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.     

Apoptosis of thyrocytes and effector cells during induction and resolution of granulomatous experimental autoimmune thyroiditis

Experimental autoimmune thyroiditis (EAT) with granulomatous histopathology (G-EAT) can be induced by cells from mouse thyroglobulin (MTg)-immunized donors activated in vitro with MTg and IL-12. G-EAT lesions reach maximum severity 18-21 days after cell transfer and, if some thyroid follicles remain, lesions almost completely resolve by day 35. CD8(+) cells are required for G-EAT resolution. To begin to determine the mechanisms involved in G-EAT resolution, apoptosis in thyroids was analyzed by TUNEL staining. Apoptotic thyrocytes and inflammatory cells were present in the thyroids of both CD8(+) and CD8-depleted recipient mice at day 19-21. By day 35, apoptotic cells were rare in thyroids of mice whose lesions had resolved; the few apoptotic inflammatory cells were generally in close proximity to thyroid follicles. Thyroids of CD8-depleted mice had ongoing inflammation at day 35 and most apoptotic cells were thyroid follicular cells. The expression of Fas and Fas ligand (FasL) mRNA in thyroids was also determined by RT-PCR in both CD8(+) and CD8-depleted recipient mice. Fas was expressed in normal thyroids and its expression was relatively constant throughout the course of disease. FasL mRNA was not expressed in normal thyroids. FasL mRNA expression generally correlated with G-EAT severity, being maximal at day 21 and diminishing as lesions resolved. However, FasL mRNA expression in thyroids of CD8-depleted mice in which resolution was delayed was decreased compared to thyroids of CD8(+) mice with comparable disease severity, suggesting that FasL expressed by CD8(+) cells may play a role in G-EAT resolution.     

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。     

The Fas death pathway controls coordinated expansions of type 1 CD8 and type 2 CD4 T cells in Trypanosoma cruzi infection

We investigated the role of the Fas ligand (FasL)/Fas death pathway on apoptosis and cytokine production by T cells in Trypanosoma cruzi infection. Anti-FasL, but not anti-TNF-alpha or anti-TRAIL, blocked activation-induced cell death of CD8 T cells and increased secretion of IL-10 and IL-4 by CD4 T cells from T. cruzi-infected mice. CD4 and CD8 T cells up-regulated Fas/FasL expression during T. cruzi infection. However, Fas expression increased earlier in CD8 T cells, and a higher proportion of CD8 T cells was activated and expressed IFN-gamma compared with CD4 T cells. Injection of anti-FasL in infected mice reduced parasitemia and CD8 T cell apoptosis and increased the ratio of CD8:CD4 T cells recovered from spleen and peritoneum. FasL blockade increased the number of activated T cells, enhanced NO production, and reduced parasite loads in peritoneal macrophages. Injection of anti-FasL increased IFN-gamma secretion by splenocytes responding to T. cruzi antigens but also exacerbated production of type 2 cytokines IL-10 and IL-4 at a late stage of acute infection. These results indicate that the FasL/Fas death pathway regulates apoptosis and coordinated cytokine responses by type 1 CD8 and type 2 CD4 T cells in T. cruzi infection.     

Expression of CD40 and apoptosis related molecules in autoimmune thyroid diseases.

