Thyroid antibodies are produced by thyroid-derived lymphocytes.

The significance of the characteristic lymphocytic infiltrate in the target organ in organ-specific autoimmune disease is unknown. We have demonstrated the production of thyroglobulin antibodies and immunoglobulins (IgG, IgM and IgA) by thyroid-derived lymphocytes in Graves' disease and Hashimoto's thyroiditis two plaque forming cell (PFC) assays. The thyroid appears to be an important site of thyroglobulin antibody production but the thyroid lymphocytes also contain many IgG PFCs of non-thyroglobulin specificity. Short-term culture and direct thyroglobulin antibody assay on micro-ELISA plates confirmed the results of the PFC assay. Therapies such as carbimazole may therefore be acting on a localized source of autoantibody production.     

Heterogeneity of immunoregulatory T cells in human thyroid autoimmunity: influence of thyroid status.

Monoclonal antibodies of the OKT series were used to identify circulating T lymphocytes (OKT3+), their helper-inducer (OKT4+) and suppressor-cytotoxic (OKT8+) subsets and cells bearing Ia antigen (OKIa+) in 75 patients with thyroid autoimmune disorders, including 14 Graves' disease, 21 myxoedema, 20 asymptomatic thyroiditis, 12 Hashimoto's thyroiditis and eight simple goitre with superimposed thyroiditis. In the whole population of patients, a negative correlation was observed between the percentage of OKT8+ cells and serum free thyroxine levels whatever the type of thyroiditis. The percentage of OKT8+ cells was decreased in Graves' disease and increased in myxoedema while it reversed after adequate treatment of the two diseases. However, a trend to a decrease in the proportion of OKT8+ cells was still observed in treated Graves' disease and in all the other groups of thyroiditis with euthyroidism. The minor modifications observed for OKT3+ and OKT4+ cells were in relation with those of OKT8+ cells. There was an increased percentage of Ia+ cells in Graves' disease and in Hashimoto's thyroiditis partly reflecting the presence of activated lymphocytes. In conclusion, these data suggest first of all a direct influence of serum T4 on the distribution of circulating OKT8+ cells in addition to documenting the heterogeneity of T cell immunoregulatory factors.     

Intrathyroidal HLA-DR expression and T lymphocyte phenotypes in Graves'' thyrotoxicosis, Hashimoto''s thyroiditis and nodular colloid goitre.

Thyroid surgical biopsies from 21 individuals were examined by a double immunoenzymatic technique with respect to HLA-DR expression and lymphocytic infiltration. HLA-DR positive thyrocytes were observed in two examined Hashimoto goitres and in nine of 11 specimens from patients with Graves' disease. HLA-DR positive thyrocytes were localized to areas harbouring infiltrating lymphocytes, whereas regions with no lymphocytes only rarely expressed HLA-DR antigens. In two specimens of nodular goitre HLA-DR positive thyrocytes were observed in the vicinity of lymphocytic infiltration. Tissues from another three nodular goitres, from one follicular adenoma and from two normal individuals contained no HLA-DR positive thyrocytes and no or only a few lymphocytes. The lymphocytic infiltrates were dominated by cells with the Leu 3a helper/inducer phenotype irrespective of underlying disease, although most pronounced in Hashimoto's thyroiditis. The results indicate that HLA-DR antigens is expressed on thyrocytes in thyroid disorder. The extent of expression correlated with lymphocytic infiltration, which suggests that the two findings are related and of importance for the development of thyroid autoimmunity.     

Tumour necrosis factor synergises with gamma interferon on the induction of mRNA for DR alpha chain on thyrocytes from Graves' disease and non toxic goitre

In both thyroid autoimmune diseases Graves' and Hashimoto's thyroiditis, the epithelial thyroid follicular cells (TFC) have been shown to express HLA class II molecules, and can restimulate autoreactive T cells cloned from the diseased tissue. This aberrant class II expression is important in the mechanism of perpetuation of the disease process, therefore we have compared the effect of interferon gamma (IFN gamma) and tumour necrosis factor (TNF alpha) on the HLA-DR alpha mRNA expression of thyroid follicular cells derived from Graves' disease (GD) and a non autoimmune disease, non toxic goitre (NTG). Our results indicate that TNF alpha synergises with IFN gamma in the induction of HLA class II mRNA. There was no consistent difference in DR alpha mRNA expression between the GD and NTG thyroid follicular cell preparations in response to induction by a combination of these lymphokines at various concentrations. Our data suggest that the differences in the level of expression of class II molecules observed in vivo in Graves' disease and non toxic goitre, which is much higher in the former, is probably due to local release of lymphokines by infiltrating T lymphocytes, although other factors may be involved.     

