Death Ligand Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Inhibits Experimental Autoimmune Thyroiditis
http://www.100md.com
《内分泌学杂志》
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0648
Abstract
The role of TNF-related apoptosis-inducing ligand (TRAIL) in autoimmune thyroiditis is unclear. We used experimental autoimmune thyroiditis to clarify the contribution of TRAIL to the development of autoimmune thyroiditis. CBA/J mice were immunized with murine thyroglobulin, and spleen cells from these mice were subsequently injected into irradiated recipient CBA/J mice. One week later, the recipient mice were treated with recombinant TRAIL or a control protein. Compared with control animals, TRAIL-treated mice developed a milder form of the disease with a significant decrease in mononuclear cell infiltration in the thyroid and less thyroid follicular destruction. Furthermore, the number of apoptotic thyrocytes and also thyroglobulin-specific T helper-1 cell responses in TRAIL-treated mice was lower than that in the control animals. This study suggests that exogenous TRAIL suppresses the development of autoimmune thyroiditis via altering the function of cells involved in the immune response. These findings may contribute toward a novel treatment autoimmune thyroiditis.
Introduction
MEMBERS OF THE TNF and TNF receptor family play key roles in the development and function of the immune system. Some members of this family, such as TNF and Fas ligand (FasL), may contribute to immune tolerance by deletion of self-reactive lymphocytes. For example, natural mouse mutants carrying mutations in the Fas and FasL genes, in which Fas-mediated cell death is impaired, exhibit lymphoproliferative and autoimmune syndrome (1). Moreover, children with Fas gene defects suffer from similar autoimmune disorders (2, 3, 4, 5). These examples suggest that the Fas-FasL system is involved in down-regulation of immune responses. Other members of TNF family, such as TNF-related apoptosis-inducing ligand (TRAIL), may also involve tolerance to self-antigens because the blockage of endogenous TRAIL exacerbates autoimmune arthritis (6). Once immune tolerance is disrupted, autoreactive lymphocytes can induce target cell apoptosis, especially in the presence of certain cytokines (7). This process is believed to contribute to the development of autoimmune diseases.
Normal thyroid glands show a low level of apoptosis, a possible result of basal thyroid cell turnover and maintenance of cell population (8, 9). In contrast, thyroid glands from patients with autoimmune thyroiditis display an increased frequency of apoptotic cells (8, 9, 10, 11). Many of the apoptotic cells in these glands are found in the areas of disrupted follicles, in proximity to infiltrating lymphoid cells (12, 13). This suggests that an increase in thyroid follicular cell apoptosis in thyroiditis may occur through a mechanism related to immune responses and inflammation. Autoimmune thyroiditis can also be induced experimentally in genetically susceptible mice by immunization with thyroid antigens (14). The mice develop autoimmune responses characterized by circulating antithyroid antibodies and infiltration of the thyroid gland by mononuclear cells. Evidence indicates that the death receptor-mediated apoptotic pathway is central to thyroid cell apoptosis in thyroiditis (15, 16). However, this pathway has been shown to be under the tight regulation of T helper (Th)1 cytokines in vitro and in vivo (7, 16, 17). Thus, it appears that the cytokine environment in the thyroid is able to influence the activation of apoptotic pathways.
As a newly identified TNF family member, TRAIL-induced apoptosis is mainly restricted to tumor cells and virus-infected cells. Recently it has been reported that TRAIL alone or in combination with other reagents has promising effects in antitumor therapy (18, 19). Although TRAIL can also block activation of human autoantigen-specific T cells (20), it is unknown whether TRAIL has an effect on immune cell function in the experimental autoimmune thyroiditis (EAT). To address this question, we used the EAT model to study the effect of TRAIL on the development of autoimmune disease.
Materials and Methods
Preparation of TRAIL protein
TRAIL protein was prepared as described by Chinnaiyan et al. (21). Briefly, it was affinity purified from bacterial lysates of cells transfected with the plasmid pET15b-His-FLAG-TRAIL. The purity of the TRAIL preparation was confirmed by sliver-stained SDS-PAGE. The endotoxin-free recombinant TRAIL was used for the experiment.
Establishment of transfer-EAT model and treatments
Eight-week-old CBA/J female mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions in our animal facility, and all animal studies were approved by our University Committee on the Use and Care of Animals. The transfer-EAT model was established as previously described (22). Briefly, CBA/J donor mice were injected twice with murine thyroglobulin (mTg) prepared from frozen mice thyroid glands and lipopolysaccharide at 10-d intervals (14, 43). The donor mice were killed 7 d after the second injection, and their spleens (5 x 106) were collected and cultured in complete culture medium with mTg (25 μg/ml) at 37 C for 72 h. The cells (1 x 107) were washed and then transferred iv into an irradiated (600 rad) CBA/J recipient. One week later, recipient CBA/J mice were treated with recombinant TRAIL or a control protein (BSA) by ip injection every other day at a dosage of 100 μg per mouse for 2 wk. At least 12–15 mice were included in each group. Thyroid function was tested by measuring serum T4 level by OptiCoat T4 enzyme immunoassay kit (Biotecx Laboratories Inc., Houston, TX).
Evaluation of thyroid infiltration
Thyroid glands were fixed in 10% formalin, embedded in paraffin, and sectioned by a standard method. Infiltration was evaluated on 5-μm-thick sections stained with hematoxylin and eosin. The severity of thyroiditis was graded on a scale of 0–4 as described by Wang et al. (7). Scoring was performed blind to the animal treatment groups.
Determination of apoptosis in thyroid gland
Apoptosis in paraffin-embedded thyroid tissue sections was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of fragmented DNA. Specific staining for apoptosis in situ was performed using the ApopTag fluorescein kit (Chemicon, Temecula, CA) according to the manufacturer’s protocol. In some instances, thyroid tissue section was dual stained with antidigioxigenin-fluorescein and phycoerythrin(PE)-conjugated cytokeratin 18 (CK 18) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Infiltrating thyroid lymphocytes (ITLs) were prepared from thyroid tissue, which was minced and digested with collagenase type IV (Sigma, St. Louis, MO) at 37 C for 2 h. The digestion was stopped by cold Hanks’ balanced salt solution and filtered through a 200-μm mesh. Isolated cells included both thyrocytes and lymphocytes. ITLs were gated out from thyrocytes by flow cytometry using PE-cys-conjugated rat antimouse CD45 monoclonal antibody (BD PharMingen, San Diego, CA). Apoptotic ITLs from TRAIL-treated or control mice were detected by flow cytometry using fluorescein diacetate (FDA) and propidium iodide (PI) stains (23).
In vitro proliferation assay
Spleen cells isolated from TRAIL-treated or control mice were cultured for 3 d in 96-well plates in the presence or absence of mTg. For the final 18–24 h of culture, 1.0 μCi [3H]thymidine (ICN Biomedicals, Costa Mesa, CA) was added to each well. The cells were then harvested onto glass fiber filters, and radioactivities were determined by a flatbed -counter.
Cytokine production in vitro
The splenocytes were incubated either with the mitogen phytohemagglutinin or mTg for 3 d, and then supernatant was harvested and measured for cytokine production. The levels of interferon (IFN), TNF, and IL-4 were detected by Quantikine M ELISA kits (R&D Systems Inc., Minneapolis, MN) according to the manufacturer’s instructions.
