Graves病和桥本病甲状腺细胞凋亡与Fas表达的研究

为了研究正常、Graves病 (GD )和桥本甲状腺炎 (HT )甲状腺细胞凋亡和凋亡相关蛋白Fas的变化特征及其临床意义 ,采用细胞培养方法和流式细胞术检测正常、GD和HT甲状腺细胞凋亡率和Fas表达量。结果发现 :(1)GD和HT甲状腺细胞凋亡率明显高于正常甲状腺细胞 (P <0 0 1)。其中 ,尤以HT甲状腺细胞凋亡增加最为显著 ;(2 )HT甲状腺细胞Fas表达阳性率明显高于正常和GD甲状腺细胞 (P <0 0 1) ,而GD与正常对照相比无统计学差异。以上结果表明 ,自身免疫性甲状腺疾病 (AITD )患者甲状腺细胞存在细胞凋亡和Fas表达的异常变化 ,尤以HT为显著 ,提示Fas介导的细胞凋亡参与AITD的发病过程 ,可能与HT甲状腺细胞破坏有关。     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.     

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.     

Up-regulated β1-integrin expression in autoimmune thyroid disorders

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, interaction with thyrocytes and extracellular matrix proteins. Recent studies have focused on the pathophysiologic role of β₁-integrins as adhesion receptors for extracellular matrix proteins and as cell-to-cell adhesion receptors. In this study, we examine by flow cytometry and immunohistochemical techniques the differences in expression of β₁-integrins in thyrocytes and EC between normal thyroids and thyroid glands from patients with Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). Remarkably, we found an up-regulated de novo expression of very late antigen (VLA)-α6 subunit in thyrocytes in close proximity to lymphocyte infiltrates in GD and HT thyroid glands, with no reactivity in control thyroids. Moreover, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α) and IL-1β produced a significant enhancement of VLA-α6 expression in vitro in thyrocytes in culture. In addition, an up-regulated expression of VLA-α5 and β1 subunits was found in thyrocytes from GD and HT glands, specifically in those areas more severely inflamed. VLA-α2 was basally expressed in middle size and large vessels in control glands, with an increased expression in vessels of all sizes in HT and GD glands. Dendritic cells in thyroid lymphoid follicles were also positive for VLA-β1, α2 and α6 subunits. These results indicate the existence of an up-regulatory process in the expression of β₁-integrins, particularly the α6 subunit, in several cell types from inflamed GD and HT thyroid glands, suggesting that these integrins could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.     

Fas/FasL mediated apoptosis of thyrocytes in Graves' disease.

We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1beta a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1beta-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1beta-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1beta-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1beta-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1beta through Fas/FasL interactions.     

Thyroid-stimulating antibody (TSAb) detected in sera of Graves'' patients using human thyroid cell cultures

As a homologous system is required to evaluate the effect of thyroid-stimulating antibody (TSAb) present in the serum of Graves' patients, primary cultures obtained from normal human thyroid gland have been used and the stimulatory effect measured as an increase of cAMP intracellular levels.

Monolayer cell cultures were stimulated by IgG purified from sera of Graves' patients or control subjects and compared to the effect of bovine TSH. Bovine TSH produced a dose-dependent increase in cAMP intracellular levels between 0·05 mU and 2·5 mU/ml, reaching a maximal value after 30 min with higher doses. While normal IgG had no effect, IgG prepared from untreated patients with frank Graves' disease elicited a significant increase in cAMP accumulation at a concentration between 0·05 and 0·5 mg/ml within 60 min in thirteen out of fourteen patients. A longer incubation period showed no further increase in cAMP values, even if in one case a higher concentration (5·0 mg/ml) of Graves' IgG had a delayed response. When the cAMP intracellular level modifications produced by Graves' IgG preparations in thyroid cell cultures were compared to those evoked in thyroid slices, an identical percentage (93%) of positive cases was obtained, without a coincidence of negative cases. Using thyroid slices the cAMP intracellular increase above basal levels was higher, if considered as a percentage, but in cultured cells a very low IgG concentration was sufficient to detect the presence of TSAb. No correlation between the two assays was found.

