Experimental autoimmune thyroiditis induced by thyroglobulin-pulsed dendritic cells

Dendritic cells (DCs), which are the most effective professional antigen-presenting cells (APCs), initiate and regulate immune responses. In this report, we examine the role of DCs in the induction of autoimmune thyroiditis. Experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin (Tg) plus adjuvant is considered to be an animal model of autoimmune thyroiditis, and is categorized as a T cell-mediated autoimmune disease. To examine the contribution of DCs to EAT, naive DCs were purified from high responder B10BR mice and pulsed with various concentrations of porcine Tg (pTg). These pTg-pulsed DCs were transferred without adjuvant to syngenic BIOBR mice to induce EAT. Mice that had received pTg-pulsed DCs showed thyroiditis, and the degree of thyroiditis induced was positively correlated to the amounts of pTg used for the incubation (pulsing) of DCs. The severity of thyroiditis was also correlated to the amounts of anti-pTg IgG2a antibodies and IFN-gamma in the recipient sera, but not to IL-4 or IL-10, indicating that Th1 cells are mainly activated by pTg-pulsed DCs and attributable to the pathogenesis of EAT.     

By-stander activation in autoimmune thyroiditis: studies on experimental autoimmune thyroiditis in the GFP+ fluorescent mouse

We have taken advantage of GFP+ fluorescent protein (GFP) tagged lymphocytes to examine by-stander activity in experimental autoimmune thyroiditis in the mouse. To generate GFP-positive EAT-susceptible CBA/J mice (H-2k) (GFP-CBA/J mice), we backcrossed CBA/J (H-2k) with heterozygous GFP+ transgenic mice (C57Bl/6; H-2b). I-Ak and GFP expression on peripheral lymphocytes was used to select the resulting progeny up to the N7 generation. Mixed lymphocyte reactions using spleen cells from N7 GFP-CBA/J mice showed negative responses to spleen cells from CBA/J confirming the inbreeding and with marked reactivity to cells from C57BL/6. Immunization with human thyroglobulin (hTg) in GFP-CBA/J mice induced thyroiditis in 50% of the animals and high titers of Tg antibodies in all the animals. In addition, priming of GFP+ spleen cells in vitro with hTg induced a marked proliferative response (mean stimulation index = 24.7), These proliferating spleen cells were then transferred to CBA/J recipients. Fourteen days after transferring 30 x 10(6) Tg-primed GFP+ spleen cells into irradiated (500 rad) normal syngeneic hosts, a GFP+ lymphocytic infiltration was seen within their thyroid glands along with a GFP- lymphocytic infiltration arising from the host. This suggested that the hTg-specific transferred cells had initiated by-stander activation of naive host lymphocytes. This model of bystander cell detection confirmed that such an effect occurs in EAT and adds weight to the importance of this phenomenon in the initiation of autoimmune thyroid disease.     

The effects of a monoclonal antibody to interferon-γ on experimental autoimmune thyroiditis (EAT): prevention of disease and decrease of EAT-specific T cells

CBA/J mice immunized with thyreoglobulin (Tg) develop an experimental autoimmune thyroiditis (EAT) with lymphocytic infiltration of the thyroid glands, autoantibodies to Tg and occurrence of EAT-specific T cells. When these mice were treated for 4 weeks after immunization with 1 mg/week of a monoclonal antibody (mAb) that neutralizes the activity of interferon-γ (IFN) a beneficial effect on the onset of EAT was observed. Characteristic features of EAT were significantly reduced, including the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to Tg. Moreover, in lymphoid organs, mAb to IFN-γ significantly reduced the percentages of Tg-specific CD8⁺ cells, labeled by the anti-clonotypic mAb AG7. These Tg-specific T cells seem responsible for thyroid damages and disease development, since EAT was simultaneously abrogated. These results show that IFN-γ plays an essential role in the pathophysiology of EAT and suggest the possibility to treat autoimmune thyroid diseases with mAb to IFN-γ or drugs able to antagonize the production and/or the action of this cytokine.     