Thyroglobulin-induced T-cell in vitro proliferation in Hashimoto's thyroiditis: identification of the responsive subset and effect of monoclonal antibodies directed to Ia antigens

Recently it was reported that the peripheral blood and thyroid gland of patients with Hashimoto's thyroiditis contain activated (Ia+ and/or MLR4+) T cells and high levels of 5/9+ ("helper") T lymphocytes. In normal individuals the 5/9 monoclonal antibody recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5/9+ and 5/9- T lymphocytes were isolated from patients with Hashimoto's disease, and the proliferative response induced by human thyroglobulin was investigated. The results show that the total thyroglobulin-induced lymphocyte DNA synthesis is confined to the 5/9+ T-cell fraction. Further subfractionation of 5/9+ into MLR4+ and MLR4- cells clearly indicates that no substantial differences exist in their proliferative capacities. Whether 5/9, MLR4, and Ia antigens, all expressed on the thyroglobulin-responsive T-cell subset, are involved in thyroglobulin-induced cell proliferation, was also analyzed. Although both 5/9 and MLR4 monoclonal antibodies had no effect, complete inhibition of antigen-induced blastogenesis was observed upon addition of monoclonal antibodies (D1/12 and BT2/9) directed to common determinants of Ia antigens. This inhibitory effect was also observed when T or non-T fractions were separately incubated with the monoclonal antibodies before culture. These results indicate that in humans, as in animals, the major histocompatibility complex may play a role in autoimmune thyroiditis. The data show that (a) the thyroglobulin-induced proliferative response is confined to a subset (5/9+) of T lymphocytes and (b) Ia antigens are involved in thyroglobulin-induced lymphocyte DNA synthesis in Hashimoto's disease.     

Studies on thyroid cell surface antigens using cultured human thyroid cells.

Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's IgG, suggesting that some of the thyroid cell surface antibodies of idiopathic myxoedema may not be detectable in other thyroid autoimmune disorders.     

Thyroglobulin synthesis of oxyphilic cells in various types of neoplastic and autoimmune thyroid diseases.

To determine the content of thyroglobulin in oxyphilic cells of the thyroid, which have been considered as non-thyroglobulin producing cells, the degree of stainability of the various oxyphilic cells for thyroglobulin was compared with that of non-oxyphilic follicular cells in either same or different lesion. A total of 13 oxyphilic lesions, including three follicular adenomas containing oxyphilic cell nodules, four pure oxyphilic cell adenomas, and six Hashimoto's thyroiditis were compared with 16 of non-oxyphilic lesions such as, seven follicular adenomas, four chronic lymphocytic thyroiditis, and five Graves' disease. Many oxyphilic cells stained positively for thyroglobulin regardless of their morphologic variation, but less intensely than the usual follicular cells in follicular adenomas, chronic lymphocytic thyroiditis, and Graves' disease. The stainability of oxyphilic cells for thyroglogulin did not show any significant correlation with morphologic features, whereas in follicular adenomas, the non-oxyphilic follicular cells forming microfollicles stained less strongly for thyroglobulin than the same cells lining large mature follicles in a reproducible way. With above findings, we concluded that oxyphilic cells maintain the functional activity in terms of thyroglobulin synthesis, although the content of the thyroglobulin is less than that of non-oxyphilic colloid forming follicular cells.     

