Thyroid Antigens Research Articles

Adhesion molecule expression by the FRTL-5 rat thyroid cell line

We have examined the expression and function of rat CD54, a homologue of human intercellular adhesion molecule-1 (ICAM-1), by the continuously growing rat thyroid cell line FRTL-5. Approximately 10% of FRTL-5 cells express CD54 under basal conditions and this is not influenced by thyrotrophin. Expression of CD54 is increased by cytokines (gamma-interferon, tumour necrosis factor, interleukin-1) and by an activator of C-kinase, phorbol 12-myristate 13-acetate. Blocking ICAM-1 with a monoclonal antibody directed against this molecule significantly (P less than 0.01) reduced the binding of splenic lymphocytes to FRTL-5 cells but inhibition was consistently greater (P less than 0.01) in the presence of antibodies against a rat homologue of lymphocyte function-associated antigen-1, the receptor on T cells for ICAM-1. In no case was complete blocking of cluster formation observed. These results show that a pure line of rat thyroid cells can express an ICAM-1 homologue and this is directly enhanced by cytokines. Expression of this homologue is partially responsible for lymphocyte adhesion to thyroid cells, which is likely to be a major event in T cell recognition of thyroid antigens in autoimmune thyroiditis.

Autoantigen recognition by thyroid-infiltrating T cells in Graves disease.

Graves disease is a common form of human autoimmune thyroiditis. It shares many pathological features and HLA associations with other, less easily studied, organ-specific autoimmune conditions such as insulin-dependent diabetes mellitus, and hence it is also a useful model for understanding these other diseases. We have previously shown that thyroid-infiltrating T cells in Graves disease that have been recently activated in vivo specifically recognize autologous thyroid epithelial cells. However, the autoantigens involved were not defined. In this study, we have made use of antigen-independent T-cell cloning techniques to show that at least three different thyroid antigens, three different epitopes on a single antigen, and two HLA class II elements are involved in this recognition process in a single individual. This demonstrates that T cells that are present and activated at the site of a human autoimmune disease may show considerable heterogeneity in their recognition of autoantigen on the target tissue. This contrasts with the limited heterogeneity recently reported in some animal models and has potentially important implications for both our understanding of the autoimmune process in humans and the design of immunotherapies to reverse it.

Reversible thyroid dysfunction during treatment with GM-CSF

To investigate whether autoimmunity against thyroid antigens is induced or exacerbated by granulocyte-macrophage colony-stimulating factor, thyroid function and thyroid autoantibodies were studied in 14 patients with advanced breast cancer and 11 with soft-tissue sarcoma who received several cycles of doxorubicin and cyclophosphamide plus GM-CSF 250 μg/m 2 intravenously daily for 10 days in every 21 day cycle. All patients had normal thyroid function before treatment. In 2 patients with pre-existing thyroid antibodies, thyroid dysfunction developed but disappeared after cessation of GM-CSF. No other autoimmune abnormalities appeared. Stimulation of antigen-presenting cells by GM-CSF may bring about this phenomenon.

Autoimmunity after Alpha-Interferon Therapy for Malignant Carcinoid Tumors

To determine the incidence of autoantibodies and autoimmune disease and their influence on therapeutic results during alpha-interferon treatment in patients with malignant midgut carcinoid tumors. Consecutive sample of patients. University hospital. One hundred thirty-five patients (70 women, 65 men; median age, 59 years) with biopsy-proven tumors, liver metastases, and no autoimmune disease. Leukocyte alpha-interferon (n = 88) or alpha-interferon 2b (n = 47) three times a week. Signs and symptoms of autoimmune disease or development of autoantibodies to thyroid antigens, nuclear antigens, or gastric parietal cells. Tumor responses were determined by reduced liver metastases or reduced urinary 5-hydroxyindole acetic acid excretion, or both. Twenty-five patients (19%) developed the following autoimmune disorders after a median of 9 months of therapy: thyroid disease (n = 18), systemic lupus erythematosus (n = 1), pernicious anemia (n = 4), and vasculitis (n = 2). Antibodies to microsomal thyroid antigen or thyroglobulin were detected in 16 patients before therapy and in another 11 patients during therapy. Antinuclear antibodies were detected in 16 patients before and in another 19 patients during therapy. Clinical thyroid disease developed in more than 60% of patients who had or developed thyroid antibodies but in only 7% of initially autoantibody-negative patients. Autoimmunity did not correlate with objective tumor response. Patients with malignant carcinoid tumors may develop autoimmune disease during alpha-interferon therapy, especially when autoantibodies are present. They should therefore be monitored for autoimmunity, which does not appear, however, to influence tumor responses.