Induction of experimental autoimmune thyroiditis by heat-denatured porcine thyroglobulin: a Tc1-mediated disease

Recent studies have shown that denatured exogenous antigens can prime cytotoxic T lymphocytes (CTL). To assess the contribution of CTL to experimental autoimmune thyroiditis (EAT), porcine thyroglobulin (pTg) was heat-denatured (hdpTg) and injected i.v. into CBA/J mice, without the aid of adjuvants. Both lymphocytic infiltrations of the thyroid glands and levels of Tg-specific CTL were similar to those found in conventional EAT induced by Tg and adjuvants. In contrast, proliferative responses could not be detected, and titers of antibodies to pTg were 20 times lower. These EAT-inducer CTL belong to the CD8+ cell subset and exerted their thyroiditogenic potential through release of IFN-gamma. We conclude that hdpTg-induced EAT is mediated by type 1 cytotoxic T cells (Tc1).     

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.     

Experimental autoimmune thyroiditis (EAT) induced by the thyroglobulin peptide (2596-2608): influence of H-2 and non H-2 genes

We have previously identified five thyroglobulin (Tg) peptides with Ak-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596-2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT--(infiltration index range=1-2)-- in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-gamma in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2q), SJL (H-2s), DBA/2J (H-2d) and C57BL/6 (H-2b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2k strains of diverse non-H-2 backgrounds but not in mice carrying H-2b, d, q or s haplotypes.     

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.     

Curative treatment of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for interleukin-10

The autoimmune response in experimental autoimmune thyroiditis (EAT) is characterized by a lymphocyte infiltration of the thyroid gland and by the appearance of circulating autoantibodies to thyroglobulin (Tg). Cytokines play a crucial role in the immunoregulation and pathology of EAT. Systemic administration of IL-10 has curative effects on EAT, but requires high doses and iterative injections due to the rapid turnover of this molecule. We have designed an original in vivo gene transfer using a mixture of liposomes and poly-L-Lysine that greatly enhanced the transfection yield, and induced a fast and long-lasting expression of IL-10 on mouse thyroid follicular cells (TFC). IL-10 expression on TFC of mice wit EAT dramatically wipe out the lymphocytic infiltration in the thyroids. A significant diminution in the proliferative anti-Tg T cell response was observed, along with a trend towards a Th2 response characterized by decreased production of IFN-gamma and by increased anti-Tg IgG1/IgG2a Ab ratios. In conclusion, local IL-10 gene therapy using non-viral vectors is a novel and promising approach for the treatment of thyroid autoimmune disorders.     

Thyroid peroxidase and the induction of autoimmune thyroid disease

Animal models of autoimmune thyroid disease are associated with thyroglobulin (Tg) as autoantigen whereas in man the autoimmune response to microsomal antigen/thyroid peroxidase (TPO) appears to play a major role in thyroiditis. Consequently, we have compared the ability of TPO and Tg to induce thyroid autoantibodies and thyroid damage in mice known to be susceptible (CBA/J) or resistant (BALB/c) to thyroiditis induced using murine Tg. Groups of three to five mice were immunized twice using Freund's complete adjuvant with 80-100 micrograms highly purified porcine (p) TPO, pTg, rat (r) Tg, human Tg, bovine serum albumin (BSA) or BSA + 0.2 micrograms pTg (the level of Tg contamination of TPO). Four weeks after immunization with TPO, plasma from CBA/J (but not BALB/c) mice contained IgG class antibodies which bound to TPO-coated tubes in the presence or absence of excess Tg (and could therefore be clearly distinguished from Tg antibodies) but there was no evidence of thyroiditis in either strain of mice. In contrast, in CBA/J mice immunized with rTg and, to a lesser extent in mice that had received pTg, thyroid tissue was infiltrated with lymphoid cells and/or neutrophils and antibodies to pTg (but not pTPO) were present. Our observations demonstrate that induction of TPO antibody alone is insufficient to lead to thyroiditis in CBA/J mice. Further, these studies emphasize the complex interactions between MHC and different thyroid antigens in the processes leading to thyroid destruction.     

Experimental Autoimmune Thyroiditis (EAT) Induced by the Thyroglobulin Peptide (2596–2608): Influence of H-2 and Non H-2 Genes

We have previously identified five thyroglobulin (Tg) peptides with A^k-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2^k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596–2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT—(infiltration index range=1–2)— in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-γ in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2^q), SJL (H-2^s), DBA/2J (H-2^d) and C57BL/6 (H-2^b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2^k strains of diverse non-H-2 backgrounds but not in mice carrying H-2^{b, d, q?or?s} haplotypes.     

