Linomide does not prevent spontaneous autoimmune thyroiditis in NOD mice

Linomide is a potent immunomodulator and has been reported to prevent type 1 diabetes mellitus in non-obese diabetic (NOD) mice and to reduce the incidence of other autoimmune diseases in animal models. The mechanisms of action seem to involve antigen expression by down regulation of macrophage activity and to antagonise the activation of Th1 cells during the cellular immune response. With the purpose to investigate the effect of Linomide on the incidence of spontaneous autoimmune thyroiditis (AIT) in female NOD mice we administered Linomide in drinking water (100 mg/kg/day) to NOD mice from 5th to 19th week of age. The mice were sacrificed at the end of week 19. None of the mice developed diabetes during the study period. The incidence of thyroiditis was evaluated on paraffin HE-stained sections and graduated on a scale from 0 to 4. Thirty-two percent of 37 mice treated with Linomide developed thyroiditis compared to 45% of 22 controls (p=0.31, chi2 =1.00). Among the mice who developed thyroiditis no difference in the degree of thyroiditis was found. Therefore no beneficial effect of Linomide on the incidence of spontaneous AIT in NOD mice could be demonstrated.     

Suppressor T cells prevent experimental autoimmune encephalomyelitis in mice.

Immune suppression (immunoprotection) in experimental autoimmune encephalomyelitis (EAE) was studied in (SJL X BALB/c)F1 mice using inocular of mouse spinal cord homogenate (MSCH), or mouse basic protein of myelin (M-BPM), in Freund's incomplete adjuvant (FIA). Such immunization specifically recruited lymphoid cells which markedly suppressed the capacity of effector lymph node cells from appropriately immunized syngeneic mice to transfer adoptively EAE. Suppression was demonstrable with transfer of bone marrow and spleen cells, but not with lymph nodes or thymus cells. Adoptively transferred suppression was maximal when cells were injected 9-30 days after the suppressive injection. Inhibition of EAE by suppressor cells was specific for the relavant antigen BPM, and required viable cells. Treatment of cells with anti-Thy-1 serum before transfer abolished their suppressor activity. After adoptive transfer of suppressor cells into syngeneic recipients subsequently immunized for EAE, there was inhibition of EAE and reduced cell-mediated immune response to BPM as judged by macrophage migration inhibition assays. Hence, in mice at least, immuno-protection against EAE is explicable by recruitment of suppressor T lymphocytes with the dual capacities of inhibiting development of effector T cells after antigenic stimulation, and of blocking their damaging effects on the antigen in the central nervous system.     

Chemokines modulate experimental autoimmune thyroiditis through attraction of autoreactive or regulatory T cells

A critical event in the pathogenesis of experimental autoimmune thyroiditis (EAT) is the entry of thyroid-specific T lymphocytes into the thyroid gland. To investigate the role of soluble mediators in that infiltration, we have assayed the expression of various chemokines in diseased thyroid glands and in cytokine-treated cultures of normal thyroid epithelial cells. MCP-1 (monocyte chemotactic protein-1) and RANTES are produced during EAT and induced in vitro by IFN-gamma, IL-10, TNF-alpha, and IL-1beta. In vitro chemotaxis experiments using immune lymph node (LN) cells showed that RANTES attracted mTg-specific responder LN cells, whereas MCP-1 attracted mTg-specific CD4(+), CD25(+) regulator cells that secreted IL-10. The in vivo transfer of LN T cells attracted in vitro either by RANTES or by MCP-1 confirmed their opposite effects on the course of EAT.     

Peptide T does not ameliorate experimental autoimmune encephalomyelitis (EAE) in Lewis rats

Peptide T has been shown to inhibit T cell activation and cytokine production and function. Moreover, it has been reported to be a safe treatment in humans. We have studied the ability of peptide T to prevent or ameliorate EAE in Lewis rats. Peptide T was administered subcutaneously at different doses and phases of the disease according to several treatment protocols, but we could not observe a consistent effect of peptide T ameliorating the disease. Lymph node cell proliferation and IL-4 and interferon-gamma production were also studied. We conclude that peptide T neither prevents nor ameliorates EAE in Lewis rats.     

Regulatory T cells induced by rAAV carrying the forkhead box P3 gene prevent autoimmune thyroiditis in mice

CD4+CD25+ regulatory T (Treg) cells are immunosuppressive and help maintain peripheral immune tolerance. The forkhead/winged helix family member, Foxp3, has been shown to be a critical regulator of CD4+CD25+ Treg cells. We demonstrate by quantitative real-time PCR that CD4+CD25+ T cells express higher levels of Foxp3 mRNA than other T cell populations. Recombinant adeno-associated virus vector carrying mouse Foxp3 gene under the control of the CMV promoter was generated and used to transduce CD4+CD25- T cells. These Foxp3-transduced T cells are similar to naturally occurring CD4+CD25+ regulatory T cells which show anergy and immunosuppressive activity in vitro. Furthermore, these Foxp3-transduced T cells prevent autoimmune thyroiditis transferred by pathogenic T cells in vivo. Our data indicates that Foxp3-transduced CD4+CD25- T cells may open new therapeutic strategies for immune disorders.     

