Induction Of Experimental Autoimmune Thyroiditis Research Articles

A Flexible Mouse Model of Autoimmune Thyroiditis Induced by Immunization with an Adenovirus Containing Full-Length Thyroglobulin cDNA.

The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models. Thyroglobulin (TG) is a key gene for autoimmune thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We also provide support protocols for evaluation of autoimmune thyroiditis including serological assessment of TG antibodies, in vitro splenocyte proliferation assay and cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g., cytokines, iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRβ1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay Support Protocol 3: Cytokine assays: measuring levels of interferon gamma (IFNγ) and interleukins IL-2, IL-4, and IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections Support Protocol 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA).

Enhanced autoimmunity associated with induction of tumor immunity in thyroiditis-susceptible mice.

Immunotherapeutic modalities to bolster tumor immunity by targeting specific sites of the immune network often result in immune dysregulation with adverse autoimmune sequelae. To understand the relative risk for opportunistic autoimmune disorders, we studied established breast cancer models in mice resistant to experimental autoimmune thyroiditis (EAT). EAT is a murine model of Hashimoto's thyroiditis, an autoimmune syndrome with established MHC class II control of susceptibility. The highly prevalent Hashimoto's thyroiditis is a prominent autoimmune sequela in immunotherapy, and its relative ease of diagnosis and treatment could serve as an early indicator of immune dysfunction. Here, we examined EAT-susceptible mice as a combined model for induction of tumor immunity and EAT under the umbrella of disrupted regulatory T cell (Treg) function. Tumor immunity was evaluated in female CBA/J mice after depleting Tregs by intravenous administration of CD25 monoclonal antibody and/or immunizing with irradiated mammary adenocarcinoma cell line A22E-j before challenge; the role of T cell subsets was determined by injecting CD4 and/or CD8 antibodies after tumor immunity induction. Tumor growth was monitored 3×/week by palpation. Subsequent EAT was induced by mouse thyroglobulin (mTg) injections (4 daily doses/week over 4 weeks). For some experiments, EAT was induced before establishing tumor immunity by injecting mTg+interleukin-1, 7 days apart. EAT was evaluated by mTg antibodies and thyroid infiltration. Strong resistance to tumor challenge after Treg depletion and immunization with irradiated tumor cells required participation of both CD4(+) and CD8(+) T cells. This immunity was not altered by induction of mild thyroiditis with our protocol of Treg depletion and adjuvant-free, soluble mTg injections. However, the increased incidence of mild thyroiditis can be directly related to Treg depletion needed to achieve strong tumor immunity. Moreover, when a subclinical, mild thyroiditis was induced with soluble mTg and low doses of interleukin-1, to simulate pre-existing autoimmunity in patients subjected to cancer immunotherapy, mononuclear infiltration into the thyroid was enhanced. Our current findings indicate that genetic predisposition to autoimmune disease could enhance autoimmunity during induction of tumor immunity in thyroiditis-susceptible mice. Thus, HLA genotyping of cancer patients should be part of any risk assessment.

Induction of experimental autoimmune thyroiditis (EAT) by heat-denaturated thyroglobulin (Tg).

Experimental Autoimmune Thyroiditis (EAT) is characterized by autoreactive T and B cell responses, assessed by a marked lymphocytic infiltration of the thyroid gland by T cells and the occurrence of circulating autoantibodies (AAb) to thyroglobulin (Tg). It was recently reported that administration of denaturated exogenous antigens primes class I-restricted cytotoxic T cells in vivo. Since cytotoxic T cells are involved in EAT development, porcine Tg (pTg) was heat-denaturated, i.v. injected into CBA/J mice and features of EAT evaluated. Simultaneously, mice were immunized with pTg and adjuvants and evaluation of EAT performed. We found that heat-denaturated pTg (hdpTg) induced EAT in recipient mice similar to native pTg/adjuvants. Surprisingly, whereas Tg-specific cytotoxic T cells were regularly found in lymph node cells from hdpTg or native pTg immunized mice, proliferative responses were only detected using T cells from native pTg immunized mice. Autoantibodies to pTg were decreased by a factor 30 in sera from mice immunized with hdpTg. These data further emphasized the role of Tg-specific cytotoxic T cells in EAT.