Induction of a TC1-Mediated Experimental Autoimmune Thyroiditis by Deglycosylated Porcine Thyroglobulin

Contribution of cytotoxic T lymphocytes (CTL) to experimental autoimmune thyroiditis (EAT) was well defined (Speidel et al., Eur. J. Immunol. 1997, 27, 2391-2399, Ref. 7). The native porcine thyroglobulin (pTg) showed high sensitivity to endo-β-N-acetylglucosaminidase F (Endo F) and its molecular weights, corresponding to about 330 kDa as a monomer and 660 kDa as a dimer, were reduced to smaller molecular weight forms by Endo F and trifluoromethanesulfonic acid (TMSF). Deglycosylated porcine Tg (dgpTg) and native pTg were injected i.v. into CBA/J mice, without the aid of adjuvants. Both lymphocytic infiltrations of the thyroid glands and levels of Tg-specific CTL were similar to those found in conventional EAT induced by Tg and adjuvants. In contrast, proliferative responses in native pTg and dgpTg-injected mice could not be detected, and titers of antibodies to pTg and dgpTg were 20 times and 30 times lower than that of pTg and adjuvants, respectively. The EAT-inducer CTL belonged to the CD8⁺ cell subset and exerted their thyroiditogenic potential through release of IFN-γ. It was concluded that dgpTg-induced EAT is mediated by type 1 cytotoxic T cells (Tcl). Also, results that EAT induction of the glycosylated pTg (gpTg) was much lower than that of dgpTg, suggested that the abberant and incomplete glycosylation of the thyroglobulin is responsible for the induction of autoimmune thyroiditis.     

Induction of a TC1-mediated experimental autoimmune thyroiditis by deglycosylated porcine thyroglobulin

Contribution of cytotoxic T lymphocytes (CTL) to experimental autoimmune thyroiditis (EAT) was well defined (Speidel et al., Eur. J. Immunol. 1997, 27, 2391-2399, Ref. 7). The native porcine thyroglobulin (pTg) showed high sensitivity to endo-o-N-acetylglucosaminidase F (Endo F) and its molecular weights, corresponding to about 330 kDa as a monomer and 660 kDa as a dimer, were reduced to smaller molecular weight forms by Endo F and trifluoromethanesulfonic acid (TMSF). Deglycosylated porcine Tg (dgpTg) and native pTg were injected i.v. into CBA/J mice, without the aid of adjuvants. Both lymphocytic infiltrations of the thyroid glands and levels of Tg-specific CTL were similar to those found in conventional EAT induced by Tg and adjuvants. In contrast, proliferative responses in native pTg and dgpTg-injected mice could not be detected, and titers of antibodies to pTg and dgpTg were 20 times and 30 times lower than that of pTg and adjuvants, respectively. The EAT-inducer CTL belonged to the CD8+ cell subset and exerted their thyroiditogenic potential through release of IFN-gamma. It was concluded that dgpTg-induced EAT is mediated by type 1 cytotoxic T cells (Tcl). Also, results that EAT induction of the glycosylated pTg (gpTg) was much lower than that of dgpTg, suggested that the abberant and incomplete glycosylation of the thyroglobulin is responsible for the induction of autoimmune thyroiditis.     

Protection from Experimental Autoimmune Thyroiditis in CBA Mice with the Novel Immunosuppressant Deoxyspergualin

The effects of the novel immunosuppressant Deoxyspergualin (DSP) on the development of experimental autoimmune thyroiditis (EAT) in CBA mice were studied. For EAT induction, the mice were immunized with 100 μg of porcine thyroglobulin (p Tg) emulsified in CFA on day 0 and in IFA, for boosting, on day 14. Twenty-eight days after primary immunization, histological and serological signs of EAT occurred in control mice treated with PBS which showed marked lymphoid infiltration of the thyroid glands along with increased serum titres of anti-pTg antibodies. Development of both these EAT features was significantly suppressed when the mice were treated with 2.5 mg/kg body weight DSP, given daily, five times a week, from day —2 to day + 28 after immunization. The effect appeared to be dose-dependent and DSP was ineffective when given under the same experimental conditions at the dose of 0.5 mg/kg body weight. No DSP-toxic effects could be observed during the experiment. These results provide further evidence for the powerful immunosuppressive properties of DSP and suggest that this drug may be used in the treatment of autoimmune thyroid diseases and other T-cell mediated autoimmune disorders in humans.     

