Granulomatous Experimental Autoimmune Thyroiditis Research Articles

Effect of Transgenic Overexpression of FLIP on Lymphocytes on Development and Resolution of Experimental Autoimmune Thyroiditis

In our previous studies, resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) was promoted when thyroid epithelial cells were protected from Fas-mediated apoptosis due to transgenic overexpression of FLIP. We hypothesized that if FLIP were overexpressed on lymphocytes, CD4(+) effector cells would be protected from Fas-mediated apoptosis, and resolution would be delayed. To test this hypothesis, we generated transgenic (Tg) mice overexpressing FLIP under the CD2 promoter. Transgenic FLIP was expressed on CD4(+) and CD8(+) T cells and B cells. Transgenic overexpression of FLIP protected cultured splenocytes from Fas-mediated, but not irradiation-induced, apoptosis in vitro. Unexpectedly, Tg(+) donor cells transferred minimal G-EAT, which was partially overcome by depleting donor CD8(+) T cells. When Tg(+) and Tg(-) donors transferred equivalent disease, G-EAT resolution was delayed in FLIP transgenic mice. However, CD2-FLIP Tg(+) donors often transferred less severe G-EAT, even after depletion of CD8(+) T cells. This influenced the rate of G-EAT resolution, resulting in little difference in G-EAT resolution between groups. Tg(+) mice always had reduced anti-mouse thyroglobulin autoantibody responses, compared with Tg(-) littermates, presumably because of FLIP overexpression on B cells. These results suggest that effects of transgenic FLIP on a particular autoimmune disease vary, depending on what cells express the transgene and whether those cells are effector cells or if they function to modulate disease.

Eosinophils infiltrate thyroids, but have no apparent role in induction or resolution of experimental autoimmune thyroiditis in interferon‐γ−/− mice

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTg)-sensitized splenocytes activated with MTg and interleukin (IL)-12. Our previous studies showed that, when used as donors and recipients, interferon (IFN)-gamma(-/-) and wild-type (WT) DBA/1 mice both develop severe G-EAT. Thyroid lesions in IFN-gamma(-/-) mice have many eosinophils and few neutrophils, while those in WT mice have extensive neutrophil infiltration and few eosinophils. Thyroid lesions in IFN-gamma(-/-) mice consistently resolve by day 40-50, whereas those in WT mice have ongoing inflammation and fibrosis persisting for more than 60 days. To determine if the extensive infiltration of eosinophils in thyroids of IFN-gamma(-/-) mice contributes to thyroid damage and/or early resolution of G-EAT, anti-IL-5 was used to inhibit migration of eosinophils to thyroids. G-EAT severity was compared at day 20 and day 40-50 in IFN-gamma(-/-) recipients given anti-IL-5 or control immunoglobulin G (IgG). Thyroids of anti-IL-5-treated IFN-gamma(-/-) mice had few eosinophils and more neutrophils at day 20, but G-EAT severity scores were comparable to those of control IgG-treated mice at both day 20 and day 40-50. Expression of chemokine (C-X-C motif) ligand 1 (CXCL1) mRNA was higher and that of chemokine (C-C motif) ligand 11 (CCL11) mRNA was lower in thyroids of anti-IL-5-treated IFN-gamma(-/-) mice. IL-5 neutralization did not influence mRNA expression of most cytokines in IFN-gamma(-/-) mice. Thus, inhibiting eosinophil migration to thyroids did not affect G-EAT severity or resolution in IFN-gamma(-/-) mice, suggesting that eosinophil infiltration of thyroids occurs as a consequence of IFN-gamma deficiency, but these cells have no apparent pathogenic role in G-EAT.

