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。     

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.     

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。     

碘化钠对人甲状腺上皮细胞TNF-α、IL-1β分泌的影响

为研究碘化钠 (NaI)对人甲状腺上皮细胞 (TEC )分泌细胞因子TNF α和IL 1β的影响 ,以探讨碘在GD病 (GD )发病中的可能机制 ,取手术切除的GD病患者甲状腺组织和甲状腺腺瘤患者瘤旁正常甲状腺组织 ,进行细胞培养。以不同浓度的NaI (10 8～ 10 3 mol/L )刺激单层培养的甲状腺细胞 ,采用放射免疫测定技术测定刺激前后甲状腺细胞培养上清液中细胞因子TNF α和IL 1β的含量。同时以透射电镜观察NaI刺激后甲状腺细胞的形态学改变。结果 :(1)正常甲状腺细胞能分泌少量的TNF α和IL 1β。GD的甲状腺细胞TNF α和IL 1β的分泌量与正常TEC相比显著增加 (P <0 0 1) ;(2 )当NaI浓度为 10 8～10 3 mol/L时 ,正常人甲状腺细胞TNF α、IL 1β的分泌量与 0mol/L组相比无显著性差异 (P >0 0 5 ) ;透射电镜示正常甲状腺细胞无损伤型改变 ;(3)当NaI浓度为 10 6～ 10 3 mol/L时 ,GD甲状腺细胞TNF α的分泌量显著增加 (P <0 0 5 ) ;当NaI浓度为 10 8～ 10 3 mol/L时 ,GD甲状腺细胞IL 1β的分泌量显著增加 (P <0 0 5 )。NaI浓度超过 10 6mol/L时 ,透射电镜示GD甲状腺细胞出现损伤型改变。表明高浓度的碘不仅造成GD甲状腺细胞的直接损伤 ,而且可以增加甲状腺细胞细胞因子TNF α和IL 1β的分泌 ,特别是在GD甲状腺细     

Modulation of Dendritic Cell Function and Cytokine Production to Prevent Thyroid Autoimmunity

Understanding autoimmune thyroid diseases provides an unique perspective on the role of various components of the immune system in the pathogenesis of organ specific autoimmune diseases, whether the effector mechanism involves autoantibodies or T cells. Hashimoto's thyroiditis (HT) is largely mediated by thyroglobulin specific T cells, while Graves' disease (GD) is mediated by thyrotropin receptor specific autoantibodies. HT is characterized by thyroid destruction mediated by infiltrating or activated resident immune cells through a variety of mechanisms. In contrast GD is characterized by excessive production of thyroid hormone with little or no glandular destruction. Irrespective of the effector mechanism involved, dendritic cells (DCs) are required for the induction of an efficient primary response and thus are the first cells involved in an autoimmune response. DCs also provide the essential link between the innate and the adaptive immune system through co-stimulatory molecules and the production of cytokines and chemokines. Furthermore, inflammatory cytokines also appear to enhance the susceptibility of thyrocytes to apoptosis. In this review, we discuss the role of innate immunity in initiating an adaptive autoimmune response against the thyroid. We will explore the role of different mechanisms involved in breaking self-tolerance to thyroid antigens. Further, we will discuss recent developments in the development of experimental therapeutics against AITD.     

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.     

Adhesion molecules from the LFA-1/ICAM-1, 3 and VLA-4/VCAM-1 pathways on T lymphocytes and vascular endothelium in Graves' and Hashimoto's thyroid glands

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, their interaction with thyrocytes and extracellular matrix proteins. A number of different ligand molecules have been identified to mediate the interaction between EC and leukocyte subpopulations. In this study, we examined by flow cytometry and immunohistochemical techniques, the expression of integrin receptors and their counter-receptors by infiltrating lymphocytes and vascular endothelium in thyroid glands from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). A high proportion of GD intrathyroidal T lymphocytes expressed the CD69 and gp95/85 (Ea2) activation antigens as well as an increased number of LFA-α_L, VLA-α1, -α4, -α5, and -β1 integrin receptors, as compared with peripheral blood T lymphocytes from the same patients. The expression of intercellular adhesion molecule (ICAM)-1 was increased in EC from GD and HT thyroids. In addition, an up-regulated de novo expression of vascular cell adhesion molecule (VCAM)-1 was found in EC in GD and HT thyroids, with no reactivity in control thyroids. Dendritic cells in thyroid lymphoid follicles were also positive for ICAM-1 and VCAM-1. In addition, most of intrathyroidal mononuclear cells expressed the ICAM-3 adhesion molecule. This enhanced expression of ICAM-1 and VCAM-1 by thyroid EC in GD and HT may reflect their ability to regulate leukocyte trafficking and activation by means of the expression of specific ligand molecules. Our data suggest that both the LFA-1/ICAM-1, ICAM-3 and VLA-4/VCAM-1 pathways could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.     