In conclusion, normal human cultured thyroid cells appeared to be a more suitable substrate when compared to human thyroid slices for detecting the presence of TSAb in Graves' disease and for studying its effect on thyroid cells. However, a 100% TSAb positivity was present in our Graves' patient series only when both assays were used.

     

The increased but non-predominant expression of Th17- and Th1-specific cytokines in Hashimoto's thyroiditis but not in Graves' disease

Hashimoto''s thyroiditis (HT) is considered to be mediated mainly by Th1 cells, but it is not known whether Graves'' disease (GD) is associated with Th1 or Th2 predominance. Th17 cells, a novel subset of Th cells, play a crucial role in the pathogenesis of various autoimmune disorders. In the present study, the expression of IL-17A and IFN-γ was investigated in patients with HT or GD. mRNA expression of IL-17A and IFN-γ in peripheral blood mononuclear cells (PBMC) from 43 patients with autoimmune thyroid disease (AITD) and in thyroid tissues from 40 AITD patients were measured by real-time quantitative PCR. The protein expression of IL-17A and IL-23p19 was examined by immunohistochemistry in thyroid tissues from 28 AITD patients. The mRNA levels of IL-17A and IFN-γ were higher in both PBMC and thyroid tissues of HT patients than in controls (mRNA levels are reported as the cytokine/β-actin ratio: IL-17 = 13.58- and 2.88-fold change and IFN-γ = 16.54- and 2.74-fold change, respectively, P < 0.05). Also, the mRNA levels of IL-17A and IFN-γ did not differ significantly in GD patients (P > 0.05). The high protein expression of IL-17A (IOD = 15.17 ± 4.8) and IL-23p19 (IOD = 16.84 ± 7.87) in HT was confirmed by immunohistochemistry (P < 0.05). The similar high levels of IL-17A and IFN-γ suggest a mixed response of Th17 and Th1 in HT, where both cells may play important roles in the destruction procedure by cell-mediated cytotoxicity.     

凋亡相关因子参与自身免疫性甲状腺疾病发病的研究

目的:了解细胞凋亡相关蛋白Fas,FasL和Bcl-2表达在自身免疫性甲状腺疾病发病机制及病理变化中的作用及意义。方法:采用免疫组织化学方法,检测20例桥本甲状腺炎,20例Graves病以及20例甲状腺腺瘤(作为对照组)患者甲状腺标本中Fas、FasL和Bcl-2表达及分布。结果:Fas在所有的标本中表达,主要分布于甲状腺滤泡细胞表面和细胞质上。除3例甲状腺瘤标本外,其余均表达FasL。Bcl-2表达于15例桥本甲状腺炎、19例Graves病以及17例甲状腺瘤滤泡细胞上。在甲状腺瘤滤泡细胞上表达中等强度Fas,很少或是没有表达FasL。在桥本甲状腺炎中Fas和FasL免疫染色强阳性甲状腺滤泡细胞多分布于浸润淋巴滤泡附近,浸润淋巴细胞中Fas、FasL免疫染色相对较弱。在Graves病中,Fas表达强度与桥本甲状腺炎类似,但FasL表达却更弱。在Graves病和甲状腺瘤组织中,Bcl-2表达两者类似。但在桥本甲状腺炎组织中,分布于浸润淋巴细胞附近的甲状腺滤泡细胞以及生发中心的淋巴细胞上,Bcl-2表达很弱。结论:Fas、FasL和Bcl-2表达在桥本甲状腺炎和Graves病中相似。FasL高表达和Bcl-2低表达可能引起桥本甲状腺炎滤泡细胞凋亡。进一步证明3种凋亡相关因子在自身免疫性甲状腺疾病发病机制中的作用。在桥本甲状腺炎中,滤泡细胞凋亡并非由浸润淋巴细胞其FasL发挥作用直接杀伤,但是它们能分泌细胞因子促进滤泡细胞自身Fas、FasL表达,从而导致滤泡细胞凋亡。     

