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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 7 2302-2308
Copyright © 1998 by The Endocrine Society

Original Studies

Thyrotropin Receptor Epitopes Recognized by Graves’ Autoantibodies Developing under Immunosuppressive Therapy

Jacobo Wortsman, Peter McConnachie, Kazuo Tahara1 and Leonard D. Kohn

Departments of Medicine (J.W.) and Microbiology Immunology (P.M.), Southern Illinois University School of Medicine, Springfield, Illinois 62701; the Second Department of Internal Medicine, Chiba University School of Medicine (K.T.), Chiba 260, Japan; and the Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (L.D.K.), Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Leonard D. Kohn, Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Building 10, Room 9C101B, National Institutes of Health, 10 Center Drive, MSC 1800, Bethesda, Maryland 20892-1800. E-mail: lenk{at}bdg10.niddk.nih.gov


   Abstract
Top
 Abstract
Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 
Abnormal modulation of the immune system is a prerequisite for the expression of Graves’ disease. Thus, when hyperthyroidism developed in a renal transplant recipient under long term immunosuppression with cyclosporine A and prednisone, we carefully evaluated the basis for her hyperthyroidism and her state of immunosuppression. Immunosuppression was confirmed by finding markedly deficient lymphocyte responses to common mitogens. Lymphocyte phenotype frequencies were those previously found in Graves’, i.e. elevated frequencies of CD3/DR, CD5/26, and CD3/25 lymphocytes. There was also reversal of the CD4/CD8 ratio due to increased CD8 frequency; this is not a typical finding in autoimmune hyperthyroidism, but has been seen in the intrathyroidal lymphocyte populations of some Graves’ patients and is associated with other forms of autoimmunity. The patient’s serum contained a broad spectrum of TSH receptor autoantibodies (TSHRAbs) characteristic of Graves’ disease. To determine whether these were an unusual population of autoantibodies, we determined their functional epitopes before and for nearly 1 yr after radioiodine therapy. Stimulating TSHRAbs that increase cAMP levels were human receptor (TSHR) specific and consistently recognized functional epitopes located on TSHR residues 90–165. Stimulating TSHRAbs that increased arachidonate release and inositol phosphate levels recognized residues 25–90, as did TSH binding inhibitory Igs present in the patient. These data demonstrate that Graves’ disease with a wide array of TSHRAbs can develop in a patient despite adequate immunosuppression. More importantly, they show that the cAMP-stimulating TSHRAb associated with disease expression in this patient had a homogeneous subtype dependent on TSHR residues 90–165. As persistence of this type of TSHRAb over time has been associated with resistance to methimazole therapy in Graves’ patients, we speculate that the development and persistence of TSHRAb with this homogeneous epitope may be linked to resistance to immunosuppressive therapy.


   Introduction
Top
 Abstract
 Introduction
Case Report
 Materials and Methods
 Results
 Discussion
 References
 
THEORIES abound as to whether Graves’ disease is primarily a disorder of immune cells, cytokine production, abnormal target tissue expression of class I or class II major histocompatibility complex (MHC) genes, all three together, or even a genetic disease (1, 2, 3). Recently, a Graves’ disease model has been produced in mice immunized with fibroblasts transfected with the TSH receptor (TSHR) and aberrantly expressed MHC class II molecules (4, 5). Thus, it appears that the acquisition of antigen-presenting ability on a target cell containing overexpressed TSHR can activate normal T and B cells and induce a disease with the major features of autoimmune Graves’ disease. Nevertheless, a prerequisite of disease expression is abnormal modulation of the immune system. Immunosuppressed patients would still not be expected to develop Graves’ disease.

In this report, we describe a patient who developed Graves’ disease while under long term immunosuppression for a renal transplant. Hyperthyroidism associated with diffuse thyroid hyperplasia and increased iodide uptake was noted, yet initial measurements of stimulating TSHR antibodies (TSHRAbs) that increase cAMP levels or TSH binding-inhibiting Igs (TBIIs) in assays based on nonhuman thyroid tissues were negative. Subsequent testing disclosed, however, the presence of multiple IgGs directed against the human (h) TSHR, consistent with hyperthyroidism and the diagnosis of Graves’ disease, as well as growth autoantibodies measurable in rat FRTL-5 thyroid cells. These studies do more, however, than establish that Graves’ disease can develop in a state of decreased immunocompetence. They define the spectrum of TSHRAbs that may result in this unusual clinical presentation. They demonstrate that the stimulating TSHRAbs present in the patient are dependent on the same homogeneous epitope (TSHR residues 90–165) as those in Graves’ patients resistant to therapy with antithyroid drugs (6, 7). In both cases they persist over a prolonged period before and after therapy. The data raise the possibility that persistence of a homogeneous population of autoantibodies directed at this epitope may be associated with resistance to immunosuppression.


