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.     

Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies

We used a strain of differentiated rat-thyroid cells in continuous culture (the FRTL-5 strain) to detect the presence of growth-promoting antibodies in serum samples from patients with autoimmune thyroid disease. We found that IgG preparations from 17 of 20 patients (85 per cent) with active Graves' disease and two of five patients (40 per cent) with Hashimoto's thyroiditis could augment thyroid-cell growth. In parallel with IgG-induced elevations in intracellular cyclic AMP levels in the same cell line, all 20 of the patients with active Graves' disease had thyroid-stimulatory antibodies. Patients' IgG preparations fell into three subclasses: those with both potent cyclic AMP stimulation and potent growth-promoting activity; those with potent cyclic AMP stimulation but low-level growth promotion; and those with potent growth promotion and low-level cyclic AMP action. Growth-promoting antibodies were not detected in patients with Graves' disease in remission (seven patients), nodular goiter (seven), subacute thyroiditis (five), or atrophic thyroiditis (one). Simultaneous assays of growth promotion and cyclic AMP stimulation may be useful in the care of patients with autoimmune thyroid disease.     

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.

     

An anti-idiotypic antibody against Graves' IgG

Rabbit antibodies were raised against Graves' IgG adsorbed onto a TSH-receptor affinity and eluted thereof by [3H]NaCl. The rabbit serum absorbed against normal human IgG (ARI) still bound to Graves', control and Hashimoto's IgG preparations but in the latter two, binding was inhibited by bTSH (10 mU/ml). In addition, ARI stimulated thyroid cell cyclic AMP accumulation in both human and rat thyroid cells. The ARI preparation may, therefore, contain an "internal image" anti-idiotype causing thyroid (Ab2) stimulation, a Graves' disease specific anti-idiotype whose binding with Ab2 inhibits its ability to bind TSH and anti-anti-idiotype (Ab3) to "internal image" Ab2. In further studies, Graves' specific cross-reactive idiotype was found in 10/11 IgGs from patients with active Graves' disease. This study emphasizes the workings of Jerne's immunologic network and the complexity of polyclonal "anti-idiotypic" antibodies.     

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema

The expression of two autoimmune thyroid diseases, GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-γ) or IL-1β for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1β or IFN-γ caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1β or IFN-γ was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1β or IFN-γ, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1β or IFN-γ. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1β or IFN-γ. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.     

Thyroid atrophy in myxedematous endemic cretinism: possible role for growth-blocking immunoglobulins.

We have examined the ability of IgGs obtained from 8 endemic cretins to inhibit TSH-stimulated thyroid cell growth in culture. Clinical and laboratory evidence for hypothyroidism was present in six subjects; the two remaining patients had borderline low serum T4, normal T3 and exaggerated TSH response to TRH. In six patients 2 mg IgG exhibited an inhibitory effect in the cellular growth expressed by a diminished incorporation of 3H-thymidine into the DNA of TSH-stimulated FRTL-5 cells (range: 26-87% inhibition). Seven patients presented clinically with thyroid atrophy of relatively small thyroid enlargements for the degree of chronic iodine deficiency that was present in the area. The remaining subject had a large multinodular goiter and IgG purified from this patient had no inhibitory effect in the FRTL-5 cellular growth. A direct relationship was noted between the degree of thyroid growth inhibition (%) and the basal serum TSH concentration. We conclude that the presence of thyroid growth inhibiting immunoglobulin may be related to the absence of thyroid growth or even thyroid atrophy in endemic cretins.     

Virus plaque assay: effective detection of virus plaque forming cells at the early stage of lymphocyte activation by mitogen and alloantigen.

Activated lymphocytes were detected quantitatively by virus plaque assay (VPA) during the course of lymphocyte cultures stimulated by mitogen or alloantigen. In Con A-stimulated cultures, the number of virus-plaque forming cells (V--PFC) was a more sensitive method of detecting the early stage of lymphocyte activation than [3H]-thymidine (3H-TdR) incorporation. This evidence was obtained by two methods of collecting cells of each stage. First, when Con A-activated lymphocytes were fractionated by velocity sedimentation at unit gravity to separate cell populations according to each cell stage, the ratio of the number of V-PFC to the radioactivity of incorporated [3H]-TdR was larger in the earlier stage of cell cycle than in the later stage. Second, when cultured lymphocytes were synchronized directly by addition of excess thymidine and colchicine, similar results were obtained. In primary mixed lymphocyte cultures, the generation of cytotoxic lymphocytes (CTL) was correlated better with the proliferative response than with V-PFC production. It was also found that both the incorporation of [3H]-TdR and the generation of CTL were abrogated by cytosine arabinoside (Ara-C) added to cultures up to one day before assay, whilst the generation of V-PFC was not so markedly affected by Ara-C. These findings suggest that V-PFC represent the number of precursor cells which require one or more generations to differentiate to CTL and not simply the number of effector lymphoyctes already exhibiting cytotoxicity.     

