Targeted expression of the human thyrotropin receptor A-subunit to the mouse thyroid: insight into overcoming the lack of response to A-subunit adenovirus immunization

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.     

Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.     

Development of an animal model of autoimmune thyroid eye disease

In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-gamma. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60-100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED.     

Exceptional hyperthyroidism and a role for both major histocompatibility class I and class II genes in a murine model of Graves' disease

Autoimmune hyperthyroidism, Graves' disease, can be induced by immunizing susceptible strains of mice with adenovirus encoding the human thyrotropin receptor (TSHR) or its A-subunit. Studies in two small families of recombinant inbred strains showed that susceptibility to developing TSHR antibodies (measured by TSH binding inhibition, TBI) was linked to the MHC region whereas genes on different chromosomes contributed to hyperthyroidism. We have now investigated TSHR antibody production and hyperthyroidism induced by TSHR A-subunit adenovirus immunization of a larger family of strains (26 of the AXB and BXA strains). Analysis of the combined AXB and BXA families provided unexpected insight into several aspects of Graves' disease. First, extreme thyroid hyperplasia and hyperthyroidism in one remarkable strain, BXA13, reflected an inability to generate non-functional TSHR antibodies measured by ELISA. Although neutral TSHR antibodies have been detected in Graves' sera, pathogenic, functional TSHR antibodies in Graves' patients are undetectable by ELISA. Therefore, this strain immunized with A-subunit-adenovirus that generates only functional TSHR antibodies may provide an improved model for studies of induced Graves' disease. Second, our combined analysis of linkage data from this and previous work strengthens the evidence that gene variants in the immunoglobulin heavy chain V region contribute to generating thyroid stimulating antibodies. Third, a broad region that encompasses the MHC region on mouse chromosome 17 is linked to the development of TSHR antibodies (measured by TBI). Most importantly, unlike other strains, TBI linkage in the AXB and BXA families to MHC class I and class II genes provides an explanation for the unresolved class I/class II difference in humans.     

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin

The thyrotropin receptor (TSHR) has a unique 50 residue (317-366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334-349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation.     

cDNA immunization of mice with human thyroglobulin generates both humoral and T cell responses: a novel model of thyroid autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis.     

T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice

We investigated Ly-1+ T lymphocyte line cells specifically reactive to thyroglobulin (Tg) that were isolated from mice primed with mouse Tg in adjuvant. Intravenous inoculation of as few as 10(5) line cells was sufficient to cause severe and prolonged thyroiditis in recipient mice that were intact, irradiated, or athymic nudes. Disease was independent of circulating Tg antibodies, suggesting that anti-Tg T lymphocytes could cause thyroiditis unaided by antibodies. Thyroiditogenic T lymphocytes could be isolated as cell lines from apparently healthy mice that had been immunized with non-thyroiditogenic bovine Tg in adjuvant, which indicates that autoimmune effector T lymphocytes may develop covertly in the course of immunization with foreign antigens. Finally, a single i.v. inoculation of anti-Tg T lymphocyte line cells attenuated by irradiation vaccinated mice completely against subsequent development of autoimmune thyroiditis produced either by active immunization to Tg or by passive transfer of intact line cells. Vaccinated mice that were protected from inflammatory lesions of thyroiditis still produced high titers of Tg antibodies in response to active immunization. Thus, vaccination specifically inhibited thyroiditogenic T lymphocytes but not helper T lymphocytes required for the production of Tg autoantibodies.     

Cooperation of invariant NKT cells and CD4+CD25+ T regulatory cells in the prevention of autoimmune myasthenia

CD1d-restricted NKT cells and CD4+CD25+ regulatory T (Treg) cells are thymus-derived subsets of regulatory T cells that have an important role in the maintenance of self-tolerance. Whether NKT cells and Treg cells cooperate functionally in the regulation of autoimmunity is not known. We have explored this possibility in experimental autoimmune myasthenia gravis (EAMG), an animal model of human myasthenia gravis, induced by immunization of C57BL/6 mice with the autoantigen acetylcholine receptor. We have demonstrated that activation of NKT cells by a synthetic glycolipid agonist of NKT cells, alpha-galactosylceramide (alpha-GalCer), inhibits the development of EAMG. alpha-GalCer administration in EAMG mice increased the size of the Treg cell compartment, and augmented the expression of foxp3 and the potency of CD4+CD25+ cells to inhibit proliferation of autoreactive T cells. Furthermore, alpha-GalCer promoted NKT cells to transcribe the IL-2 gene and produce IL-2 protein. Depletion of CD25+ cells or neutralization of IL-2 reduced the therapeutic effect of alpha-GalCer in this model. Thus, alpha-GalCer-activated NKT cells can induce expansion of CD4+CD25+ Treg cells, which in turn mediate the therapeutic effects of alpha-GalCer in EAMG. Induced cooperation of NKT cells and Treg cells may serve as a superior strategy to treat autoimmune disease.     