Antithyroglobulin (Tg) antibody
Sera collected before immunization, 7 d after immunization, and before time of death were used to determine the anti-Tg antibody by solid-phase ELISA. Briefly, plastic microtiter plates were coated with 100 μl mTg (10 μg/ml), and sera from individual mice in 1:1,000, 1:2,500, 1:5,000, 1:7,500, and 1:10,000 dilutions were added to each well. An alkaline phosphatase-conjugated, sheep antimouse IgG (Sigma) was added to determine anti-mTg IgG. In some instances, mTg-specific isotypes (IgG1, IgG2a, IgG2b, and IgG3) were also determined. Antimouse IgG1, IgG2a, IgG2b, and IgG3 that were conjugated to alkaline phosphatase were obtained from Rockland Immunochemicals for Research (Gilbertsville, PA).
Statistical analysis
All values were expressed as mean ± SE. The statistical significance of the differences between control and experimental groups was analyzed with Wilcoxon matched pair test and Student’s test using the software Stat View (Abacus Concepts, Inc., Berkeley CA). P < 0.05 was taken as statistically significant.
Results
TRAIL treatment inhibits transfer EAT
Based on our prior work, we found that transfer-induced EAT produces a massive mononuclear cell infiltration into the thyroid gland. This allows studies on how TRAIL affects ITLs. Seven days after CBA/J mice were injected with mTg-activated splenocytes, the mice were treated with either TRAIL or BSA once every other day for 2 wk. As shown in Fig. 1, TRAIL-treated mice developed a milder form of the disease when compared with control animals. The reduction was evidenced primarily by a decrease in the average disease score from 3.5 ± 0.2 (n = 12) to 2.1 ± 0.1 (n = 15), showing both decreased mononuclear cell infiltration in the thyroid and reduced thyroid follicular destruction in TRAIL-treated mice. TRAIL treatment did not alter the thyroid hormone level during the period of our study because the level of T4 in TRAIL-treated mice did not change significantly [2.74 ± 0.42 μg/dl, compared with the values before spleen cell transfer (3.0 ± 0.57 μg/dl) and 21 d after the transfer (2.49 ± 0.40 μg/dl)].
TRAIL decreases thyroid cell apoptosis but does not affect apoptosis of ITLs
To determine whether TRAIL treatment specifically promotes thyrocyte apoptosis, thyroid sections from EAT mice were evaluated for cell death by in situ TUNEL staining. After 2 wk of TRAIL treatment, the number of apoptotic cells in the thyroid glands was markedly decreased in the EAT mice when compared with mice without treatment. This was demonstrated by the number of TUNEL-positive cells (in green), which are shown to be decreased in Fig. 2A, right panel). To confirm that apoptotic cells were indeed thyroid cells, we applied CK18, an epithelial cell marker, to evaluate the cell type. After thyroid section was dual stained with antidigioxigenin-fluorescein and PE-conjugated CK18 antibodies, we found that the fluorescent staining cells were also positive for CK18, indicating that those apoptotic cells were thyroid cells in nature.
Cell viability/death was determined by flow cytometry using FDA and PI staining (24). A total of 10,000 cells were stained with PE-cys, FDA, and PI. To our surprise, after ITLs were gated from the thyroid cell population, the percentages of apoptotic ITLs in TRAIL-treated and control mice were similar, being 7.1 and 8.4%, respectively (Fig. 2B). This indicated that the reduction of infiltrating mononuclear cells in the thyroid gland seen in TRAIL-treated mice (Fig. 1) did not result from apoptotic mechanism in this model of thyroiditis.
Characteristics of the immune response of EAT mice treated with TRAIL
Tg-specific proliferation of immune cells (mainly lymphocytes) in the presence of TRAIL was measured. The proliferation index in response to Tg was decreased in TRAIL-treated EAT mice (Fig. 3A). This demonstrated that TRAIL inhibited the proliferation of lymphocytes that were stimulated by Tg. The production of IFN, TNF, and IL-4 in supernatants of these Tg-stimulated spleen cells was determined by ELISA. Spleen cells from mice injected with TRAIL or control protein produced similar levels of TNF and IL-4. In contrast, splenocytes from the TRAIL-treated mice produced a significantly lower level of IFN (Fig. 3B), a Th1 cytokine, which is required to sensitize thyroid cells to apoptosis probably via removing the inhibitor of apoptosis (7, 18).
The impact of TRAIL on the humoral response to mTg was studied in the sera of mice with ongoing EAT. Seven days after spleen cell transfer, anti-mTg IgG was markedly higher, compared with its level before immunization (Fig. 4). After 7 d of TRAIL treatment, the level of anti-mTg IgG continued to rise in both the TRAIL and BSA groups. However, compared with the BSA group, the increase in anti-mTg IgG was significantly inhibited in the TRAIL group at 2 wk after the experiment (Fig. 4), suggesting an inhibitory role of TRAIL on anti-mTg IgG production. Further analyses of the anti-mTg IgG subclass showed that the levels of anti-mTg IgG1, IgG2b, and IgG3 were not affected by TRAIL, and the overall levels of IgG2b and IgG3 were very low in the sera of mice, regardless of whether the mice were treated with TRAIL. Most important, we observed that the anti-mTg IgG2a response was significantly diminished in the sera of mice treated by TRAIL, compared with that treated by BSA (Fig. 4). It was thus believed that this decrease was the major factor contributing to the reduction of anti-mTg IgG.
Discussion
As a newly identified TNF family member, TRAIL has been reported to be a promising agent in antitumor therapy (18, 19). In contrast to TRAIL, the systemic administration of FasL is limited because FasL can cause an animal’s death by widely inducing apoptosis in animal liver cells (1). A therapeutic role for TRAIL has been proposed in antiautoimmune diseases (6, 25). In a current study, we demonstrate that exogenous TRAIL inhibits EAT in a transfer model of thyroiditis. Although TRAIL treatment did not obviously alter the level of serum T4, it did significantly reduce mononuclear cell infiltrations in the thyroid glands. This reduction was correlated with a decrease in apoptosis of thyroid cells. These findings are in line with the fact that infiltrating lymphocytes play a vital role in the development of autoimmune thyroiditis by secreting inflammatory cytokines and stimulating the apoptosis in thyrocytes (7, 26, 27). Therefore, any mediation that is able to reduce the infiltrating lymphocytes in thyroid, such as the one described herein, should have potential as a novel treatment for autoimmune thyroiditis.
The transfer EAT was developed in irradiated susceptible recipients by the transfer of in vitro Tg-stimulated spleen cells obtained from Tg-immunized donors. This EAT model introduces an enormous number of infiltrated lymphoid cells, including CD4+ and CD8+ T cells, into the thyroid gland. Using this EAT model, we demonstrated that TRAIL could significantly decrease the number of infiltrated lymphoid cells in thyroid. It is possible that the reduction of infiltrated lymphoid cells in thyroid is caused by an increase in apoptosis of lymphocytes because TRAIL is initially discovered as an apoptotic molecule. However, to our surprise, the apoptotic rate of infiltrated lymphoid cells in thyroid in both TRAIL-treated and nontreated mice remained similar in our experiment. Therefore, it is unlikely that the increased apoptosis is a significant factor contributing to the decreased infiltrated lymphoid cells in thyroid seen in the present study. However, it is noted that the effect of TRAIL on inflammatory cytokine production may be responsible for the reduction of infiltrated mononuclear cells in thyroid in our EAT model.