Heterogeneity of thyroid autoantigens identified by immunoblotting

Autoimmune thyroid disease in man is commonly associated with autoantibodies against thyroglobulin, microsomes, and the TSH receptor, and the character and specificity of these antithyroid antibodies have been extensively utilized in investigating these conditions. In the present study we have asked whether other thyroid-related antigens exist, against which autoantibodies may be directed. A crude thyroid extract was separated by polyacrylamide gel electrophoresis followed by immunoblotting with serum obtained from patients with Graves' disease or Hashimoto's thyroiditis. Antibodies in sera from patients with Graves' disease and Hashimoto's thyroiditis reacted with many antigenic determinants in immunoblots of the thyroid membrane preparation (2000g supernatant). These determinants were disease specific in that sera from normals and patients with Addison's disease and rheumatoid arthritis did not react, but there was no difference between the patterns of reactivity with Graves' disease or Hashimoto's thyroiditis sera. Thyroglobulin produced two predominant bands of reactivity at 320 and 200 kDa, whereas purified microsomal antigen produced a triplet of bands around 105 kDa, when these preparations were reacted with appropriate autoimmune sera. Nonetheless, some sera produced additional bands with the microsomal antigen blots, indicating that some of the antigens which were detected using crude thyroid membrane remained in the microsome preparation to produce multiple antibody binding reactivities. We were unable to inhibit any of the antibody binding with TSH. Purification of individual thyroid antigens on the basis of their molecular weights should standardize current antibody assays and permit more detailed evaluation of the cellular immune responses in Graves' disease and Hashimoto's thyroiditis.     

Studies on the immunoregulation of thyroid autoantibody production in vitro.

The spontaneous production (without mitogen or antigen) of antithyroglobulin and antimicrosomal antibodies by peripheral (PBL) and thyroid-derived lymphocytes from patients with Hashimoto's thyroiditis (HT) has been studied with particular emphasis on the regulation of this phenomenon. Based on studies of DNA and protein synthesis, kinetic studies and B/T reconstitution experiments, in most HT patients, spontaneous production by PBL is accounted for by secretion of preformed antithyroglobulin (termed Type 1 patients), whereas active production is observed in a small minority (termed Type 2). In none of 24 HT patients could active antimicrosomal antibody production by PBL be detected. Conversely, thyroid-derived lymphocytes produced both autoantibodies by an active process. Pokeweed mitogen (PWM) stimulation enhanced antibody production by PBL in the Type 1 group but not in Type 2 or thyroid-derived lymphocytes. T lymphocytes were required for antibody synthesis in both thyroid antigen-driven and peripheral PWM-driven cultures. By separating T lymphocytes into T4+ (helper) and T8+ (suppressor) subsets with monoclonal antibodies, T-cell modulation of autoantibody production in both systems was studied. In a PWM-induced system, both thyroid and peripheral T-cell subsets were capable of modulating peripheral antibody production. In the thyroid lymphocyte antigen-specific system, further addition of thyroid derived T8+ cells alone caused partial suppression of antibody production but not with peripheral T8+ cells. Of interest was the partial decrease of antibody production by the thyroid lymphocytes by added peripheral T4+ cells. The fact that the production of thyroid autoantibodies by thyroid-derived mononuclear cells (which included T suppressor, T helper and B lymphocytes) could be reduced by the addition of more suppressor T lymphocytes suggests that an antigen-specific defect in the T4+/T8+ thyroid cell balance may account for the in vivo production of these antibodies in patients with Hashimoto's thyroiditis.     

Thyroid autoantibody synthesis by cultures of thyroid and peripheral blood lymphocytes. I. Lymphocyte markers and response to pokeweed mitogen

Thyroid lymphocytes from Graves' and Hashimoto patients have been investigated and compared with lymphocytes from the peripheral blood. Considerably more lymphocytes (20-30 X 10(6)/g) could be isolated from Hashimoto thyroids than from Graves' tissue (1-5 X 10(6)/g) but the cell suspensions extracted from Hashimoto and Graves' glands were similar in terms of cell surface markers and the ability to synthesize immunoglobulin. Thyroid lymphocytes contained a lower proportion of T cells (OKT3+ cells) and in some cases more B cells than the peripheral blood but the ratio of helper to suppressor T cells (OKT4+:OKT8+ cells) was similar to the values obtained for blood lymphocytes. Further, thyroid lymphocytes (unlike blood lymphocytes) synthesized relatively large amounts of microsomal and/or thyroglobulin antibody when cultured in medium only and these levels were significantly decreased by the addition of pokeweed mitogen. The results of this study provide further evidence for the role of the thyroid as a major site of thyroid autoantibody synthesis and emphasize the importance of characterizing the cells infiltrating the gland in autoimmune thyroid disease.     

