Yersinia enterocolitica provides the link between thyroid-stimulating antibodies and their germline counterparts in Graves' disease.

Graves' disease results from thyroid-stimulating Abs (TSAbs) activating the thyrotropin receptor (TSHR). How TSAbs arise from early precursor B cells has not been established. Genetic and environmental factors may contribute to pathogenesis, including the bacterium Yersinia enterocolitica. We developed two pathogenic monoclonal TSAbs from a single experimental mouse undergoing Graves' disease, which shared the same H and L chain germline gene rearrangements and then diversified by numerous somatic hypermutations. To address the Ag specificity of the shared germline precursor of the monoclonal TSAbs, we prepared rFab germline, which showed negligible binding to TSHR, indicating importance of somatic hypermutation in acquiring TSAb activity. Using rFab chimeras, we demonstrate the dominant role of the H chain V region in TSHR recognition. The role of microbial Ags was tested with Y. enterocolitica proteins. The monoclonal TSAbs recognize 37-kDa envelope proteins, also recognized by rFab germline. MALDI-TOF identified the proteins as outer membrane porin (Omp) A and OmpC. Using recombinant OmpA, OmpC, and related OmpF, we demonstrate cross-reactivity of monoclonal TSAbs with the heterogeneous porins. Importantly, rFab germline binds recombinant OmpA, OmpC, and OmpF confirming reactivity with Y. enterocolitica. A human monoclonal TSAb, M22 with similar properties to murine TSAbs, also binds recombinant porins, showing cross-reactivity of a spontaneously arising pathogenic Ab with Y. enterocolitica. The data provide a mechanistic framework for molecular mimicry in Graves' disease, where early precursor B cells are expanded by Y. enterocolitica porins to undergo somatic hypermutation to acquire a cross-reactive pathogenic response to TSHR.

[Association of TNF-α gene polymorphisms with Graves disease susceptibility and early course thyroid stimulating hormone receptor antibody level in Chinese Han population in Anhui region].

OBJECTIVE: To assess the association of tumour necrosis factor-α (TNF-α) gene polymorphisms at positions -863C/A, -857C/T, -238G/A and Graves disease (GD) susceptibility in Chinese Han population in Anhui region. METHODS: The polymorphisms of TNF-α gene were determined by polymerase chain reaction with specific primers in 254 patients affected with GD and 212 healthy controls. Allelic and genotypic frequencies in GD group and normal controls as well as in different genders were compared. The allelic and genotypic frequencies for different thyroid stimulating hormone receptor antibody (TRAb) levels (TRAb > 12 U/L; ≤12 U/L) were also compared among patients with earlier onset GD. RESULTS: (1) The A allele at -863C/A locus in GD group (16.73%) was significantly greater than that of the control group (11.79%) (P< 0.05, OR = 1.503); the frequency of AA+CA genotype of -863C/A locus in GD group (32.68%) was significantly greater than that of control group (23.58%) (P< 0.05, OR = 1.573). There was no significant difference (P> 0.05) in the allelic and genotypic frequencies of -857C/T, -238G/A loci between the two groups. (2) There was no significant difference (P> 0.05) in the allelic and genotypic frequencies of -863C/A, -857C/T, -238G/A loci between patients of different genders. (3) There was no significant difference (P>0.05) in such frequencies between patients with earlier onset GD and different TRAb levels (TRAb > 12 U/L; ≤12 U/L). CONCLUSION: (1) The -863 A allele of TNF-α gene may contribute to the development of GD in Chinese Han population in Anhui, whilst -857C/T, -238G/A alleles may not. (2) There is no association between TNF-α gene -863C/A, -857C/T, -238G/A polymorphisms and development of GD in different genders. (3) There was no association between above polymorphisms and TRAb levels in patients with earlier onset GD.

PDGF enhances orbital fibroblast responses to TSHR stimulating autoantibodies in Graves' ophthalmopathy patients.