Administration of Thyroxine in Treated Graves' Disease

Antibodies to thyroid-stimulating hormone (TSH) receptors that stimulate the thyroid gland cause hyperthyroidism in patients with Graves' disease, and their production during antithyroid drug treatment is an important determinant of the course of the disease. One factor that might contribute to the persistent production of antibodies to TSH receptors is stimulation of the release of thyroid antigens by TSH during antithyroid drug therapy. We therefore studied the effect of the suppression of TSH secretion by thyroxine on the levels of antibodies to TSH receptors after thyroid hormone secretion had been normalized by methimazole. The levels of antibodies to TSH receptors were measured during treatment with methimazole, either alone or in combination with thyroxine, in 109 patients with hyperthyroidism due to Graves' disease. The patients first received 30 mg of methimazole daily for six months. All were euthyroid after six months, and their mean (+/- SD) level of antibodies to TSH receptors decreased from 64 +/- 9 percent to 25 +/- 15 percent (P less than 0.01; normal, 2.9 +/- 1.4 percent). Sixty patients then received 100 micrograms of thyroxine and 10 mg of methimazole and 49 received placebo and 10 mg of methimazole daily for one year. In the thyroxine-treated group, the mean serum thyroxine concentration increased from 108 +/- 16 nmol per liter to 145 +/- 11 nmol per liter (P less than 0.01), and the level of antibodies to TSH receptors decreased from 28 +/- 10 percent to 10 +/- 3 percent after one month of combination therapy. In the patients who received placebo and methimazole, the mean serum thyroxine concentration decreased and the level of antibodies to TSH receptors did not change. Methimazole, but not thyroxine or placebo, was discontinued in each group 1 1/2 years after the beginning of treatment. The level of antibodies to TSH receptors further decreased (from 6.6 +/- 3.2 percent at the time methimazole was discontinued to 2.1 +/- 1.2 percent one year later) in the patients who continued to receive thyroxine, but it increased (from 9.1 +/- 4.8 percent to 17.3 +/- 5.8 percent during the same period) in the patients who received placebo. One patient in the thyroxine-treated group (1.7 percent) and 17 patients in the placebo group (34.7 percent) had recurrences of hyperthyroidism within three years after the discontinuation of methimazole. The administration of thyroxine during antithyroid drug treatment decreases both the production of antibodies to TSH receptors and the frequency of recurrence of hyperthyroidism.

Concordant Graves' Disease after Bone Marrow Transplantation: Implications for Pathogenesis

The current working hypothesis on the pathogenesis of autoimmune disease focuses on the interactions between susceptibility genes and environmental stimuli. In Graves' disease it is postulated that aberrant expression of HLA class II antigens on thyroid epithelial cells permits the presentation of specific thyroid antigen to activated lymphocytes. Evidence suggests that thyrocyte HLA-DR expression is secondary to the production of cytokines by presensitized T-lymphocytes. A 20-yr-old woman and her 18-yr-old brother presented with classical findings of Graves' disease with ophthalmopathy within a year of each other. Diagnosis was confirmed by demonstration of elevated serum levels of T4 and T3, strongly positive titers of TSH binding inhibitory immunoglobulins, and histological examination after subtotal thyroidectomy. Eight years previously, acute life-threatening aplastic anemia in the brother led to therapeutic transplantation of bone marrow from his sister. After the procedure, 100% of his peripheral leucocytes were genotype 46,XX. HLA typing performed before transplantation and 2 months after thyroidectomy in the female indicated complete identity with her brother's leukocytes for class I and class II antigens. Thyroid autoantibodies at this time were weakly positive. Although the concordance of thyroid disease in these patients could be due to chance, the patients were of different sexes, the family history was negative, and neither the probands nor the first degree relatives bore the HLA-DR3/B8 antigens. We propose that the male passively acquired a clone of programmed or activated lymphocytes from his sister and that his hyperthyroidism was not primarily dependent on exposure to specific thyroid-derived antigen.

