Case no. 1 was diagnosed to have Hashimoto's thyroiditis and was treated with L-thyroxine (L-T4)' With the treatment, serum free T 4 (FT 4)normalized with a decline in the serum TSH concentration. The other patient diagnosed to have Hashimoto's thyroiditis (Case no. 2) remained euthyroid even without supplemental thyroid hormone therapy and the serum concentrations of FT4 and TSH stayed within the nor-
mal range. The third is a case of subacute thyroiditis (Case no. 3) with a typical clinical course of the disease. She had the anti-bTSH antibodies on her first outpatient visit. Serial examination of her sera disclosed the antibody titers to be on the same range over the 28 months after the onset of the symptoms. The fourth is a patient with SLE who had been treated with steroid (alternative day therapy of 40 mg/day prednisolone). Titers of the anti-bTSH antibodies spontaneously declined to the negative level 5 months later. None of the four cases had antibodies against human TSH a-subunit of bovine LH and asubunit of bovine FSH. Except for the SLE patient whose anti-bTSH antibodies disappeared spontaneously, the rest of the three patients persistently showed the antibodies suggesting a continuous antigenic stimulation by bTSH or bTSH-like molecule.
INTRODUCTION
stimulatory effect of anti-TSH receptor autoantibodies (8, 9, 10), some investigators postulate that these autoantibodies could be the anti-idiotypic antibodies of the TSH receptor antibodies (11, 12). Two recent articles, however are against the opinion that anti-TSH antibodies are anti-idiotype of TSH receptor antibodies (4,5). We have experienced unusually negative TRAb values in four cases, two with Hashimoto's thyroiditis, one with subacute thyroiditis and another with SLE under steroid treatment. The addition of 12sl-bovine TSH (bTSH) to their sera, followed by precipitation with either polyethylene glycol (PEG) or rabbit anti-human IgG confirmed the presence of anti-bTSH antibodies in all of them. However, a similar experiment using 1251-humanTSH (hTSH), 12sl-bovine LHa (bLH- a) and 12sl-bovine FSH-a (bFSH-a) failed to show significantly high binding with serum gamma globulin. Since the presence of anti-TSH antibodies in Hashimoto's thyroiditis is a rare condition (13), and no case has yet been reported on the pres-
The finding of inappropriately high radioimmunoassay (RIA) results of TSH by Chaussain et al. (1), in four patients with hypopituitarism led to the first discovery of the presence of anti-hTSH antibodies in the serum. This report was followed by another case with similar finding (2). The recent availability of anti-TSH receptor antibody (TRAb) kit had shown that the antibody prevalence in patients with Graves' disease is about 0.32 -1.30% (3-5), although others have reported that it could be as high as 17.5% (6) or 41.5% (7). Since the etiology of Graves' disease is considered to be the overproduction of thyroid hormones secondary to the
Key-words.' Hashimoto's thyroiditis, subacute thyroiditis, SLE, anti-bovine TSH antibodies. Correspondence: Dr. Shigeki Sakata, The Third Department of Internal Medicine, Gifu University School of Medicine, Gifu SOO, Japan.
Received April
9, 1990; accepted November 29,1990.
123
S. Sakata, K. Nagai, H Takuno et a/.
ence of these antibodies in subacute thyroiditis or SLE, this report will give additional information on the possible etiology as well as the clinical significance of the production and the presence of antiTSH antibodies.
bTSH for 24 h at 25 C, followed by the addition of 500 III of in-house made rabbit antihuman IgG antiserum and incubated for another 24 h at 25 C and then centrifuged. Radioactivity of the precipitate was counted and titers of anti-bTSH antibodies were expressed as the % of bound 1251-bTSH to serum gamma glubulin.
MATERIALS AND METHODS Evaluation of thyroid function in each patient
Detection of antibody to a-subunit of bLH, and bFSH Serum antibody against bLH- a and bFSH, were measured according to the method previously reported (14). Briefly, each labelled hormone preparation was incubated with 10 III of the test serum from each patient in a total volume of 1 ml containing Tris/NaCI plus 1% bovine serum albumin. After an overnight incubation at 4 C, 50 III of goat antiserum to human gamma globulin was added, and the incubation continued overnight at 4 C. Bound and free fractions were separated by centrifugation at 2,000 xgfor 30 min, and the pellets were washed twice with assay buffer to reduce nonspecific binding.