Serum Levels of Soluble Fas in Patients with Multinodular Goiter

Objective: Fas is a cell-surface receptor responsible for induction of apoptosis in human thyrocytes upon interaction with Fas Ligand. Fas protein expression on thyroid cells and Fas-mediated apoptosis is decreased in multinodular goiter (MNG) resulting in thyroid cell proliferation. The soluble form of Fas (sFas) produced by alternative mRNA splicing may inhibit Fas-Fas Ligand binding and apoptosis. The aim of this study was to examine whether sFas is differentially expressed in multinodular goiter (MNG), which is associated with decreased Fas-mediated apoptosis. Method: We determined serum sFas levels using enzyme-linked immunosorbent assay (ELISA) in 42 patients with MNG and 23 normal controls. Results: Serum sFas levels were increased in patients with MNG (7.47 ± 2.55 ng/ml) compared to normal controls (2.26 ± 0.9 ng/ml). Levels of sFas were not significantly correlated with age, sex or clinical parameters, such as serum levels of FT4 or TSH. Discussion: Increased sFas in MNG may indicate increased expression of alternatively spliced Fas mRNA variant and decreased expression of cell-surface Fas protein, and may enhance thyroid cell proliferation by protecting thyroid cells from Fas-mediated apoptosis.     

Analysis of the expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disorders

To investigate the expression of apoptosis-related protein (Fas, FasL, and Bcl-2) in the pathogenesis of autoimmune thyroid disorders (ATDs), immunohistochemical staining was performed on 20 Hashimoto‘s thyroiditis (HT), 20 Graves‘ disease (GD), and 20 thyroid follicular adenoma (TFA, as control). All the cases expressed Fas, mainly on the cell surface and cytoplasm. FasL was found in 17 cases of the TFA. Bcl-2 was detected in 15 cases of HT, 19 of GD and 17 of TFA. In T FA, a moderate Fas expression and a minimal or no FasL expression was detected on follicular cells. In HT, the follicles adjacent to infiltrating lymphocytes showed increased levels of Fas and FasL expression. A weaker staining of Fas and FasL was exhibited on infiltrating lymphocytes than on thyrocytes. In a comparison of GD with HT, thyrocytes and lymphocytes showed similar Fas staining, but for FasL the staining was rather weaker in HT. The expression of Bcl-2 was nearly identical in GD and TFA, but much weaker on the follicular cells in vicinity of lymphocytes and on the lymphocytes located in germinal centers of HT tissues. The expression of Fas, FasL, Bcl-2 in Hashimoto‘s thyroiditis and Graves‘ disease were almost same. FasL strong expression and Bcl-2 weak expression on the follicles in HT may induce apoptosis. These results provided evidence for expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disease. The lymphocytes seem not to be directly engaged in the process v/a their own FasL, but they may provide some cytokines that, in turn, upregulate Fas and/or FasL expression to induce apoptosis.     

A novel hypothesis for the etiology of Graves' disease: TSAb may be thyroid stimulating animal IgG-like hormone and TBAb may be the precursor of TSAb

There are doubtful points about the theory that autoimmunity with auto-antibody (Ab) to TSH receptor (R) causes hyperthyroidism in Graves' disease (GD). A main doubtful point is no curative effect of corticosteroid on Graves' hyperthyroidism in spite of curative effect of corticosteroid for all autoimmune diseases. Recently we demonstrated the immunological similarity of TSAb and TBAb-IgG to animal IgGs, except for human (h)IgG, by neutralization and purification of TSAb and TBAb-IgG using (1) heterophilic Ab to animal IgG in GD sera and (2) experimentally generated anti-animal IgG Abs [such as dog (d), bovine (b), porcine (p), and rabbit (rb)]. Furthermore, greater immunological similarity of Fab- and F(ab')(2)-portion of TSAb- and TBAb-IgG to bovine Fab, compared to hFab, was demonstrated using goat anti-bovine F(ab')(2) Ab. Existence of b and p TSH-like portions in the LATS-IgG molecule (probably Fab portion) was suggested by a previous report of neutralization of LATS activity by anti-b- or anti-p-TSH Ab. We suggested the existence of a mammalian animal-TSH-like structure, excepting hTSH, in the TSAb-IgG molecule (probably Fab portion), by discovery of anti-mammalian TSH Ab (such as d, b, p, guinea-pig, rat, whale, except h) in sera of GD. Lately, similar TSHR binding of H- and L-chain of human stimulating monoclonal TSHR Ab (M22)-Fab with TSH-α and-β subunit was reported. This evidence suggests that Fab portion of TSAb has a structure like mammalian TSH, but not hTSH. IgG-λ type of d, horse, b, p, goat, ovine is 95% and IgG-κ type is 5%, while human κ and λ chain is 60:40. Previous report that LATS (TSAb)-IgG composed of predominant λ type is supporting evidence that TRAb-IgG has immunological similarity with these animal IgGs compared to hIgG. We speculate that TSAb-IgG may be referred as a mermaid consisted in face (Fab) and trunk-leg (Fc). Face may be a kind of hormone with animal TSH-like structure and trunk-leg has animal IgG-like structure (in spite of no antibody function). There are many reports for co-existence of TSAb and TBAb-IgG in sera of GD. We reported conversion from TBAb (non-thyroid stimulating type IgG) to TSAb by co-incubation of anti-hIgG Ab (containing anti-animal IgG Ab as a cross-reaction) with TBAb-bound porcine thyroid cells. Thus, we suggest that TBAb may be the precursor form of TSAb.     