The Regulatory Role of Human Helper T-Cell Clones on Antithyroid Antibody Production by Peripheral B-Cells*

The helper effects of thyroid antigen-specific T-cell clones (TCC) on antibody production by peripheral B-cells were studied and compared with similar effects of self major histocompatibility complex II (MHC-II)-reactive TCC as well as uncloned CD4+ cells. Ten TCC were derived from thyroid tissue or peripheral blood mononuclear cells (PBMC) in patients with Graves' disease. Uncloned CD4+ cells were also obtained from PBMC in patients with autoimmune thyroid disease. All TCC were CD3+/CD4+. B-Cells from patients with mainly high serum levels of microsomal antibodies (McAb) were cultured alone and with either TCC or uncloned CD4+ cells in the presence or absence of thyroid antigens [microsomal antigen/thyroid peroxidase (McAg/TPO) and thyroglobulin (Tg)] or pokeweed mitogen (PWM). Total immunoglobulin G (IgG) and specific thyroid antibodies were measured by enzyme-linked immunosorbent assay. Self MHC-II-reactive TCC induced B-cell production of total IgG and even McAb independent of antigens or PWM. Specific TCC required thyroid antigens to induce antibodies. The optimal McAg/TPO or Tg concentration was 10 ng/mL for total IgG production and 1 ng/mL McAg/TPO for McAb synthesis. The addition of PWM did not affect McAb production, but enhanced total IgG synthesis by B-cells under the influence of some specific TCC. Uncloned CD4+ cells induced both total IgG and McAb synthesis in the presence of PWM. With thyroid antigens, uncloned CD4+ cells induced total IgG synthesis at levels comparable to those of specific TCC, but induced smaller quantities of McAb in the presence of McAg/TPO. Our antigen-specific TCC could, therefore, stimulate specific B-cells to produce thyroid antibodies in vitro. Self MHC-II-reactive TCC could also induce specific antibodies by B-cells. Both self MHC-II-reactive CD4+ cells and antigen-specific CD4 cells may play an important role in the pathogenesis and/or perpetuation of autoimmune thyroid disease.

Patients with Endocrine Ophthalmopathy not Associated with Overt Thyroid Disease Have Multiple Thyroid Immunological Abnormalities*

Twenty-two apparently euthyroid patients with endocrine ophthalmopathy not associated with goiter, antithyroid microsomal or antithyroglobulin antibodies, or overt thyroid disease (so-called ophthalmic Graves' disease) were tested for subclinical hyperthyroidism or hypothyroidism. We measured 131I uptake and scan, serum T3 (by RIA), and serum TSH using a sensitive (by immunoradiometric assay) assay. Three patients were found to be hyperthyroid, and 1 was hypothyroid. The remaining 18 patients, who remained euthyroid throughout the study period, were investigated for evidence for antibody-mediated immunity against thyroid antigens. We measured antibody-dependent cell-mediated cytotoxicity against fresh thyroid cells using a 51chromium release assay, thyroid membrane-reactive antibodies in an enzyme-linked immunosorbent assay incorporating solubilized thyroid membranes, and TSH receptor-binding antibodies using a RRA and carried out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with patient sera for antibodies reactive with 64 and 110 kDa (thyroid peroxidase) membrane proteins. Bands were demonstrated, on SDS-PAGE, at 64 or 110 kDa in 13 patients, antibody-dependent cell-mediated cytotoxicity tests were positive in 7 patients, and enzyme-linked immunosorbent assay was positive in 4 of the 17 patients tested. In addition, TSH receptor antibody tests were positive in 5 patients, none of whom had other evidence for hyperthyroidism. Finally, significant lymphocyte infiltration was demonstrated on aspiration biopsy in 3 patients. All 18 patients had positive tests in at least 1 of the immunological assays. We believe that these data support the hypothesis that endocrine ophthalmopathy always occurs in patients with overt or subclinical Graves' hyperthyroidism, Hashimoto's thyroiditis, or thyroid immunological abnormalities. Those patients previously described as having euthyroid Graves' disease should, thus, be considered to have associated thyroid immunological abnormalities even though histological confirmation (from aspiration needle biopsy) may be obtained in only a minority of the patients. The possibility that the mechanism for this close association is cross-reactivity of cytotoxic antibodies against a thyroid/eye muscle cell surface shared antigen is discussed in the context of recent evidence from the authors' laboratory.