Experimental autoimmune thyroiditis in mice chronically treated from birth with anti-IgM antibodies

The role of T lymphocytes in the pathogenesis of experimental autoimmune thyroiditis in mice is well established while the role of B lymphocytes is unclear. Mice with thyroid lesions have thyroglobulin antibodies whereas these antibodies can occur in mice immunized with Tg that do not develop thyroid lesions. To determine whether thyroglobulin antibodies are necessary for the development of the thyroid infiltrates with mononuclear cells, which are characteristic for experimental autoimmune thyroiditis, AKR mice chronically treated from birth with goat anti-mouse IgM antibodies were immunized with mouse thyroglobulin in Freund's complete adjuvant when they were 7 weeks old. Control mice, similarly immunized, were chronically injected from birth with normal goat gamma-globulin. Three weeks after immunization, all mice were sacrificed, thyroglobulin antibodies in the serum were measured by hemagglutination assay and enzyme-linked immunosorbent assay, and thyroid pathology was assessed. The serum concentration of IgG and IgM, the percentage of B and T lymphocytes in the spleen (flow cytometry), and the in vitro proliferative response of spleen lymphocytes to stimulation by PHA, LPS, and Tg were also measured. All mice treated with anti-IgM antibodies did not have detectable thyroglobulin antibodies but 63% of these mice and 88% of control mice (all of which had thyroglobulin antibodies) had thyroid lesions. Mice treated with anti-IgM antibodies that did not have thyroid lesions had a more pronounced depression of B lymphocytes than similarly treated mice that had thyroid lesions. These experiments suggest that thyroglobulin antibodies are not necessary for the development of thyroid infiltrates with mononuclear cells. B lymphocytes could still participate in the production of experimental autoimmune thyroiditis by presenting thyroglobulin to helper T lymphocytes.     

Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts

Controversy continues about whether, and to whatlevels of abundance, thyroid-stimulating hormone receptors (TSHR) arefound in human tissues other than the thyroid gland. Restrictedexpression to the thyroid and orbit would suggest that TSHR representsthe target autoantigen in thyroid-associated ophthalmopathy. A more generalized pattern of tissue expression would be inconsistent withTSHR acting as the autoantigen that is solely responsible forselectively targeting the immune system to the orbit. We have detectedTSHR mRNA in human abdominal adipose tissue by Northern blot analysis.TSHR protein was also detected, by immunoblotting with two differentantibodies, in preadipocytes isolated from human abdominal subcutaneousand omental adipose tissue and in derivative adipocytes differentiatedin primary culture. Preadipocytes treated with thyroid-stimulatinghormone (TSH) exhibited a sevenfold increase in the activity of p70 S6kinase, a serine/threonine kinase recently recognized as a downstreamtarget of TSHR in thyroid cells. Activation of p70 S6 kinase by TSH wasalso observed in orbital fibroblasts. Thus TSHR protein expression isfound in fibroblasts from several anatomic locations, suggesting thatfactors other than site-limited TSHR expression must be involved inrestricting the distribution of Graves' disease manifestations.Furthermore, the presence of functional TSHR in preadipocytes raisesthe possibility of a novel role for TSHR signaling in adipose tissue development.

     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

The effect of aging on the induction of experimental autoimmune thyroiditis

The effect of age on the ability to elicit the various immune functions comprising experimental autoimmune thyroiditis in mice has been examined. Compared with young mice (2 to 3 mo), CBA/CaJ and A/J aged mice (20 to 30 mo) show a drastic reduction in their ability to develop circulating antibody after injection of mouse thyroglobulin (MTg) or mouse thyroid extract (MTE) in complete Freund's adjuvant (CFA). Delayed-type hypersensitivity responses were also depressed, as well as the ability of aged lymph node cells to proliferate in vitro to antigen and the ability of aged splenic T cells to function as helper cells for in vitro antibody production. However, after injection of these thyroid antigens in CFA, aged mice developed thyroid lesions either comparable to or only slightly less intense than those observed in young mice. The disparity between the levels of immune responses and thyroid lesions observed in aged mice can be explained by the greater susceptibility of aged thyroids to tissue damage, since transfer of identical numbers of Con A-activated MTE-primed young splenocytes to young and aged recipients results in a more severe thyroiditis in the aged recipients. Priming mice to MTE in CFA at 9 mo of age, at which time mice are responsive to MTE, did not enhance either T or B cell responsiveness to injection of MTE in CFA at 24 mo of age. Lymphocytes from MTE-injected aged mice also failed to transfer thyroiditis to young recipients after in vitro activation of the lymphocytes with Con A.     

Disruption of CD40/CD40-ligand interactions in a retinal autoimmunity model results in protection without tolerance

We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.     