   Case Report
Top
 Abstract
 Introduction
 Case Report
Materials and Methods
 Results
 Discussion
 References
 
In July 1996, a 64-yr-old female Caucasian was referred to the Endocrinology Service because of hyperthyroidism. For the previous 3 months she had been experiencing progressive weight loss (~4 kg) with normal or increased appetite, diarrhea, and marked fatigue. Thyroid function tests performed elsewhere had shown the following: serum T4 of 14.7 µg/dL (reference range, 4.5–12), free T4 index of 26.3 U (reference range, 3.3–15.4), and TSH of 0.2 µU/mL (reference range, 0.2–4.0). The patient had polycystic kidney disease and received a cadaver renal graft in 1985. Subsequently, she had been receiving continuous immunosuppressive therapy consisting of prednisone (17 mg on alternate days) and cyclosporine A (75 mg daily). Additional therapy included diphenoxylate HCl (Lomotil; G. D. Searle, Chicago, IL), metroprolol (Lopressor; CibaGeneva, Summit, NJ), ranitidine HCl (Zantac; Glaxo Wellcome, Research Triangle Park, NC), and amlodipine (Norvasc; Pfizer, New York, NY). Her renal function was stable (serum creatinine, 1.7 mg/dL; blood urea nitrogen, 29 mg/dL). The past medical history included a mastectomy for breast cancer performed in July 1990, removal of a squamous cell carcinoma from the skin of her chest in September 1994, and a subtotal parathyroidectomy in February 1996. The operative report from the latter surgery did not note any thyroid pathology. There was no history of autoimmune or thyroid disorders in her family.

On physical examination, there was slight bilateral exophthalmos (22 mm; normal, <18), and the thyroid gland appeared enlarged, although it was difficult to palpate. The results of initial thyroid function tests performed on August 1, 1996 are listed in Table 1Go. Stimulating TSHRAbs, measured at Mayo Laboratories (Rochester, MN) using FRTL-5 cells and cAMP as the end point, were negative at less than 1.0 (Grave’s disease, >1.3). TBIIs were measured using solubilized porcine thyroid membranes and were also negative, with only 3.2% inhibition (Graves’ disease, >10%). The 131I thyroid uptake was 27.4% at 6 h (4–9%) and 67.4% at 24 h (11–33%). The thyroid scan showed diffuse glandular enlargement to approximately twice normal size with homogeneous isotope uptake. Fine needle aspiration cytology of the thyroid showed benign follicular cells and colloid. A computed tomography scan of the orbits showed bilateral exophthalmos. The humoral tumor markers CA 15-3, CA 125, {alpha}-fetoprotein, carcinoembryonic antigen (CEA), and hCGß were within the normal range. The blood cyclosporine A concentration was 90 ng/mL, which was within the therapeutic range (i.e. 80–140).


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  		Table 1. Thyroid function tests results

 
The patient was treated with 12 mCi 131I on August 16, 1996 (Table 1Go). Three weeks later, on September 5, 1996 (Table 1Go), the patient was still thyrotoxic. At that time the concentrations of Igs were: IgG, 1313 mg/dL (range, 690-1400); IgA, 258 mg/dL (range, 70–370); and IgM, 443 mg/dL (range, 40–240). The diarrhea almost completely disappeared 1 month after 131I therapy. Three months after treatment there was significant weight gain (~3 kg), the thyroid gland was not palpable, and thyroid function tests were indicative of hypothyroidism (Table 1Go). L-T4 therapy (0.112 mg daily) was started, and the patient has remained well through her most recent visit to the clinic on July 24, 1997 (Table 1Go).

Because of the discrepancy between the florid clinical picture of Graves’ disease and the apparent absence of TSHR autoantibodies, we reevaluated the possible presence of the latter using a recently developed hTSHR-hTSHR/LH-CG receptor (LH/CGR) chimera bioassay and hTBII membrane binding system as well as a broader array of rat FRTL-5 cell assays.