Serum free T3' free T4 (Amerlex FT3' FT4' Amersham International, Tokyo, normal range: FT 3; 4.1 - 9.1 pmol/I (2.7 - 5.9 pg/ml), FT4 11 - 26 pmol/I (0.85 2.0 ng/dl). T3, and T4 (Eiken RIA kit T3 and T4, Eiken Co, normal range: T3; 1.2 - 3.2 nmol/I (80 - 210 ng/dl), T4; 58 - 167 nmol/I (4.5 - 13.0 1l9/dl)) were measured by commercially available radioimmunoassay (RIA) kits. Two kinds of TSH determination was performed; by immunoradiometric assay (IRMA; Spac TSH, Daiichi Isotope Co., Tokyo, Japan, normal range 0.2 -5 .0 mU/I) and by double antibody radioimmunoassay (TSH kit Daiichi , Daiichi Isotope Co., Tokyo, normal range less than 8 mU/I). Serum concentration of thyroxine-binding globulin (TBG) was measured by a single antibody RIA kit (RIA-gnost TBG, Hoechst, normal range: 16 - 22 mg/I). Serum TRAb were measured by a commercially available radioreceptor assay (RRA) kit (Baxter, normal range: -10 - + 10%). Serum antithyroglobulin and anti microsomal antibodies were measured by hemagglutination technique (TGHA, Thyroid test and MCHA, Microsome test, both Fujizoki, Tokyo). 1231-uptake of the thyroid was done by the standard 24 hour method.
Case reports Case no 1: A 36-year-old female with a chief complaint of an enlarged thyroid gland consulted at our outpatient clinic for the evaluation of thyroid function on May 27, 1988. Her past and family histories were unremarkable. On physical examination, she was 151 cm tall and weighed 50 kg. Her temperature and pulse rate were 36 .6 C and 76/min, respectively. She had an enlarged non-tender thyroid gland which was rubbery hard with no palpable nodules. Relaxation time of her Achilles jerk was delayed . Thyroid function tests showed low serum T3' T4' FT3' and FT 4' being 1.4 nmol/I (94 ng/dl), 39 nmol/I (3 .0 1l9/dl), 5.2 pmol/I (3.4 pg/ml), and 5.5 pmol/I (0.43 ng/dl), respectively and her serum TSH level was high, being 51.1 mU/I. She had positive antithyroglobulin (102, TGHA) and antimicrosomal antibodies (202, MCHA). A diagnosis of Hashimoto's thyroiditis was made and treatment was started with 25 Ilg/day of synthetic L-T4' The clinical course of the patient is shown in Figure 1. Her TRAb values fluctuated between -43.3 - -64.6% which suggested the presence of anti-TSH antibodies in her serum. Case no. 2: An 82-year-old female consulted in our outpatient clinic because of an enlarged thyroid gland since October 1985. She was diagnosed to have hypertension in 1983 and gastric ulcer and gall stone in 1984. She denied any history of general malaise and cold intolerance. On physical examination, she was 148 cm tall and weighed 46 kg. Her
Reagents Purified hTSH, bLH- a, and bFSH- a were obtained from UCB-Bioproducts (Brussels). 1251-bTSH was from TRAb assay kit Measurement of anti-b TSH or anti-h TSH antibodies Serum anti-bTSH antibodies were measured by both single and double antibody RIAs . For single antibody RIA, 50 III of serum obtained from each patient was incubated with 1251-bTSH or 1251-hTSH for 24 h at 25 C. This procedure was further followed by the addition of an equal amount of 25% of polyethylene glycol (PEG, PEG 6,000, Wako Pure Chemicals, Osaka) and centrifugation. Radioactivity of the preCipitate was counted and titers of antibTSH or anti-hTSH antibodies were expressed as the % of bound 1251-TSH to serum gamma globulin. For double antibody RIA, 50 III of serum obtained from each patient was incubated with 125 1_
124
TSH antibodies in thyroid diseased and SLE
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Anti-bovine thyrotropin autoantibodies in patients with Hashimoto's thyroiditis, subacute thyroiditis, and systemic lupus erythematosus S. Sakata*, H. Takuno*, K. Nagai**, Y. Kimata*, H. Maekawa*, M. Yamamoto*, N. Takeda*, Y. Ochi***, and K. Miura* *Third Department of Internal Medicine, Gifu University School of Medicine, Gifu 500, **Department of Internal Medicine, Takayama Red Cross Hospital, Takayama 506, and ***Central Clinical Laboratory, Shiga University of Medical Science, Ohtsu 520-21 Japan.