A critical role for TRAIL in resolution of granulomatous experimental autoimmune thyroiditis

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTG)-sensitized splenocytes activated in vitro with MTG and IL-12. Thyroid lesions reach maximal severity 20 days after cell transfer, and usually resolve or progress to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Our previous studies indicated that neutralization of TNF-alpha or FasL had no effect on G-EAT induction, but neutralization of TNF-alpha promoted, while neutralization of FasL inhibited, G-EAT resolution. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. This study was undertaken to define the role of endogenous TRAIL in G-EAT development and/or resolution. Neutralization of endogenous TRAIL had little effect on G-EAT induction, but significantly inhibited G-EAT resolution and increased thyroid fibrosis. This correlated with higher expression of pro-inflammatory cytokines and preferential expression of the pro-apoptotic molecule TRAIL, and anti-apoptotic molecules FLIP and Bcl-xL on inflammatory cells in thyroids of anti-TRAIL-treated recipients. The results suggest that endogenous TRAIL is not required for G-EAT development in recipients, but is critical for G-EAT resolution. Endogenous TRAIL might promote resolution, at least in part, through modulation of the balance between pro- and anti-inflammatory cytokines, and the expression pattern of pro- and anti-apoptotic molecules of thyroid epithelial cells (TECs) and inflammatory cells.

Interleukin-10 Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized splenocytes activated in vitro with mouse thyroglobulin and interleukin (IL)-12. Thyroid lesions reach maximal severity 20 days after cell transfer, and inflammation either resolves or progresses to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Depletion of CD8(+) T cells inhibits G-EAT resolution. Our previous studies indicated that IL-10 was generally higher in G-EAT thyroids that resolved. Using both wild-type and IL-10(-/-) CBA/J mice, this study was undertaken to determine whether G-EAT resolution would be inhibited in the absence of IL-10. The results showed that either depletion of CD8(+) T cells or IL-10 deficiency increased fibrosis and inhibited resolution of inflammation. We also found a correlation between higher expression levels of proinflammatory cytokines and preferential expression levels of proapoptotic molecules, such as FasL and TRAIL, and antiapoptotic molecules, such as FLIP and Bcl-xL, in inflammatory cells from thyroids of both CD8-depleted and IL-10-deficient mice. Furthermore, many of the CD8(+) T cells were also IL-10(+). These results suggest that IL-10 plays an important role in G-EAT resolution and might promote resolution, at least in part, through its production in CD8(+) T cells. Further understanding of the mechanisms that promote the resolution of inflammation will facilitate the development of novel strategies for treating autoimmune diseases.

Cultured Murine Thyroid Epithelial Cells Expressing Transgenic Fas-Associated Death Domain-Like Interleukin-1β Converting Enzyme Inhibitory Protein Are Protected from Fas-Mediated Apoptosis

The antiapoptotic molecule Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP) inhibits Fas-mediated apoptosis by blocking activation of caspase-8. We previously showed that expression of transgenic FLIP on thyroid epithelial cells (TECs) of DBA/1 and CBA/J mice promoted earlier resolution of granulomatous experimental autoimmune thyroiditis in vivo. This study was undertaken to directly determine whether transgenic FLIP expressed on cultured TECs can protect TECs from Fas-mediated apoptosis in vitro. The results indicate that cultured TECs from DBA/1 and CBA/J mice can be sensitized in vitro by interferon-gamma and TNF-alpha to undergo Fas-mediated apoptosis. Transgenic overexpression of FLIP protected cultured TECs of FLIP transgene (Tg)+ DBA/1 and CBA/J mice from Fas-mediated apoptosis, and FLIP small interfering RNA transfection of cultured TECs of FLIP Tg+ DBA/1 and CBA/J mice abolished the protective effect. These in vitro results are consistent with our previous in vivo studies using DBA/1 and CBA/J FLIP Tg+ mice and provide direct support for the hypothesis that transgenic expression of FLIP promotes resolution of granulomatous experimental autoimmune thyroiditis by protecting TECs from apoptosis.

