Jeannine Charreire

In vivo beneficial effects of cyclosporin A and 1,25-dihydroxyvitamin D3 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 D3 (1,25(OH)2D3). 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)2D3 (0.1 and 0.2 microgram/kg/day, ip) for 21 days. Control mice that were given a placebo preparation orally and the vehicle of vitamin D3 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 less than 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)2D3 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)2D3. 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.

Immune mechanisms in autoimmune thyroiditis.

Publisher Summary This chapter mainly focuses on autoimmune thyroiditis in man and animals. Discussion made in the chapter has shared thoughts from an extensive review on Graves disease published in 1985. Autoimmune thyroid diseases result from the persistence of forbidden B cell clones. In this hypothesis, the contact of antibody (Ab)-forming cells with their respective antigens (Ags) during fetal life leads to the destruction of the corresponding clones. In this way, self-reactive clones, so-called forbidden clones, would be deleted unless they originated later in life by somatic mutation of lymphocytes. Numerous experiments have failed to verify this hypothesis for autoreactive B cells as well as for autoreactive T cells; these cells are readily detected in high frequencies in normal animals and healthy humans. Immunization with individual synthetic peptides has revealed additional T cell determinants not seen following immunization with the native molecule. The lack of responsiveness to these determinants cannot be explained by an absence of T cells in the repertoire or a failure to bind a particular major histocompatibility complex (MHC) molecule. Understanding the pathogenesis of the naturally occurring syndromes and the experimental autoimmune thyroid diseases has been a central theme n the experimental immunology of autoimmunity. Experimental models and genetic control of thyroiditis and cellular immune responses during thyroiditis have been discussed well in the chapter. The field of immunogenetics promises answers to how a given MHC gene regulates or facilitates thyroiditis and how it cooperates with genes coding for Tg, A-Abs, cytokines, or other products. The powerful transgenic mouse model appears to be one of the most appropriate tools to answer these questions.

Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN-γ receptor gene

To investigate the role of interferon‐γ (IFN‐γ) in experimental autoimmune thyroidits (EAT), H‐2k mice with a disrupted IFN‐γ receptor (IFN‐γ R) gene were immunized with porcine thyroglobulin (pTg). We observed that EAT occurred on day 19 and remitted on day 35 in IFN‐γ R‐ deficient (IFN‐γR0/0) mice, whereas in wild‐type mice, EAT occurred on day 21 and remitted on day 42u2009–u200949. Moreover, EAT in the mutant mice was attenuated and accompanied by diminished Tg‐specific cytotoxic and proliferative responses and decreased titers of anti‐Tg antibodies, notably of the IgG2a and IgG2b isotypes. In contrast, Tg‐specific IgG1 was increased in the IFN‐γ R0/0 mice. In supernatants from T cells further stimulated in vitro by Tg, IFN‐γ levels were higher in IFN‐γ R0/0 than in wild‐type mice throughout the course of the dis ease, whereas interleukin‐10 was transiently increased prior to EAT onset in both groups of mice. Finally, using IFN‐γ R0/0 mice, we demonstrate that induction of EAT does not require an intact IFN‐γ system, while progression to full‐blown disease depends on the action of IFN‐γ.

Experimental autoimmune thyroiditis induced by recombinant interferon-γ

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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 Tu2004cell response was observed, along with a trend towards a Th2 response characterized by decreased production of IFN‐γ 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.

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‐γ, IL‐10, TNF‐α, and IL‐1β. 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.

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells. VII. Generation of thyroid-specific cytotoxic effector cells.

We observed T lymphoblast generation after three days of culture of normal spleen lymphocytes on monolayers of syngeneic thyroid epithelial cells. It appears that only these T lymphoblasts sensitized on thyroid monolayers are specifically labelled by fluorescein-conjugated thyroglobulin. In this study, the role of thyroglobulin, and the manner in which it is presented to syngeneic T cells bearing receptors for thyroglobulin were investigated. It appears that thyroglobulin plays a key role in primary syngeneic sensitization of spleen cells in vitro, but its action is not exclusive. Proteins, which are encoded by a gene located in the I-A subregion level of the major histocompatibility complex, are capable of inducing a primary proliferative signal when the presentation of syngeneic class II antigens by syngeneic thyroid epithelial cells is simultaneous. In addition, thyroglobulin-pulsed macrophages are not able to do so. In contrast, once this primary syngeneic T cell proliferation has been accomplished, soluble thyroglobulin is sufficient to induce a secondary response by these syngeneic T cells.