ABSTRACT. We report four cases found to have anti-bovine thyrotropin (bTSH) antibodies, two with Hashimoto's thyroiditis and the other two, each with subacute thyroiditis and systemic lupus erythematosus (SLE). The unusually high negative titers of anti-TSH receptor antibodies (Case no. 1, -43.1 %; Case no. 2, -34.9%; Case no. 3, -55.2%; Case no. 4, -59.9%) led to the incidental finding of the presence of anti-bovine (bTSH) antibodies in each patient. Case no. 1 was diagnosed to have Hashimoto's thyroiditis and was treated with L-thyroxine (L-T4)' With the treatment, serum free T 4 (FT 4)normalized with a decline in the serum TSH concentration. The other patient diagnosed to have Hashimoto's thyroiditis (Case no. 2) remained euthyroid even without supplemental thyroid hormone therapy and the serum concentrations of FT4 and TSH stayed within the nor-
mal range. The third is a case of subacute thyroiditis (Case no. 3) with a typical clinical course of the disease. She had the anti-bTSH antibodies on her first outpatient visit. Serial examination of her sera disclosed the antibody titers to be on the same range over the 28 months after the onset of the symptoms. The fourth is a patient with SLE who had been treated with steroid (alternative day therapy of 40 mg/day prednisolone). Titers of the anti-bTSH antibodies spontaneously declined to the negative level 5 months later. None of the four cases had antibodies against human TSH a-subunit of bovine LH and asubunit of bovine FSH. Except for the SLE patient whose anti-bTSH antibodies disappeared spontaneously, the rest of the three patients persistently showed the antibodies suggesting a continuous antigenic stimulation by bTSH or bTSH-like molecule.
INTRODUCTION
stimulatory effect of anti-TSH receptor autoantibodies (8, 9, 10), some investigators postulate that these autoantibodies could be the anti-idiotypic antibodies of the TSH receptor antibodies (11, 12). Two recent articles, however are against the opinion that anti-TSH antibodies are anti-idiotype of TSH receptor antibodies (4,5). We have experienced unusually negative TRAb values in four cases, two with Hashimoto's thyroiditis, one with subacute thyroiditis and another with SLE under steroid treatment. The addition of 12sl-bovine TSH (bTSH) to their sera, followed by precipitation with either polyethylene glycol (PEG) or rabbit anti-human IgG confirmed the presence of anti-bTSH antibodies in all of them. However, a similar experiment using 1251-humanTSH (hTSH), 12sl-bovine LHa (bLH- a) and 12sl-bovine FSH-a (bFSH-a) failed to show significantly high binding with serum gamma globulin. Since the presence of anti-TSH antibodies in Hashimoto's thyroiditis is a rare condition (13), and no case has yet been reported on the pres-
The finding of inappropriately high radioimmunoassay (RIA) results of TSH by Chaussain et al. (1), in four patients with hypopituitarism led to the first discovery of the presence of anti-hTSH antibodies in the serum. This report was followed by another case with similar finding (2). The recent availability of anti-TSH receptor antibody (TRAb) kit had shown that the antibody prevalence in patients with Graves' disease is about 0.32 -1.30% (3-5), although others have reported that it could be as high as 17.5% (6) or 41.5% (7). Since the etiology of Graves' disease is considered to be the overproduction of thyroid hormones secondary to the
Key-words.' Hashimoto's thyroiditis, subacute thyroiditis, SLE, anti-bovine TSH antibodies. Correspondence: Dr. Shigeki Sakata, The Third Department of Internal Medicine, Gifu University School of Medicine, Gifu SOO, Japan.
Received April
9, 1990; accepted November 29,1990.
123
S. Sakata, K. Nagai, H Takuno et a/.
ence of these antibodies in subacute thyroiditis or SLE, this report will give additional information on the possible etiology as well as the clinical significance of the production and the presence of antiTSH antibodies.
bTSH for 24 h at 25 C, followed by the addition of 500 III of in-house made rabbit antihuman IgG antiserum and incubated for another 24 h at 25 C and then centrifuged. Radioactivity of the precipitate was counted and titers of anti-bTSH antibodies were expressed as the % of bound 1251-bTSH to serum gamma glubulin.