It is known that both apoptosis and recruiting mononuclear cells into thyroid cells is under the tight control of inflammatory cytokines (15, 16). Cytokines can regulate the expression of apoptotic signaling components and inhibitors in target cells (16, 26, 27). Several publications (28, 29, 30, 31) suggested a crucial role for cytokines in the pathogenesis of EAT. It has been shown that T cell clones isolated from intrathyroidal mononuclear infiltrates of Hashimoto’s thyroiditis produce high levels of IFN (32, 33). As we have demonstrated previously, both IFN and TNF were required for sensitizing a specific (Fas) pathway in the thyroid cells (7, 17). Although the level of TNF did not change by TRAIL treatment, IFN production was significantly decreased. This could lead to inactivation of the cell death pathway in thyroid cells. Indeed, a decrease of thyroid cell apoptosis was observed in the EAT mice treated with TRAIL. This is possibly due to the alteration of the cytokine environment because TRAIL can cause a reduction of IFN production. In addition to inactivating apoptosis of thyrocytes, the decreased IFN level by TRAIL can also lead to reduced mononuclear cell infiltration in thyroid. IFN is a key molecule in regulation of production of chemokine production, such as CCL1–5 and CXCL 10. These chemokines motivate lymphocytes/mononuclear cells into thyroid (34, 35). Therefore, it is reasonable to assume that the inhibition of IFN production can cause reduction of mononuclear cell infiltration in thyroid, which is supported by our current study and also in agreement with a report that TRAIL-R(–/–) mice are associated with increased levels of IFN (36). Studies from our group have shown that thyroid cells are known to express TRAIL receptors such as TRAIL receptor death receptor 4 and death receptor 5 (37, 38). However, the detailed pathway leading to reducing IFN level by TRAIL needs further investigation. Nevertheless, our data suggest TRAIL inhibits transfer EAT by inactivating apoptosis of thyrocytes and decreasing the number of mononuclear cells into thyroid.
Both spontaneous and induced thyroiditis in rats and mice appear to be mediated by cellular immune elements because thyroid inflammation can be passively transferred between animals by T lymphocytes and thyroid antigen-specific T cell clones (39, 40, 41, 42) but not with antibodies. Also, thymectomy has been shown to prevent the development of the disease (39). T cells from animals with EAT have been shown to be cytotoxic to cultured thyrocytes in vitro (43), and CD8+ cells have been demonstrated to proliferate in response to thyroglobulin (15). Therefore, it is important to ascertain whether TRAIL could inhibit cellular immune response to autoantigen. We showed that the antigen-specific lymphocyte function was decreased by TRAIL. The impaired lymphocyte function was reflected by a decrease in lymphocyte proliferation and mTg antibody production. A decrease in anti-mTg IgG2a was particularly obvious. Furthermore, the production of IFN-, a Th1 cytokine, was also reduced. Hence, our study suggested that the Tg-specific immune response was significantly reduced in EAT mice treated with TRAIL. Such a reduction of Tg special immune response may contribute to the inhibition of autoimmune thyroiditis development and also bear a resemblance to the scenario in which the TRAIL system functions as a negative regulator of innate immune cell responses (36).
Based on the above information, it is clear that the infiltrating mononuclear cells and the cytokine microenvironment in the thyroid are central to the autoimmune destruction of thyrocytes in EAT. Hashimoto’s thyroiditis appears to involve a defect in the mechanism against mononuclear cell infiltration in thyroid, which leads to the loss of tolerance to self-antigen (44, 45). Therefore, reduction and functional inhibition of thyroid infiltrating lymphocytes could open a new approach to protection against thyroid autoimmunity.
In conclusion, we have found that the down-regulation of mTg-specific, antoreactive immune cell function, at least in part by TRAIL, is responsible for the inhibition of EAT. This leads to alteration of the thyroid cytokine environment, inactivates the apoptotic pathway, and protects thyroid cell apoptosis under immune disorder. To the best of our knowledge, this is the first report of an inhibitory effect of TRAIL on EAT. Based on this, it is important to further investigate the detailed mechanism of how TRAIL is involved in the inhibition of autoimmune responses.
Acknowledgments
We gratefully acknowledge Drs. R. J. Koenig and Y. M. Kong for helpful discussions and Dr. James Bretz for help with TRAIL preparation.
Footnotes
This work was supported by National Institutes of Health Grant 5 R01 AI037141-10.
Abbreviations: CK 18, Conjugated cytokeratin 18; EAT, experimental autoimmune thyroiditis; FasL, Fas ligand; FDA, fluorescein diacetate; IFN, interferon; ITL, infiltrating thyroid lymphocyte; mTg, murine Tg; PE, phycoerythrin; PI, propidium iodide; Tg, thyroglobulin; Th, T helper; TRAIL, TNF-related apoptosis-inducing ligand; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
References
Nagata S, Golstein P 1995 The Fas death factor. Science 267:1449–1456
Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, Puck JM 1995 Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935–946
Rieux-Laucat F, Le Deist F, Hivroz C, Roberts IA, Debatin KM, Fischer A, de Villartay JP 1995 Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268:1347–1349
Drappa J, Vaishnaw AK, Sullivan KE, Chu JL, Elkon KB 1996 Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. N Engl J Med 335:1643–1649
Le Deist F, Emile JF, Rieux-Laucat F, Benkerrou M, Roberts I, Brousse N, Fischer A 1996 Clinical, immunological, and pathological consequences of Fas-deficient conditions. Lancet 348:719–723
Song K, Chen Y, Gke R, Wilmen A, Seidel C, Gke A, Hilliard B, Chen Y 2000 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J Exp Med 191:1095–1104
Wang SH, Bretz JD, Phelps E, Mezosi E, Arscott PL, Utsugi S, Baker Jr JR 2002 A unique combination of inflammatory cytokines enhances apoptosis of thyroid follicular cells and transforms non-destructive to destructive thyroiditis in experimental autoimmune thyroiditis. J Immunol 168:2470–2474
Dremier S, Golstein J, Mosselmans R, Dumont JE, Galand P, Robaye B 1994 Apoptosis in dog thyroid cells. Biochem Biophys Res Commun 200:52–58
Okayasu I, Saegusa M, Fujiwara M, Hara Y, Rose NR 1995 Enhanced cellular proliferative activity and cell death in chronic thyroiditis and thyroid papillary carcinoma. J Cancer Res Clin Oncol 121:746–752
Tanimoto C, Hirakawa S, Kawasaki H, Hayakawa N, Ota Z 1995 Apoptosis in thyroid diseases: a histochemical study. Endocr J 42:193–201
Bagnasco M, Venuti D, Paolieri F, Torre G, Ferrini S, Canonica GW 1989 Phenotypic and functional analysis at the clonal level of infiltrating T lymphocytes in papillary carcinoma of the thyroid: prevalence of cytolytic T cells with natural killer-like or lymphokine-activated killer activity. J Clin Endocrinol Metab 69:832–836