Apoptosis is responsible for the loss of thyrocytes in autoimmune thyroiditis. Recent investigations into the pathogenesis of apoptosis have revealed that the important roles of suicide molecules expression on both thyrocytes and cytotoxic T-lymphocytes. To study the mechanism of thyrocyte loss in various forms of thyroiditis, we evaluated in situ expression patterns of CD40, Fas, and Fas-L on thyrocytes and infiltrating inflammatory cells by immunohistochemical staining of thyroid samples obtained from 49 patients (Graves' disease, n=10: Hashimoto's thyroiditis, n=14; nonspecific lymphocytic thyroiditis, n=11; subacute granulomatous thyroiditis, n=11; normal, n=3). The role of cytotoxic T-lymphocytes was also evaluated by analyzing the expression of granzyme B along with their phenotypic characteristics. CD40 was not expressed on thyrocytes of normal controls while they showed a diffuse expression of Fas and a scattered focal expression of Fas-L. The plump thyrocytes proximal to the inflammatory infiltrates showed more intense expressions of these three molecules in various forms of thyroiditis and a close correlation was found between CD40 and Fas-L expression on thyrocytes. Unlike Fas, which was expressed on infiltrating lymphocytes in all groups, Fas-L was not expressed on infiltrating lymphocytes, except those in subacute granulomatous thyroiditis. Granzyme B expressing activated cytotoxic T-lymphocytes occupied a negligible proportion of CD8+ T-lymphocytes in various forms of thyroiditis, and no difference was found in terms of their proportions according to the type of thyroiditis. These results show the acquisition of CD40, Fas and Fas-L molecules on thyrocytes proximal to inflammatory cell aggregates and the negligible expression of granzyme B and Fas-L on the infiltrating lymphocytes, and suggest that Fas and Fas-L mediated apoptosis of thyrocytes (fratricide) may be more important than T cell-mediated cytotoxicity in various forms of thyroiditis.     

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.     

Intercellular adhesion molecule-1 (ICAM-1) in Graves' disease: contrast between in vivo and in vitro results.

We have reassessed the possible role of the adhesion molecule ICAM-1 in the pathogenesis of thyroid autoimmunity. In order to do that, we have investigated its expression in eight Graves' thyroids both in vivo (i.e. on cryostat sections and on cell suspensions), and in vitro (i.e. on cells cultured in monolayers for 3 days), and the results were compared with those obtained with similar preparations from four normal glands. On cryostat sections, the expression of ICAM-1, and for comparison that of HLA Class I and Class II molecules, was studied by immunofluorescence (IFL), but the former were also assessed by a distinct immunohistochemical technique. ICAM-1 was not detected in thyrocytes in vivo of both normal and Graves' glands, but solely in endothelial cells and antigen-presenting cells (APC). This selective reaction was confirmed by a four-layer technique using specific markers which identify endothelial cells and thyrocytes. HLA Class II molecules were confirmed to be inappropriately expressed in thyrocytes of Graves' glands, but there was no co-expression of these products and ICAM-1 in the same cells. In contrast, ICAM-1 appeared de novo in a proportion of Graves' and normal thyrocytes soon after the attachment and spreading of these cells in monolayer cultures (36-48 h). Graves' thyrocytes showed a quantitatively higher degree of expression compared with that detected on normal thyroid cells (40-70% versus 12-20%). Under these experimental conditions, the four-layer staining with thyroid microsomal antibodies confirmed that thyrocytes were indeed the positive cells which expressed ICAM-1. Blocking experiments with cultured thyrocytes from two Graves' glands and MoAbs to tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) did not prevent the occurrence of ICAM-1 expression. As a result of our study, we failed to demonstrate that Graves' thyrocytes express ICAM-1 in vivo. The unexpected case of inducing ICAM-1 on thyroid cells under certain in vitro conditions remains intriguing. The phenomenon could be the simple consequence of a mechanical effect rather than exerted by specific biological processes. Further investigations are, therefore, needed to establish whether ICAM-1 is really involved in the pathogenesis of Graves' disease.     

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.     

Fas ligand-mediated depletion of CD4 and CD8 lymphocytesby monomeric HIV-1-gp120

Summary.  In order to determine in what condition and by what mechanism gp 120 can deplete not only CD4 but also CD8 T cells, an in vitro system was established in which peripheral blood lymphocytes from healthy donors were treated with recombinant gp 120. We found that gp 120 can deplete both CD4 and CD8 T cells when they have recently been activated and are exposed to IL-2-deficient conditions. Bioassay of the Fas ligand (FasL) demonstrated augmented expression and release of soluble FasL by CD4 T cells in the supernatant of this culture. The administration of anti-FasL mAb and anti-Fas mAb, both of which exhibit neutralizing activity, completely abolished the depletion of these two T cell populations in culture. Based on these findings, we concluded that FasL depletes Fas antigen expressing CD4 and CD8 T cells by programmed cell death. Received December 18, 1996 Accepted May 2, 1997     