H-2 gene products influence susceptibility of target thyroid gland to damage in experimental autoimmune thyroiditis

The restriction of the pathogenesis of experimental autoimmune thyroiditis (EAT) by H-2 gene products was investigated. EAT was induced by injecting thyroglobulin extract plus adjuvant into F₁, hybrid mice that had been implanted under the kidney capsules with thyroid glands originating from either the EAT-susceptible or -resistant parental strain mice. We found relative H-2 restriction of thyroid damage to those glands originating from the H-2-susceptible parental strain. H-2 restriction of damage at the level of the target thyroid gland implicates cytotoxic effector T lymphocytes as a pathogenic agent of EAT.     

Autoimmune response to thyroglobulin. Proliferative response to thyroglobulin fragments in low responder mice]

Thyroglobulin (Tg) is one of the major thyroid autoantigens involved in autoimmune thyroiditis. The immune response of mice to Tg is genetically controlled by H-2-linked genes. To elucidate the regulation mechanism of autoimmune response to Tg in low responder mice, we studied the proliferative response of lymph node cells (LNC) to mouse Tg (MTg) and enzyme-digested MTg fragments. MTg was treated with Staphylococcus aureus V8 protease followed by separation of the fragments into 6 fractions (Fr1-Fr6: 264,000-17,000) by high performance liquid chromatography (HPLC), LNC from MTg immunized CBA/N (H-2k) mice, a high responder strain, proliferated in response to MTg and all fractions (Fr1-Fr6) of MTg fragments in vitro. In contrast, LNC from MTg immunized BALB/c (H-2d) and B10 (H-2b) mice, low responder strains, did not respond to native Tg but responded well to some smaller Tg fractions (Fr3, 4, 5). In addition, when BALB/c mice were immunized with MTg Fr4 with a molecular weight of 63,000, LNC from BALB/c mice proliferated in response to MTg as well as MTg Fr4. These findings suggest that T cells which are capable of responding to Tg do exist even in low responder mice and that the activation of these autoreactive T cells is suppressed by a regulatory cell subpopulation in low responder mice.     

Pathogenicity of a human thyroglobulin peptide (2340-2359) in mice with high or low genetic susceptibility to thyroiditis

We have previously identified a 20-mer peptide of human thyroglobulin (hTg), p2340 (aa2340-2359), which induced experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) and HLA-DR3 transgenic mice. In this study, we investigated the thyroiditogenic potential of p2340 in 'high responder' CBA/J (H-2(k)) and SJL/J (H-2(s)) or 'low responder' C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Mice were immunized subcutaneously with 100 nmol of p2340 in complete Freund's adjuvant (CFA) and both the proliferative capacity of their lymph node cells in the presence of p2340 or intact Tg and the production of peptide-specific antibodies were investigated. The p2340 peptide was found to contain B-cell and non-dominant T-cell epitope(s) in all strains tested. Moreover, it elicited EAT in CBA/J (2/6, infiltration index (I.I.) 1) and SJL/J (5/5, I.I. 1-3) mice after direct challenge and in BALB/c (4/7, I.I. 1) and C57BL/6 (1/5, I.I. 1) after adoptive transfer of p2340-primed lymph node cells. P2340 is the first Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction in a genetically resistant host.     

Autoantibody specific for a thyroglobulin epitope inducing experimental autoimmune thyroiditis or its anti-idiotype correlates with the disease.

In order to elucidate the role of anti-thyroglobulin (Tg) autoantibodies (A-Ab) in experimental autoimmune thyroiditis (EAT), a kinetic study was conducted in EAT-susceptible (CBA/J) and non-susceptible (C57BL/6) strains of mice. From day 0 to 70 post-Tg immunization, titers of A-Ab to Tg and to the linear 5-10-kDa Tg tryptic fragment inducing EAT as well as anti-idiotypic A-Ab representing the internal image of the thyroidogeneic antigen were measured. EAT onset, development and recovery correlate with the presence of two subsets of A-Ab only in susceptible strains of mice. First, with the presence of anti-Tg A-Ab to one determinant borne by the linear 5-10-kDa Tg tryptic fragment, and second with the presence of anti-idiotypic A-Ab specific for the monoclonal anti-Tg A-Ab (3B8G9) paratope which binds to Tg determinant inducing EAT.     