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.     

Phenotypic characteristics of cells involved in induced suppression to murine experimental autoimmune thyroiditis

Suppression of induced experimental autoimmune thyroiditis can be consistently transferred with spleen cells to syngeneic recipients, provided they are first treated with 200 rads irradiation. Treatment with anti-Thy-1 in vivo immediately prior to transfer abrogates the suppression, while depleting B cells has no effect. The in situ induced tolerance can be prevented by treatment with monoclonal antibodies to the Ly-1 or L3T4 molecules either prior to or post tolerization. Anti-Ly-2 treatment has no effect. In the transfer, again treatment of donors with anti-L3T4 prior to transfer prevents the demonstration of suppression in the recipients, while anti-Ly-2 does not affect suppression. These data suggest that the suppression is being mediated either by a CD4+ T suppressor cell or a CD4+ suppressor inducer cell. Preliminary experiments do not support the possibility of a CD8+ suppressor cell being activated in the recipient.     

Autoimmune murine thyroiditis. VIII. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis.

Mice of the C57Br strain, which are susceptible to the induction of autoimmune thyroiditis with mouse thyroglobulin, and C57Bl mice, which are resistant, were immunized with human and rabbit thyroglobulins in Freund's complete adjuvant. Susceptible strain C57Br developed higher degrees of thyroid infiltration than the resistant strain. The results indicate that the responses to xenogeneic (foreign) thyroglobulins parallel allogeneic and syngeneic (mouse) thyroglobulin. BSVS mice, which are highly susceptible to thyroiditis, were immunized with mouse thyroid extract from five different mouse strains including syngeneic antigen. Recipients of C57Bl and DBA thyroid extracts showed lower indices of pathology than recipients of similar extracts from C3H, BSVS and non-inbred CF-1 mice. The results suggest that there is a difference in the immunogenicity of mouse thyroid extracts from different strains. Purified thyroglobulin was prepared from congenic strains B10.D2 (H-2d, resistant) and B10Br (H-2k, susceptible). H-2k thyroglobulin gave a greater response in both H-2k and H-2d mice than H-2d thyroglobulin.     

T cell competence to heterologous and homologous thyroglobulins during the induction of experimental autoimmune thyroiditis

The ability of T cells to respond to homologous vs. heterologous thyroglobulins (Tg) has been evaluated using different models for the induction of experimental autoimmune thyroiditis. Substantial levels of T cell activation could be demonstrated to heterologous Tg following immunization with heterologous Tg in complete Freund's adjuvant, whereas only minimal levels of T cell activation to homologous Tg could be obtained following immunization with homologous Tg in complete Freund's adjuvant. Using this immunization protocol, heterologous and homologous Tg induced equivalent levels of serum antibody to the immunizing Tg. However, when injected in incomplete Freund's adjuvant, homologous Tg induced less antibody than heterologous Tg. Even greater differences in serum antibody levels to heterologous and homologous Tg, were apparent following immunization with soluble Tg. These thyroiditis differences are attributed to the presence of only a minimal level of T cell competence for homologous Tg, which is capable of inducing experimental autoimmune thyroiditis with stringent immunization protocols, but not with weaker immunization regimens.     

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.     

Late-term fetal thymectomy does not prevent the development of gut-homing T cells after birth.

Tissue-specific circulation of T cells is a critical element in the integration of systemic immune responses. Current models of T-cell migration suggest that homing specificities of T cells for tissues such as gut and skin are generated outside the thymus as a result of activation of virgin T cells by antigen in lymph nodes. We have used the sheep fetus (which is immunologically virgin and contains no memory or effector T-cell subsets) to examine the migration of 51Cr-labelled T cells in vivo. We report that gut-homing T cells are not present in the fetus and that gut-homing T cells from postnatal lambs home normally to fetal gut. Fetal thymectomy performed immediately prior to birth failed to prevent the development of gut-homing T cells in postnatal life. Gut-homing specificities on T cells are thus acquired extrathymically.     

Regulatory T cells in experimental autoimmune disease

During the past 10 years, CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) have been extensively studied for their function in autoimmune disease. This review summarizes the evidence for a role of Treg in suppression of innate and adaptive immune responses in experimental models of autoimmunity including arthritis, colitis, diabetes, autoimmune encephalomyelitis, lupus, gastritis, oophoritis, prostatitis, and thyroiditis. Antigen-specific activation of Treg, but antigen-independent suppressive function, emerges as a common paradigm derived from several disease models. Treg suppress conventional T cells (Tcon) by direct cell contact in vitro. However, downmodulation of dendritic cell function and secretion of inhibitory cytokines such as IL-10 and TGF-β might underlie Treg function in vivo. The final outcome of autoimmunity vs tolerance depends on the balance between stimulatory signals (Toll-like receptor engagement, costimulation, and antigen dose) and inhibitory signals from Treg. Whereas most experimental settings analyze the capacity of Treg to prevent onset of autoimmune disease, more recent efforts indicate successful treatment of ongoing disease. Thus, Treg are on the verge of moving from experimental animal models into clinical applications in humans.     