Treatment of lupus in NZB/W F1 mice with monoclonal antibody against Fas ligand

Since Fas ligand (FasL) can induce apoptosis of Fas-bearing cells, Fas/FasL interactions can play a critical role in maintaining self-tolerance. Fas/FasL interactions in lupus-like autoimmune disease have been well characterized in studies using either Fas or FasL mutant mice. However, the effect of the defective FasL-mediated signaling on the establishment of lupus in other mouse strains, such as NZB/W (B/W) F1, remains uncertain. In the present study, we examined the effect of anti-FasL monoclonal antibody (mAb) on the development of lupus. Treatment of B/W F1 mice with anti-FasL mAb augmented IgG1- and IgG2a-type anti-dsDNA Ab production. However, treatment of B/W F1 mice with anti-FasL mAb also significantly prevented the development of lupus nephritis. These results indicate that Fas/FasL interactions not only regulate IgG-type autoantibody production, but also influence the development of lupus nephritis in B/W F1 mice.     

Synergy in antigen presentation by thyroid epithelial cells and monocytes from patients with graves'' disease

The present study was undertaken to examine the ability of thyrocytes from Graves' patients to present purified protein derivative (PPD) to autologous peripheral blood T cells. Normal human thyrocytes which were pre-cultured with interferon-gamma were able to induce the proliferation of T cells in response to PPD antigen, but unstimulated thyrocytes failed to do. Thyrocytes from Graves' patients on which HLA-DR antigens were expressed have an ability to induce the proliferation of T cells. Thyrocytes from Graves' patients which were pulsed with PPD antigen for 4 h were capable of stimulating proliferation of the T cells. However, the stimulation index of T cells co-cultured with thyrocytes and PPD were significantly lower than that of T cells co-cultured with monocytes and PPD. Sub-optimal numbers of monocytes which by themselves were unable to support T-cell proliferation synergistically augmented antigen presentation by thyrocytes. These results suggest that cellular interactions among thyrocytes, monocytes and T cells may perpetuate immune or autoimmune responses in thyroid tissues from Graves' patients.     

Expression of intercellular adhesion molecule-1 in thyroid follicular cells in autoimmune, non-autoimmune and neoplastic diseases of the thyroid gland: discordance with HLA.

The presence of intercellular adhesion molecule-1 (ICAM-1) on epithelial cells facilitates their recognition by specific T lymphocytes. To assess the possible role of ICAM-1 in the recognition of thyroid follicular cells by T cells in thyroid autoimmune disease, we investigated the expression of ICAM-1 in thyrocytes from thyroid glands affected by Graves' disease, in glands with non-autoimmune pathology and normal glands using immunofluorescence staining on cryostat sections and on dispersed cell preparations. Sequential tissue sections from glands affected by Graves' disease (n = 15), multinodular goitre (MNG, n = 26), benign nodules (n = 11), primary carcinomas (n = 12) and control thyroid glands (n = 5) were stained for ICAM-1, HLA class I, HLA class II, CD3 and thyroid peroxidase (TPO). Weak and patchy ICAM-1 expression was found in the thyrocytes of 4/15 (27%) Graves' disease and of 1/26 (4%) multinodular goitre glands. In contrast, ICAM-1 expression was detected in the thyrocytes of 5/11 (45%) benign nodules and of 8/12 (67%) thyroid carcinomas in which it was sometimes strong. Thyrocytes in the five control glands were negative. These results correlated well with flow cytometry data from 23 of these glands which showed that ICAM-1 expression in thyrocytes from Graves' patients was, when present, 'dull', while in some malignant thyrocytes it was 'bright'. In preparations of thyrocytes from Graves' disease glands we found a striking discordance between the high levels of expression of HLA class I and HLA class II and the low expression of ICAM-1. This is surprising since in vitro the expression of these three molecules is equally induced by IFN-gamma and TNF-alpha. These results suggest that additional factors are involved in the induction of the inappropriate HLA class II expression observed in the thyrocytes of glands affected by Graves' disease.     

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.     