Statement of retraction

The following article has been retracted from publication in Immunopharmacology and Immunotoxi-cology:Kang BS, Chung TW, Moon JY, Lim JK, Shon YH, Nam KS, Kim DS, Jeon BH, Kim CH. Induction of a TC1-Mediated Experimental Autoimmune Thyroiditis by Deglycosylated Porcine Thyroglobulin. Immunopharmacol Immunotoxicol. 2004, 26(3):355–72This article has been found to reproduce content, without appropriate attribution or acknowledgement, from the following original article:Brazillet MP, Batteux F, Abehsira-Amar O, Nicoletti F, Charreire J. Induction of experimental auto-immune thyroiditis by heat-denatured porcine thyroglobulin: a Tc1-mediated disease. Eur J Immunol. 1999, 29(4):1342–52.The journal’s policy in this respect is clear: Immunopharmacology and Immunotoxicology considers all manuscripts on the strict condition that they have been submitted only to Immunopharmacology and Immunotoxicology, that they have not been published already, nor are they under consideration for publication or in press elsewhere.Immunopharmacology and Immunotoxicology published this article in good faith, and on the basis of legal warranties made by the corresponding author regarding the originality of their work. The article is withdrawn from all print and electronic editions.

Experimental autoimmune thyroiditis in the mouse.

Experimental autoimmune thyroiditis (EAT) in mice is an excellent model for Hashimoto's thyroiditis (HT). It is induced with thyroglobulin (Tg), a known thyroid autoantigen that is common to both mouse and human and for which several conserved, thyroiditogenic epitopes have been identified. This unit describes induction and evaluation of EAT using thyroid histology and in vitro proliferative response assays. An ELISA is presented to detect the level of antibody to mouse thyroglobulin (MTg). To induce EAT, either bacterial lipopolysaccharide (LPS) or supplemented complete Freund's adjuvant (CFA) can be used as adjuvant. A support protocol for preparing MTg is included. The T cell proliferation assay can be used to examine the antigenicity of synthetic peptides derived from MTg or heterologous Tg. EAT can be adoptively transferred utilizing cells that have been expanded in vitro, as described. A protocol is provided for inducing tolerance using deaggregated MTg; induction of tolerance requires larger amounts of MTg but efficiently suppresses EAT development. Also included is a protocol to demonstrate the role of regulatory T cells in mediating tolerance. A protocol to delineate HLA association with HT is illustrated using HLA class II transgenic mice.

Maturation of dendritic cells by necrotic thyrocytes facilitates induction of experimental autoimmune thyroiditis

Dendritic cell (DC) maturation is required for efficient presentation of autoantigens leading to autoimmunity. In this report, we have examined whether release of tissue antigens from necrotic thyroid epithelial cells can trigger DC maturation and initiation of a primary anti-self response. DC were cocultured with either viable (VT/DC) or necrotic (NT/DC) thyrocytes, and their phenotypic and functional maturation as well as immunopathogenic potential were assessed. Significant up-regulation of surface MHC class II and costimulatory molecule expression was observed in NT/DC but not in VT/DC. This was correlated with a functional maturation of NT/DC, determined by IL-12 secretion. Challenge of CBA/J mice with NT/DC, but not with VT/DC, elicited thyroglobulin (Tg)-specific IgG as well as Tg-specific CD4(+) T-cell responses and led to development of experimental autoimmune thyroiditis. These results support the view that thyroid epithelial cell necrosis may cause autoimmune thyroiditis via maturation of intrathyroidal DC.

Animal models for autoimmune myocarditis and autoimmune thyroiditis.

This chapter describes four murine models of autoimmune diseases: two related to autoimmune myocarditis and two related to autoimmune thyroiditis. The first model, Coxsackie virus B3 (CB3)-induced myocarditis, results in the development of acute myocarditis in susceptible as well as resistant mouse strains, whereas chronic myocarditis develops only in genetically susceptible mice. CB3-induced myocarditis closely resembles the course of human myocarditis, which is believed to be initiated by viral infection. Mouse cardiac myosin heavy chain has been identified as the major antigen associated with the late chronic phase of viral myocarditis. The second model is cardiac myosin-induced experimental autoimmune myocarditis (EAM) and, in a modification, cardiac alpha-myosin heavy chain peptide-induced myocarditis. In the EAM model, cardiac myosin or the relevant peptide in Freund's complete adjuvant (FCA) is injected subcutaneously into mice. The immune response, the histological changes, and the genetic susceptibility seen in EAM are similar to those of CB3-induced myocarditis. The third model is experimental autoimmune thyroiditis (EAT). EAT can be induced in genetically susceptible strains of mice by immunization with mouse thyroglobulin in FCA or lipopolysaccharide. Mice susceptible to EAT have the H-2A(k), H-2A(s), or H-2A(q) alleles. We describe here a standard technique for the induction of EAT; it was developed in our laboratory and is widely used as a model for studying Hashimoto's thyroiditis. The fourth model presented in this chapter is that of spontaneous autoimmune thyroiditis in NOD.H2h4 mice. These mice express the H-2A(k) allele on an NOD genetic background and develop spontaneous thyroiditis, which is exacerbated with dietary iodine.