   Materials and Methods
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
Results
 Discussion
 References
 
Lymphocyte function

Lymphocytes were separated from whole blood using standard methods (8) and were cultured for 3 days with the following mitogens (9): phytohemagglutinin, concanavalin A, and pokeweed. The lymphocyte proliferative capacity was determined by [3H]thymidine incorporation.

Lymphocyte phenotype frequencies (CD3, CD4, CD8, CD16/56, CD19, CD25, CD26, and DR) were measured in 0.1-mL aliquots of whole blood using two-color combinations of phycoerythrin- or fluorescein isothiocyanate-labeled monoclonal antibodies (9). Stained samples were lysed and fixed (Qprep, Coulter, Hialeah, FL), then analyzed in an XL flow cytometer (Coulter).

IgG preparation

IgGs were extracted from the patient’s serum by affinity chromatography with a protein A-Sepharose CL-4B column (5, 6, 10). The IgG containing fraction was dialyzed against distilled water, centrifuged to remove denatured protein, lyophilized, and stored at -20 C until assay. Normal pooled IgG was prepared from the sera of 20 healthy subjects without thyroid disease. The purified IgGs were dissolved in assay buffer immediately before use.

TSHRAb assays in FRTL-5 rat thyroid cells or in Chinese hamster ovary cells stably transfected with hTSHR or hTSHR-LH/CGR chimeras

FRTL-5 cells (Interthyr Research Foundation, Baltimore, MD; CRL 8305, American Type Culture Collection, Rockville, MD) were the F1 subclone and exhibited properties of fresh phenotype cells during the course of these studies (5, 10, 11). Cells were fed every third day and were passaged every 6–9 days. For the assay of stimulating TSHRAb activity, cells were fed every 3 days with medium lacking TSH (5H medium) and were maintained therein for 7 days (5, 10, 11).

Assays with the hTSHR used CHO cells stably transfected with the hTSHR, the Mc2, Mc1+2, or Mc4 chimeras (5, 10, 11, 12, 13, 14). In Mc1+2, residues 8–165 of the TSHR were replaced by residues 10–166 of the LH/CGR. In Mc2, residues 90–165 of the TSHR were replaced by residues 91–166 of the LH/CGR. In Mc4, TSHR residues 261–370 were replaced by residues 261–329 of the LH/CGR. Cells were maintained in F-12 medium containing 10% FCS with 1 mg/mL geneticin. Assays were typically performed with 5–6 x 105 confluent cells/well.

Stimulating TSHRAb activity was measured as previously detailed (5, 6, 10, 11, 12, 13, 14). Cells were incubated with the patient’s IgG (5 g/L), 1 x 10-10 mol/L/L bovine TSH, a standard Graves’ IgG with stimulating TSHRAb activity (positive control), or normal IgG (negative control). After incubation, the supernatants were frozen and stored at -20 C until the cAMP content was measured using a commercial RIA. Conversion assays were performed using Mc2-transfected CHO cells with a procedure previously described (10, 14). Cultured cells were incubated with patient’s IgG for 30 min at 37 C, washed with the same buffer, then incubated with goat antihuman IgG (Fab fragment specific) for 30 min at 4 C, then for 3 h at 37 C.

Total inositol phosphate (IP) was measured in 12-well plates as previously described (10, 15). In brief, cells were incubated overnight in inositol-free DMEM with 10% FCS and 2 µCi/mL myo-[2-N-3H]inositol. IP formation was determined using Dowex AG1-X8 (Bio-Rad, Richmond, CA) columns. Values in each well were corrected for cell protein and total tritiated inositol incorporated. Arachidonic acid release was determined on cells preincubated overnight with 20 µCi [3H]arachidonic acid/10-cm diameter dish (10, 16). After being washed with NaCl-free Hanks’ Balanced Salt Solution, cells were incubated in the same buffer for 30 min with TSH, IgG, or 10 µmol/L A23187; the released radioactivity was then measured.

[3H]Thymidine incorporation into FRTL-5 thyroid cells was performed as previously described (10, 11, 17). Incubation with IgG plus 0.1 µCi tritiated thymidine was allowed to proceed for 72 h. Controls were 1 x 10-10 mol/L bovine TSH, known Graves’ IgG with stimulating TSHRAb activity as a positive control, and normal IgG as a negative control.