Kotani T, Aratake Y, Hirai K, Fukazawa Y, Sato H, Ohtaki S 1995 Apoptosis in thyroid tissue from patients with Hashimoto’s thyroiditis. Autoimmunity 20:231–236
Hammond LJ, Lowdell MW, Cerrano PG, Goode AW, Bottazzo GF, Mirakian R 1997 Analysis of apoptosis in relation to tissue destruction associated with Hashimoto’s autoimmune thyroiditis. J Pathol 182:138–144
Kong Y, David CS, Giraldo AA, El Rehewy M, Rose NR 1979 Regulation of autoimmune response to mouse thyroglobulin: influence of H-2D-end genes. J Immunol 123:15–18
Arscott PL, Knapp J, Rymaszewski M, Bartron JL, Bretz JD, Thompson NW, Baker Jr JR 1997 Fas (APO-1, CD95)-mediated apoptosis in thyroid cells is regulated by a labile protein inhibitor. Endocrinology 138:5019–5027
Stassi G, Di Liberto D, Todaro M, Zeuner A, Ricci-Vitiani L, Stoppacciaro A, Ruco L, Farina F, Zummo G, De Maria R 2000 Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins. Nat Immunol 1:483–488
Bretz JD, Arscott PL, Myc A, Baker Jr JR 1999 Inflammatory cytokine regulation of fas-mediated apoptosis in thyroid follicular cells. J Biol Chem 274:25433–25438
Wang SH, Mezosi E, Wolf JM, Cao Z, Utsugi S, Gauger PG, Doherty GM, Baker Jr JR 2004 IFN sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression. Oncogene 23:928–935
Kagawa S, He C, Gu J, Koch P, Rha SJ, Roth JA, Curley SA, Stephens LC, Fang B 2001 Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. Cancer Res 61:3330–3338
Kayagaki N, Yamaguchi N, Nakayama M, Kawasaki, A, Akiba H, Okumura K, Yagita H 1999 Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity. J Immunol 162:2639–2647
Chinnaiyan AM, Prasad U, Shankar S, Hamstra DA, Shanaiah M, Chenevert TL, Ross BD, Rehemtulla A 2000 Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc Natl Acad Sci USA 97:1754–1759
Mignon-Godefroy K, Brazillet MP, Rott O, Charreire J 1995 Distinctive modulation by IL-4 and IL-10 of the effector function of murine thyroglobulin-primed cells in "transfer-experimental autoimmune thyroiditis." Cell Immunol 162:171–177
Killinger Jr WA, Dorofi DB, Tinsley Jr EA, Keagy BA, Johnson Jr G 1992 Flow cytometric analysis of organ preservation-induced endothelial cell membrane damage. Ann Thorac Surg 53:472–476
Bartkowiak D, Hogner S, Baust H, Nothdurft W, Rottinger EM 1999 Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate. Cytometry 37:191–196
Hilliard B, Wilmen A, Seidel C, Liu TS, Goke R, Chen Y 2001 Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis. J Immunol 166:1314–1319
Mezosi E, Wang SH, Utsugi S, Bajnok L, Bretz JD, Gauger PG, Thompson NW, Baker Jr JR 2005 Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells. Mol Endocrinol 19:804–811
Wang SH, Baker Jr JR 2001 Autoimmune thyroid disease. Clinical Immunology 81:1–20
Alimi E, Huang S, Brazillet MP, Charreire J 1998 Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN- receptor gene. Eur J Immunol 28:201–208
Ajjan RA, Watson PF, Weetman AP 1996 Cytokines and thyroid function. Adv Neuroimmunol 6:359–386
Frohman M, Francfort JW, Cowing C 1991 T-depend destruction of thyroid isografts exposed to IFN-. J Immunol 146:2227–2234
Barin JG, Afanasyeva M, Talor MV, Rose NR, Burek CL, Caturegli P 2003 Thyroid-specific expression of IFN- limits experimental autoimmune thyroiditis by suppressing lymphocyte activation in cervical lymph nodes. J Immunol 170:5523–5529
Kawakami Y, Kuzuya N, Watanabe T, Uchiyama Y, Yamashita K 1990 Induction of experimental thyroiditis in mice by recombinant interferon administration. Acta Endocrinol 122:41–48
Hamilton F, Black M, Farquharson MA, Stewart C, Foulis AK 1991 Spatial correlation between thyroid epithelial cells expressing class II MHC molecules and interferon--containing lymphocytes in human thyroid autoimmune disease. Clin Exp Immunol 83:64–68
Kimura H, Kimura M, Rose NR, Caturegli P 2004 Early chemokine expression induced by interferon- in a murine model of Hashimoto’s thyroiditis. Exp Mol Pathol 77:161–167
Antonelli A, Rotondi M, Fallahi P, Romagnani P, Ferrari SM, Buonamano A, Ferrannini E, Serio M 2004 High levels of circulating CXC chemokine ligand 10 are associated with chronic autoimmune thyroiditis and hypothyroidism. J Clin Endocrinol Metab 89:5496–5499
Diehl GE, Yue HH, Hsieh K, Kuang AA, Ho M, Morici LA, Lenz LL, Cado D, Riley LW, Winoto A 2004 TRAIL-R as a negative regulator of innate immune cell responses. Immunity 21:877–889
Bretz JD, Rymaszewski M, Arscott PL, Myc A, Ain KB, Baker Jr JR, Thompson NW 1999 TRAIL death pathway expression and induction in thyroid follicular cells. J Biol Chem 274:23627–23632
Bretz JD, Mezosi E, Giordano TJ, Gauger PG, Thompson NW, Baker Jr JR2002 Inflammatory cytokine regulation of TRAIL-mediated apoptosis in thyroid epithelial cells. Cell Death Diff 9:274–286
Okayasu I 1985 Transfer of experimental autoimmune thyroiditis to normal syngeneic mice by injection of mouse thyroglobulin-sensitized T lymphocytes after activation with concanavalin A. Clin Immunol Immunopathol 36:101–109
Charreire J, Michel-Bechet M 1982 Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells. III. Induction of thyroiditis by thyroid-sensitized T lymphoblasts. Eur J Immunol 12:421–425
Romball CG, Weigle WO 1987 Transfer of experimental autoimmune thyroiditis with T cell clones. J Immunol 138:1092–1098
Maron R, Zerubavel R, Friedman A, Cohen IR 1983 T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice. J Immunol 131:2316–2322
Creemers P, Rose NR, Kong YM 1983 Experimental autoimmune thyroiditis. In vitro cytotoxic effects of T lymphocytes on thyroid monolayers. J Exp Med 157:559–571
Lenardo M, Chan KM, Hornung F, McFarland H, Siegel R, Wang J, Zheng L 1999 Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev Immunol 17:221–253
Pender MP 1999 Activation-induced apoptosis of autoreactive and alloreactive T lymphocytes in the target organ as a major mechanism of tolerance. Immunol Cell Biol 77:216–223(Su He Wang, Zhengyi Cao, )
Abstract
The role of TNF-related apoptosis-inducing ligand (TRAIL) in autoimmune thyroiditis is unclear. We used experimental autoimmune thyroiditis to clarify the contribution of TRAIL to the development of autoimmune thyroiditis. CBA/J mice were immunized with murine thyroglobulin, and spleen cells from these mice were subsequently injected into irradiated recipient CBA/J mice. One week later, the recipient mice were treated with recombinant TRAIL or a control protein. Compared with control animals, TRAIL-treated mice developed a milder form of the disease with a significant decrease in mononuclear cell infiltration in the thyroid and less thyroid follicular destruction. Furthermore, the number of apoptotic thyrocytes and also thyroglobulin-specific T helper-1 cell responses in TRAIL-treated mice was lower than that in the control animals. This study suggests that exogenous TRAIL suppresses the development of autoimmune thyroiditis via altering the function of cells involved in the immune response. These findings may contribute toward a novel treatment autoimmune thyroiditis.