Th1 and Th2 subsets equally undergo Fas-dependent and -independent activation-induced cell death

Stimulation of previously activated T cells results in apoptosis, termed activation-induced cell death (AICD). Recent analysis revealed that the Fas/Fas ligand (FasL) interaction is predominantly involved in AICD of T cells. Furthermore, based on the analysis of various T cell clones and lines, it has been reported that FasL is expressed mainly in Th1 but not in Th2 cells. However, the exact expression pattern of FasL and its function in normal activated T cells has not been determined. In the present study, by utilizing completely differentiated Th1 and Th2 cell populations obtained from ovalbumin-specific T cell receptor (TCR)-transgenic mice, the FasL expression on Th1 and Th2 was determined. Furthermore, involvement of Fas-FasL interaction in AICD of Th1 and Th2 cells was analyzed by two approaches: one was the inhibition of AICD by anti-FasL monoclonal antibodies, and the other AICD of Th1/Th2 subsets from TCR-transgenic mice backcrossed to lpr mice. We demonstrated that Th2 cells express FasL on the cell surface at a level similar to that expressed by Th1 cells, and that both subsets were equally susceptible to the Fas-mediated AICD. These observations suggest not only that the expression of FasL is not always correlated with Th subsets as defined by the cytokine-producing profile, but also that the responses of both Th1 and Th2 subsets are regulated by Fas-mediated AICD. Finally, analysis of the kinetics of AICD revealed a novel Fas/FasL-independent pathway in its initial stage. These findings revealed the precise function of Fas/FasL-mediated as well as Fas/FasL-independent AICD in the regulation of helper T cell responses.     

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.     

IL-10 is necessary for FasL-induced protection from experimental autoimmune thyroiditis but not for FasL-induced immune deviation

Ectopic expression of FasL on thyrocytes confers immune privilege status to the thyroid by inducing apoptosis of Fas-expressing autoimmune effector T cells and anti-thyroglobulin (Tg) immune deviation away from the T1 type. Fas-mediated apoptosis of lymphoid cells leads to rapid production of anti-inflammatory cytokines such as IL-10. On the other hand, cytokines play a crucial role in the immunoregulation and pathology of experimental autoimmune thyroiditis (EAT), and systemic and local administration of IL-10 has a curative effect on EAT. To test the effect of endogenous IL-10 production in EAT, and to find out whether IL-10 production could be involved in FasL-induced protection, EAT was induced in IL-10(-/-) and in IL-10(-/-)xFasL-transgenic CBA/J mice.The results demonstrated that wild-type and IL-10 knockout (KO) animals developed similar EAT. In contrast, lack of endogenous IL-10 abolished the protective effect of FasL. Polymorphonuclear cells were observed significantly more frequently in the inflammatory cell infiltrates from IL-10(-/-)xFasL animals compared to IL-10(-/-) animals, but they were never detected in wild-type or IL-10(+/+)/FasL-transgenic mice. A shift away from T1 response was observed in FasL-transgenic mice irrespective of their IL-10 status, demonstrating that in our model, endogenous IL-10 plays no part in the T1-towards-T2 anti-Tg immune balance induced by FasL. In summary, endogenous IL-10 is not essential in EAT, or for the immune deviation induced by thyroid FasL expression, whereas it is necessary for the immune privilege status of the thyroid conferred by FasL expression on thyrocytes.     

IFN-gamma regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions

Fas and Fas Ligand (FasL) expression, activation-induced cell death (AICD) and mycobacterial antigen-specific cytotoxicity of peripheral T cells from patients with complete inherited IFN-gamma receptor 1 binding chain deficiency (IFN-gammaR1-/-) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti-Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN-gamma signaling, IFN-gamma induced surface FasL export and their Fas-dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN-gammaR1 (IFN-gammaR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II-matched, mycobacterial antigen-pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN-gammaR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN-gamma signaling in the control of mycobacterial infection in humans.     