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.     

On the mechanisms of thyrocyte proliferation and death in autoimmune thyroid diseases

Lymphocytes isolated from diffuse toxic goiter (Graves' disease, GD) stimulate the proliferation of "normal" thyrocytes (isolated from euthyroid goiter) in primary culture, and give them the properties of GD-thyrocytes (loss of sensitivity to the growth-promoting factors of FCS and lesser capacity of binding antibodies from GD patients' serum). The complement-free sera of GD patients (but not the sera of patients with Hashimoto's thyroiditis, HT) induce the death of "normal" thyrocytes more rarely than full-complement sera do. Both types of serum cytotoxicity are manifested on GD-thyrocytes much more rarely than on "normal" cells. The Fas-receptor on GD-thyrocytes in situ is expressed less than on "normal" and especially on HT-cells. The level of soluble Fas-ligand in the serum of some complement-free patients was found to be increased. These sera induce apoptosis in "normal" thyrocytes, but not in GD-cells nor in human skin fibroblasts. In the authors' opinion, the proliferation of GD-thyrocytes in situ is stimulated by intrathyroid lymphocytes, which directly stimulate this process and induce the loss of receptors which mediate the cytotoxic effects of serum factors.     

Effect of carbimazole treatment on specific and non-specific immunological parameters in patients with Graves' disease.

Twenty-two patients with newly diagnosed Graves' disease (GD) were treated with carbimazole (CBZ) for 6 months. Thyroid stimulating immunoglobulins (TSI) and T cell subsets were studied prior to treatment and after 3 and 6 months of therapy. TSI were measured on human thyroid epithelial cell monolayers by the cAMP production after the addition of highly purified GD IgG. Before treatment, serum IgG from 20 out of the 22 patients (91%) stimulated cAMP production significantly compared to normal IgG. The mean index of cAMP production (GD IgG relative to normal IgG) was 2.15. After 3 months of CBZ treatment, a non-significant decrease of the mean cAMP production index was observed, whereas it was significantly decreased at the end of the 6 month course of therapy. Before treatment, total T cells (OKT3+), helper/inducer T cells (OKT4+) and suppressor/cytotoxic T cells (OKT8+) were all significantly decreased compared with controls. After 3 or 6 months of CBZ therapy, both total T cells and helper/inducer T cells returned to a normal level, while cytotoxic/suppressor T cells remained at the same low level. Taken together, these data indicate that treatment with CBZ leads to an early increase of helper/inducer T cells followed 3 months later by a decrease of the TSI level with no change in decreased suppressor/cytotoxic T cells. The disappearance of TSI following the increased helper/inducer T cell level suggests that an anti-idiotypic reaction may have occurred, and the persistent decrease of the suppressor/cytotoxic T subset that CBZ therapy does not act upon the underlying autoimmune disease.     

Heterogeneity of thyrotropin binding inhibitor immunoglobulins in serum from untreated patients with hyperthyroid Graves' disease.

We have previously established an assay for the simultaneous assessment of thyrotropin (TSH) binding inhibitor immunoglobulin (TBII) and thyroid stimulating autoantibody activities in cultured rat thyroid cells (FRTL-5 cell), and found a discrepancy in some patients with untreated Graves' disease between the activities of TBII measured in FRTL-5 cells (TBII-rc) and in solubilized thyroid membranes (TBII-pm). In three selected patients with untreated Graves' disease, the different dose-response relationship between TBII-rc and TBII-pm clearly indicated the heterogeneous populations of TBII-pm in patients' sera, with different binding affinities for TSH receptor in intact cells.     