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.     

Modulation of dendritic cell function and cytokine production to prevent thyroid autoimmunity

Understanding autoimmune thyroid diseases provides an unique perspective on the role of various components of the immune system in the pathogenesis of organ specific autoimmune diseases, whether the effector mechanism involves autoantibodies or T cells. Hashimoto's thyroiditis (HT) is largely mediated by thyroglobulin specific T cells, while Graves' disease (GD) is mediated by thyrotropin receptor specific autoantibodies. HT is characterized by thyroid destruction mediated by infiltrating or activated resident immune cells through a variety of mechanisms. In contrast GD is characterized by excessive production of thyroid hormone with little or no glandular destruction. Irrespective of the effector mechanism involved, dendritic cells (DCs) are required for the induction of an efficient primary response and thus are the first cells involved in an autoimmune response. DCs also provide the essential link between the innate and the adaptive immune system through co-stimulatory molecules and the production of cytokines and chemokines. Furthermore, inflammatory cytokines also appear to enhance the susceptibility of thyrocytes to apoptosis. In this review, we discuss the role of innate immunity in initiating an adaptive autoimmune response against the thyroid. We will explore the role of different mechanisms involved in breaking self-tolerance to thyroid antigens. Further, we will discuss recent developments in the development of experimental therapeutics against AITD.     

HLA-DMB expression by thyrocytes: indication of the antigen-processing and possible presenting capability of thyroid cells

Expression of HLA class II molecules on thyrocytes is a characteristic feature of autoimmune thyroid disease and may lead the thyroid cells to present autoantigens to CD4+ T lymphocytes. Since HLA-DM is a critical molecule in class II-restricted antigen processing and presentation, we assessed the expression of HLA-DMB, -invariant chain (Ii), class II transactivator (CIITA) and DRA in an untransformed, pure thyrocyte strain HTV-59A. Here we report that both HLA-DMB mRNA and the protein are expressed in thyrocytes and that CIITA expression is enhanced by interferon-gamma (IFN-gamma) treatment and occurs before DMB, Ii and DRA up-regulation, suggesting CIITA expression is a requirement for antigen processing in thyrocytes. These results indicate that thyrocytes are capable of antigen processing and possibly antigen presentation to T cells.     

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.     

Costimulatory molecules and autoimmune thyroid diseases

At least two signals for proliferation and cytokine secretion by T-cells are required. The first signal is delivered through the interaction of the T-cell receptor with major histocompatibility complex (MHC) molecules expressed on the surface of antigen-presenting cells (APC). The second or costimulatory signal is delivered by cell surface molecules expressed by APC. The interaction of B7.1/B7.2 with CD28 provide the most potent costimulatory signal for T-cell activation. CD40 antigen and its ligand (CD40L) have been shown to play a major role in regulating both humoral and cellular immune responses. In autoimmune thyroid diseases autoantigen presentation could be provided by "professional" APC, such as dendritic cells, as well as "nonprofessional" APC, such as thyroid follicular cells (TFC). In fact, these cells aberrantly express MHC class II molecules in Graves' disease (GD) and Hashimoto's thyroiditis (HT), together with large amounts of MHC class I antigens: moreover, the expression of CD40 on TFC, has been demonstrated. On the other hand B7.1 has been demonstrated in HT, but not in GD TFC. This could provide in HT a local costimulatory signal for T-cell differentiation towards a type 1 cytokine secretion pattern and also result in rescue from apoptosis of infiltrating lymphocytes. The presence of ICAM-1 on the surface of HT TFC may further strengthen contact and facilitate cross-signaling between T-cells and TFC. In contrast, the absence of B7 and ICAM-1 antigens in most GD TFC may more easily be associated with anergy and apoptosis of infiltrating T-cells, preventing the perpetuation and expansion of a "destructive" autoimmune reaction.     