Antigen-specific T(h)1 cells as direct effectors of Propionibacterium acnes-primed lipopolysaccharide-induced hepatic injury

T(h)1 cells are cytotoxic effector cells that utilize Fas ligand (FasL) and tumor necrosis factor. The physiological roles of cytotoxic T(h)1 cells are considered to be immunoregulation by eliminating autoreactive lymphocytes or hyper-activated foreign antigen-specific lymphocytes. Their pathological roles, however, remain to be clarified. To investigate whether T(h)1 cells can destroy organs, we generated a Propionibacterium acnes-specific T(h)1 clone from C57BL/6 mice and tested whether the clone could serve as an effector in a P. acnes-primed lipopolysaccharide (LPS)-induced hepatic injury system, one of the septic shock models. B6SMN:C3H-FasL(gld) (B6-gld) mice, which were deficient in functional FasL, were resistant to P. acnes/LPS-induced hepatic shock. The T(h)1 clone rendered B6-gld mice sensitive to the hepatic shock after the i.v. transfer. The hepatic injury in the clone-transferred B6-gld mice, which was evaluated by both biochemical and histological examination, was inhibited by an anti-FasL mAb that we developed. These results suggested that bacterial antigen-specific T(h)1 cells like this clone can participate in organ destruction in vivo as one of the cytotoxic effectors and play a critical role in endotoxin-induced hepatic injury.     

Activation through CD40 ligation induces functional Fas ligand expression by Langerhans cells

Langerhans cells (LC) are professional antigen-presenting cells of dendritic cell (DC) lineage and are critical for the induction of primary immune responses in skin. Following antigenic stimulation, LC migrate to regional lymph nodes and induce antigen-specific activation of T cells. After primary expansion, the majority of T cells undergo Fas/Fas ligand (FasL)-mediated apoptotic cell death, thereby suppressing their excessive expansion. Although recent investigations have indicated an immunoregulatory function for DC, whether LC could be involved in Fas/FasL-mediated suppression of activated T cells is still unclear. In this study, we found that LC express FasL after activation triggered through CD40 molecules on their surface, but not by stimulation with LPS or IFN-gamma. The functional significance of FasL expression by LC was demonstrated using two different assays for apoptosis induced in Jurkat cells. The apoptosis in Jurkat cells was completely blocked by anti-FasL blocking antibody, suggesting a Fas/FasL-mediated mechanism. These results indicate a new feedback mechanism to down-regulate T cell activation by LC through the interaction of the TNF receptor/ligand superfamily, CD40/CD40L and Fas/FasL.     

Decreasing TNF-alpha results in less fibrosis and earlier resolution of granulomatous experimental autoimmune thyroiditis

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced in DBA/1 mice by adoptive transfer of mouse thyroglobulin (MTg)-primed spleen cells. TNF-alpha is an important proinflammatory cytokine and apoptotic molecule involved in many autoimmune diseases. To study its role in G-EAT, anti-TNF-alpha mAb was given to recipient mice. Disease severity was comparable between mice with or without anti-TNF-alpha treatment at days 19-21, the time of maximal severity of G-EAT, suggesting TNF-alpha is not essential for development of thyroid inflammation. However, thyroid lesions resolved at day 48 in anti-TNF-alpha-treated mice, while thyroids of rat Ig-treated controls had fibrosis. These results suggested that reducing TNF-alpha contributed to resolution of inflammation and inhibited fibrosis. Gene and protein expression of inflammatory molecules was examined by RT-PCR and immunostaining, and apoptosis was detected using TUNEL staining and an apoptosis kit. Thyroids of anti-TNF-alpha-treated controls had reduced proinflammatory and profibrotic molecules, e.g., IFN-gamma, IL-1beta, IL-17, inducible NOS and MCP-1, at day 19 compared with thyroids of rat Ig-treated mice. There were more apoptotic thyrocytes in rat Ig-treated controls than in anti-TNF-alpha-treated mice. The site of expression of the anti-apoptotic molecule FLIP also differed between rat Ig-treated and anti-TNF-alpha-treated mice. FLIP was predominantly expressed by inflammatory cells of rat Ig-treated mice and by thyrocytes of anti-TNF-alpha-treated mice. These results suggest that anti-TNF-alpha may regulate expression of proinflammatory cytokines and apoptosis in thyroids, resulting in less inflammation, earlier resolution, and reduced fibrosis.