HLA and H2 class II transgenic mouse models to study susceptibility and protection in autoimmune thyroid disease

Using single H2 and HLA class II transgenic mice, in the absence of endogenous H2 class II molecules, we have studied the permissiveness of class II molecules for experimental autoimmune thyroiditis (EAT). Resistant strains expressing susceptible class II molecules, H2Ak or HLA-DR3, developed EAT, clearly demonstrating the importance of class II gene inheritance. Polymorphism for HLA-DRB1 was observed, as DR3, but not DR2 or DR4, molecules were permissive for EAT induction with either mouse (m) or human (h) thyroglobulin (Tg). HLA-DQ polymorphism was also detectable, as hTg-induced EAT developed in DQ8+, but not DQ6+, mice. Class II gene interactions leading to reduced EAT severity were observed in H2 transgenic mice, when H2E transgene was expressed in H2A+ mice or H2A molecules were introduced into our novel H2A- E+ transgenic model. Similarly, in DR3+ mice, only the DQ8 transgene reduced EAT severity, depending on both background genes (C57BL/10 or NOD) and Tg species. Based on computer-predicted, class II-binding motifs, potential pathogenic Tg peptides, either unique to hTg (H2A- E+ model) or shared between mTg and hTg (HLA-DR3+ model), were identified. We have also developed a Graves' disease model by immunizing DR3+ mice with TSH receptor DNA. Thus, transgenic models are excellent tools to study human autoimmune thyroid diseases in the context of murine EAT.     

A Thyroxine-containing Thyroglobulin Peptide Induces both Lymphocytic and Granulomatous Forms of Experimental Autoimmune Thyroiditis

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg can induce two histologically distinct forms of experimental autoimmune thyroiditis (EAT). MTg-sensitized cells activated with MTg alone induce a mild chronic form of EAT in which the thyroid infiltrate consists primarily of lymphocytes and other mononuclear cells (lymphocytic EAT). The same donor cells activated with MTg and anti-IL2R mAb induce a more severe and acute form of EAT with a thyroid inflammatory lesion having granulomatous histopathological features. A thyroxine-containing (T4) peptide, corresponding to positions 2549–2560 of human Tg, was shown by others to activate spleen cells of mouse thyroglobulin (MTg)-sensitized CBA/J mice to induce lymphocytic EAT. To determine if the CD4⁺effector T cells that induce granulomatous EAT can respond to the same T-cell epitope, the present study was undertaken to determine if both forms of EAT could be induced by the 2549–2560 thyroxine (T4)-containing peptide. This peptide was very effective for activation of T cells from MTg-primed CBA/J donors to induce granulomatous EAT but, in contrast to MTg, did not activate T cells from AKR/J or DBA/1 mice to induce granulomatous EAT. The T4 peptide did not apparently activate peptide-specific B cells in vivo but did activate MTg-primed B cells in vitro to produce anti-MTg autoantibody in recipient mice. These results demonstrate that a single 12-amino-acid thyroxine-containing peptide can activate T cells from CBA/J mice to induce both lymphocytic and granulo-matous EAT. However, this peptide does not activate T cells from some other EAT-susceptible strains of mice, suggesting that MTg contains multiple epitopes able to activate T cells to induce granulomatous EAT.     

Spontaneous autoimmune thyroiditis in NOD.H-2h4 mice

NOD.H-2h4 mice, which express I-Ak on the NOD genetic background, spontaneously develop autoimmune thyroiditis (SAT) and anti-mouse thyroglobulin (MTg) autoantibodies. The incidence of SAT is nearly 100% in mice of both sexes 6-8 weeks after administration of 0.05% NaI in the drinking water. After reaching maximum severity, inflammation is chronic over the next 3-4 months. All mice that develop thyroid lesions also produce MTg-specific IgG1 and IgG2b autoantibodies. Thyroid lesions and anti-MTg autoantibodies did not develop in CBA/J (H-2(k)) or NOD.SWR(H-2(q)) mice after administration of NaI water. Both CD4(+)and CD8(+)T cells are involved in the initial development of SAT. Depletion of CD4(+), but not CD8(+), T cells after thyroid lesions have developed also markedly reduced SAT severity, indicating that CD4(+)T cells are required for both developing and maintaining SAT. Analysis of cytokine gene expression indicated that both Th1 and Th2 cytokines were expressed in thyroids of NOD.H-2h4 mice with SAT. Th1 and proinflammatory cytokines were maximally expressed 4-6 weeks after mice began receiving NaI water, while Th2 cytokine gene expression was greatest at 8-15 weeks, when lesions had reached maximal severity and were in the chronic phase. TGF-beta was highly expressed in NOD.H-2h4 thyroids, irrespective of whether the mice had received NaI water or had thyroid lesions.