Costimulatory Molecules and Autoimmune Thyroid Diseases

At least two signals for proliferation and cytokine secretion by T-cells are required. The first signal is delivered through the interaction of the T-cell receptor with major histocompatibility complex (MHC) molecules expressed on the surface of antigen-presenting cells (APC). The second or costimulatory signal is delivered by cell surface molecules expressed by APC. The interaction of B7.1/B7.2 with CD28 provide the most potent costimulatory signal for T-cell activation. CD40 antigen and its ligand (CD40L) have been shown to play a major role in regulating both humoral and cellular immune responses. In autoimmune thyroid diseases autoantigen presentation could be provided by "professional" APC, such as dendritic cells, as well as "nonprofessional" APC, such as thyroid follicular cells (TFC). In fact, these cells aberrantly express MHC class II molecules in Graves' disease (GD) and Hashimoto's thyroiditis (HT), together with large amounts of MHC class I antigens: moreover, the expression of CD40 on TFC, has been demonstrated. On the other hand B7.1 has been demonstrated in HT, but not in GD TFC. This could provide in HT a local costimulatory signal for T-cell differentiation towards a type 1 cytokine secretion pattern and also result in rescue from apoptosis of infiltrating lymphocytes. The presence of ICAM-1 on the surface of HT TFC may further strengthen contact and facilitate cross-signaling between T-cells and TFC. In contrast, the absence of B7 and ICAM-1 antigens in most GD TFC may more easily be associated with anergy and apoptosis of infiltrating T-cells, preventing the perpetuation and expansion of a "destructive" autoimmune reaction.     

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.     

Vascular endothelial growth factor (VEGF), VEGF receptors expression and microvascular density in benign and malignant thyroid diseases

Angiogenesis is critical for the growth and metastatic spread of tumours. Vascular endothelial growth factor (VEGF) is the most potent inducer of neovasculature, and its increased expression has been related to a worse clinical outcome in many diseases. The purpose of this study was to evaluate the relation between VEGF, its receptors (VEGFR-1 and VEGFR-2) and microvessel density (MVD) in thyroid diseases. Immunostaining for VEGF and VEGF receptors was performed in 66 specimens of thyroid tissue, comprising 17 multinodular goitre (MNG), 14 Graves' disease, 10 follicular adenoma, 8 Hashimoto's thyroiditis, 7 papillary carcinoma and 10 normal thyroid specimens. Thyrocyte positivity for VEGF and VEGF receptors was scored 0-3. Immunohistochemistry for CD31, and CD34 on the same sections was performed to evaluate MVD. Immunohistochemical staining of VEGF in thyrocytes was positive in 92% of all the thyroid tissues studied. Using an immunostaining intensity cut off of 2, increased thyrocyte staining was seen in follicular adenoma specimens, MNG and normal thyroids compared with Hashimoto's thyroiditis and Graves' disease (P < 0.05). Similarly, VEGF thyrocyte expression in Graves' disease was less than other pathologies (P < 0.05). VEGFR-1 expression and the average MVD score did not differ between the different thyroid pathologies. VEGF expression was lower in autoimmune pathologies compared to autonomous growth processes. Conversely, both VEGFR-1 and VEGFR-2 were widely expressed in benign and neoplastic thyroid disease, suggesting that the up-regulation of VEGF and not its receptors occurs as tissue becomes autonomous. There was no clear relationship between MVD measurement and thyroid pathology.     