Analysis of T-cell receptor usage in activated T-cell clones from Hashimoto's thyroiditis and Graves' disease

Rearrangements to the T-cell receptor (TcR) beta and gamma gene loci were studied in T cells derived from the thyroid glands of a patient with Hashimoto's (HT) and another with Graves' (GD) autoimmune thyroiditis. The cells studied were freshly isolated mononuclear cells, T-cell lines grown in the presence of anti-CD3 and IL-2 and T-cell clones. Numerous different rearrangements to the constant regions of TcR beta and TcR gamma and in the variable gene region of TcR beta were observed. These findings indicate that the T-cell response in autoimmune thyroiditis is multiclonal and may have implications for the epitopes recognized by autoreactive T cells and for the mechanisms of the disease.     

CD4+ T-cell clones from autoimmune thyroid tissue cannot be classified according to their lymphokine production

In order to define whether CD4+ T cells from autoimmune and non-autoimmune thyroid tissue could be classified according to their mediator production, lymphokine production was studied in 63 thyroid-derived CD4+ T-cell clones from four patients with Graves' disease, one with Hashimoto's thyroiditis, and one with non-toxic goitre (9-12 clones per patient). The production of interleukin 2 (IL-2), gamma interferon (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), lymphotoxin (LT), interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) was assessed at the mRNA level by slot-blot analysis in unstimulated clones as well as after activation with monoclonal anti-CD3 (OKT3) and IL-2. No lymphokine production was found in unstimulated clones, whereas 56% of the clones produced all six lymphokines simultaneously after stimulation. In the remaining 44% usually not more than one lymphokine was missing from the complete panel. Lymphokine mRNA concentrations varied between different clones and different patients, but, in this small sample, not between the diseases from which the clones were originated. There was a significant correlation between IL-6, LT, and IL-2 mRNA levels and T-cell helper function, which was estimated by the stimulation of thyroid microsomal autoantibody production using autologous peripheral B cells. TGF-beta and IFN-gamma mRNA expression was unrelated to T-cell help. The results demonstrate that intrathyroid T cells from autoimmune and non-autoimmune thyroid disorders cannot be classified according to their lymphokine production, unlike some results with in vitro-induced mouse T-cell clones, where two populations, Th1 and Th2, have been described. Single T cells are capable of producing a whole panel of lymphokines and thus are capable of triggering a multitude of different processes.     

Clinical value of a bispecific antibody binding to thyroglobulin and thyroperoxidase (TGPO-aAb) in various thyroid diseases.

TGPO-aAb is a bispecific antibody which binds to thyroglobulin as well as thyroid peroxidase. It is supposed to be raised in some patients with autoimmune thyroid disease. We investigated 205 patients suffering from Graves' disease (n = 81), Hashimoto's thyroiditis (n = 36), toxic nodular goitre (n = 50), differentiated carcinoma of the thyroid (n = 10), and autoimmune thyropathy of unknown origin (n = 28). An immunoradiometric assay was used to measure serum TGPO-aAb. Eighty-nine of 205 patients had elevated titres of TGPO-aAb. If TGPO-aAb were raised then autoantibodies against thyroglobulin and thyroid peroxidase were always raised, too. This was, however, not true vice versa. We found TGPO-aAb in 61% of patients with Hashimoto's, 49% of patients with Graves', 64% of patients with autoimmune thyropathy, but only in 12% of patients with toxic nodular goitre. In patients with thyroid carcinoma TGPO-aAb was found only if there was evidence of paraneoplastic autoimmune thyroiditis. We re-examined 16 of 36 patients with Hashimoto's thyroiditis after 1 year: 8 patients had retained their raised TGPO-aAb, 4 patients showed no TGPO-aAb on both occasions, and 4 patients had 'lost' their previously raised TGPO-aAb on follow-up. We conclude that TGPO-aAb may provide additional information in Hashimoto's thyroiditis. Determination of TGPO-aAb does not allow to distinguish between various forms of autoimmune thyroid disease. Nevertheless, the presence of TGPO-aAb and its variation during the natural course of autoimmune thyroid disease remains to be understood which would give a better insight into its clinical significance.     