Absence of IL-4, and not suppression of the Th2 response,prevents development of experimental autoimmune Graves' disease

In autoimmune Graves' disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) and cause hyperthyroidism. We studied the effects of fms-like tyrosine kinase receptor 3 ligand (Flt3-L) or GM-CSF treatment on the development of experimental autoimmune GD (EAGD) in mice, a slowly progressing Ab-mediated organ-specific autoimmune disease of the thyroid induced by immunization with syngeneic cells expressing TSHR. Flt3-L and GM-CSF treatment resulted in up-regulation of CD8a(+) and CD8a(-) dendritic cells, and skewing of cytokine and immune responses to TSHR in favor of Th1 and Th2, respectively. However, this skewing did not persist until the later stages, and thus failed to affect the course or severity of the disease. To determine whether the total absence of either IL-4 or IFN-gamma could affect the development of EAGD, we immunized wild-type, IFN-gamma(-/-) and IL-4(-/-) BALB/c mice with TSHR. Nearly 100% of the wild-type and IFN-gamma(-/-) mice developed EAGD with optimal TSHR-specific immune responses, while IL-4(-/-) mice completely resisted disease and showed delayed and suboptimal pathogenic Ab response. These data demonstrated that skewing immune responses to TSHR, using either Flt3-L or GM-CSF, in favor of Th1 or Th2, respectively, may not be sufficient to alter the course of the disease, while the complete absence of IL-4, but not IFN-gamma, can prevent the development of EAGD.     

Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

The murine B cell repertoire is severely selected against endogenous cellular prion protein

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnp o/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnp o/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnp o/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnp o/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnp o/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.     

Induction of type 1 immune pathology in the brain following immunization without central nervous system autoantigen in transgenic mice with astrocyte-targeted expression of IL-12.

IL-12, a cytokine produced by microglia, may regulate cellular immunity at a localized level in the CNS. To investigate this further, we examined the consequences of peripheral immune stimulation without specific autoantigen in wild-type or transgenic (termed GF-IL12) mice with astrocyte production of the bioactive IL-12 p75 heterodimer. Active immunization with CFA and pertussis toxin, a procedure known to stimulate a robust type 1-biased immune response, produced CNS immune pathology from which GF-IL12 but not wild-type mice developed signs of clinical disease consisting of loss of activity, piloerection, mild tremor, and motor change. All immunized mice had some degree of mononuclear cell infiltration into the brain; however, the severity of this was markedly increased in GF-IL12 mice where leukocytes accumulated in perivascular and parenchymal locations. Accumulating cells consisted of CD4(+) and CD8(+) T cells and macrophage/microglia. Moreover, expression of cytokines (IFN-gamma and TNF), chemokines (IFN-inducible protein-10 and RANTES), the immune accessory molecules, MHC class II, B7.2, ICAM-1 and VCAM-1, and NO synthase-2 was induced in the CNS of the GF-IL12 mice. Therefore, peripheral immunization of GF-IL12 but not wild-type mice can provoke active type 1 immunity in the brain-a process that does not require CNS-specific immunizing autoantigen. These findings indicate that the cytokine milieu of a tissue can dramatically influence the development of intrinsic immune responses and associated pathology.     

Altered expression level of a systemic nuclear autoantigen determines the fate of immune response to self.

One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA) transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest that the expression level of systemic autoantigens influences the outcome of the immune response to self.     

Detection of the novel autoantibody (anti-UACA antibody) in patients with Graves' disease

Uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) is an autoantigen in patients with panuveitis such as Vogt-Koyanagi-Harada disease. The prevalence of IgG anti-UACA antibodies in patients with uveitis is significantly higher than healthy controls, suggesting its potential role as an autoantigen. Originally, UACA was cloned from dog thyroid tissue following TSH stimulation. So, we presumed UACA could be a novel autoantigen in autoimmune thyroid diseases. We measured serum anti-UACA antibody titer using ELISA in patients with autoimmune thyroid diseases (Graves' disease, Hashimoto's thyroiditis, subacute thyroiditis, and silent thyroiditis). The prevalence of anti-UACA antibodies in Graves' disease group was significantly higher than that in healthy group (15% vs. 0%). Moreover, the prevalence of anti-UACA antibodies in Graves' ophthalmopathy was significantly higher than that in Graves' patients without ophthalmopathy (29% vs. 11%). Especially, 75% of severe ocular myopathy cases showed high UACA titer. Immunohistochemical analysis revealed that UACA protein is expressed in eye muscles as well as human thyroid follicular cells. Taken together, UACA is a novel candidate for eye muscle autoantigens in thyroid-associated ophthalmopathy.     

Announcements

The effect of high doses of PHA in autoimmune thyroiditis was studied. The autoimmune response was elicited by the injection of syngeneic thyroglobulin (Tg) followed 3 hr later by LPS on days 0 and 7. PHA was injected 48 and 24 hr before the first antigen administration. The antibody titer to thyroglobulin and the degree of cellular infiltration in the thyroid were determined thereafter. It was found that PHA reduces the humoral response to Tg and completely suppresses the infiltration in the thyroid. However, this suppression is no longer observed 7 weeks later. By testing this effect with CFA instead of LPS, no PHA-induced suppressive effect was seen. The highest suppression is found when PHA is given close to the antigen injection.