NADH─细胞色素b_5还原酶在甲状腺过氧化氢生物合成中的作用

应用高香草酸荧光分析技术及NADH─高铁氰化钾还原酶法对正常和Graves病甲状腺过氧化氢(H2O2)和NADH─细胞色素b5还原酶(b5R)进行测定, 发现Graves病甲状腺b5R活性和H2O2水平均明显高于正常, 而H2O2酶活性在Graves病和正常甲状腺间无显著差异。加b5R 抑制剂对氯汞苯甲酸后,Graves病和正常甲状腺b5R活性降低近85%,同时H2O2降低50％。b5R活性和H2O2水平两者呈显著正相关关系。以上结果表明b5R参与甲状腺内H2O2的生物合成,是甲状腺内H2O2生成的重要酶系。     

Immunological features of sporadic multinodular goiter

Summary The origin of sporadic multinodular goiter is still uncertain. To obtain information on a number of unexplored immunological features, the distribution and characterization of T, B, and natural killer lymphocyte subsets were studied in the peripheral blood of 15 patients with multinodular goiter; 8 patients with Graves' disease (for reference purposes with a well-characterized autoimmune disease) and 29 age- and sex-matched healthy controls, combining double-staining immunofluorescence technique with monoclonal antibodies and flow cytometry. Although in both thyroid diseases increased CD3⁺ HLA-DR⁺ activated T cells (P < 0.01) were detected, in Graves' disease this was associated with decreased numbers of CD8⁺ cells (P<0.05) and an increased CD4/CD8 ratio (P < 0.01). These abnormalities were absent in multinodular goiter, which displayed increased CD8⁺ CD57⁺ cytotoxic/suppressor cells (P < 0.01). There was an increase in the percentage of natural killer cells expressing CD16 and CD57 antigens in multinodular goiter but not in Graves' disease. The B-cell associated antigens CD 19 and CD19⁺ CD5⁺ were significantly increased in Graves' disease (P < 0.01), while the multinodular goiter patients exhibited only an increased number of B cells coexpressing the CD5 antigen (CD19⁺ CD5⁺), which was unrelated to the titers of antimicrosomal and antithyroglobulin autoantibodies. Our results point to the presence of several abnormalities of peripheral T, B, and natural killer lymphocytes in sporadic multinodular goiter, with a distribution pattern quite different from that observed in Graves' disease. These results support the notion that, in contrast to Graves' disease, in sporadic multinodular goiter different suppressor and/or cytotoxic mechanisms are set up by the immune system, reflecting either pathogenic mechanisms of the disease or an immune response to pathological growing tissue.Abbreviations FITC fluorescein isothiocyanate - NK natural killer - PE phycoerythrin - T₃ triiodothyronine - T₄ thyroxine - TBII thyroid binding inhibitory immunoglobulin - TSH thyroid-stimulating hormone     

The Operation of Immunological Networks in Graves'' Disease

Association between Graves' disease and HLA--B8 has been previously documented, as have been associations between 1 gG heavy chain allotype markers (Gm). We found a significant increase in the phenotype fnb/fb (ie. positivity for fb) in patients with Graves' disease compared to controls, raising the possibility of allotypic restriction of thyroid stimulating antibodies thought to be causally related to the disease. The influence of fb on the susceptibility to Graves' disease was found to be independent of HLA--B8 status suggesting that the immunological network operated by the Histocompatibility-linked genes is independent of that centered around IgG allotypes. It is postulated that, whereas the former genes determine the level of helper T lymphocyte function in the production of thyroid stimulating antibodies in Graves' disease, a person who also happens to carry the Gm marker fb would be assured of the production of IgG antibodies with thyroid stimulatory activity.     

Concanavalin A stimulation of mouse lymphocytes at low concentration. I. The effect of peritoneal exudate cells.

The results presented here show that concanavalin A (Con A) stimulated mouse spleen lymphocytes cultured at 1/10th optimal concentration incorporated less [3H]-TdR than those cultured at the optimal concentration. It was possible to increase the response of 1/10th optimal concentration lymphocytes by supplementing the cultures with macrophage-rich peritoneal exudate cell (PEC) preparations. This effect was dependent on the PEC concentration. [3H]-TdR incorporation by lymphocytes at optimal concentration was inhibited by the addition of PEC to the cultures. Thymocyte cultures at 1/10th optimal concentration also showed increased [3H]-TdR incorporation when supplemented with PEC but there was no inhibition effect at optimal concentration. PEC which had been pretreated with fresh mitomycin C to prevent cell division were as effective as controls at promoting the stimulation of 1/10th optimal concentration lymphocytes. PEC which had been killed were much less effective than controls.     