Relationship Between Thyroid Autoantibody Spectrotype and IgG Subclass

The relationship between spectrotype and IgG subclass of autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) has been investigated using sera from Hashimoto and Graves' patients. Isoelectric focussing (IEF) was carried out in gels over the range pI 3.5-9.5 followed by transblotting to nitrocellulose and probing the filters using 125I-labelled TPO and Tg. As has been shown previously for Tg antibodies, TPO antibody focussed over different pI values in different patients but the spectrotypes for individuals were constant over 2-5 years. Further, the pI values for TPO and Tg autoantibodies appeared to be related to IgG subclass, for example IgG1 Tg antibodies tended to focus nearer the cathode (pI 8.0-9.5) than IgG4 antibodies (pI 6.5-8.5) while antibodies of subclasses IgG1 + 2 + 4 produced spectrotypes covering a broad pI range (5.7-9.5). Consequently, it seems likely that the characteristic spectrotypes described by others for Tg autoantibodies, and those we now report for TPO antibodies, reflect the IgG subclass "fingerprints" which we suggest may be a measure of the ability of an individual to respond to different epitopes on these two thyroid antigens.

Thyroid Auto Antibodies in Black and in White Children and Adolescents with Type 1 Diabetes Mellitus and Their First Degree Relatives

Genetic susceptibility is an important issue in understanding the mechanism of the autoimmune endocrinopathies and in assessing the risk of these conditions in pediatric patients. To this end, we evaluated autoantibodies to thyroid antigens, thyroglobulin (TgA) and microsomal antigen (TMA), in white and in American black juvenile patients with Type I diabetes mellitus (DM) to determine the predictive value of thyroid autoantibodies for the development of autoimmune thyroid disease. Sera from 159 patients (77 black and 82 white) with Type I DM were evaluated. A greater number of whites (41/82 or 50%) than blacks (12/72 or 16%) had thyroid autoantibodies (p less than 0.01). Fourteen patients (4 black and 10 white) exhibited hypothyroidism, and all had both TgA and TMA. Three patients (all black) had Graves' disease, one of whom had both TgA and TMA. Families of each racial group that had a diabetic child (proband) with thyroid autoantibodies (seropositive) or without thyroid autoantibodies (seronegative) were assessed for TgA and TMA as well as autoimmune thyroid disease. The prevalence of thyroid autoantibodies among siblings of seropositive probands was significantly greater than among the siblings of seronegative probands (p less than 0.01). The white sibling population showed a closer association of thyroid autoantibody prevalence with increasing age (p less than 0.05) than the blacks. Significantly more parents of probands than control parents exhibited thyroid autoantibodies (p less than 0.01). The general pattern of inheritance of either racial group showed that if one or both parents had thyroid autoantibodies, their progeny developed a significantly higher prevalence of thyroid autoantibodies than those of the seronegative parents. While there was no increase in overt thyroid disease among siblings of seropositive probands, a risk of developing autoimmune thyroid disease is probably imparted to these siblings by virtue of the thyroid autoantibodies.

Chapter 19 Immunologic disorders of the endocrine system

Autoimmune thyroid disease is one of the most common immunologic disorders of the endocrine system. Hashimoto’s thyroiditis was the first of these diseases to be identified as an autoimmune disorder. This disease manifests by lymphocytic infiltration of the thyroid gland with progressive glandular destruction. A hallmark of Hashimoto’s disease is serum autoantibodies against thyroid antigens including thyroglobulin and microsomal antigen (now known to be the thyroid peroxidase). In addition, cellular sensitization to thyroid extracts is demonstrated by lymphocytic proliferation to thyroid membrane extracts. Clinically, these patients have goiter and local adenopathy, which is sometimes tender. The patient can progress to hypothyroidism due to chronic inflammatory destruction of the thyroid gland. These individuals need thyroid hormone replacement. On occasion, however, hyperthyroidism is seen either from the breakdown and release of thyroid hormone or from stimulating antibodies, as in Graves’ disease.