Expression of Transgenic FLIP on Thyroid Epithelial Cells Inhibits Induction and Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in CBA/J Mice

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of thyroglobulin-primed in vitro activated splenocytes. Thyroid lesions reach maximal severity 20 d later, and inflammation resolves or progresses to fibrosis by d 60, depending on the extent of thyroid damage at d 20. Depletion of CD8+ T cells inhibits G-EAT resolution. We showed that expression of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP) transgene (Tg) on thyroid epithelial cells (TECs) of DBA/1 mice had no effect on G-EAT induction but promoted earlier resolution of G-EAT. However, when CBA/J wild-type donor cells were transferred to transgenic CBA/J mice expressing FLIP on TECs, they developed less severe G-EAT than FLIP Tg- littermates. Both strains expressed similar levels of the FLIP Tg, but endogenous FLIP was up-regulated to a greater extent on infiltrating T cells during G-EAT development in DBA/1 compared with CBA/J mice. After transient depletion of CD8+ T cells, FLIP Tg+ and Tg- CBA/J recipients both developed severe G-EAT at d 20. Thyroid lesions in CD8-depleted Tg+ recipients were resolving by d 60, whereas lesions in Tg- littermates did not resolve, and most were fibrotic. FLIP Tg+ recipients had increased apoptosis of CD3+ T cells compared with Tg- recipients. The results indicate that transgenic FLIP expressed on TECs in CBA/J mice promotes G-EAT resolution, but induction of G-EAT is inhibited unless CD8+ T cells are transiently depleted.

Expression of transgenic FLIP on thyroid epithelial cells in CBA/J mice inhibits induction and promotes resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) (129.17)

Abstract G-EAT is induced by transfer of thyroglobulin-primed in vitro activated splenocytes. In EAT susceptible DBA/1 and CBA/J mice, inflammation either resolves or progresses to fibrosis by day 60 depending on the extent of damage at day 20. Depletion of CD8+ T cells inhibits G-EAT resolution. We previously showed that FLIP transgene (Tg) expressed on thyroid epithelial cells (TEC) of DBA/1 mice had no effect on induction, but promoted earlier resolution of G-EAT. However, when Tg+ CBA/J mice expressing FLIP on TEC were used as recipients of WT donor splenocytes, they developed less severe G-EAT than FLIP Tg− littermates (average severity score of 1.3 vs. 4.5 at day 20), although both strains had similar levels of FLIP transgene. Following transient depletion of CD8+ T cells, FLIP Tg+ and Tg− CBA/J recipients both developed severe G-EAT (severity score of 4.4) at day 20. Lesions in CD8-depleted Tg+ recipients were resolving by day 60, whereas lesions in Tg− littermates did not resolve and most were fibrotic. FLIP and FasL were mainly expressed by TEC in FLIP Tg+ recipients and by inflammatory cells in Tg− recipients. mRNA expression of IL-10 and IL-13 was higher, and IFN-γ and TNF-α were lower, in FLIP Tg+ compared to Tg− recipients. The results support the hypothesis that transgenic FLIP expressed on TEC in CBA/J mice promotes G-EAT resolution, but induction of G-EAT is inhibited unless CD8+ T cells are depleted (NIH Grant DK35527).

Murine FLIP Transgene Expressed on Thyroid Epithelial Cells Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in DBA/1 Mice

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized splenocytes activated in vitro with mouse thyroglobulin and interleukin-12. In wild-type (WT) DBA/1 recipients of WT donor splenocytes, thyroid lesions reach maximal severity at day 20, with ongoing inflammation and extensive fibrosis at day 60. Our previous studies indicated the site of expression of FLIP and Fas ligand [thyroid epithelial cells (TECs) versus inflammatory cells] differed in mice when lesions would resolve or progress to fibrosis. To test the hypothesis that expression of FLIP by TECs would promote earlier G-EAT resolution in DBA/1 mice, transgenic (Tg) DBA/1 mice expressing FLIP on TECs were generated. In FLIP Tg(+) and Tg(-) littermate recipients of WT donor splenocytes, G-EAT severity was comparable at day 20, but fibrosis was decreased, and many lesions resolved by day 60 in Tg(+) but not Tg(-) recipients. FLIP and Fas ligand were primarily expressed by TECs in Tg(+) recipients and by inflammatory cells in Tg(-) recipients at day 60. Apoptosis of inflammatory cells was greater, and expression of proinflammatory cytokines was decreased in thyroids of Tg(+) compared with Tg(-) recipients. These results are consistent with the hypothesis that transgenic expression of FLIP on thyroid epithelial cells promotes earlier resolution of granulomatous experimental autoimmune thyroiditis.