[125I]TSH binding to membranes containing the hTSHR or TSHR/LH-CGR chimeras, as a measurement of TBII activity

TBIIs were measured using either a commercial kit with solubilized porcine thyroid membranes (TRAK assay, BRAHMS, Berlin, Germany) or solubilized membranes from CHO cells transfected with wild-type TSHR or Mc2 or Mc1+2 chimeras (10, 14). TBII activity was expressed as the percent inhibition of [125I]TSH binding to the TSHR by comparison to pooled normal IgG. TBII values that exceeded 12%, which is greater than 2 SD above the mean value from 20 normal samples, were considered positive.

Statistical analysis

One-way ANOVA was used to determine the level of significance for the differences between groups. Spearman’s rank correlation coefficient was used to validate the correlation between two series of data.


   Results
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
Discussion
 References
 
Immune status of the patient

Tests performed on August 16, 1996, before 131I therapy, showed that lymphocyte responses to three common mitogens were consistently deficient (Table 2Go). Repeat mitogen stimulation tests, performed when the patient was hypothyroid, gave results essentially unchanged from those observed during the thyrotoxic phase (Table 2Go).


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  		Table 2. Lymphocyte responses to mitogens

 
Determination of lymphocyte phenotype frequencies in the same sample showed that CD3 (T helper), CD19 (B cells), and CD16/56 (natural killer cells) were within the reference ranges. In contrast, the frequency of CD8 (cytotoxic T cells) was slightly elevated to 37.7% (reference range, 17–31), and the CD4/CD8 ratio was markedly decreased at 0.9:1 (reference range, 1.1–2.6:1). Also, the frequencies of activated T cells were elevated: CD3/CD25 (interleukin-2 receptor), 33.3% (reference range, 0.9–8.4); CD3/DR, 9% (reference range, 0–6.4); and CD26/CD5, 24.5% (reference range, 6.1–8.6).

TSHR autoantibodies

In the August 16, 1996 serum sample, the patient’s IgG failed to increase cAMP levels in FRTL-5 cells (Table 3Go, column 2), consistent with earlier data from the commercial assay. Subsequent to her radioiodine therapy, a very slight stimulation in the same cell system was, however, noted (Table 3Go, column 2).


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  		Table 3. cAMP-stimulating and growth-promoting activities of patient IgG as a function of time

 
In contrast, assays using CHO cells transfected with the hTSHR clearly showed cAMP-stimulating TSHRAb activity before 131I treatment (Table 3Go, column 3). The cAMP-stimulating TSHRAb activity was wholly located on the N-terminus of the extracellular domain, as activity was lost in both the Mc2 and Mc1+2 chimeras (Table 3Go, columns 4 and 5), with, respectively, residues 90–165 and 8–165 substituted by comparable LH/CGR residues. Unlike Mc2 and Mc1+2 assays, stimulating activity was retained in assays using the Mc4 chimera (Table 3Go, column 6). For example, in the serum sample from September 5, 1996, patient’s IgG increased cAMP levels 272 ± 14% above control values in Mc4 assays vs. 293 ± 19% in the wild-type TSHR assays (Table 3Go, column 6 vs. column 3). Normal IgG values were in both cases less than 128% of the control value (Table 3Go, column 6 vs. column 3).

After radioactive iodine treatment, the titer of stimulating antibody increased, consistent with rising antigen load from glandular destruction. Although this was only a trend with no statistical significance in assays, using the wild-type hTSHR-CHO cell assay (Table 3Go, column 3), there was a statistically significant (P < 0.05) increase in cAMP-stimulating TSHRAb activity that could be measured in FRTL-5 cells (Table 3Go, column 2). This was also evident in assays of growth IgG activity (Table 3Go, bottom; see below). Nevertheless, the stimulating TSHRAb activities in these cAMP assays maintained an absolute requirement for residues 90–165 over time (Table 3Go). This phenomenon has been termed retention of a homogeneous epitope and is seen in Graves’ patients resistant to antithyroid drug therapy (6, 7). Almost 1 yr after 131I therapy, autoantibody activity was still detected, albeit with a decreasing titer indicative of glandular destruction with decreased antigen load.