Multiple fragments of human TG are capable of inducing oral tolerance to whole human TG.

Oral tolerance is the mechanism by which the immune system remains unresponsive to orally administered soluble antigens. Mice immunized with human TG (hTG), resulting in the induction of experimental autoimmune thyroiditis (EAT), provide an ideal in vivo system in which to examine oral tolerance to hTG. In the present study, we characterize epitopes of hTG that are capable of inducing oral tolerance. hTG is a large homodimeric protein, 660 Kd. The limited proteolysis of hTG using trypsin (TR) generates several smaller fragments of hTG ranging in size from 29 Kd to 145 Kd. Using hTG fragments h1TR (residues 1-521), h4bisTR (residues 2513-2713), h6TR (residues 522-1626), and h7TR (residues 1627-2512), prepared from both iodine rich and iodine poor hTG, we investigated the ability of these fragments to induce oral tolerance. The oral administration of iodine rich h6TR or h7TR suppresses hTG specific immune responses in a manner similar to whole hTG. In contrast, the oral administration of iodine rich h1TR or h4bisTR exacerbates hTG specific immune responses. Unlike iodine rich h1TR or h4bisTR, the oral administration of iodine poor h1TR or h4bisTR fails to augment hTG specific immune responses. In fact, h4bisTR suppresses hTG specific immune responses. These results indicate that hTG contains multiple epitopes that differentially affect oral tolerization. Tolerogenic epitopes reside within fragments h6TR and h7TR. The removal of iodine, and presumably hormone, from h4bisTR converts an immunogenic epitope to a tolerogenic epitope.

Tumour necrosis factor-alpha is a fundamental cytokine in autoimmune thyroid disease induced by thyroglobulin and lipopolysaccharide in interleukin-12 p40 deficient C57BL/6 mice.

Experimental autoimmune thyroiditis (EAT) is inducible in mice by immunization with mouse thyroglobulin (mTg) together with adjuvant, either lipopolysaccharide (LPS) or complete Freund's adjuvant (CFA). The severity of the disease is dependent on the mouse strain and on the adjuvant used. We have previously shown that interleukin (IL)-12 deficient C57BL/6 mice immunized with mTg and CFA develop a significantly less severe thyroid infiltration in comparison to wild type C57 BL/6 mice. This result indicated a pivotal role for IL-12 in the development of thyroiditis induced with CFA and mTg. In the present study we demonstrate that IL-12 deficiency does not impair EAT induction when LPS is used as adjuvant. We also demonstrate that peritoneal exudate cells from IL-12-deficient mice stimulated in vitro either with LPS or IL-18 secrete high levels of tumour necrosis factor (TNF). Together the results emphasize the difference between the use of CFA and LPS in the induction of EAT, the importance of TNF-alpha for the pathogenesis of LPS-induced EAT, and also show the capacity of IL-12-deficient mice to develop a competent response to LPS.

Immune intervention effects on the induction of experimental autoimmune thyroiditis.

To explore immune intervention effects of the combined use of cycloporin A (CsA) and 1, 25-dihydroxyvitamin D3[1,25(OH)2D3] at low doses on experimental autoimmune thyroiditis (EAT), porcine thyroglobulin (pTG) was injected into a CBA mouse at the dose of 100 micrograms on day 0 and day 14 to establish the model of EAT. The immune prevention group from day 0 to day 28, and treatment group from day 10 to day 38 were daily administered CsA (10 mg/kg) intragastrically and/or 1,25(OH)2D3 (0.2 microgram/kg) i.p. After immunized by pTG, the mice were sacrificed on day 28 and day 38 to examine their thyroid gland pathologically, and to check the levels of serum porcine thyroglobulin antibodies (pTGAb), porcine thyromicrosomal antibodies (pTMAb). The incidences of EAT in the immune prevention group and treatment group, with administration of low dose of CsA and 1,25(OH)2D3, were decreased respectively by 44.44% and 37.50%. Those of severe disease in the two groups were decreased respectively by 71.43% and 60.32%. The levels of serum pTGAb and pTMAb in the immune prevention group were lower than those of the positive control group. It was concluded that combined use of CsA and 1,25(OH)2D3 at low doses could effectively prevent EAT with a synergic effect.