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

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

Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis

Severe granulomatous experimental autoimmune thyroiditis (G-EAT) in DBA/1 or CBA/J wild type (WT) mice at day 19 progresses to fibrosis by day 35, but severe G-EAT in DBA/1 interferon (IFN)-gamma-/- mice or less-severe G-EAT at day 19 in WT mice resolves by day 35. To study the role of chemokines in autoimmune diseases and fibrosis, profiles of chemokines and chemokine receptors were analyzed in DBA/1 WT versus IFN-gamma-/- and CBA/J thyroids, which have distinct outcomes of autoimmune inflammation. Gene expression of CXC chemokine ligand 1 (CXCL1) and CXC chemokine receptor 2 (CXCR2) paralleled neutrophil infiltration and thyrocyte destruction in DBA/1 WT or CBA/J thyroids, and gene expression of CC chemokine ligand 11 (CCL11), CCL8, and CC chemokine receptor 3 paralleled eosinophil infiltration in IFN-gamma-/- thyroids. Gene and protein expression of CXCL10, CXCL9, and CXCR3 was significantly lower in IFN-gamma-/- compared with DBA/1 WT thyroids. Moreover, immunostaining showed that CXCL10 was expressed by thyrocytes and inflammatory cells, and strong expression of CXCL10 by thyrocytes was as early as day 7. High expression of CCL2 was only observed in severely destroyed DBA/1 WT or CBA/J thyroids, which would develop fibrosis. Thus, the differential expression of chemokines may direct distinct cellular populations in DBA/1 WT versus IFN-gamma-/- thyroids. Up-regulation of CXCL10 by thyrocytes suggests its role in regulating the recruitment of specific subsets of activated lymphocytes to the thyroid during autoimmune inflammation. The early expression of CXCL1, CXCL10, and CCL2 may suggest their involvement in the initiation and perpetuation of disease in severe G-EAT thyroids, which progress to fibrosis.

Fas Ligand Is Required for Resolution of Granulomatous Experimental Autoimmune Thyroiditis

We previously suggested that CD8(+) T cells promoted resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) at least in part through regulation of Fas ligand (FasL) expression on thyroid epithelial cells. To directly evaluate the role of the Fas pathway in G-EAT resolution, Fas- and FasL-deficient mice on the NOD.H-2h4 background were used as recipients of activated G-EAT effector cells. When MTg-primed wild-type (WT) donor splenocytes were activated and transferred to WT recipients, thyroid lesions reached maximal severity on day 20 and resolved on day 50. Fas, FasL, and FLIP were up-regulated, and many apoptotic inflammatory cells were detected in recipient thyroids on day 20. Fas was predominantly expressed by inflammatory cells, and FasL and FLIP were mainly expressed by thyroid epithelial cells. After depletion of CD8(+) T cells, G-EAT resolution was delayed, FLIP and FasL were predominantly expressed by inflammatory cells, and few inflammatory cells were apoptotic. When WT donor splenocytes were transferred to gld recipients, disease severity on day 20 was similar to that in WT recipients, but resolution was delayed. As in CD8-depleted WT recipients, there were few apoptotic inflammatory cells, and FLIP and FasL were expressed primarily by inflammatory cells. These results indicated that the expression of functional FasL in recipient mice was critical for G-EAT resolution. WT cells induced minimal disease in lpr recipients. This was presumably because donor cells were eliminated by the increased FasL on lpr recipient cells, because donor cells were not eliminated, and the mice developed G-EAT if lpr recipients were given anti-FasL mAb.

Balance of proliferation and cell death between thyrocytes and myofibroblasts regulates thyroid fibrosis in granulomatous experimental autoimmune thyroiditis (G-EAT)