Chronic lymphocytic thyroiditis in a cynomolgus macaque (Macaca fascicularis)

Chronic lymphocytic thyroiditis characterized by multifocal follicular lymphoid cell infiltrates with germinal centers, thyroid acinar atrophy and pituitary cell hyperplasia/hypertrophy of the adenohypophysis was detected in a vehicle control, 4-year-old female Cynomolgus macaque in a routine toxicology study. Lymphoid cells of germinal centers were positive for the B-cell marker CD20 by immunohistochemistry (IHC), while remaining lymphocytes were positive for the T-cell marker CD3. Hypertrophied/hyperplastic pituitary cells were positive for thyroid stimulating hormone (TSH) by IHC, consistent with an adaptive response due to removal of hormonal negative feedback from the diseased thyroid gland. Features of this case are similar to chronic lymphocytic thyroiditis in humans, an autoimmune disorder also known as Hashimoto's disease. Chronic lymphocytic thyroiditis with compensatory pituitary changes may occur spontaneously in young, clinically normal cynomolgus macaques and its presence in drug treated animals should be interpreted with caution.     

Detection of HLA-DR antigens in paraffin-embedded thyroid epithelial cells with a monoclonal antibody.

The human Class II major histocompatibility (MHC) antigens, or Ia antigens, which are thought to regulate immune cell interaction, can be detected in paraffin-embedded tissues by immunoperoxidase staining with a recently developed monoclonal antibody (LK8D3). HLA-DR antigens were observed in lymphoid tissues, Langerhans cells of the skin, some epithelial cells, and pulmonary alveolar macrophages. The expression of HLA-DR antigens was analyzed in formalin-paraffin sections by immunoperoxidase in 86 normal and abnormal thyroid epithelial tissues. All patients with Hashimoto's disease (8/8) and most patients with Graves' disease (6/8) expressed HLA/DR antigens in the thyroid epithelial cells and in adjacent inflammatory cells. Most papillary carcinomas (12/18), including 3 of 5 follicular variant of papillary thyroid carcinomas, had HLA-DR antigens detected in epithelial cells; whereas medullary thyroid carcinomas (0/5), follicular carcinomas (0/5), and multinodular goiters (0/4) did not have detectable HLA-DR immunoreactivity. A few other thyroid lesions had HLA-DR antigens detected in epithelial cells, including anaplastic carcinomas (2/5), Hurthle-cell tumors (1/16), and thyroid lymphomas (2/2). Monoclonal antibody LK8D3 and two other commercially available monoclonal antibodies against HLA-DR-stained tissues equally well in cryostat sections, but only antibody LK8D3 was effective in formalin-fixed paraffin-embedded tissue sections. These results indicate that epithelial cells from thyroids of patients with autoimmune diseases commonly express HLA-DR antigens. The presence of HLA-DR antigens in most papillary thyroid carcinomas may be helpful diagnostically in cases of follicular variants of papillary carcinomas. The role of HLA-DR expression in autoimmune thyroid disease and in papillary thyroid carcinoma remains to be determined.     

T-cell receptors and autoimmune thyroid disease--signposts for T-cell-antigen driven diseases