Autoimmune thyroid disease induced by thyroglobulin and lipopolysaccharide is inhibited by soluble TNF receptor type I

Experimental autoimmune thyroiditis (EAT) is inducible in mice by immunization with thyroglobulin and adjuvant. Previous studies have shown that EAT is an autoimmune Th1-mediated disease but its characteristics differ with the adjuvant. Granulomatous lesions with marked follicular disruption develop following administration of thyroglobulin (Tg) and complete Freund's adjuvant (CFA) whereas when lipopolysaccharide (LPS) is used as the adjuvant only focal infiltrates of mononuclear cells are observed. The pro-inflammatory cytokine, TNF-α, is associated with Th1 autoimmune-mediated conditions. Cytokine antagonists have been used as potential therapeutic agents in several experimental autoimmune models. Soluble cytokine receptors belong to this category and may naturallybe shed from cell membranes to inhibit cytokine activity. We show that the administration of the soluble TNF receptor type I (sTNFR I) in the induction of EAT has very different effects on the two models of induced autoimmune thyroiditis. sTNFR I treatment inhibits the induction of EAT only when mouse Tg is given with LPS not with CFA, suggesting an important difference in the pathogenic processes.

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).

The Kinetics of Cytokine Gene Expression in the Thyroids of Mice Developing Granulomatous Experimental Autoimmune Thyroiditis

To study the potential roles of cytokines in development and resolution of granulomatous experimental autoimmune thyroiditis (EAT), the kinetics ofin vivoexpression of cytokine genes in thyroid infiltrates was analysed using reverse transcriptase-PCR (RT-PCR). Both Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines as well as TGF-βTNF-αIL-12 and IL-1β were detected in thyroids during both the initial phase and peak of granulomatous EAT. Maximal expression of cytokine genes generally occurred 11–14 days after cell transfer, prior to maximal EAT severity, which occurred 19–21 days after cell transfer. The relative ratios of Th1:Th2 cytokines and mouse thyroglobulin-(MTg)-specific IgG1 and IgG2a autoantibody levels were similar during both the initial phase and peak of EAT. Depletion of CD8+T cells did not decrease the severity of EAT but delayed resolution of lesions. Cytokine gene expression in thyroids was not decreased by anti-CD8 treatment. Together, these data indicate that both Th1 and Th2 cytokines produced by CD4+T cells are involved in induction and development of granulomatous EAT, and CD8-dependent resolution of granulomatous EAT is apparently not mediated by these cytokines.

Recent developments in the relevance of animal models to Hashimotoʼs thyroiditis and Gravesʼ disease

The study of animal models of autoimmune thyroid disease has just entered an exciting era, with major thrusts into new avenues of research. First, the HLA-DRB1 polymorphism has been shown to be a determinant of mouse thyroglobulin (mTg)-induced experimental autoimmune thyroiditis (EAT). Similar to murine class II transgene in conferring EAT susceptibility on resistant mice, the HLA-DRB1*0301 (DR3) transgene permits the induction of EAT with either mTg or human thyroglobulin (hTg). Second, Tg as a potential initiator of autoimmunity is reintroduced by the hTg induction of EAT in mice with a DR3-selected T-cell receptor (TCR) repertoire and by the appearance of anti-Tg in three spontaneous autoimmune thyroiditis models. Third, the completed mTg sequence will enable in-depth study of conserved and unique epitopes on mTg and hTg. Fourth, the importance of appropriate class II genes over other genetic and environmental factors is reemphasized by the secondary role of iodine residues in Tg immunogenicity and the flexibility of the TCR repertoire. Individual DR or DQ transgenes can now be tested without the complication of linkage disequilibrium. Finally, the induction of Graves' disease-like syndrome by immunizing mice with human thyroid-stimulating hormone receptor-transfected cells offers the possibility of establishing a Graves' disease model.

Intravenous administration of deaggregated mouse thyroglobulin suppresses induction of experimental autoimmune thyroiditis and expression of both Th1 and Th2 cytokines.