THE INFLUENCE OF ANTITHYROID DRUGS AND IODINE ON THYROID CELL MHC CLASS II ANTIGEN EXPRESSION

We have investigated the influence of antithyroid drugs (methimazole and propylthiouracil) and sodium iodide on the expression of major histocompatibility (MHC) class II antigen expression in human and rat thyroid cells. While methimazole and propylthiouracil significantly inhibited lectin-induced MHC class II (HLA-DR) antigen in crude human thyroid monolayer preparations these drugs had no influence on gamma-interferon induction of class II antigens in similar cell preparations. Hence, antithyroid drugs probably had a direct effect on thyroid monolayer T cells which are known to effect the lectin induction of MHC class II antigen via T-cell secretion of the cytokine, gamma-interferon (IF). Sodium iodide, similarly, had no direct influence on human thyroid cell MHC class II antigen expression induced by gamma-IF. However, iodide significantly inhibited the gamma-IF-induced expression of MHC class II antigens in a proliferating rat thyroid cell clone derived from FRTL-5 cells (clone 1B-6). In the rat thyroid cell clone this antagonistic action appeared to be exerted via inhibition of TSH-induced proliferation. These data add further support to the multifactorial nature of the profound immunosuppressive influence of antithyroid drugs in autoimmune thyroid disease and experimental thyroiditis. In contrast, iodides may have a major, and often overlooked, influence on thyroid cell proliferation and antigen expression.

Cross-reaction of eye muscle antibodies with thyroid tissue in thyroid-associated ophthalmopathy.

The nature of the association of ophthalmopathy with autoimmune thyroid disease is not understood. Serum autoantibodies to eye muscle have previously been identified and in this study we have explored the hypothesis that there may be shared antigenic determinants between orbital and thyroid tissues. Sera were obtained from patients in whom eye muscle antibodies (EMAb) had been detected by an enzyme-linked immunosorbent assay (ELISA); the sera were preincubated with membrane preparations of thyroid or eye muscle, hepatic membranes being used as control. Tissue-binding antibodies were removed from serum by centrifugation and the supernatant serum was analysed using an indirect ELISA and by immunoblotting. In the ELISA, all sera gave a positive response for EMAb. In one serum, the binding was entirely non-specific. All sera showed significant neutralization of EMAb by eye muscle. In six sera there was reduction of EMAb after exposure to thyroidal antigens, indicative of cross-reaction. Western blotting confirmed the non-specific nature of the binding in one serum. In five of the remaining nine sera, protein bands were identified which interacted specifically with eye muscle and, in two of these, the same determinants were neutralized by preincubation with thyroid tissue. The Western blots confirmed the findings in the ELISA. The determinants recognized by IgG were variable between patients and no common antigen could be identified. This study demonstrates that, in some cases of thyroid-associated ophthalmopathy, there is cross-reaction of EMAb with thyroidal antigens, but this is variable and not found in every case. This may explain the association of the disease with autoimmune thyroid disease, at least in some cases.

Affinity purification of IgG subclasses and the distribution of thyroid auto-antibody reactivity in Hashimoto's thyroiditis.

To delineate accurately the IgG subclass distribution of thyroid auto-antibodies, sera from nine patients with Hashimoto's thyroiditis were fractionated into IgG subclasses by complete depletion of the other IgG subclasses on affinity columns. All IgG subclass fractions contained thyroglobulin and microsomal (or thyroid peroxidase) antibody activity, although when compared to the total serum concentrations of IgG subclasses, IgG4 antibodies were overrepresented. However, in contrast to recent studies, this particular subclass never predominated--IgG4 antibody levels being exceeded by those of the IgG1 and IgG2 subclasses; it seems likely that these differences relate to varying sensitivity for different subclasses in previously used assay methods. This pattern of subclass activity differed from that of tetanus toxoid antibodies, which were found in six subjects. There was no light chain restriction within any subclass, showing that the overproduction of IgG4 thyroid antibodies is not of monoclonal origin. The functional affinity of subclasses for both thyroid antigens varied between patients, but IgG2 subclass fractions showed the highest functional affinity in the majority of samples. We also found that IgG2 subclass thyroid antibodies were ineffective in eliciting antibody-dependent cell-mediated cytotoxicity, as distinct from the other three subclasses. Our results show that thyroid antibodies are less restricted in their IgG subclass distribution and patients are less heterogeneous than previously described. Moreover, IgG2 thyroid antibodies are quantitatively important and differ in relative functional affinity and effector function from IgG1 and IgG4 thyroid antibodies.