Besides increasing cAMP levels, the patient’s IgG exhibited growth-promoting activity in rat FRTL-5 thyroid cells, measured as increased tritiated thymidine uptake, both before and after radioiodine therapy. The patient’s IgG was also able to increase arachidonic acid release and inositol phosphate levels in both FRTL-5 cells and CHO cells transfected with the wild-type hTSHR (Table 4Go, columns 2 and 3). In both cases the activity was retained in the Mc2 chimera but was lost in the Mc1+2 chimera (Table 4Go, columns 4 and 5, respectively), suggesting that the epitope for these TSHRAb(s) was localized to residues 25–90 on the N-terminus of the extracellular domain. This is in contrast to the cAMP-stimulating TSHRAb, whose activity required residues 90–165.


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  		Table 4. Other stimulating TSHRAb activity of the patient IgG (sample of 9/5/96)

 
We confirmed the absence of significant TBII activity noted in the commercial assay using porcine thyroid membranes (Table 5Go). TBII activity, however, was clearly detected in solubilized membranes from CHO cells transfected with the hTSHR (Table 5Go). The TBII activity was slightly decreased in assays using membranes from cells transfected with the Mc2 chimera, but was completely lost in assays using membranes from cells transfected with the Mc1+2 chimera (Table 5Go). The TBII activity was retained in assays using membranes from Mc4 cells (Table 5Go, column 6), whereas the TBII activity of the IgG from the control myxedema patient was lost in these assays (Table 5Go, column 6 vs. columns 2–5). These results indicated the TBII was a Graves’ TBII, not a TBII associated with blocking TSHRAb activity and hypothyroidism (10, 12, 13, 14). These results additionally suggest that the Graves’ TBII activity required an epitope on residues 25–90, similar to the stimulating TSHRAbs that increased arachidonic acid release and IP levels (Table 4Go). This type of Graves’ TBII is associated with conversion activity, i.e. the ability of antihuman IgG to allow the expression of stimulating TSHRAb activity in the Mc2 chimera assay (10, 14). This was true in the case of this patient. Thus, in the absence of antihuman IgG, patient IgG did not increase cAMP levels in assays using the Mc2 chimera (Table 3Go). In contrast, in the presence of antihuman IgG, cAMP levels increased to 152 ± 16% (normal pooled IgG, <128% of control; P < 0.05).


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  		Table 5. TBII activities over time

 
In summary, as is the case in most patients with Graves’ disease, the marked clinical hyperthyroidism of the present case was associated with the presence of multiple stimulating TSHRAbs (1, 2, 3, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18) (Fig. 1Go). The patient had stimulating TSHRAbs that increased cAMP levels and stimulated the phosphoinositol phosphate-arachidonic acid signal transduction system. The sum of these activities was the cause of enhanced thyroid metabolism with increased thyroid hormone synthesis; their combined activities probably also caused the thyroid growth-promoting activity (3, 16, 17). The cAMP-stimulating activity was dependent on an epitope between residues 90–165, whereas the phosphoinositide/Ca2+/arachidonate signal transducing activities were dependent on an epitope localized between residues 25–90. These IgGs were, therefore, distinct (Fig. 1Go). There was also Graves’ TBII activity directed at residues 25–90 of the extracellular domain of the TSHR (Fig. 1Go). It is not certain whether the TBII was also responsible for the stimulation of both arachidonate release and increased IP levels, as reported in a recent monoclonal TSHRAb study (10).



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  		Figure 1. Putative three-dimensional model of the TSHR localizing the TSHRAb epitopes of the present patient. The model is derived from work reviewed in Ref. 3. Determinants important for the action of TSH are presumed to comprise the blocking TSHRAb epitope, the stimulating TSHRAb epitopes, and the Graves’ TBII epitopes (3 6 7 10 14 ). The expression of hypothyroidism has been associated with the functional epitope for blocking TSHRAbs. The loop between residues 303 and 382 is x-marked and separated from the remainder of the external domain, because residues within it can be deleted without loss of receptor function (3 ). Most Graves’ sera react with the immunodominant peptide located on residues 352–366 of this loop (3 ). In this patient, the functional epitope for the cAMP-stimulating TSHRAb was associated with residues 90–165, whereas the epitopes for the TBII and the stimulating TSHRAbs increasing arachidonate release or inositol phosphate levels were associated with residues 25–90. Blocking TSHRAbs of the type seen in hypothyroid patients with idiopathic myxedema were not detected.