Experimental autoimmune thyroiditis (EAT) can be induced by transfer of mouse thyroglobulin (MTg) and lipopolysaccharide (LPS)-immunized, MTg-activated spleen cells into syngeneic recipients. Recipients of MTg-activated T cells develop lymphocytic EAT, whereas recipients of cells activated by MTg and anti-IL-2R antibody develop a more severe and histologically distinct granulomatous form of EAT. Intravenous administration of deaggregated MTg (dMTg) 7 days before and 5 days after the first immunization of donor mice with MTg/LPS suppresses the induction of both forms of EAT. Thyroid infiltration is significantly decreased in recipients of effector cells from tolerant donors. MTg-specific T cell proliferation is partially suppressed in tolerant mice. Both IgG1 and IgG2a subclasses of anti-MTg autoantibodies are markedly inhibited in both tolerant donor mice and in recipients of tolerant spleen cells. Expression of T cell cytokine gene transcripts (IL-2, IFN gamma, IL-4, IL-10, tumor necrosis factor-alpha and transforming growth factor-beta) are all decreased in spleen cells of donor mice given dMTg. CD8+ T cells were not required for expression of tolerance since depletion of CD8+ T cells in vivo before tolerance induction or in vitro before MTg re-stimulation did not abrogate tolerance induction. Taken together, these results suggest that i.v. administration of dMTg can induce MTg-specific tolerance in both Th1- and Th2-like EAT effector cell precursors, and therefore prevent induction of adoptively transferred EAT.

Identification of a thyroiditogenic sequence within the thyroglobulin molecule.

Thyroglobulin (Tg)-specific T cells are important in the induction of experimental autoimmune thyroiditis (EAT), but the nature and the number of the Tg T cell epitopes involved in the disease process are unknown. Through the use of computerized algorithms that search for putative T cell epitopes, a 17-mer peptide (TgP1) was identified within the known portion of the rat Tg sequence (corresponding to amino acids 2495 to 2511 of the human Tg sequence) that induced strong mononuclear cell infiltration of the thyroid in classic EAT-susceptible murine strains such as SJL, C3H, and B10.BR and low or undetectable infiltration in EAT-resistant strains such as BALB/c and B10. TgP1 appears to be phylogenetically conserved since it is completely homologous to its bovine counterpart and differs at a single amino acid position from its human analogue. After priming with TgP1 in vivo, significant proliferative T cell responses to TgP1 in vitro were observed only with lymphocytes from susceptible (high responder) strains, thus correlating proliferative capacity with EAT induction. TgP1-primed T cells did not respond to intact mouse Tg (MTg) or rat Tg in vitro and, conversely, T cells primed in vivo with MTg or rat Tg did not respond to TgP1 in culture, suggesting that TgP1 is comprised of non-immunodominant T cell determinants. TgP1 was defined as a serologically nonimmunodominant epitope as well, since in vivo priming of all strains with MTg led to strong MTg-specific IgG responses but no TgP1-specific responses in ELISA assays. This was not due to lack of immunogenic B cell determinants on TgP1, however, because peptide challenge of EAT-susceptible strains elicited TgP1-specific IgG that also cross-reacted with MTg and rat, human, bovine, and porcine Tg. The data demonstrate that TgP1 delineates nonimmunodominant but highly immunogenic determinants at both the T and B cell level, which may play an important role in the development of autoimmune thyroiditis.

Thyroid-specific T cells in the normal Wistar rat: I. Characterization of lymph node T cell reactivity to syngeneic thyroid cells and thyroglobulin

Wistar rats are susceptible to the induction of experimental autoimmune thyroiditis (EAT) and recently a cloned Wistar thyroid epithelial cell line (WRT) has become available. In this study we have examined the in vitro proliferative response of Wistar rat lymphoid cells to the WRT cells as a model for better understanding thyroid cell-T cell interactions. Lymph node (LN) cells from normal Wistar rats showed an early and vigorous response to syngeneic thyroid cells (WRT). Further examination of the Wistar phenomenon revealed that unpurified lymphoid cells were also able to mount a proliferative response to rat thyroglobulin. The Wistar LN cell response to WRT cells consisted of T cell proliferation that was both MHC class I and class II restricted. There was a greater proliferation of CD8+ than CD4+ T cells, but WRT-stimulated lymphoblast cells were not cytotoxic to 51Cr-labeled WRT cell targets. These data suggest that normal Wistar rats have circulating thyroid-specific T cells that may be related to their known susceptibility to EAT. Furthermore, the interaction between such thyroid-specific T cells and cloned syngeneic thyroid cells offers a unique model for the further investigation of thyroid-immune interactions and the escape from tolerance induction.