Autologous CD8-positive cells suppress T cell proliferation in response to thyroid antigens in Hashimoto's thyroiditis

To assess whether there is a defect in thyroid antigen-specific suppressor cells in Hashimoto's thyroiditis (HT) and whether such cells actively prevent thyroid autoreactivity in control subjects, T cell proliferation was measured before and after removal of CD8-positive cells from the lymphocyte population. CD8 depletion significantly increased the proliferation of HT lymphocytes to soluble thyroglobulin and to thyroid microsomal antigen immunoblotted onto nitrocellulose; some of these cultures also reacted with an unidentified thyroid antigen, mol wt approx 16 kDa. However, CD8 depletion did not permit normal lymphocytes to respond to thyroid antigens. Peripheral blood CD8 cells were also found to suppress proliferation of a thyroid T cell line, derived from a patient with HT, in response to autologous, Ia-positive thyroid follicular cells. These results do not support the existence of a defect in antigen-specific suppressor cells in HT, nor the active suppression of thyroid autoreactivity by such cells in normal subjects. The data suggest that there may be an imbalance in T cell helper and suppressor influences in thyroiditis, but in some patients a new balance is achieved, so that T cell sensitization to thyroid autoantigens can only be identified by removal of CD8-positive cells.

Natural course of iodine-induced thyrotoxicosis (Jodbasedow) in endemic goiter area: a 5 year follow-up.

In the Balsas region of North Brazil (85% prevalence of goiter), 1876 goitrous subjects (1663 with goiter grade I and II and 103 with grade III multinodular goiter) were treated with 1 ml of iodized oil im (l-oil). From the population with grade III goiter we were able to follow-up for 5 yr 13 euthyroid goitrous patients (group 1) and 8 goitrous individuals (group 2) who developed iodine-induced thyrotoxicosis (IIT, 0.42% of the total population or 7.76% of the multinodular goiter subjects). The two groups were matched for age and goiter size, and had no significant differences in the baseline levels of thyroid hormone concentration, T3/T4 ratio or mean serum TSH. However, group 2 had a higher concentration of serum Tg, and 48 h after challenge with 10 U of bovine TSH (bTSH) had a significantly higher absolute release of T3 and Tg than group 1. In 4 patients IIT was transitory and resolved by 12 months. Four other subjects, however, maintained a mild clinical form of IIT that only normalized at 50 months. Only one patient needed treatment with methylmercaptoimidazol. There was no evidence of autoantibodies (TSH-receptor inhibiting antibody, anti-Tg or anti-microsomal antibodies) against thyroid antigens in group 2 patients. All subjects, including those with thyrotoxicosis, showed a remarkable shrinkage of the goiter by 60 months which was reflected by a significantly lower absolute response of T3 and Tg after bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)

REOVIRUS INDUCTION OF MHC CLASS II ANTIGEN IN RAT THYROID CELLS

We have previously demonstrated that cultured rat thyroid cells do not exhibit constitutive expression of major histocompatability (MHC) class II antigens. Using reovirus types 1 and 3, we infected 1B-6 cells (a cloned derivative of the Fisher rat cell line FRTL-5), and found a dose-dependent induction of thyroid cell MHC class II antigen expression as determined by laser flow cytometry and FITC-labelled OX-6 anti-RTl.B. As the number of viral particles/cell used for the infections increased from 5 to 100, the number of antigen positive cells increased, in reovirus type 3 infections to 50%, and in reovirus type 1 infections to 15%. Kinetic studies indicated that MHC class II antigen expression continues to increase three days after infection. Viral infection and the resulting MHC class II antigen expression may allow presentation of thyroid antigen to the immune system and participate in the initiation of autoimmune thyroid disease.

Lack of effect of methimazole on thyrocyte cell-surface antigen expression.

The nature of the immunosuppressive effect of antithyroid drugs has been a subject of controversy. It has been claimed that these agents exert a direct effect on the immune system, although we and others have suggested that the drugs affect the thyroid cells primarily with consequent reduced thyrocyte-immunocyte signalling. This may occur from reduced thyroid hormone production and/or reduced antigen presentation by the thyrocytes to local T lymphocytes. Using a cytotoxicity assay system, with chromium-51 labelling, monoclonal antibodies against thyroperoxidase (TPO) and HLA-DR, and complement, we have measured the expression of TPO and HLA-DR on cultured normal human thyroid cells; we have also measured thyroglobulin (Tg) release by radioimmunoassay into the medium of the cultured cells. The thyroid cells were stimulated with TSH or thyrotropin binding inhibitory immunoglobulin (TBII) for 48 hours before measuring for TPO induction, and with interferon gamma (IFN-gamma) (with or without TSH or TBII) for thyrocyte HLA-DR expression. A dosage of 1.6 milliunits per ml of TSH resulted in a significant increase in TPO expression on thyrocytes when compared with control unstimulated thyroid cells (p less than 0.001). The concentrations of Tg released into the medium with TSH or TBII were also significantly higher than those of the control thyrocytes. IFN-gamma at 200 units per ml induced HLA-DR expression, but did not induce thyrocyte TPO expression, or Tg release. Addition of the antithyroid drug, methimazole (MMI), at different concentrations, in addition to the other stimulators, IFN-gamma, TSH, or TBII, did not result in any inhibition of TPO, Tg release, or HLA-DR expression on the thyroid cells. It would thus appear that the pathways for stimulation for the expression of TPO and HLA-DR appear to be different. Finally, MMI does not cause its immunosuppressive effect by any reduction of thyroid antigen expression or release.