Application of mouse monoclonal antibodies for identification of antigen regions of human thyroid peroxidase in adolescents with Graves' disease and non-toxic multinodular goiter by flow cytometry

Thyroid peroxidase (TPO) is the major thyroid autoantigen recognized by serum autoantibodies from patients with Graves' disease (GD) or Hashimoto's thyroiditis directed to two immunodominant conformational regions termed A and B. The epitopes of human TPO have been defined using a panel of mouse monoclonal antibodies (mAbs). The aim of this study was to estimate the expression of chosen surface antigen regions of TPO (1, 18, 30, 64 epitopes) on thyroid cells in 15 patients with non-toxic multinodular goiter (NTMG) and 15 patients with GD. The thyrocytes were identified by indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')(2) with FITC. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus. The percentages of thyrocytes with expression of epitopes 1, 18, 30, 64 TPO were measured in relation to the respective anti-TPO concentrations: 50-1600 microg/ml. The analysis of epitopes located in immunodominant regions (IDR) of TPO revealed higher percentages of thyrocytes in cases with GD in comparison to NTNG. The most predominant difference was observed for mAb 64 epitope (48 vs 7%, p < 0.019; 39 vs 5%, p < 0.017) at the concentration of 100-200 microg/ml mAbs. The expression of 18 epitope on thyrocytes was also statistically higher in Graves' patients than in the NTMG (14 vs 6%, p < 0.025) at concentration of 400 microg/ml mAbs. However, this expression was much less pronounced. In all the cases, the percentages of thyrocytes with epitopes 1 and 30 were in low detection (8-15% of positive cells). In conclusion, our findings suggest that the elevated expression of TPO epitopes 18 and 64 in young patients with thyroid autoimmune diseases increase stimulation and activation of thyroid cells during inflammatory reaction within the thyroid gland. In addition, predominant expression of 64 TPO epitope that recognizes B domain in GD patients could be a useful marker of the immune process in the thyroid gland.     

Characterization of lymphoid cells in the thyroid of patients with Graves'' disease.

The distribution and function of lymphoid cells has been investigated in thyroid glands obtained at operation from 16 patients with Graves' disease (GD) using a peroxidase technique to enumerate total T and B lymphocytes as well as helper and suppressor T cell subsets in tissue sections. A spectrum of lymphocytic infiltration was observed and the increase from minimal numbers of immune cells in some GD thyroids to focal thyroiditis in others appeared to be due to a rise in all the lymphoid cell types analysed and was not the result of major change in any one lymphoid compartment. T cells were diffusely distributed whereas B cells tended to occur in aggregates. Small numbers of OKT6+ cells (possibly antigen presenting cells) were observed although these were less numerous than in lymphoid organs such as tonsil. Lymphoid cell suspensions prepared from the thyroid tissue of five of seven GD individuals treated pre-operatively with propranolol synthesized thyroid autoantibodies spontaneously in culture and this synthesis was decreased in the presence of pokeweed mitogen. Since the OKT8+ T cell subset has been shown to suppress immunoglobulin production by lymphocyte cultures containing mitogen, it appears that the suppressor T cells, which are readily demonstrable in GD thyroid sections, are functional. It seems unlikely, therefore, that a defect in this type of suppression is responsible for the initiation or perpetuation of the autoimmune response to thyroid antigens in GD.     

Negative correlation between the conversion of thyrotropin receptor-bound blocking type thyrotropin receptor antibody to the stimulating type by anti-human IgG antibodies and the biological activity of blocking type thyrotropin receptor antibody.

It has been reported that receptor-bound blocking type TSH receptor antibody (TRAb) can be converted to the stimulating type by anti-human IgG antibodies. To evaluate the relationship between the conversion of receptor-bound blocking type TRAb to the stimulating type and the biological activity of blocking type TRAb, we compared converting activities of blocking type TRAb from 10 patients with primary nongoitrous hypothyroidism with both the doses of blocking type TRAb which show 50% inhibition of 125I-bTSH binding to the TSH receptor and those which show 50% inhibition of TSH-stimulated cAMP production in cultured rat thyroid cells (FRTL-5). The additions of anti-human IgG antibody to FRTL-5 cell-bound blocking IgGs resulted in the increase in cAMP production in a dose-dependent manner and the converting activities (percent increase of cAMP production) also depended on the doses of blocking IgGs. The converting activities were significantly correlated with the doses of blocking IgGs which showed 50% inhibition of 125I-bTSH binding to the TSH receptor (r = 0.71, p = 0.011). And these converting activities were also significantly correlated with the doses of blocking IgGs which showed 50% inhibition of TSH-stimulated cAMP increase (r = 0.81, p = 0.002), and were negatively correlated with thyroid stimulation blocking antibody activities (r = 0.58, p = 0.02). We have demonstrated that all cell-bound blocking type TRAb were converted to the stimulating type by anti-human IgG antibody and the degree of conversion was negatively correlated with the biological activity of blocking type TRAb.(ABSTRACT TRUNCATED AT 250 WORDS)