Genetic control of the humoral response to cryptococcal capsular polysaccharide in mice

Cryptococcus neoformans is responsible for opportunistic infections in patients with cellular immune defects. However, C. neoformans-specific capsular polysaccharide antibodies have been shown to participate in host defenses during cryptococcosis. We investigated the humoral response after primary immunization in various inbred strains of mice and the genetic control. Our data strengthen the arguments for the T-independent type-2 nature of cryptococcal antigen, since CBA/N mice were unable to produce specific antibodies. They show that the influence of the genetic background is predominant for the good response with at least four independent autosomal genes governing this response, including an Igh control as reported for other polysaccharides. Immunization of intra-H-2 recombinant mice on a B10 background allowed us to identify a major histocompatibility complex control located in the subregion Eα. The genetic control of antibody production following immunization with cryptococcal polysaccharide might explain the high variability of humoral responses during cryptococcosis.

Influenza A virus hemagglutinin is a B cell-superstimulatory lectin

Influenza A viruses display T cell-independent polyclonal B cell-activating properties which are mediated by the B cell-superstimulatory envelope glycoprotein hemagglutinin (HA). In this report, the receptor-binding requirements for B cell activation by influenza viruses were expected. Neuraminidase treatment of resting mature B cells from BALB/c mice abrogated late (proliferation/immunoglobulin synthesis), early (up-regulation of cell surface markers, including CD25, B220, and B7-1) and veryearly events (homotypic adhesion) in virus-responding B lymphocytes. Similarly, pretreatment of murine responder cells with different inhibitors ofN-glycosylation (tunicamycin, deoxymannojirimycin) significantly suppressed subsequent B lymphocyte activation by HA, but not control responses to lipopolysaccharide or anti-μ. Assays with chimeric HA transfectants, expressing the loop region of epitope B (amino acids 155–160) of the globular head of H2 (high B cell-stimulatory subtype) or H3 (medium-stimulatory subtype) on the protein backbone of a low-stimulatory subtype (H1) failed to alter the B cell-stimulatory activity of the virus, suggesting that the hypervariable loop region is not crucial in determining the B cell-activating properties of the protein. Collectively, our results imply that the B cell-superstimulatory function of influenza virus HA is not mediated by a direct protein/protein interaction, but via binding of HA to terminal sialic acid residues on cell surface receptor glycoproteins. These findings identify the influenza virus HA glycoprotein as the first viral lectin with lymphocyte-activating properties.

B-Cell Activation by Superstimaulatory Influenza Virus Hemagglutinin: A Pathogenesis for Autoimmunity?

Viruses have been implicated in the pathogenesis of almost every kind of autoimmune disease. Indeed, a great number of sporadic reports in the hterature points to the possible involvement of so-called banal viral infectiotis in the pathogenesis of autoimmune diseases, notably in the context of autoimmune-mediated endocrinopathies. Accordingly, many different mechanisms have been postulated in which virus infections could lead to pathology and/or disease, including molecular mimicry, induction of cytokine gene expression, disturbance of regulatory lymphocyte circuits, up-regulation of major histocompatibility complex (MHC) elements, incorporation of host antigens into viral envelopes and. most recently, the expression of T-cell superantigenic antigens by viruses. Here we add another possible mechanism: a polyclonal mitogenic B-cell activation by viral protein. The B-cell mitogenicity of many viral proteins has been studied for years, but the consequence of such a viral characteristic on breakage of immune tolerance, i.e. autoimmune reaction, is poorly known. In this review, we present our latest results with a certain B-cell mitogenic virus (infiuenza virus) that might induce an autoimmune pathogenesis. Virus infections are often accompanied by transiently appearing self-reactive Tcell responses, which might be high enough to cause destruction of self-components, i.e. to manifest clinical signs of an autoimmune disease. In fact, experimental models of vims-mediated autoimmune diseases are still the only satisfying