 

   Discussion
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
References
 
The present case illustrates the rare occurrence of Graves’ disease in an environment of continuous immunosuppression. Recently, in a setting similar to that of our patient, the reoccurrence of autoimmune diabetes mellitus was reported in two recipients of cadaver pancreas grafts (19). While those two patients appeared to represent persistence of a preexisting condition, the autoimmune thyroid disease of the present subject occurred de novo. This assumption is based on the absence of visible thyroid abnormalities during the parathyroidectomy performed 5 months previously as well as the rapid clinical course of the hyperthyroidism.

Adequate immunosuppression was suggested by the absence of graft rejection episodes and by the history of a skin cancer known to be associated with immunosuppression (20, 21); it was confirmed by the deficient mitogen proliferative response observed during her disease course. As a state of significant immunosuppression was associated with the apparent absence of the signature Graves’ IgGs measured in initial assays using nonhuman assay systems, the possibility of an autoimmune disease was considered unlikely at first. A search for clinical or humoral evidence of neoplasia, in particular hCG, an occasional mediator of hyperthyroidism associated with choriocarcinoma (22) was, however, negative. This prompted reevaluation of the possibility that the patient had Graves’ disease despite adequate immunosuppression.

The higher than control frequencies of CD3/CD25-, CD26/CD5-, and CD3/DR-activated T lymphocytes were consistent with previous observations in Graves’ disease (23). An increased frequency of CD26 lymphocytes has also been described in patients with autoantibodies or autoimmunity (18, 24). An increased frequency of the CD8 cytotoxic/suppressor population and decreased CD4/CD8 ratio are unusual in Graves’ disease (1); however, these changes were noted in the intrathyroidal lymphocyte populations of Graves’ patients (25), and an elevated CD8 frequency may occur in other situations of autoimmunity (26). These phenomena may, therefore, be related both to her Graves’ disease and to an underlying immune reaction that is the consequence of her renal transplant. Nevertheless, the additional immunological studies performed with her purified IgG unequivocally confirmed the diagnosis of Graves’ and provided an explanation for the absence of cAMP-stimulating TSHRAbs or TBIIs in the nonhuman assay systems.

Thus, the cAMP-stimulating TSHRAb and TBII in this patient displayed species specificity in the test systems, exhibiting activity only when tested on CHO cells expressing wild-type hTSHR. Such pathogenic IgGs fall in the small fraction (~10%) of cases in which IgGs are better measured with a human TSHR-based assay (27). These data do not, however, imply that TSHRAbs are better measured in human rather than nonhuman test systems. First, the patient IgG did have activity against FRTL-5 cells in assays measuring IP accumulation, arachidonic acid release, and growth-promoting activity. Growth-promoting activity in FRTL-5 cells has been suggested as a means to detect TSHRAbs in Graves’ patients whose IgGs do not increase cAMP levels in FRTL-5 cells (6, 27) and, in retrospect, may reflect the activity of stimulating TSHRAbs that increase the IP/arachidonate signal in the patients (10, 16). Second, in some patients with bonafide clinical Graves’ disease, cAMP-stimulating TSHRAb activity is better measured in rat FRTL-5 than in human receptor-containing cells (6, 27). Thus, when there is clinical evidence of Graves’ disease, failure to detect autoantibody activity in a single assay system may be overcome by measuring TSHRAbs in human and nonhuman test systems.

Unfortunately, we have no definitive explanation for the apparent species specificity of this patient’s cAMP-stimulating TSHRAbs, only speculation. Species specificity is not exhibited for her growth antibodies, which are expressed functionally in FRTL-5 cells, or for her stimulating TSHRAbs, which increase arachidonate release or IP levels. As the Mc1+2, but not the Mc2, chimera loses the latter activities, and the growth antibodies may be related to the arachidonate/IP changes, the species specificity of the cAMP-stimulating TSHRAb may be linked to the Mc2 region, residues 90–165. This is consistent with the observation that the functional epitope of the cAMP-stimulating TSHRAbs detected in this patient requires residues 90–165. The regions between residues 90–165 in rat and human TSHR are highly homologous; nevertheless, amino acid differences do exist. Mutation studies involving these different residues may, therefore, be revealing in resolving this problem.