Introduction
MEMBERS OF THE TNF and TNF receptor family play key roles in the development and function of the immune system. Some members of this family, such as TNF and Fas ligand (FasL), may contribute to immune tolerance by deletion of self-reactive lymphocytes. For example, natural mouse mutants carrying mutations in the Fas and FasL genes, in which Fas-mediated cell death is impaired, exhibit lymphoproliferative and autoimmune syndrome (1). Moreover, children with Fas gene defects suffer from similar autoimmune disorders (2, 3, 4, 5). These examples suggest that the Fas-FasL system is involved in down-regulation of immune responses. Other members of TNF family, such as TNF-related apoptosis-inducing ligand (TRAIL), may also involve tolerance to self-antigens because the blockage of endogenous TRAIL exacerbates autoimmune arthritis (6). Once immune tolerance is disrupted, autoreactive lymphocytes can induce target cell apoptosis, especially in the presence of certain cytokines (7). This process is believed to contribute to the development of autoimmune diseases.
Normal thyroid glands show a low level of apoptosis, a possible result of basal thyroid cell turnover and maintenance of cell population (8, 9). In contrast, thyroid glands from patients with autoimmune thyroiditis display an increased frequency of apoptotic cells (8, 9, 10, 11). Many of the apoptotic cells in these glands are found in the areas of disrupted follicles, in proximity to infiltrating lymphoid cells (12, 13). This suggests that an increase in thyroid follicular cell apoptosis in thyroiditis may occur through a mechanism related to immune responses and inflammation. Autoimmune thyroiditis can also be induced experimentally in genetically susceptible mice by immunization with thyroid antigens (14). The mice develop autoimmune responses characterized by circulating antithyroid antibodies and infiltration of the thyroid gland by mononuclear cells. Evidence indicates that the death receptor-mediated apoptotic pathway is central to thyroid cell apoptosis in thyroiditis (15, 16). However, this pathway has been shown to be under the tight regulation of T helper (Th)1 cytokines in vitro and in vivo (7, 16, 17). Thus, it appears that the cytokine environment in the thyroid is able to influence the activation of apoptotic pathways.
As a newly identified TNF family member, TRAIL-induced apoptosis is mainly restricted to tumor cells and virus-infected cells. Recently it has been reported that TRAIL alone or in combination with other reagents has promising effects in antitumor therapy (18, 19). Although TRAIL can also block activation of human autoantigen-specific T cells (20), it is unknown whether TRAIL has an effect on immune cell function in the experimental autoimmune thyroiditis (EAT). To address this question, we used the EAT model to study the effect of TRAIL on the development of autoimmune disease.
Materials and Methods
Preparation of TRAIL protein
TRAIL protein was prepared as described by Chinnaiyan et al. (21). Briefly, it was affinity purified from bacterial lysates of cells transfected with the plasmid pET15b-His-FLAG-TRAIL. The purity of the TRAIL preparation was confirmed by sliver-stained SDS-PAGE. The endotoxin-free recombinant TRAIL was used for the experiment.
Establishment of transfer-EAT model and treatments
Eight-week-old CBA/J female mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions in our animal facility, and all animal studies were approved by our University Committee on the Use and Care of Animals. The transfer-EAT model was established as previously described (22). Briefly, CBA/J donor mice were injected twice with murine thyroglobulin (mTg) prepared from frozen mice thyroid glands and lipopolysaccharide at 10-d intervals (14, 43). The donor mice were killed 7 d after the second injection, and their spleens (5 x 106) were collected and cultured in complete culture medium with mTg (25 μg/ml) at 37 C for 72 h. The cells (1 x 107) were washed and then transferred iv into an irradiated (600 rad) CBA/J recipient. One week later, recipient CBA/J mice were treated with recombinant TRAIL or a control protein (BSA) by ip injection every other day at a dosage of 100 μg per mouse for 2 wk. At least 12–15 mice were included in each group. Thyroid function was tested by measuring serum T4 level by OptiCoat T4 enzyme immunoassay kit (Biotecx Laboratories Inc., Houston, TX).
Evaluation of thyroid infiltration
Thyroid glands were fixed in 10% formalin, embedded in paraffin, and sectioned by a standard method. Infiltration was evaluated on 5-μm-thick sections stained with hematoxylin and eosin. The severity of thyroiditis was graded on a scale of 0–4 as described by Wang et al. (7). Scoring was performed blind to the animal treatment groups.
Determination of apoptosis in thyroid gland
Apoptosis in paraffin-embedded thyroid tissue sections was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of fragmented DNA. Specific staining for apoptosis in situ was performed using the ApopTag fluorescein kit (Chemicon, Temecula, CA) according to the manufacturer’s protocol. In some instances, thyroid tissue section was dual stained with antidigioxigenin-fluorescein and phycoerythrin(PE)-conjugated cytokeratin 18 (CK 18) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Infiltrating thyroid lymphocytes (ITLs) were prepared from thyroid tissue, which was minced and digested with collagenase type IV (Sigma, St. Louis, MO) at 37 C for 2 h. The digestion was stopped by cold Hanks’ balanced salt solution and filtered through a 200-μm mesh. Isolated cells included both thyrocytes and lymphocytes. ITLs were gated out from thyrocytes by flow cytometry using PE-cys-conjugated rat antimouse CD45 monoclonal antibody (BD PharMingen, San Diego, CA). Apoptotic ITLs from TRAIL-treated or control mice were detected by flow cytometry using fluorescein diacetate (FDA) and propidium iodide (PI) stains (23).
In vitro proliferation assay
Spleen cells isolated from TRAIL-treated or control mice were cultured for 3 d in 96-well plates in the presence or absence of mTg. For the final 18–24 h of culture, 1.0 μCi [3H]thymidine (ICN Biomedicals, Costa Mesa, CA) was added to each well. The cells were then harvested onto glass fiber filters, and radioactivities were determined by a flatbed -counter.
Cytokine production in vitro
The splenocytes were incubated either with the mitogen phytohemagglutinin or mTg for 3 d, and then supernatant was harvested and measured for cytokine production. The levels of interferon (IFN), TNF, and IL-4 were detected by Quantikine M ELISA kits (R&D Systems Inc., Minneapolis, MN) according to the manufacturer’s instructions.