The ultrastructure of thyroid in chronic autoimmune thyroiditis

Summary In an investigation of autoimmune thyroiditis, two cases of chronic asymptomatic thyroiditis, two cases of nodular goitre with focal thyroiditis and two cases of Hashimoto goitre were studied with the electron microscope. The ultrastructural observations were similar in the six cases. Cytological alterations of the follicle cells were found, some of which suggested an increased metabolic activity. Lymphocytes and plasmocytes established close relationships with the thyroid cells: emperipolesis was encountered.

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Die Ultrastruktur der chronischen Autoimmun-Thyreoiditis

Zusammenfassung 6 Fälle von Autoimmunthyreoiditis (2 Fälle von chronischer asymptomatischer Thyreoiditis, 2 Fälle von Knotenstruma mit herdförmiger Thyreoiditis und 2 Fälle von Hashimoto-Thyreoiditis) werden licht- und elektronenoptisch untersucht. Die ultrastrukturellen Veränderungen stimmen in allen Fällen überein. Die cytologischen Veränderungen in den Follikelzellen machen eine erhöhte Stoffwechselaktivität wahrscheinlich. Als Besonderheit wird das aktive Eindringen von Lymphocyten und Plasmazellen in die Follikelepithelien (Emperipolese) hervorgehoben.

     

Activation of cytotoxic T cells and effector cells in experimental autoimmune thyroiditis by shared determinants of mouse and human thyroglobulins

Previous studies have shown that T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro and differentiate into cells cytotoxic for syngeneic thyroid monolayers. To examine further the effector cells involved in pathogenesis and the determinants on MTg responsible for their activation, spleen cells (SC) and lymph node cells (LNC) from mice immunized with MTg or human (H) Tg, and adjuvant (complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS] were cultured in vitro with MTg or HTg. Control cultures were incubated with concanavalin A (Con A) or purified protein derivative (PPD). The in vitro-activated cells which proliferated in response to MTg, HTg, or Con A adoptively transferred thyroiditis to normal recipients, whereas cells transferred directly without in vitro culture were very ineffective. The capacity to transfer EAT was abrogated by irradiation (1500 R), and SC from CFA-immunized control mice which responded in vitro to PPD stimulation did not transfer thyroiditis. The serum titers of MTg autoantibodies were uniformly low and were not correlated with severity of disease. The localization of EAT-effector (precursor) cells depended upon the site of immunization; they were found in the spleens after inguinal (subcutaneous) or systemic (intravenous) immunizations, but were present in the popliteal lymph nodes after hind footpad injections. Both homologous MTg and heterologous HTg functioned as in vivo sensitizing antigen and in vitro activating antigen for each other; such cultured cells transferred thyroiditis in vivo and became cytotoxic for thyroid monolayers in vitro. These findings show that shared determinants are autoantigenic and thyroiditogenic, and support the hypothesis that EAT-effector cells responsible for initiating thyroid damage include cytotoxic cells.     

Transgenic overexpression of human Bcl-2 in islet beta cells inhibits apoptosis but does not prevent autoimmune destruction

Insulin-dependent diabetes mellitus results when > 90% ofthe insulin-producing ß cells in the pancreatic isletsare killed as a result of autoimmune attack by T cells. Duringthe progression to diabetes, islet ß cells die as a resultof different insults from the immune system. Agents such asperforin and granzymes, CD95 ligand and tumor necrosis factor-,or cytokines and free-radicals have all been shown to causeß cell apoptosis. The anti-apoptotic protein, Bcl-2, mightprotect against some of these stimuli. We have therefore generatedtransgenic mice expressing human Bcl-2 in their islet ßcells. Although Bcl-2 was able to prevent apoptosis inducedby cytotoxic agents against ß cells in vitro, Bcl-2 alonecould not prevent or ameliorate cytotoxic or autoimmune ßcell damage in vivo.     

Immunomorphological transformation of the thyroid gland in autoimmune thyroiditis]

In parallel immunochemical, immunohistochemical, and histopathological studies of blood sera and biopsies of the thyroid gland tissue the autoimmune nature of thyroiditis was proved in 11 female patients. The detected antithyroid autoantibody was classified as complement-dependent immunoglobulin. Immunocytochemical data are presented confirming the histiocytic derivation of macrophages and non-epithelial nature of B- and C-cells. Transformation of colloid and cells ans metabollsm of monoclonal immunoglobulins (IgG, IgA, IgM) permit the judgement on the duration of autoimmune thyroiditis. The diffuse lymphoid variant is typical of the first 5 years, and focal fibrinolymphoid variant-for later peroids of autoimmune thyroiditis.     

The role of the spleen in experimental autoimmune thyroiditis

We have investigated the role of the spleen in the humoral and cellular immune response of rats with experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin and Freund's complete adjuvant. Animals subjected to splenectomy within 4 days of immunization developed lower thyroglobulin antibody levels and less severe thyroiditis compared to sham operated controls. There was no impairment in the ability of the animals to recover spontaneously from the disease after splenectomy. Together with the results obtained using splenocyte infusions, this suggests that suppressor cell production within the spleen plays only a small part in the normal immunological control which is presumably responsible for spontaneous regression of the disease.     

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.