Identification of apoptotic proteins in thyroid gland from patients with Graves' disease and Hashimoto's thyroiditis

Apoptosis, i.e. natural programmed cell death, is a physiological phenomenon indispensable for normal functioning of the organism. The signal to apoptosis can be started practically in any cell. Disturbances in the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of the study was to assess the expression of Fas/FasL and caspase eight in the tissues of the thyroid gland in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). The analysis of Fas/FasL expression was performed by western blot and immunohistochemical investigation with DAB-visualization and Mayer's hematoxylin staining. Caspase-8 expression in thyroid follicular cells was assayed by western blot method. Identification of the proapoptotic proteins FasL and Fas exhibited their pronounced expression in the thyroid tissue in GD patients (++; ++) and HT (+++; +++) as compared to the NTNG group (0/+; 0/+). Among the study groups, the expression of caspase-8 was revealed in band 55 kDa from patients with autoimmune thyroid diseases. In GD patients, the percentage of thyrocytes with FasL expression correlated positively with TRAb (R = 0.58, p < 0.02). However, no such correlations were noted in HT or non-toxic multinodular goiter. There were no significant correlations between thyroid hormones and the percentage of thyrocytes with Fas and FasL expression. In conclusion, our findings suggest that the changes in the expression of apoptotic molecules on the surface of T lymphocytes and thyroid follicular cells in patients with autoimmune thyroid disorders reflect their substantial involvement in the pathogenesis of GD and HT. In addition, analysis of Fas/FasL and caspase-8 expression in thyroid tissue may indicate the disease activity and immunological phenotype.     

Prognostic value of thyroid stimulating antibodies and TSH-binding inhibiting immunoglobulins in the follow-up of Graves' disease

Summary The prognostic value of the determinations of autoantibodies in Graves' disease is still questionable. So far, the role of different assay procedures used has not been intensively investigated. We simultaneously applied two different techniques, a radioreceptor assay and a T3 releasing in vitro assay, in the follow-up of patients with Graves' disease to directly compare the course of the antibody activities determined by these assays and to find out a prognostic significance of the composition of the antibody spectrum present. The initial activities of thyroid stimulating antibodies (TSAb) and TSH-binding inhibiting immunoglobulins (TBII) were not significantly correlated in patients before treatment. During a 12-month antithyroid medication antibody titres showed a concordant course in the majority of patients. In 6 of 25 patients, however, a discordant behaviour was clearly documented including dose-response curves. At the end of treatment, the patients could be divided into three groups: group I included 5 patients positive for both TSAb and TBII, group II 6 patients positive for TBII and negative for TSAb and group III 14 patients negative for both of them. During the following survey of 18 months all patients of group I, 2 patients of group II and 6 patients of group III experienced a relapse of hyperthyroidism. In conclusion, TSAb and TBII activities dissociate in some patients during antithyroid drug therapy. For the individual patient, the disappearance of both TSAb and TBII was no certain indicator for a longstanding remission of Graves' hyperthyroidism. The persistence of TSAb seems to be more reliably associated with persisting or rapidly relapsing disease than the persistence of TBII.Abbreviations cAMP Cyclic Adenosine Monophosphate - GD Graves' disease - T3 Triiodothyronine - T4 Tetraiodothyronine - TBII TSH-binding inhibiting immunoglobulins - TRH TSH releasing hormone - TSAb Thyroid stimulating antibodies - TSH Thyroid stimulating hormone     

Immunology of the thyrotropin receptor.