Antithyroglobulin (Tg) antibody
Sera collected before immunization, 7 d after immunization, and before time of death were used to determine the anti-Tg antibody by solid-phase ELISA. Briefly, plastic microtiter plates were coated with 100 μl mTg (10 μg/ml), and sera from individual mice in 1:1,000, 1:2,500, 1:5,000, 1:7,500, and 1:10,000 dilutions were added to each well. An alkaline phosphatase-conjugated, sheep antimouse IgG (Sigma) was added to determine anti-mTg IgG. In some instances, mTg-specific isotypes (IgG1, IgG2a, IgG2b, and IgG3) were also determined. Antimouse IgG1, IgG2a, IgG2b, and IgG3 that were conjugated to alkaline phosphatase were obtained from Rockland Immunochemicals for Research (Gilbertsville, PA).
Statistical analysis
All values were expressed as mean ± SE. The statistical significance of the differences between control and experimental groups was analyzed with Wilcoxon matched pair test and Student’s test using the software Stat View (Abacus Concepts, Inc., Berkeley CA). P < 0.05 was taken as statistically significant.
Results
TRAIL treatment inhibits transfer EAT
Based on our prior work, we found that transfer-induced EAT produces a massive mononuclear cell infiltration into the thyroid gland. This allows studies on how TRAIL affects ITLs. Seven days after CBA/J mice were injected with mTg-activated splenocytes, the mice were treated with either TRAIL or BSA once every other day for 2 wk. As shown in Fig. 1, TRAIL-treated mice developed a milder form of the disease when compared with control animals. The reduction was evidenced primarily by a decrease in the average disease score from 3.5 ± 0.2 (n = 12) to 2.1 ± 0.1 (n = 15), showing both decreased mononuclear cell infiltration in the thyroid and reduced thyroid follicular destruction in TRAIL-treated mice. TRAIL treatment did not alter the thyroid hormone level during the period of our study because the level of T4 in TRAIL-treated mice did not change significantly [2.74 ± 0.42 μg/dl, compared with the values before spleen cell transfer (3.0 ± 0.57 μg/dl) and 21 d after the transfer (2.49 ± 0.40 μg/dl)].
TRAIL decreases thyroid cell apoptosis but does not affect apoptosis of ITLs
To determine whether TRAIL treatment specifically promotes thyrocyte apoptosis, thyroid sections from EAT mice were evaluated for cell death by in situ TUNEL staining. After 2 wk of TRAIL treatment, the number of apoptotic cells in the thyroid glands was markedly decreased in the EAT mice when compared with mice without treatment. This was demonstrated by the number of TUNEL-positive cells (in green), which are shown to be decreased in Fig. 2A, right panel). To confirm that apoptotic cells were indeed thyroid cells, we applied CK18, an epithelial cell marker, to evaluate the cell type. After thyroid section was dual stained with antidigioxigenin-fluorescein and PE-conjugated CK18 antibodies, we found that the fluorescent staining cells were also positive for CK18, indicating that those apoptotic cells were thyroid cells in nature.
Cell viability/death was determined by flow cytometry using FDA and PI staining (24). A total of 10,000 cells were stained with PE-cys, FDA, and PI. To our surprise, after ITLs were gated from the thyroid cell population, the percentages of apoptotic ITLs in TRAIL-treated and control mice were similar, being 7.1 and 8.4%, respectively (Fig. 2B). This indicated that the reduction of infiltrating mononuclear cells in the thyroid gland seen in TRAIL-treated mice (Fig. 1) did not result from apoptotic mechanism in this model of thyroiditis.
Characteristics of the immune response of EAT mice treated with TRAIL
Tg-specific proliferation of immune cells (mainly lymphocytes) in the presence of TRAIL was measured. The proliferation index in response to Tg was decreased in TRAIL-treated EAT mice (Fig. 3A). This demonstrated that TRAIL inhibited the proliferation of lymphocytes that were stimulated by Tg. The production of IFN, TNF, and IL-4 in supernatants of these Tg-stimulated spleen cells was determined by ELISA. Spleen cells from mice injected with TRAIL or control protein produced similar levels of TNF and IL-4. In contrast, splenocytes from the TRAIL-treated mice produced a significantly lower level of IFN (Fig. 3B), a Th1 cytokine, which is required to sensitize thyroid cells to apoptosis probably via removing the inhibitor of apoptosis (7, 18).
The impact of TRAIL on the humoral response to mTg was studied in the sera of mice with ongoing EAT. Seven days after spleen cell transfer, anti-mTg IgG was markedly higher, compared with its level before immunization (Fig. 4). After 7 d of TRAIL treatment, the level of anti-mTg IgG continued to rise in both the TRAIL and BSA groups. However, compared with the BSA group, the increase in anti-mTg IgG was significantly inhibited in the TRAIL group at 2 wk after the experiment (Fig. 4), suggesting an inhibitory role of TRAIL on anti-mTg IgG production. Further analyses of the anti-mTg IgG subclass showed that the levels of anti-mTg IgG1, IgG2b, and IgG3 were not affected by TRAIL, and the overall levels of IgG2b and IgG3 were very low in the sera of mice, regardless of whether the mice were treated with TRAIL. Most important, we observed that the anti-mTg IgG2a response was significantly diminished in the sera of mice treated by TRAIL, compared with that treated by BSA (Fig. 4). It was thus believed that this decrease was the major factor contributing to the reduction of anti-mTg IgG.
Discussion
As a newly identified TNF family member, TRAIL has been reported to be a promising agent in antitumor therapy (18, 19). In contrast to TRAIL, the systemic administration of FasL is limited because FasL can cause an animal’s death by widely inducing apoptosis in animal liver cells (1). A therapeutic role for TRAIL has been proposed in antiautoimmune diseases (6, 25). In a current study, we demonstrate that exogenous TRAIL inhibits EAT in a transfer model of thyroiditis. Although TRAIL treatment did not obviously alter the level of serum T4, it did significantly reduce mononuclear cell infiltrations in the thyroid glands. This reduction was correlated with a decrease in apoptosis of thyroid cells. These findings are in line with the fact that infiltrating lymphocytes play a vital role in the development of autoimmune thyroiditis by secreting inflammatory cytokines and stimulating the apoptosis in thyrocytes (7, 26, 27). Therefore, any mediation that is able to reduce the infiltrating lymphocytes in thyroid, such as the one described herein, should have potential as a novel treatment for autoimmune thyroiditis.
The transfer EAT was developed in irradiated susceptible recipients by the transfer of in vitro Tg-stimulated spleen cells obtained from Tg-immunized donors. This EAT model introduces an enormous number of infiltrated lymphoid cells, including CD4+ and CD8+ T cells, into the thyroid gland. Using this EAT model, we demonstrated that TRAIL could significantly decrease the number of infiltrated lymphoid cells in thyroid. It is possible that the reduction of infiltrated lymphoid cells in thyroid is caused by an increase in apoptosis of lymphocytes because TRAIL is initially discovered as an apoptotic molecule. However, to our surprise, the apoptotic rate of infiltrated lymphoid cells in thyroid in both TRAIL-treated and nontreated mice remained similar in our experiment. Therefore, it is unlikely that the increased apoptosis is a significant factor contributing to the decreased infiltrated lymphoid cells in thyroid seen in the present study. However, it is noted that the effect of TRAIL on inflammatory cytokine production may be responsible for the reduction of infiltrated mononuclear cells in thyroid in our EAT model.