Single-cell analysis of intrathyroidal lymphocytes shows differential cytokine expression in Hashimoto's and Graves' disease

Most human organ-specific autoimmune diseases such as Hashimoto's thyroiditis (HT) are considered to be Th1 mediated, and a quantitative dominance of Th1 cells in thyroid infiltrates from both Graves' disease (GD) and HT affected glands has been reported. However, Th2 dominance would be expected in GD, where thyroid hyperfunction induced by stimulating antibodies predominates over tissue destruction. We have analyzed the interleukin-4 (IL-4), interferon-γ (IFN-γ) production by T cells at the single-cell level, both in infiltrating lymphocytes isolated from digested GD and HT thyroid glands and in derived T cell lines, by direct intracellular cytokine detection. Results showed a heterogeneous pattern of cytokine production in bulk GD infiltrates and derived T cell lines, and a similar pattern was observed in the much larger HT infiltrates. Both type 1 and type 2 cytokines were simultaneously produced by the infiltrating populations, and T cells with both patterns as well as intermediate patterns similar to ThO cells could be detected ex vivo. However, the larger T lymphocytes, presumably activated and responsible for the autoimmune damage, predominantly produced IL-4 in GD and IFN-γ in HT. The specificity of the Th2 responses in GD was suggested by the enrichment in IL-4 production after antigen-specific expansion of two oligoclonal T cell lines. These data show that both type 1 and type 2 cytokines are produced in the thyroid glands affected by autoimmunity and that the difference between diseases may be the effect of a functionally dominant population at a given time. This in vivo chronically activated antigen-specific population, producing type 1 or type 2 cytokines locally, may be responsible for the effect finally leading to one of the disease states.     

The expression and distribution of S-100 protein and CD 83 in thyroid tissues of autoimmune thyroid diseases

To investigate the expression and distribution of S-100 protein and CD83 in the thyroid tissues of autoimmunethyroid diseases(ATDs),and to study the role of the dendritic cells in the pathogenesis of ATDs,immunohistochemical staining was used on pathological tissues of 20 patients with Hashimoto's thyroiditis(HT)and 20 patients with Graves' disease(GD) to check the expression and distribution of S-100 protein and CD83.Compared with control group(20 cases of thyroid follicular adenoma,TFA),the higher expressions of S-100 inHT(139.38±5.92 vs 59.47±11.69) and GD(119.42±14.48 vs 59.47±11.69) were observed respectively(p<0.001).The increased positive expressions of CD83 which is known as a marker of mature and activated DCs inHT(22.58±13.96 vs 5.19±8.08) and GD(29.92±14.43 vs 5.19±8.08) were also found respectively(p<0.001).Serum TPO antibody(TPO-Ab,67.3±11.6%) and Tg antibody(Tg-Ab,59.8±10.1%) in HT were higher thanthose in GD(28.4±5.7%,23.1±4.9%) and TFA(6.1±3.4%,7.2±4.6%)(p<0.01).Serum TR-Ab in GD(16.3±5.6 U/L) was higher than those in HT(4.8±2.3 U/L) and TFA(2.5±1.2 U/L)(p<0.01).Our findings suggestthat the high expression of DCs' markers may be related to the pathogenesis of HT and GD.The upregulationof both the number and the matured functions of DCs,may lead to present more antigens and to produce moreauto-antibodies(such as Tg-Ab and TPO-Ab in HT,TR-Ab in GD),which may be involved in pathogenesis ofthe autoimmune thyroid diseases.Cellular & Molecular Immunology.2004;1(5):378-382.     

Graves病和桥本甲状腺炎患者外周血T细胞表面及血清中Fas/FasL的表达特点

目的探讨Graves病(GD)和桥本甲状腺炎(HT)患者外周血中Fas+、FasL+细胞占总T淋巴细胞的比例(Fas%、FasL%)以及血清中sFas、sFasL、TGAb、TPOAb等指标的变化及意义。方法选择GD患者36例、HT患者32例、对照组20例。用流式细胞术检测外周血T细胞表面Fa(sCD95)、FasL(CD178)的表达特点,用双抗体夹心法(ELISA)测定血清中可溶性Fas及FasL的含量,用化学发光法测定相关抗体TGAb、TPOAb的含量。结果 GD及HT患者外周血Fas%均高于对照组(P<0.05),且以HT组更为显著,而各组均未检测到FasL的表达;GD及HT患者血清中sFas含量均高于对照组,尤以GD组显著;各组间均可检测到sFasL,但差异无统计学意义(P>0.05)。结论 Fas及其配体介导的凋亡在GD和HT的自身免疫反应过程中起着重要的作用。流式细胞术的应用,可为探讨AITD的发病机制提供新的方法。