Significant immunosuppression to prevent graft rejection was induced by cyclosporine A/prednisone therapy in this patient. Cyclosporine A is known to inhibit T and B cell proliferation induced by Ca2+; interleukin-3, -4, or -5; and interferon-{gamma} (28). It also decreases cytotoxic T cell exocytosis and class II MHC expression by monocytes (28). Prednisone causes marked depression in the number of circulating T and B cells and inhibition of interleukin-1 and -2 synthesis (24) and suppresses MHC class I gene expression in thyrocytes (29). Interestingly, glucocorticoid-induced immunosuppression also prevents autoimmune thyroid disease, as shown in patients with Cushing’s syndrome due to adrenal adenoma (30). In these patients removal of the excess cortisol source has led to exacerbation of autoimmune thyroid disease (30). Nevertheless, there is also evidence indicating that Graves’ disease can override the effect of moderate iatrogenic immunosuppression resulting from prednisone administration (31, 32, 33, 34), even with the addition of cyclosporine (34).

In the present work we confirmed that a lymphocyte phenotype pattern of autoimmunity can coexist with deficient lymphocyte responses to mitogens. Such an occurrence is, however, rare. When contacted, the United Network for Organ Sharing Scientific Registry (Richmond VA; phone communication) stated that there were no records on the development of Graves’ disease in patients with renal transplants. This patient, therefore, afforded a rare opportunity to evaluate the epitopes of the TSHRAbs forming despite adequate immunosuppression. It is noteworthy that the cAMP-stimulating TSHRAbs of our patient were consistently of the homogeneous subtype, dependent on residues 90–165, throughout the follow-up period. The persistence of homogeneous subtype antibodies of this type before and after therapy has been associated with resistance to therapy with methimazole (7, 8). Methimazole has been considered to have an immunosuppressive action (35, 36, 37, 38). This is most recently evidenced by its ability to decrease MHC class I and II gene expression in thyrocytes (39, 40) and to mimic the action of a class I knockout in preventing or treating other autoimmune diseases (41, 42, 43, 44, 45, 46). Obviously, the spectrum of cAMP-stimulating TSHRAbs detected in this patient must be measured in other Graves’ patients with clearly documented immunosuppression. If the same phenomenon is measured in these rare occurrences, it is reasonable to speculate that the development and persistence of TSHRAbs with this type of epitope may be linked to resistance to immunosuppressive therapy. Studies in the Graves’ model have now demonstrated the importance of residues 90–165 to the development of stimulating TSHRAbs (47). They may additionally provide us with information concerning the sensitivity of the homogeneous epitope to immunosuppressive therapy and the basis for the phenomenon.


   Footnotes
 
1 Deceased. Back

Received January 26, 1998.

Revised March 25, 1998.

Accepted April 6, 1998.


   References
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burman KD, Baker JR. 1985 Immune mechanisms in Graves’ disease. Endocr Rev. 6:183–232.[Medline]
  2. Weetman AP, McGregor AM. 1994 Autoimmune thyroid disease: further developments in our understanding. Endocr Rev. 15:788–830.[Abstract]
  3. Kohn LD, Giuliani C, Montani V, et al. 1995 Antireceptor immunity. In: Rayner D, Champion B, eds. Thyroid immunity. Austin/Georgetown: Landes; 115–170.
  4. Shimojo N, Kohno Y, Yamaguchi K-I, et al. 1996 Induction of Graves’-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA. 93:11074–11079.[Abstract/Free Full Text]
  5. Yamaguchi K-I, Shimojo N, Kikuoka S, et al. 1997 Genetic control of anti-thyrotropin receptor antibody generation in H-2k mice immunized with thyrotropin receptor-transfected fibroblasts. J Clin Endocrinol Metab. 82:4266–4269.[Abstract/Free Full Text]
  6. Kim WB, Cho BY, Park, HY, et al. 1996 Epitopes for thyroid stimulating antibodies in Graves’ sera: a possible link of heterogeneity to differences in response to antithyroid drug treatment. J Clin Endocrinol Metab. 81:1758–1767.[Abstract]
  7. Kim WB, Chung HK, Lee HK, Kohn LD, Tahara K, Cho BY. 1997 Changes in epitopes for thyroid stimulating antibodies in Graves’ sera during treatment of hyperthyroidism: therapeutic implications. J Clin Endocrinol Metab. 82:1953–1988.[Abstract/Free Full Text]
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