Severe granulomatous experimental autoimmune thyroiditis (G-EAT), which progresses to fibrosis, is induced in DBA/1 mice by adoptive transfer of mouse thyroglobulin-primed and -activated spleen cells. There is extensive destruction of thyrocytes and inflammatory cell infiltration including T cells, macrophages, neutrophils, and myofibroblasts (myofbs). Suppression of transforming growth factor-beta (TGF-beta) and deficiency of interferon-gamma (IFN-gamma) inhibit fibrosis, and inflammation eventually resolves. Thyrocyte destruction in wild-type (WT) mice was a result of apoptosis, as many deoxynucleotide triphosphate nick-end labeling + apoptotic thyrocytes were present in these thyroids. The balance of apoptosis and proliferation between thyrocytes and myofbs may be important factors determining the outcome of inflammation to fibrosis versus resolution. Apoptosis and proliferation in thyrocytes versus myofbs were evaluated by dual-staining of cell-proliferating marker (Ki-67) or in situ cell death and cytokeratin or alpha-smooth muscle actin and were analyzed by confocal microscopy. Apoptotic and antiapoptotic molecules in G-EAT thyroids were detected by immunostaining. In WT thyroids, which develop fibrosis, only a few myofbs were apoptotic, and many myofbs were Ki-67+, Fas-associated death domain protein-like interleukin-1beta-converting enzyme-like inhibitory protein (FLIP)+, and Bcl-XL+. In contrast, proliferation was predominant on thyrocytes of IFN-gamma-/- mice or anti-TGF-beta-treated WT mice. These results indicate that apoptosis of inflammatory cells and regeneration of thyrocytes in IFN-gamma-/- mice and anti-TGF-beta-treated WT mice may limit development of fibrosis, whereas excessive proliferation of myofbs and loss of thyrocytes in WT mice may contribute to fibrosis.

FLIP and FasL expression by inflammatory cells vs thyrocytes can be predictive of chronic inflammation or resolution of autoimmune thyroiditis

Spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice is a model of chronic inflammation of the thyroid, while granulomatous experimental autoimmune thyroiditis (G-EAT) is a model with spontaneous resolution of inflammation. In chronic inflammation (SAT), Fas, FasL, and FLIP were upregulated and predominant in inflammatory cells. There were few apoptotic cells, and low expression of active caspase-8 and -3. In resolving G-EAT in CBA/J and NOD.H-2h4 mice, FasL and FLIP were predominantly expressed by thyrocytes. There were many apoptotic inflammatory cells, and increased expression of active caspase-8 and -3. Depletion of CD8+ T cells inhibited G-EAT resolution and resulted in chronic inflammation. FLIP was expressed predominantly by inflammatory cells, and apoptosis of inflammatory cells and expression of active caspase-3 was reduced as in chronic SAT. Thus, differences in expression of pro- or antiapoptotic molecules in SAT or G-EAT were apparently related to the acute vs chronic nature of the inflammatory response rather than the method of disease induction. Upregulation of FLIP by inflammatory cells may block Fas-mediated apoptosis, contributing to chronic inflammation, whereas increased FLIP expression by thyrocytes in resolving G-EAT may protect thyrocytes from apoptosis, and FasL expression by thyrocytes may induce apoptosis of inflammatory cells, contributing to resolution.

Inhibition of TGFbeta1 by anti-TGFbeta1 antibody or lisinopril reduces thyroid fibrosis in granulomatous experimental autoimmune thyroiditis.

In this study, a murine model of granulomatous experimental autoimmune thyroiditis (G-EAT) was used to determine the role of TGFbeta1 in fibrosis initiated by an autoimmune inflammatory response. The fibrotic process was evaluated by staining thyroid tissue for collagen, alpha-smooth muscle actin, TGFbeta1, and angiotensin-converting enzyme (ACE), and measuring serum thyroxine in mice given anti-TGFbeta1 or the ACE inhibitor lisinopril. The role of particular inflammatory cells in fibrosis was tested by depletion experiments, and the cytokine profile in thyroids was examined by RT-PCR. Neutralization of TGFbeta1 by anti-TGFbeta1 or lisinopril resulted in less collagen deposition and less accumulation of myofibroblasts, and levels of active TGFbeta1 and ACE were reduced in thyroids of treated mice compared with those of untreated controls. Other profibrotic molecules, such as platelet-derived growth factor, monocyte chemotactic protein-1, and IL-13, were also reduced in thyroids of anti-TGFbeta1- and lisinopril-treated mice compared with those of controls. Confocal microscopy showed that CD4(+) T cells and macrophages expressed TGFbeta1. Fibrosis was reduced by injection of anti-CD4 mAb on day 12, when G-EAT was very severe (4-5+). Together, these results suggest a critical role for TGFbeta1 in fibrosis initiated by autoimmune-induced inflammation. Autoreactive CD4(+) T cells may contribute to thyroid fibrosis through production of TGFbeta1. This G-EAT model provides a new model to study how fibrosis associated with autoimmune damage can be inhibited.