MATERIALS AND METHODS Evaluation of thyroid function in each patient
Detection of antibody to a-subunit of bLH, and bFSH Serum antibody against bLH- a and bFSH, were measured according to the method previously reported (14). Briefly, each labelled hormone preparation was incubated with 10 III of the test serum from each patient in a total volume of 1 ml containing Tris/NaCI plus 1% bovine serum albumin. After an overnight incubation at 4 C, 50 III of goat antiserum to human gamma globulin was added, and the incubation continued overnight at 4 C. Bound and free fractions were separated by centrifugation at 2,000 xgfor 30 min, and the pellets were washed twice with assay buffer to reduce nonspecific binding.
Serum free T3' free T4 (Amerlex FT3' FT4' Amersham International, Tokyo, normal range: FT 3; 4.1 - 9.1 pmol/I (2.7 - 5.9 pg/ml), FT4 11 - 26 pmol/I (0.85 2.0 ng/dl). T3, and T4 (Eiken RIA kit T3 and T4, Eiken Co, normal range: T3; 1.2 - 3.2 nmol/I (80 - 210 ng/dl), T4; 58 - 167 nmol/I (4.5 - 13.0 1l9/dl)) were measured by commercially available radioimmunoassay (RIA) kits. Two kinds of TSH determination was performed; by immunoradiometric assay (IRMA; Spac TSH, Daiichi Isotope Co., Tokyo, Japan, normal range 0.2 -5 .0 mU/I) and by double antibody radioimmunoassay (TSH kit Daiichi , Daiichi Isotope Co., Tokyo, normal range less than 8 mU/I). Serum concentration of thyroxine-binding globulin (TBG) was measured by a single antibody RIA kit (RIA-gnost TBG, Hoechst, normal range: 16 - 22 mg/I). Serum TRAb were measured by a commercially available radioreceptor assay (RRA) kit (Baxter, normal range: -10 - + 10%). Serum antithyroglobulin and anti microsomal antibodies were measured by hemagglutination technique (TGHA, Thyroid test and MCHA, Microsome test, both Fujizoki, Tokyo). 1231-uptake of the thyroid was done by the standard 24 hour method.
Case reports Case no 1: A 36-year-old female with a chief complaint of an enlarged thyroid gland consulted at our outpatient clinic for the evaluation of thyroid function on May 27, 1988. Her past and family histories were unremarkable. On physical examination, she was 151 cm tall and weighed 50 kg. Her temperature and pulse rate were 36 .6 C and 76/min, respectively. She had an enlarged non-tender thyroid gland which was rubbery hard with no palpable nodules. Relaxation time of her Achilles jerk was delayed . Thyroid function tests showed low serum T3' T4' FT3' and FT 4' being 1.4 nmol/I (94 ng/dl), 39 nmol/I (3 .0 1l9/dl), 5.2 pmol/I (3.4 pg/ml), and 5.5 pmol/I (0.43 ng/dl), respectively and her serum TSH level was high, being 51.1 mU/I. She had positive antithyroglobulin (102, TGHA) and antimicrosomal antibodies (202, MCHA). A diagnosis of Hashimoto's thyroiditis was made and treatment was started with 25 Ilg/day of synthetic L-T4' The clinical course of the patient is shown in Figure 1. Her TRAb values fluctuated between -43.3 - -64.6% which suggested the presence of anti-TSH antibodies in her serum. Case no. 2: An 82-year-old female consulted in our outpatient clinic because of an enlarged thyroid gland since October 1985. She was diagnosed to have hypertension in 1983 and gastric ulcer and gall stone in 1984. She denied any history of general malaise and cold intolerance. On physical examination, she was 148 cm tall and weighed 46 kg. Her
Reagents Purified hTSH, bLH- a, and bFSH- a were obtained from UCB-Bioproducts (Brussels). 1251-bTSH was from TRAb assay kit Measurement of anti-b TSH or anti-h TSH antibodies Serum anti-bTSH antibodies were measured by both single and double antibody RIAs . For single antibody RIA, 50 III of serum obtained from each patient was incubated with 1251-bTSH or 1251-hTSH for 24 h at 25 C. This procedure was further followed by the addition of an equal amount of 25% of polyethylene glycol (PEG, PEG 6,000, Wako Pure Chemicals, Osaka) and centrifugation. Radioactivity of the preCipitate was counted and titers of antibTSH or anti-hTSH antibodies were expressed as the % of bound 1251-TSH to serum gamma globulin. For double antibody RIA, 50 III of serum obtained from each patient was incubated with 125 1_
124
TSH antibodies in thyroid diseased and SLE
50
60
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