The human autoimmune thyroid diseases (AITDs) are characterized by profuse infiltrates of both CD4+ and CD8+ T cells. The intrathyroidal T-cell-receptor repertoire in Graves' disease, more than in Hashimoto's disease, has been shown to be biased as evidenced by phenotypic analysis and by the use of a restricted T-cell-receptor variable (V) gene repertoire seen in both TCR alpha and beta chains. Evidence for a bias in the T-cell repertoire has also been observed in animal models of induced and spontaneous autoimmune thyroiditis. We found a similar phenomenon of autoimmune thyroid-related T-cell bias in thyroid-humanized scid mice. In these studies we transplanted lymphocyte-depleted thyrocytes and autologous peripheral lymphocytes from AITD patients with a basement membrane preparation which allowed the formation of an artificial thyroid which we have called an "organoid". T-cell clonal expansion was present in these artificial mixed-cell organoids which appeared to mimic the in vivo process. Such clonal expansion was suggestive of an antigen-driven immune response and could also be identified in thyroid tissue from patients with Graves' disease. Our data on scid mice grafted with human mixed-cell thyroid organoids, therefore, suggested that the major antigens driving T-cell selection in patients with AITD were most likely to be thyroid specific. These antigens include thyroglobulin, thyroid peroxidase, and the receptor for thyroid stimulating hormone (TSHR) on the surface of thyroid epithelial cells and we found significant T-cell proliferation to synthetic TSHR peptides in patients with AITD as compared with normals. Our search for a TCR recognition motif for the autoantigen TPO did not reveal any specific sequence motifs. Instead, analysis of the physico-chemical characteristics i.e. hydrophobicity of the amino acids in the CDR3 (N) region of the TCR alpha chain, revealed a strong negative linear correlation between strength of stimulation and the average hydrophobicity of N-region amino acids. This led us to hypothesize that lower affinity T-cell clones were commonly more hydrophobic in their CDR3 alpha region amino acids in keeping with potential crossreactivity of such T cells as a consequence of promiscuous, hydrophobic CDR3 regions. This phenomenon would be analogous to polyreactive, natural autoantibodies which tend to be crossreactive and 'sticky'. Thus, the physico-chemical characteristics of the TCR alpha CDR3 region supported the interaction with antigen/MHC by potentially cross-reactive T cells of low affinity. It would seem likely that such low-affinity autoreactive T-cell populations serve as a pool of potentially pathogenetic cells. These cells would be able to respond to an insult which, via a number of possible mechanisms such as molecular mimicry, would initiate a thyroid lymphocytic infiltration in an antigen-driven fashion with intrathyroidal T-cell expansion and a marked bias in the utilization of T-cell-receptor V genes.     

The expression and role in T cell adhesion of LFA-3 and ICAM-2 on human thyroid cells.

Thyroid follicular cells from patients with Graves' disease and Hashimoto's thyroiditis express intercellular adhesion molecule-1 (ICAM-1) and this is in part responsible for T cell adherence in vitro. To assess the potential role of other adhesion molecules in autoimmune thyroiditis, we investigated the expression and function of lymphocyte function-associated antigen-3 (LFA-3) and ICAM-2 on thyroid cells. Under basal culture conditions, a mean of 22.7% of Graves' thyroid cells (n = 8) expressed LFA-3 and this was enhanced by a mixture of T cell-derived cytokines and by IL-1, but not by TSH. LFA-3 was also demonstrated on Graves' (n = 4) and Hashimoto (n = 2) thyroid cells by immunohistochemical staining ex vivo. A small number of thyroid cells (mean 5.5%, n = 5) expressed ICAM-2 by flow cytometry but this was not altered by cytokines, and ICAM-2 could only be demonstrated on endothelial cells by immunohistochemical staining. It seems likely that contamination of primary thyroid cultures by such cells accounted for the small number of ICAM-2+ cells found using flow cytometry. Almost all of the cultured cells expressing LFA-3 or ICAM-2 also expressed ICAM-1, as assessed by dual staining. Blocking LFA-1, LFA-3, and ICAM-1 with monoclonal antibodies inhibited the adherence of T cells to thyroid follicular cells in assays of cell clustering; antibodies against ICAM-2 had no effect. These results show that two important adhesion receptor ligands, ICAM-1 and LFA-3, are expressed by thyroid cells in autoimmune thyroiditis and that these are likely to have functional importance in allowing T cells to bind to thyroid cell targets. This may play an important role in the initiation and maintenance of Graves' disease and Hashimoto's thyroiditis.     