Characterization of thyroid fibrosis in a murine model of granulomatous experimental autoimmune thyroiditis

This study was initiated to identify and characterize thyroid fibrosis in a murine model of granulomatous experimental autoimmune thyroiditis (G-EAT) and determine if TGF-beta1 might be involved in fibrosis. G-EAT was induced by transfer of mouse thyroglobulin-sensitized spleen cells activated in vitro with thyroglobulin, anti-IL-2R, and IL-12. There was almost complete destruction of thyroid follicles, leading to fibrosis of the gland and reduced serum T4 levels. Fibrosis was confirmed by staining for collagen and alpha smooth-muscle actin, a marker of myofibroblasts. Kinetic studies characterized the onset and development of thyroid fibrosis. TGF-1beta was increased at mRNA and protein levels, and expression of TGF-beta1 protein paralleled G-EAT severity. Comparison of staining patterns showed that TGF-beta1 was expressed in areas of myofibroblast and collagen accumulation, implying that TGF-beta1 may play a role in fibrosis in G-EAT. Further studies demonstrated that myofibroblasts, macrophages, and thyrocytes contributed to TGF-beta1 production. This provides an excellent model to study the mechanisms of fibrosis associated with autoimmune damage.

Adoptive Transfer Murine Model of Granulomatous Experimental Autoimmune Thyroiditis

Experimental autoimmune thyroiditis (EAT) is a chronic inflammatory autoimmune disease that can be induced in genetically susceptible animals by immunization with mouse thyroglobulin (MTg) in an appropriate adjuvant or by the adoptive transfer of MTg-sensitized donor spleen cells, activated in vitro with MTg, into naive recipients. In the adoptive transfer model used in our laboratory, donor cells activated with MTg alone induce a relatively mild chronic lymphocytic form of EAT (L-EAT), in which the thyroid infiltrate consists primarily of mononuclear cells, and the thyroid inflammation persists for several months. When the same donor cells are activated with MTg together with anti-IL-2R and/or IL-12, a more severe and histologically distinct granulomatous form of EAT is induced in recipient mice. In addition to having distinct histopathologic features, granulomatous EAT (G-EAT) differs from L-EAT in that granulomatous thyroid lesions are not chronic. After reaching maximal severity 21 days after cell transfer, G-EAT thyroid lesions either resolve or the thyroids become atrophic and fibrotic by day 35. In this review, the histopathologic features of G-EAT and L-EAT are described, and our studies with the adoptive transfer G-EAT model which have focused on the mechanisms involved in induction of G-EAT in mice, and the evolution of G-EAT lesions to resolution of inflammation or fibrosis, are reviewed.

CD40L is Necessary for the Priming of Effector Cells for Lymphocytic and Granulomatous Experimental Autoimmune Thyroiditis

The interaction of CD40 on antigen presenting cells (APC) with CD40L on mouse thyroglobulin (MTg)-specific T cells may deliver an essential signal for the development of CD4+experimental autoimmune thyroiditis (EAT) effector cells and anti-MTg producing B cells. To determine the requirement for CD40–CD40L interactions in G-EAT, donor mice were injected with an anti-CD40L monoclonal antibody (mAb) on days −1, 0, and +1 relative to immunization with MTg and adjuvant. Recipients of spleen cells from MTg-primed donor mice injected with anti-CD40L did not develop EAT, while spleen cells from similarly immunized hamster Ig-treated donors transferred severe G-EAT. Although the decreased EAT severity was accompanied by increased IL-4 mRNA expression by CD4+T cells from anti-CD40L-treated donors, the increased IL-4 wasnotnecessary for suppression of EAT, since anti-CD40L treatment prevented EAT in IL-4-deficient mice. Addition of MTg-primed B cells duringin vitroactivation of spleen cells from anti-CD40L-treated donors did not induce EAT in recipients, suggesting that anti-CD40L suppresses EAT by preventing the sensitization of EAT effector cells. Addition of anti-CD40L duringin vitroactivation of MTg-primed spleen cells or treatment of recipients with anti-CD40L had no effect on EAT severity, indicating that CD40–CD40L interactions are not required after EAT effector cells are primed to MTg.