PURPOSE: Thyroid-stimulating hormone receptor (TSHR) stimulating autoantibodies are associated with Graves' ophthalmopathy (GO), the orbital manifestation of Graves' disease (GD). TSHR autoantibody levels and orbital TSHR expression levels correlate positively with GO disease activity. Platelet-derived growth factors (PDGF) are increased in GO and potently activate orbital fibroblast effector functions. We investigated the possible relationship between PDGF and TSHR expression on orbital fibroblasts and how that influences the immunopathological effects of TSHR autoantibodies on orbital fibroblast activity. METHODS: Orbital fibroblasts were stimulated with PDGF-AA, PDGF-AB, and PDGF-BB, and TSHR expression was determined by flow cytometry. Stimulatory effects of bovine TSH and GD immunoglobulins on orbital fibroblasts (with or without PDGF-BB preincubation) were determined by IL-6, IL-8, chemokine (C-C motif) ligand (CCL)-2, CCL5, CCL7, and hyaluronan ELISA. The TSHR blocking antibody K1-70 and the cAMP inhibitor H89 were used to determine involvement of TSHR signaling. RESULTS: PDGF-AB and PDGF-BB stimulation increased TSHR expression on orbital fibroblasts, whereas PDGF-AA did not. Furthermore, stimulation with bovine TSH and immunoglobulins from GD patients induced IL-6, IL-8, CCL2, and hyaluronan production by orbital fibroblasts, and PDGF-BB preincubation enhanced this response of orbital fibroblasts. Blocking studies with a TSHR blocking antibody and a cAMP inhibitor inhibited these effects, indicating the involvement of TSHR signaling and thus of TSHR stimulating autoantibodies herein. CONCLUSIONS: These findings indicate that PDGF-B containing PDGF isoforms amplify the immunopathological effects of TSHR-stimulating autoantibodies in GO patients by stimulating TSHR expression on orbital fibroblasts.

Immunoglobulin heavy chain variable region genes contribute to the induction of thyroid-stimulating antibodies in recombinant inbred mice.

Graves' hyperthyroidism is an autoimmune disease occurring spontaneously in humans and caused by autoantibodies that stimulate the thyrotropin receptor. In mice, inducing Graves'-like hyperthyroidism requires in vivo expression of the thyrotropin receptor using plasmid or adenovirus vectors. However, mice with different genetic backgrounds vary markedly in their susceptibility to induced hyperthyroidism. Further, in some strains major disparities exist between the induction of hyperthyroidism and detection of thyroid-stimulating antibodies. To break tolerance, virtually all Graves' mouse models involve immunization with human thyrotropin-receptor DNA and the standard thyroid-stimulating antibody bioassay uses cells expressing the human thyrotropin receptor. We hypothesized, and now report, that disparities between hyperthyroidism and thyroid-stimulating antibody bioactivity are explained, at least in part, by differential antibody recognition of the human vs the mouse thyrotropin receptor. The genetic basis for these species differences was explored using genotyped, recombinant-inbred mouse strains. We report that loci in the immunoglobulin heavy chain variable region as well as in the major histocompatibility complex region contribute in a strain-specific manner to the development of antibodies specific for the human or the mouse thyrotropin receptor. The novel finding of a role for immunoglobulin heavy chain variable region gene involvement in thyroid-stimulating antibody epitopic specificity provides potential insight into genetic susceptibility in human Graves' disease.

Clonal relationships between thyroid-stimulating hormone receptor-stimulating antibodies illustrate the effect of hypermutation on antibody function.

Graves' disease is characterized by production of agonist antibodies to the thyroid-stimulating hormone receptor (TSHR), but knowledge of the genetic and somatic events leading to their aberrant production is limited. We describe the genetic analysis of two monoclonal antibodies (mAbs) with thyroid-stimulating activity (TSAb) obtained from a single mouse with experimental Graves' disease. The mAbs were class switched, but used the same rearrangement of immunoglobulin heavy chain, variable region (IGHV) and immunoglobulin light chain, variable region (IGLV) germline genes, implying a clonal relationship and derivation from a single precursor B-cell clone. The IGHV-region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity-determining regions (CDRs) and the framework regions. The cloned IGHV and IGLV genes were confirmed to have TSAb properties in experiments in which they were expressed as recombinant Fabs (rFabs). In other experiments, we swapped the IGLV genes with IGHV genes by constructing chimeric rFabs and showed that the chimeras retained TSAb activities, confirming the close functional relatedness of the V-region genes. Importantly, the IGLV genes in chimeric rFabs had a dominant stimulatory effect at low concentrations, while the IGHV genes had a dominant effect at higher concentrations. Our findings demonstrate that, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves' disease.

Attenuation of induced hyperthyroidism in mice by pretreatment with thyrotropin receptor protein: deviation of thyroid-stimulating to nonfunctional antibodies.