Nontoxic Nodular Goiter and Papillary Thyroid Carcinoma Are Not Associated With Peripheral Blood Lymphocyte Sensitization to Thyroid Cells*

We tested the claim that uni- and multinodular goiter (UNG and MNG) and papillary carcinoma (PC) of the thyroid are autoimmune thyroid diseases (AITD) similar to Graves' disease (GD) and Hashimoto's thyroiditis (HT). The expression of HLA-DR on cultured thyroid epithelial cells (thyrocytes) from UNG, MNG, and PC after coculture with autologous peripheral blood mononuclear cells (PBMC) was compared with that on GD and HT cells. The thyrocytes also were cultured with interferon-gamma (IFN gamma) alone. A cytotoxicity assay involving 51Cr-labeled thyrocytes, anti-HLA-DR, and complement was used to determine HLA-DR expression. Stimulation of thyrocytes with 200 U/mL IFN gamma induced HLA-DR (expressed as a cytotoxicity index) equally well on all thyrocytes [AITD (n = 6): IFN gamma, 23.8 +/- 7.7 (+/- SD); unstimulated, 3.6 +/- 2.0; UNG (n = 6), MNG (n = 9), and PC (n = 5): IFN gamma, 22.5 +/- 4.7; unstimulated, 4.0 +/- 3.0]. When cocultured with autologous PBMC, the values were: AITD, 24.9 +/- 10.1; UNG, MNG, and PC, 3.8 +/- 3.7 (P less than 0.001). The supernatants from the AITD cocultures had higher IFN gamma concentrations (by RIA) than those from the other cocultures. We conclude that in UNG, MNG, and PC, the peripheral blood helper T-lymphocytes are not sensitized to thyrocyte membrane antigen(s); consequently, little if any IFN gamma is produced in cocultures, and hence, there is no increase in thyrocyte HLA-DR expression, unlike the situation in AITD (GD and HT). Thus, UNG, MNG, and PC are not primarily autoimmune in nature, as defined by a lack of sensitization of the PBMC of such patients to thyroid antigen(s).

5 Human autoimmune thyroid disease: Cellular and molecular aspects

We have reviewed the impact of cellular and molecular biology on our understanding of the immune system and thyroid autoimmune disease and presented the evidence that MHC, TCR and immunoglobulin genes are involved in susceptibility to such disease. At the level of the target thyroid epithelial cell, the identification of the genes for Tg and TPO (the microsomal antigen) using recombinant DNA techniques are evidence of dramatic progress. On the humoral side of the immune response, the investigation of restricted clonality is still hampered by the technical difficulties in obtaining immortal B cell lines producing thyroid antigen specific high affinity IgG antibodies, although the advent of tools to sequence the immunoglobulin V genes from small quantities of DNA will overcome this difficulty (e.g. by polymerase chain reactions). T cells have also begun to be characterized both phenotypically, thanks to the advent of ever better characterized monoclonal antibodies, and functionally at the clonal level, thanks to refinement of cell culture techniques. Future studies in this area will also need to focus on cell immortalization and maintenance of antigen-specific responses although, major strides in the direct sequencing of the TCR are taking place and investigation of T cell receptors in antigen-specific T cells should be feasible. MHC associations with thyroid autoimmune disease, as studied by analysis of RFLP patterns, have not significantly improved already established serological HLA associations and direct MHC gene sequencing will be required. Analysis of the organ-specific regulation of MHC class II gene expression has led to a better understanding of the functional role of MHC class II genes in thyroid autoimmune disease at the target cell level. Such studies have pointed out important local immune responses within the thyroid gland but have not yet provided the initiating factor or factors for human autoimmune thyroid disease in genetically susceptible individuals.