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.

IFN-γ-Deficient Mice Develop Severe Granulomatous Experimental Autoimmune Thyroiditis with Eosinophil Infiltration in Thyroids

AbstractTo study the role of IFN-γ in the development of granulomatous experimental autoimmune thyroiditis (EAT), DBA1 mice with a disrupted IFN-γ gene were used for adoptive EAT induction. Effector cells from either IFN-γ+/+ or IFN-γ−/− donor mice activated with mouse thyroglobulin and anti-IL-2R mAb induced severe granulomatous EAT. A predominant infiltration of the thyroid by eosinophils was observed in recipients of IFN-γ−/− effector cells but not in recipients of IFN-γ+/+ cells. Compared with wild-type mice, thyroids of recipients of IFN-γ−/− effector cells had decreased expression of mRNA for Th1 cytokines and inducible nitric oxide synthetase. Expression of Th2 cytokine mRNA was comparable to that of IFN-γ+/+ mice, and expression of eotaxin was increased in the thyroids of recipients of IFN-γ−/− effector cells. Activation of cells from either IFN-γ+/+ or IFN-γ−/− donors in the presence of IL-12 also induced severe granulomatous EAT. Eosinophil infiltration in recipients of IFN-γ−/− cells was unaffected when effector cells were activated with IL-12, and thyroids expressed predominantly Th2 cytokines. The extent of fibrosis of recipient thyroids was generally greater when donor IFN-γ+/+ and IFN-γ−/− cells were activated with IL-12. Compared with IFN-γ+/+ mice, IFN-γ−/− mice produced lower levels of mouse thyroglobulin-specific autoantibodies after immunization with MTg and LPS. These results indicate that cells from both IFN-γ+/+ and IFN-γ−/− donors can induce severe granulomatous EAT. However, damage of thyroid follicles by IFN-γ−/− and that by IFN-γ+/+ cells appear to involve different mediators of inflammation.

Induction of Granulomatous Experimental Autoimmune Thyroiditis in IL-4 Gene-Disrupted Mice

To study the role of IL-4 in development of granulomatous experimental autoimmune thyroiditis (EAT), IL-4 gene-disrupted mice expressing the EAT-susceptible H-2k haplotype were generated and used for EAT induction. Spleen cells from mouse thyroglobulin (MTg) and LPS-primed IL-4(+/+) and IL-4(-/-) donors could induce severe granulomatous EAT when spleen cells were activated with MTg and anti-IL-2R mAb in the presence of IL-12. Thyroid lesions had extensive follicular cell proliferation, large numbers of histiocytes, polymorphonuclear leukocytes, and multinucleated giant cells, in addition to lymphocytes and other mononuclear cells. Expression of IFN-gamma gene mRNA and production of IFN-gamma by effector spleen cells stimulated with MTg and IL-12 were similar for both IL-4(+/+) and IL-4(-/-) mice. Although IL-4 was undetectable in IL-4(-/-) mice, expression of mRNA for IL-5, IL-10, and IL-13 and production of IL-5 by both MTg-activated spleen cells and anti-CD3-activated CD4+ T cells were comparable for cells from IL-4(+/+) and IL-4(-/-) mice, indicating that the absence of IL-4 did not prevent production of other Th2 cytokines. Production of MTg-specific IgG1 was very low or undetectable in IL-4(-/-) mice. IL-4 gene mRNA and MTg-specific IgG1 could be detected in IL-4(+/+) or IL-4(-/-) recipients only when they received effector cells from IL-4(+/+) donor mice, indicating that IL-4- and IgG1-secreting cells are of donor origin. These results demonstrate that IL-4 is not essential for development of granulomatous EAT.