It is known that both apoptosis and recruiting mononuclear cells into thyroid cells is under the tight control of inflammatory cytokines (15, 16). Cytokines can regulate the expression of apoptotic signaling components and inhibitors in target cells (16, 26, 27). Several publications (28, 29, 30, 31) suggested a crucial role for cytokines in the pathogenesis of EAT. It has been shown that T cell clones isolated from intrathyroidal mononuclear infiltrates of Hashimoto’s thyroiditis produce high levels of IFN (32, 33). As we have demonstrated previously, both IFN and TNF were required for sensitizing a specific (Fas) pathway in the thyroid cells (7, 17). Although the level of TNF did not change by TRAIL treatment, IFN production was significantly decreased. This could lead to inactivation of the cell death pathway in thyroid cells. Indeed, a decrease of thyroid cell apoptosis was observed in the EAT mice treated with TRAIL. This is possibly due to the alteration of the cytokine environment because TRAIL can cause a reduction of IFN production. In addition to inactivating apoptosis of thyrocytes, the decreased IFN level by TRAIL can also lead to reduced mononuclear cell infiltration in thyroid. IFN is a key molecule in regulation of production of chemokine production, such as CCL1–5 and CXCL 10. These chemokines motivate lymphocytes/mononuclear cells into thyroid (34, 35). Therefore, it is reasonable to assume that the inhibition of IFN production can cause reduction of mononuclear cell infiltration in thyroid, which is supported by our current study and also in agreement with a report that TRAIL-R(–/–) mice are associated with increased levels of IFN (36). Studies from our group have shown that thyroid cells are known to express TRAIL receptors such as TRAIL receptor death receptor 4 and death receptor 5 (37, 38). However, the detailed pathway leading to reducing IFN level by TRAIL needs further investigation. Nevertheless, our data suggest TRAIL inhibits transfer EAT by inactivating apoptosis of thyrocytes and decreasing the number of mononuclear cells into thyroid.
Both spontaneous and induced thyroiditis in rats and mice appear to be mediated by cellular immune elements because thyroid inflammation can be passively transferred between animals by T lymphocytes and thyroid antigen-specific T cell clones (39, 40, 41, 42) but not with antibodies. Also, thymectomy has been shown to prevent the development of the disease (39). T cells from animals with EAT have been shown to be cytotoxic to cultured thyrocytes in vitro (43), and CD8+ cells have been demonstrated to proliferate in response to thyroglobulin (15). Therefore, it is important to ascertain whether TRAIL could inhibit cellular immune response to autoantigen. We showed that the antigen-specific lymphocyte function was decreased by TRAIL. The impaired lymphocyte function was reflected by a decrease in lymphocyte proliferation and mTg antibody production. A decrease in anti-mTg IgG2a was particularly obvious. Furthermore, the production of IFN-, a Th1 cytokine, was also reduced. Hence, our study suggested that the Tg-specific immune response was significantly reduced in EAT mice treated with TRAIL. Such a reduction of Tg special immune response may contribute to the inhibition of autoimmune thyroiditis development and also bear a resemblance to the scenario in which the TRAIL system functions as a negative regulator of innate immune cell responses (36).
Based on the above information, it is clear that the infiltrating mononuclear cells and the cytokine microenvironment in the thyroid are central to the autoimmune destruction of thyrocytes in EAT. Hashimoto’s thyroiditis appears to involve a defect in the mechanism against mononuclear cell infiltration in thyroid, which leads to the loss of tolerance to self-antigen (44, 45). Therefore, reduction and functional inhibition of thyroid infiltrating lymphocytes could open a new approach to protection against thyroid autoimmunity.
In conclusion, we have found that the down-regulation of mTg-specific, antoreactive immune cell function, at least in part by TRAIL, is responsible for the inhibition of EAT. This leads to alteration of the thyroid cytokine environment, inactivates the apoptotic pathway, and protects thyroid cell apoptosis under immune disorder. To the best of our knowledge, this is the first report of an inhibitory effect of TRAIL on EAT. Based on this, it is important to further investigate the detailed mechanism of how TRAIL is involved in the inhibition of autoimmune responses.
Acknowledgments
We gratefully acknowledge Drs. R. J. Koenig and Y. M. Kong for helpful discussions and Dr. James Bretz for help with TRAIL preparation.
Footnotes
This work was supported by National Institutes of Health Grant 5 R01 AI037141-10.
Abbreviations: CK 18, Conjugated cytokeratin 18; EAT, experimental autoimmune thyroiditis; FasL, Fas ligand; FDA, fluorescein diacetate; IFN, interferon; ITL, infiltrating thyroid lymphocyte; mTg, murine Tg; PE, phycoerythrin; PI, propidium iodide; Tg, thyroglobulin; Th, T helper; TRAIL, TNF-related apoptosis-inducing ligand; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
References
Nagata S, Golstein P 1995 The Fas death factor. Science 267:1449–1456
Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, Puck JM 1995 Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935–946
Rieux-Laucat F, Le Deist F, Hivroz C, Roberts IA, Debatin KM, Fischer A, de Villartay JP 1995 Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268:1347–1349
Drappa J, Vaishnaw AK, Sullivan KE, Chu JL, Elkon KB 1996 Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. N Engl J Med 335:1643–1649
Le Deist F, Emile JF, Rieux-Laucat F, Benkerrou M, Roberts I, Brousse N, Fischer A 1996 Clinical, immunological, and pathological consequences of Fas-deficient conditions. Lancet 348:719–723
Song K, Chen Y, Gke R, Wilmen A, Seidel C, Gke A, Hilliard B, Chen Y 2000 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J Exp Med 191:1095–1104
Wang SH, Bretz JD, Phelps E, Mezosi E, Arscott PL, Utsugi S, Baker Jr JR 2002 A unique combination of inflammatory cytokines enhances apoptosis of thyroid follicular cells and transforms non-destructive to destructive thyroiditis in experimental autoimmune thyroiditis. J Immunol 168:2470–2474
Dremier S, Golstein J, Mosselmans R, Dumont JE, Galand P, Robaye B 1994 Apoptosis in dog thyroid cells. Biochem Biophys Res Commun 200:52–58