Antibodies to the thyrotropin receptor appear to be responsible for hyperthyroidism in Graves' disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves' disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Thyroid function tests

About 80% of thyroid disease consists of thyroid-specific autoimmune diseases, Hashimoto's disease and Grave's disease. To diagnose thyroid diseases, testings for (1) thyroid function and (2) pathogenetic autoantibodies are indispensable. To assess thyroid function, serum hormone concentrations, such as TSH, FT4 and FT3 are measured. Among these hormones, serum TSH concentrations are the most reliable and informative regarding thyroid function, correcting indicating a hyperthyroid, euthyroid or hypothyroid state. Therefore, TSH measurement appears to be the first choice in selecting the hormone determination. Reference intervals for normal healthy subjects of TSH are around 0.4-5.0 microU/ml. The second choice for thyroid function assessment are FT4 which supersedes total T4(TT4). TT4 is affected by changes in serum thyroid hormone binding proteins(TBG, TTR, Albumin). For example, euthyroid pregnant women whose serum TBG are physiologically higher than those of non-pregnant women show augmentation of TT4. However, FT4 depicts within reference intervals, although measurement of FT4 alone is unable to detect any abnormality of thyroid hormone binding proteins. According to its plasma concentration and binding affinity, FT3 measurement deserves no more significance than T3. Another important test for thyroid diseases is to detect serum autoantibodies against thyroid tissues, such as TgAb, TPOAb. Much more important is TSH receptor antibody which differentiates Graves' disease from Hashimoto's thyroiditis. In patients who show hyperthyroidism and some very uncommon hypothyroidism, TSH receptor antibodies should be measured. Three indicators are available as routine tests; TRAb measured by radioreceptor assay; TSAb determined by bioassay using cultured porcine thyroid cells. Usually, TRAb activity clinically correlates well with TSAb. TSBAb was initially discovered in patients with severe hypothyroidism with atrophic thyroid gland. TSBAb blocks thyroid stimulating activity of TSH and consequently causes severe hypothyroidism. TRAb and TSAb are very useful to diagnose and follow patients with Grave's disease.     

Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins

After autoimmune inflammation, interactions between CD95 and its ligand (CD95L) mediate thyrocyte destruction in Hashimoto's thyroiditis (HT). Conversely, thyroid autoimmune processes that lead to Graves' disease (GD) result in autoantibody-mediated thyrotropin receptor stimulation without thyrocyte depletion. We found that GD thyrocytes expressed CD95 and CD95L in a similar manner to HT thyrocytes, but did not undergo CD95-induced apoptosis either in vivo or in vitro. This pattern was due to the differential production of TH1 and TH2 cytokines. Interferon gamma promoted caspase up-regulation and CD95-induced apoptosis in HT thyrocytes, whereas interleukin 4 and interleukin 10 protected GD thyrocytes by potent up-regulation of cFLIP and Bcl-xL, which prevented CD95-induced apoptosis in sensitized thyrocytes. Thus, modulation of apoptosis-related proteins by TH1 and TH2 cytokines controls thyrocyte survival in thyroid autoimmunity.     

Generation of a transgenic animal model of hyperthyroid Graves' disease

Graves' disease (GD) is an organ-specific autoimmune disease characterized by hyperthyroidism. Agonistic anti-thyrotropin receptor antibodies (thyroid-stimulating antibodies, TSAb), which mimic the thyrotropin (TSH) action, are thought to cause GD. The precise immunological mechanism of TSAb production, however, remains elusive. Previous immunization approaches using TSH receptor led to transient hyperthyroidism, but did not seem sufficient for comprehensive understanding of the development of autoimmune responses. To create GD-related autoimmunity in mice, we here generated TSAb-transgenic mice in which a patient-derived TSAb is expressed in B cells. Expression of the human TSAb in mice resulted in various manifestations of hyperthyroidism including increased free thyroxine levels with concomitantly decreased TSH levels, increased thyroid uptake of technetium pertechnetate, hyperthermia and thyroid hyperplasia. We found a correlation between the serum levels of human TSAb immunoglobulin and free thyroxine. In addition, conventional B cells expressing the TSAb were partially deleted in the periphery while B1 cells expressing the TSAb persisted and accumulated in the peritoneal cavity, a finding consistent with previous demonstrations that the maintenance of B1 cells plays an important role in the development of autoimmune diseases. Thus, our transgenic mouse may provide a novel and useful animal model for elucidating the pathogenesis and pathophysiology of GD.     