IMMUNOGENETIC ASPECTS OF HUMAN THYROGLOBULIN‐REACTIVE T CELL LINES AND HYBRIDOMAS

The in vitro proliferative response to T cells primed with human thyroglobulin (Tg) was compared in 11 independent haplotypes on B10 background. B10.K and B10.S mice were the most responsive, whereas, with the exception of B10.PL (H-2u), all other B10 congenics were intermediate responders. The two best responders to in vitro challenge with human Tg, of the k and s haplotype, are the same as those showing H-2-linked susceptibility to induction of experimental autoimmune thyroiditis (EAT) with mouse Tg. Since shared epitopes on human and mouse Tgs have been shown to be thyroiditogenic by adoptive transfer studies in CBA (H2k) mice, the findings indicate that shared epitopes may be studied in appropriate (i.e. EAT-susceptible) strains of mice. Therefore, we proceeded to develop methods to produce T-cell lines and hybridomas to human Tg in B10.K and B10.S mice, test their cross-reactivity to heterologous Tgs and their Ia restriction patterns. By using antigen-presenting cells from recombinant strains, we identified restriction elements encoded by the I-A subregion alone and a combinatorial molecule from the I-A/I-E subregions.

In vivo beneficial effects of cyclosporin A and 1,25-dihydroxyvitamin D 3 on the induction of experimental autoimmune thyroiditis

In a recent work, we provided evidence that the in vitro inhibitory effect of cyclosporin A (CsA) was potentiated by the addition of another immunosuppressive molecule, the 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). In the present study, we investigated the in vivo influence of the association of both drugs administered at infratherapeutic doses, using an experimental model of autoimmune thyroiditis in CBA mice. Treatment regimen of the animals was initiated at priming with thyroglobulin (Tg) and consisted of daily administration of CsA (10 and 20 mg/kg/day, intragastrically) and/or 1,25(OH) 2D 3 (0.1 and 0.2 μg/kg/day, ip) for 21 days. Control mice that were given a placebo preparation orally and the vehicle of vitamin D 3 metabolite ip developed a severe disease as assessed by histological examination on Day 28 postimmunization and detection of circulating anti-Tg antibodies. Treatment with either drug administered alone at the doses mentioned above did not affect the incidence of thyroiditis and only reduced by up to 26% the severity of histological lesions. In contrast, the mice treated simultaneously with both drugs exhibited a lower incidence of thyroid pathology and developed a significantly milder disease ( P < 0.001) as compared to controls. However, there was no alteration in the levels of anti-Tg antibodies. This in vivo beneficial effect of low doses of CsA and 1,25(OH) 2D 3 was not due to an accumulation of CsA in the blood of treated mice since the levels of CsA were similar, regardless of the administration of 1,25(OH) 2D 3. Our data suggest that these two immunomodulatory agents used together at low doses may be an effective therapy of autoimmune disorders with fewer side effects.

Long-term observation and effect of age on induction of experimental autoimmune thyroiditis in susceptible and resistant mice

After induction of experimental autoimmune thyroiditis (EAT) in mice, long-term observation on both the severity of thyroiditis and antibody formation against mouse thyroglobulin (MTg) was carried out over a period of 18 months. In EAT-susceptible strains (CBA, C3H Anf ), both the severity of thyroiditis and MTg antibody production continued for a long term. In EAT-resistant mice ( BALB c ), thyroiditis remained minimal, if present, and the low antibody response to MTg became negligible. On the other hand, aging had a prominent effect on the EAT induction in susceptible mice: both MTg antibody response and thyroid infiltration were markedly reduced. These data were well correlated with reduced antibody formation against sheep red blood cells. Young and old mice were further compared as donors or recipients in adoptive transfer of concanavalin A-stimulated cells. Lymph node cells from either young or old MTg-immunized mice transferred appreciable thyroiditis to normal young and old recipient mice. However, similarly treated spleen cells from MTg-immunized old mice responded more poorly to in vitro stimulation with mitogens or MTg and transferred thyroiditis to very few normal young or old mice.