Okayasu I, Saegusa M, Fujiwara M, Hara Y, Rose NR 1995 Enhanced cellular proliferative activity and cell death in chronic thyroiditis and thyroid papillary carcinoma. J Cancer Res Clin Oncol 121:746–752
Tanimoto C, Hirakawa S, Kawasaki H, Hayakawa N, Ota Z 1995 Apoptosis in thyroid diseases: a histochemical study. Endocr J 42:193–201
Bagnasco M, Venuti D, Paolieri F, Torre G, Ferrini S, Canonica GW 1989 Phenotypic and functional analysis at the clonal level of infiltrating T lymphocytes in papillary carcinoma of the thyroid: prevalence of cytolytic T cells with natural killer-like or lymphokine-activated killer activity. J Clin Endocrinol Metab 69:832–836
Kotani T, Aratake Y, Hirai K, Fukazawa Y, Sato H, Ohtaki S 1995 Apoptosis in thyroid tissue from patients with Hashimoto’s thyroiditis. Autoimmunity 20:231–236
Hammond LJ, Lowdell MW, Cerrano PG, Goode AW, Bottazzo GF, Mirakian R 1997 Analysis of apoptosis in relation to tissue destruction associated with Hashimoto’s autoimmune thyroiditis. J Pathol 182:138–144
Kong Y, David CS, Giraldo AA, El Rehewy M, Rose NR 1979 Regulation of autoimmune response to mouse thyroglobulin: influence of H-2D-end genes. J Immunol 123:15–18
Arscott PL, Knapp J, Rymaszewski M, Bartron JL, Bretz JD, Thompson NW, Baker Jr JR 1997 Fas (APO-1, CD95)-mediated apoptosis in thyroid cells is regulated by a labile protein inhibitor. Endocrinology 138:5019–5027
Stassi G, Di Liberto D, Todaro M, Zeuner A, Ricci-Vitiani L, Stoppacciaro A, Ruco L, Farina F, Zummo G, De Maria R 2000 Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins. Nat Immunol 1:483–488
Bretz JD, Arscott PL, Myc A, Baker Jr JR 1999 Inflammatory cytokine regulation of fas-mediated apoptosis in thyroid follicular cells. J Biol Chem 274:25433–25438
Wang SH, Mezosi E, Wolf JM, Cao Z, Utsugi S, Gauger PG, Doherty GM, Baker Jr JR 2004 IFN sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression. Oncogene 23:928–935
Kagawa S, He C, Gu J, Koch P, Rha SJ, Roth JA, Curley SA, Stephens LC, Fang B 2001 Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. Cancer Res 61:3330–3338
Kayagaki N, Yamaguchi N, Nakayama M, Kawasaki, A, Akiba H, Okumura K, Yagita H 1999 Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity. J Immunol 162:2639–2647
Chinnaiyan AM, Prasad U, Shankar S, Hamstra DA, Shanaiah M, Chenevert TL, Ross BD, Rehemtulla A 2000 Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc Natl Acad Sci USA 97:1754–1759
Mignon-Godefroy K, Brazillet MP, Rott O, Charreire J 1995 Distinctive modulation by IL-4 and IL-10 of the effector function of murine thyroglobulin-primed cells in "transfer-experimental autoimmune thyroiditis." Cell Immunol 162:171–177
Killinger Jr WA, Dorofi DB, Tinsley Jr EA, Keagy BA, Johnson Jr G 1992 Flow cytometric analysis of organ preservation-induced endothelial cell membrane damage. Ann Thorac Surg 53:472–476
Bartkowiak D, Hogner S, Baust H, Nothdurft W, Rottinger EM 1999 Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate. Cytometry 37:191–196
Hilliard B, Wilmen A, Seidel C, Liu TS, Goke R, Chen Y 2001 Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis. J Immunol 166:1314–1319
Mezosi E, Wang SH, Utsugi S, Bajnok L, Bretz JD, Gauger PG, Thompson NW, Baker Jr JR 2005 Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells. Mol Endocrinol 19:804–811
Wang SH, Baker Jr JR 2001 Autoimmune thyroid disease. Clinical Immunology 81:1–20
Alimi E, Huang S, Brazillet MP, Charreire J 1998 Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN- receptor gene. Eur J Immunol 28:201–208
Ajjan RA, Watson PF, Weetman AP 1996 Cytokines and thyroid function. Adv Neuroimmunol 6:359–386
Frohman M, Francfort JW, Cowing C 1991 T-depend destruction of thyroid isografts exposed to IFN-. J Immunol 146:2227–2234
Barin JG, Afanasyeva M, Talor MV, Rose NR, Burek CL, Caturegli P 2003 Thyroid-specific expression of IFN- limits experimental autoimmune thyroiditis by suppressing lymphocyte activation in cervical lymph nodes. J Immunol 170:5523–5529
Kawakami Y, Kuzuya N, Watanabe T, Uchiyama Y, Yamashita K 1990 Induction of experimental thyroiditis in mice by recombinant interferon administration. Acta Endocrinol 122:41–48
Hamilton F, Black M, Farquharson MA, Stewart C, Foulis AK 1991 Spatial correlation between thyroid epithelial cells expressing class II MHC molecules and interferon--containing lymphocytes in human thyroid autoimmune disease. Clin Exp Immunol 83:64–68
Kimura H, Kimura M, Rose NR, Caturegli P 2004 Early chemokine expression induced by interferon- in a murine model of Hashimoto’s thyroiditis. Exp Mol Pathol 77:161–167
Antonelli A, Rotondi M, Fallahi P, Romagnani P, Ferrari SM, Buonamano A, Ferrannini E, Serio M 2004 High levels of circulating CXC chemokine ligand 10 are associated with chronic autoimmune thyroiditis and hypothyroidism. J Clin Endocrinol Metab 89:5496–5499
Diehl GE, Yue HH, Hsieh K, Kuang AA, Ho M, Morici LA, Lenz LL, Cado D, Riley LW, Winoto A 2004 TRAIL-R as a negative regulator of innate immune cell responses. Immunity 21:877–889
Bretz JD, Rymaszewski M, Arscott PL, Myc A, Ain KB, Baker Jr JR, Thompson NW 1999 TRAIL death pathway expression and induction in thyroid follicular cells. J Biol Chem 274:23627–23632
Bretz JD, Mezosi E, Giordano TJ, Gauger PG, Thompson NW, Baker Jr JR2002 Inflammatory cytokine regulation of TRAIL-mediated apoptosis in thyroid epithelial cells. Cell Death Diff 9:274–286
Okayasu I 1985 Transfer of experimental autoimmune thyroiditis to normal syngeneic mice by injection of mouse thyroglobulin-sensitized T lymphocytes after activation with concanavalin A. Clin Immunol Immunopathol 36:101–109
Charreire J, Michel-Bechet M 1982 Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells. III. Induction of thyroiditis by thyroid-sensitized T lymphoblasts. Eur J Immunol 12:421–425
Romball CG, Weigle WO 1987 Transfer of experimental autoimmune thyroiditis with T cell clones. J Immunol 138:1092–1098
Maron R, Zerubavel R, Friedman A, Cohen IR 1983 T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice. J Immunol 131:2316–2322
Creemers P, Rose NR, Kong YM 1983 Experimental autoimmune thyroiditis. In vitro cytotoxic effects of T lymphocytes on thyroid monolayers. J Exp Med 157:559–571
Lenardo M, Chan KM, Hornung F, McFarland H, Siegel R, Wang J, Zheng L 1999 Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev Immunol 17:221–253
Pender MP 1999 Activation-induced apoptosis of autoreactive and alloreactive T lymphocytes in the target organ as a major mechanism of tolerance. Immunol Cell Biol 77:216–223(Su He Wang, Zhengyi Cao, )