The expression of the microsomal/peroxidase autoantigen in human thyroid cells is thyrotrophin-dependent.

In the present report the mechanisms responsible for the expression of the thyroid microsomal autoantigen (M-Ag) were studied in primary cultures of human thyroid cells prepared from Graves' or non-toxic goitres. The indirect immunofluorescence (IFL) technique using human sera positive for anti-microsomal antibody (anti-MAb) was employed to detect M-Ag. Studies were performed to ascertain whether M-Ag recognized by anti-MAb could be identified with thyroid peroxidase (TPO). Preabsorption experiments showed that, similarly to solubilized thyroid microsomes, purified human TPO abolished the binding of anti-MAb to thyrocytes, while no inhibition was obtained with control human tissues. The identity of M-Ag and TPO was also demonstrated using a double layer IFL technique which allowed a simultaneous staining of the antigen(s) recognized by anti-MAb and by a monoclonal anti-TPO antibody. After 5-15 days of TSH withdrawal from the culture medium the M/TPO-Ag disappeared from the surface and the cytoplasm of human thyroid cells. Readdition of TSH (0.1-100 mU/ml) to cells lacking M/TPO-Ag elicited its reappearance within 48-72 h. This effect of TSH was prevented by 10 microM cycloheximide but not by methimazole (0.1-2 mM). Two stimulators of the adenylate cyclase-cAMP system, cholera toxin and forskolin, and 8-bromo-cAMP mimicked TSH in inducing M/TPO-Ag. Thyroid stimulating antibody (TSAb) of Graves' disease also reproduced the effect of TSH on M/TPO-Ag reexpression in human thyroid cells. By contrast, epidermal growth factor, oestradiol or NaI were ineffective in inducing M/TPO-Ag. The present data indicate that: (i) the expression of M/TPO-AG in human thyroid cells is dependent on TSH stimulation, through pathways which involve cAMP production and protein synthesis, (ii) TSAb reproduces this effect of TSH; (iii) oestradiol and NaI have no direct influence on the expression of M/TPO-Ag.     

Identification of apoptotic proteins in thyroid gland from patients with Graves' disease and Hashimoto's thyroiditis

Apoptosis, i.e. natural programmed cell death, is a physiological phenomenon indispensable for normal functioning of the organism. The signal to apoptosis can be started practically in any cell. Disturbances in the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders.

The aim of the study was to assess the expression of Fas/FasL and caspase eight in the tissues of the thyroid gland in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). The analysis of Fas/FasL expression was performed by western blot and immunohistochemical investigation with DAB-visualization and Mayer's hematoxylin staining. Caspase-8 expression in thyroid follicular cells was assayed by western blot method.

Identification of the proapoptotic proteins FasL and Fas exhibited their pronounced expression in the thyroid tissue in GD patients (++; ++) and HT (+++; +++) as compared to the NTNG group (0/+; 0/+). Among the study groups, the expression of caspase-8 was revealed in band 55 kDa from patients with autoimmune thyroid diseases.

In GD patients, the percentage of thyrocytes with FasL expression correlated positively with TRAb (R = 0.58, p < 0.02). However, no such correlations were noted in HT or non-toxic multinodular goiter. There were no significant correlations between thyroid hormones and the percentage of thyrocytes with Fas and FasL expression.

In conclusion, our findings suggest that the changes in the expression of apoptotic molecules on the surface of T lymphocytes and thyroid follicular cells in patients with autoimmune thyroid disorders reflect their substantial involvement in the pathogenesis of GD and HT. In addition, analysis of Fas/FasL and caspase-8 expression in thyroid tissue may indicate the disease activity and immunological phenotype.     

Immunological of the Thyrotropin Receptor

Antibodies to the thyrotropin receptor appear to he responsible for hyperthyroidism in Graves disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.