Proliferation of T8-positive cytolytic T lymphocytes in response to thyroglobulin in human autoimmune thyroiditis: analysis of cell interactions and culture requirements

These experiments were designed to analyze the involvement of T-lymphocyte subpopulations in autoimmune thyroid disorders such as Graves' Disease (GD) and Hashimoto's Disease (HD). In a first set of experiments, lymphocytes isolated from thyroid infiltrates or from peripheral blood of GD and HD patients were analyzed for the expression of various surface antigens. While HLA-DR + T cells were numerous among thyroid infiltrating T lymphocytes in both groups of patients, the proportions of T8 + cells (as defined by their reactivity with the B 9.4 monoclonal antibody specific for T8 surface molecule) were strikingly different in HD and GD. In the latter group of patients only 19% of infiltrating T cells were T8 +, whereas these cells represented approximately 50% in four HD patients. Given the previous demonstration that all T cells expressing T8 antigen are cytolytic T lymphocytes (CTL) or their precursors (CTL-P) in conjunction with the fact that lymphocytes from HD or GD patients are known to proliferate in vitro in response to human tg (Htg), we further analyzed the T-cell subset(s) responsible for in vitro proliferation to Htg. In these experiments, peripheral blood T lymphocytes purified from patients with GD or HD were cultured with 1 microgram/ml Htg and irradiated autologous T-depleted mononuclear cells as the source of antigen presenting cells (APC). The proportions of T8 + cells declined considerably during culture in GD patients, but at Days 6 to 9, T8 + cells represented as much as 51% of cultured T lymphocytes from patients with HD. Moreover, the majority of T8 + cells were medium-large size lymphoblasts. Removal of Htg at Day 6 resulted in both abrogation of proliferative responsiveness and in decreases of T8 + percentages. Further analysis of the cell interactions leading to T8 + cell proliferation in response to Htg showed that helper/inducer T cells, as defined by 5/9 antigen expression, were strictly required. Collectively, these features are reminiscent of the T-cell involvement in experimental autoimmune thyroiditis of mice and stress for the first time the potential role of CTL in tissue damage occurring in Hashimoto's thyroiditis.     

Analysis of intrathyroidal cytokine production in thyroid autoimmune disease: thyroid follicular cells produce interleukin-1 alpha and interleukin-6.

Cytokine production was studied in thyroid tissue from patients with Graves' disease, Hashimoto's thyroiditis and non-toxic goitre. The expression of interferon gamma, tumour necrosis factor alpha and beta, interleukin-1 alpha and beta, interleukin-6 and platelet-derived growth factor A chain was assessed by slot-blot analysis of the respective mRNA in freshly isolated tissue samples. All seven cytokines were detected in patients of all groups. Although the respective mRNA levels were, in general, higher in thyroid autoimmune disorders, this appeared to relate to the degree of the lymphocytic infiltration of the thyroid gland at the time of surgery. Purified thyroid follicular cells expressed high levels of interleukin-1 alpha and interleukin-6 mRNA and when established in primary culture, purified thyroid follicular cells from Graves' disease as well as non-toxic goitre produced interleukin-1 alpha and interleukin-6 bioactivity spontaneously. In the case of interleukin-1 this could be further augmented by addition of lipopolysaccharide to the thyroid follicular cell cultures. These results demonstrate that the lymphocytic infiltrate found in autoimmune and non-autoimmune thyroid disorders is associated with cytokine production. Additionally we have shown that intrathyroidal cytokine production is not restricted to thyroid-infiltrating mononuclear cells, but may also involve thyroid follicular cells both in vivo and in vitro. The cytokines produced by thyroid follicular cells may have an important role in stimulating autoantigen specific T cells in vivo as both interleukin-1 and interleukin-6 facilitate T cell activation.     

Post-partum thyroiditis and goitrous (Hashimoto's) thyroiditis are associated with HLA-DR4

Using new panels of HLA-DR typing sera, we found an increase in the prevalence of HLA-DR4 in 21 patients with Hashimoto's thyroiditis (57%) and 32 patients with post-partum thyroiditis (53%) compared to controls (21%). Hashimoto's thyroiditis was previously found to be associated with HLA-DR5 and Dw5. We feel that the doubts raised by this study warrant the study of DR antigens in thyroid autoimmune disease using large panels of specific sera.