Graves'-like hyperthyroidism is induced by immunizing BALB/c mice with adenovirus expressing the thyrotropin receptor (TSHR) or its A-subunit. Nonantigen-specific immune strategies can block disease development and some reduce established hyperthyroidism, but these approaches may have unforeseen side effects. Without immune stimulation, antigens targeted to the mannose receptor induce tolerance. TSHR A-subunit protein generated in eukaryotic cells binds to the mannose receptor. We tested the hypothesis that eukaryotic A-subunit injected into BALB/c mice without immune stimulation would generate tolerance and protect against hyperthyroidism induced by subsequent immunization with A-subunit adenovirus. Indeed, one sc injection of eukaryotic, glycosylated A-subunit protein 1 wk before im A-subunit-adenovirus immunization reduced serum T(4) levels and the proportion of thyrotoxic mice decreased from 77 to 22%. Prokaryotic A-subunit and other thyroid proteins (thyroglobulin and thyroid peroxidase) were ineffective. A-subunit pretreatment reduced thyroid-stimulating and TSH-binding inhibiting antibodies, but, surprisingly, TSHR-ELISA antibodies were increased. Rather than inducing tolerance, A-subunit pretreatment likely expanded B cells that secrete nonfunctional antibodies. Follow-up studies supported this possibility and also showed that eukaryotic A-subunit administration could not reverse hyperthyroidism in mice with established disease. In conclusion, glycosylated TSHR A-subunit is a valuable immune modulator when used before immunization. It acts by deviating responses away from pathogenic toward nonfunctional antibodies, thereby attenuating induction of hyperthyroidism. However, this protein treatment does not reverse established hyperthyroidism. Our findings suggest that prophylactic TSHR A-subunit protein administration in genetically susceptible individuals may deviate the autoantibody response away from pathogenic epitopes and provide protection against future development of Graves' disease.

Development of peptide microarrays for epitope mapping of antibodies against the human TSH receptor.

Accurate characterization of the antigen binding region of antibodies is of great value in many fields of research, assay development and clinical diagnostics. Up to now, there is an unmet clinical need to use antibodies as diagnostic markers for the prediction of both prognosis and therapeutic response. To this end, comprehensive but differentiated immunoassays need to be generated. We have developed a peptide microarray for the diagnosis and epitope mapping of anti-thyrotropin receptor antibodies. The primary sequence of the human thyrotropin receptor (hTSHR) was represented by a library of 251 synthetic peptides. The peptides were site-specifically immobilized in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde coated glass surface. The technology was used to map the epitopes of seven commercially available murine monoclonal antibodies specific for the human TSH receptor (mTSHRAb). A previously unknown epitope recognized by mTSHRAb 4C1 was identified at amino acids (AA) 379 through 384 and the epitope recognized by mTSHRAb A9 was also localized (AA 214-222). Previously identified epitopes recognized by mTSHRAbs 2C11 (AA 349-360), 28 (AA 34-39), 49 (AA 289-297), A7 (AA 406-411) and A10 (AA 34-39) were confirmed. The peptide microarray exhibited excellent performance in single and multiplex antibody analysis and high specificity. This technology may have potential as a multi-determinate in vitro diagnostic assay for the differential analysis of a heterogeneity of antibodies involved in the pathogenesis of autoimmune diseases.

Insight into Graves' hyperthyroidism from animal models.

Graves' hyperthyroidism can be induced in mice or hamsters by novel approaches, namely injecting cells expressing the TSH receptor (TSHR) or vaccination with TSHR-DNA in plasmid or adenoviral vectors. These models provide unique insight into several aspects of Graves' disease: 1) manipulating immunity toward Th1 or Th2 cytokines enhances or suppresses hyperthyroidism in different models, perhaps reflecting human disease heterogeneity; 2) the role of TSHR cleavage and A subunit shedding in immunity leading to thyroid-stimulating antibodies (TSAbs); and 3) epitope spreading away from TSAbs and toward TSH-blocking antibodies in association with increased TSHR antibody titers (as in rare hypothyroid patients). Major developments from the models include the isolation of high-affinity monoclonal TSAbs and analysis of antigen presentation, T cells, and immune tolerance to the TSHR. Studies of inbred mouse strains emphasize the contribution of non-MHC vs. MHC genes, as in humans, supporting the relevance of the models to human disease. Moreover, other findings suggest that the development of Graves' disease is affected by environmental factors, including infectious pathogens, regardless of modifications in the Th1/Th2 balance. Finally, developing immunospecific forms of therapy for Graves' disease will require painstaking dissection of immune recognition and responses to the TSHR.

Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions.

Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8-9 degrees C improved thermal stability and 0.8-0.9 M improved stability for guanidinium chloride were found after 4-5 enrichment cycles. The most stable mutants selected contained either Lys(H)66Arg or Asn(H)52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.

Prevention of autoantibody-mediated Graves'-like hyperthyroidism in mice with IL-4, a Th2 cytokine.

Graves' hyperthyroidism has long been considered to be a Th2-type autoimmune disease because it is directly mediated by autoantibodies against the thyrotropin receptor (TSHR). However, several lines of evidence have recently challenged this concept. The present study evaluated the Th1/Th2 paradigm in Graves' disease using a recently established murine model involving injection of adenovirus expressing the TSHR (AdCMVTSHR). Coinjection with adenovirus expressing IL-4 (AdRGDCMVIL-4) decreased the ratio of Th1/Th2-type anti-TSHR Ab subclasses (IgG2a/IgG1) and suppressed the production of IFN-gamma by splenocytes in response to TSHR Ag. Importantly, immune deviation toward Th2 was accompanied by significant inhibition of thyroid-stimulating Ab production and reduction in hyperthyroidism. However, in a therapeutic setting, injection of AdRGDCMVIL-4 alone or in combination with AdCMVTSHR into hyperthyroid mice had no beneficial effect. In contrast, coinjection of adenoviruses expressing IL-12 and the TSHR promoted the differentiation of Th1-type anti-TSHR immune responses as demonstrated by augmented Ag-specific IFN-gamma secretion from splenocytes without changing disease incidence. Coinjection of adenoviral vectors expressing IL-4 or IL-12 had no effect on the titers of anti-TSHR Abs determined by ELISA or thyroid-stimulating hormone-binding inhibiting Ig assays, suggesting that Ab quality, not quantity, is responsible for disease induction. Our observations demonstrate the critical role of Th1 immune responses in a murine model of Graves' hyperthyroidism. These data may raise a cautionary note for therapeutic strategies aimed at reversing Th2-mediated autoimmune responses in Graves' disease in humans.

The influence of the exon 1 polymorphism of the cytotoxic T lymphocyte antigen 4 gene on thyroid antibody production in patients with newly diagnosed Graves' disease.

Increasing evidence supports the genetic susceptibility for thyroid antibody (TAb) production in patients with autoimmune thyroid disease, and recently, it has been shown that the cytotoxic T lymphocyte antigen 4 (CTLA-4) gene is most likely a major TAb susceptibility gene. To assess the relationship between exon 1 CTLA-4 gene polymorphism and TAb production, we genotyped 67 patients with newly diagnosed Graves' disease. Free thyroid hormones and TAb were measured, including thyroglobulin antibodies (TgAb), thyroid peroxidase antibodies (TPOAb), and thyroid-stimulating antibodies (TSAb). AA genotype was found in 25 patients, AG genotype in 34 patients, and GG genotype in 8 patients. G allele carrying genotypes showed significantly higher frequency of positive TPOAb (p < 0.005) and TgAb (p < 0.05) compared to AA genotype. Furthermore, the median values of TPOAb were significantly higher in the group with G allele (p < 0.002). However, the median values of TgAb and TSAb did not differ significantly between both groups and similarly, CTLA-4 genotype showed no association with serum free thyroxine (T(4)) and Graves' ophthalmopathy. In conclusion, our findings suggest that G allele carrying genotype of the CTLA-4 gene influences higher production of TPOAb and TgAb, and therefore, support the hypothesis that CTLA-4 gene plays a major role in TAb production.

Molecular analysis of stimulatory anti-thyrotropin receptor antibodies (TSAbs) involved in Graves' disease. Isolation and reconstruction of antibody genes, and production of monoclonal TSAbs.

Anti-thyrotropin (TSH) receptor autoantibodies (TRAbs) have been known to be involved in Graves' disease. To understand the molecular mechanism for pathogenesis of TSAbs in Graves' disease, we isolated and reconstituted the Ig genes of EBV-transformed B cell clones producing monoclonal thyroid stimulating Ab (TSAb) obtained from patients with Graves' disease. The V region genes of Ig heavy (H) and light (L) chains of two TSAb clones, IgG clone B6B7 and IgM clone 101-2, were isolated by the PCR. Nucleotide sequencing analysis revealed that germ-line VH and VK segments widely used for autoantibodies including the previously isolated TRAbs were utilized in the two clones. A significant number of somatic mutations were found in V regions of both clones, indicating the involvement of somatic mutations for the TSAb specificity. Reconstituted Ig H and L chain genes of the two clones were stably introduced into myeloma cells for IgG1 production. IgGs purified from cultured supernatants of both transfectants exhibited significant TSAb activities, while they did not inhibit TSH binding to the receptor. The successful expression of recombinant TSAbs in eukaryotic cells will provide opportunities to apply them to various pathophysiologic, diagnostic and therapeutic investigations in autoimmune thyroid diseases.