Differential Susceptibility of BALB/c and BALB/cBy mice to Graves' hyperthyroidism.

BALB/c mice are susceptible to the induction of Graves' hyperthyroidism. To investigate the susceptibility of BALB/c substrains of mice to the induction of hyperthyroidism, we immunized BALB/cJ and BALB/cByJ mice with an adenovirus expressing amino acid residues 1-289 of thyrotropin receptor (TSHR). The data presented in this article showed that 17 of 26 (65%) BALB/c and only 4 of 30 (13%) BALB/cBy mice developed hyperthyroidism.

Differential requirement of signal transducer and activator of transcription-4 (Stat4) and Stat6 in a thyrotropin receptor-289-adenovirus-induced model of Graves' hyperthyroidism.

T helper type 1 (Th1) and Th2 cells have critical roles in the development of cell-mediated and humoral immune responses, respectively. This division of function predicts that Th1 cells mediate inflammatory diseases and Th2 cells promote antibody (Ab)-mediated autoimmunity. Our previous studies using HEK-293 cells expressing the extracellular domain of the TSH receptor (TSHR) showed that Stat4-/- mice, which lack Th1 cells, are susceptible, whereas Stat6-/- mice, which lack Th2 cells, are resistant to the induction of Graves' hyperthyroidism.

Signal transducer and activator of transcription (Stat)-6-dependent, but not Stat4-dependent, immunity is required for the development of autoimmunity in Graves' hyperthyroidism.

The role of T helper (Th) cells in experimental models of Graves' hyperthyroidism is still somewhat controversial. To further investigate the role of Th1- and Th2-dependent immunity during the development of Graves' hyperthyroidism, we tested mice with targeted deletion of signal transducer and activator of transcription-4 (Stat4) or Stat6 genes that, respectively, have impaired Th1 and Th2 immunity. We immunized wild-type BALB/c, Stat4(-/-), or Stat6(-/-) mice with human embryonic kidney cells (293 cells) expressing the extracellular domain of human TSH receptor (293-TBP cells).

Animal models of Graves' hyperthyroidism.

An animal model of Graves' disease (GD) will help us to clearly understand the role of thyroid-stimulating hormone receptor (TSHR)-specific T cells and TSHR-Abs during the development of GD and to develop TSHR-specific immunotherapy. This review focuses on four different recent approaches towards the development of an animal model of GD. These approaches are: (1) Immunization of AKR/N mice with fibroblasts coexpressing syngeneic major histocompatibility complex (MHC) class II and TSHR.

Relevance of differential immunogenicity of human and mouse recombinant desmoglein-3 for the induction of acantholytic autoantibodies in mice.

Pemphigus vulgaris (PV) is an antibody-mediated autoimmune disease of the skin and mucous membranes. Desmoglein-3 (dsg-3) expressed in the suprabasal layer of the skin serves as an autoantigen in PV. Passive transfer of sera, either from patients with PV or from experimental animals immunized with a recombinant human dsg3 (hdsg3) into neonatal BALB/c mice results in blister formation, suggesting strongly that there is significant cross-reactivity between the mouse dsg3 (mdsg3) and the hdsg3.

Studies using recombinant fragments of human TSH receptor reveal apparent diversity in the binding specificities of antibodies that block TSH binding to its receptor or stimulate thyroid hormone production.

Patients with Graves' disease have autoantibodies that bind to the TSH receptor and stimulate the thyroid, leading to hyperthyroidism. Earlier studies have shown that the ectodomain of the glycosylated human TSH receptor contains epitopes that could adsorb these pathogenic antibodies. Further studies with mutated cDNAs, chimeric proteins, peptides, and antipeptide antibodies suggested that alterations in the conformation of the protein could lead to loss of reactivity, and that thyroid-stimulating antibodies interact with the N-terminal region of the TSH receptor.

Regulation of thyrotropin receptor protein expression in insect cells.

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE.

Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins.

The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated.

Comparison of immune responses to extracellular domains of mouse and human thyrotropin receptor.

The mouse and human thyrotropin receptors show greater than 87% homology in their amino acid sequences. However, glycosylated extracellular domains of mouse (mET-gp) and human (hET-gp) thyrotropin receptors showed differences in their ability to react with patient autoantibodies to thyrotropin receptor (TSHR). To test for potential differences in their immunogenicity, we immunized BALB/c mice with either gel pure non-glycosylated ectodomain of human TSHR (ETSHR II), or hET-gp (hET-gp III), or mET-gp (mET-gp III).

Neuroendocrine-induced synthesis of bone marrow-derived cytokines with inflammatory immunomodulating properties.

Although cytokines and other soluble regulators of immunity are known to be involved in hematopoiesis, little is known about the signals that induce the synthesis of those mediators locally. Based on recent studies linking the neuroendocrine hormone thyrotropin [thyroid-stimulating hormone (TSH)] to immune cell function in other tissues, we investigated the capacity of TSH to activate cytokine responses from bone marrow cells. These studies reveal that stimulation of the TSH receptor on bone marrow cells-using highly purified or recombinant TSH or by direct stimulation with anti-TSH receptor antibodies-rapidly induces the synthesis of cytokines from bone marrow cells that are classically used in the regulation of inflammatory responses.

Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes.

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities.

Thyrotropin-receptor-mediated diseases: a paradigm for receptor autoimmunity.

Autoantibodies to the thyrotropin receptor (TSHR) can act as thyrotropin agonists or antagonists, or can cause thyroid hypertrophy. Neither the autoantibody-binding sites on the TSHR nor the intracellular mechanisms by which the autoantibodies mediate their diverse functional effects are completely understood. This article reviews how cloning of the TSHR has contributed to our understanding of its structure and function, and has allowed induction of experimental autoimmunity to the TSHR.

Flow cytometric analyses of antibody binding to Chinese hamster ovary cells expressing human thyrotropin receptor.

To develop a method that can be used to directly detect binding of antibodies to TSH receptor (TSHr), we employed Chinese hamster ovary (CHO) cells permanently transfected with a human TSHr complementary DNA (CHOR). These cells showed increased cAMP production when treated with either human TSH or thyroid-stimulating antibodies and decreased TSH-mediated cAMP production when treated with stimulation-blocking antibodies. We employed flow cytometry and rabbit antibodies against the extracellular domain of the TSHr (ETSHr) to test whether these cells can be used to directly detect and quantitate the binding of anti-TSHr antibodies.

Differential reactivities of recombinant glycosylated ectodomains of mouse and human thyrotropin receptors with patient autoantibodies.

We expressed the extracellular domain of the mouse TSH receptor (mET-gp) using the baculovirus expression system. The recombinant protein was identified as mET-gp by immunoblotting and N-terminal amino acid sequencing. Carbohydrate analysis of the recombinant protein showed that the protein is glycosylated.

Requirement of glycosylation of the human thyrotropin receptor ectodomain for its reactivity with autoantibodies in patients' sera.

To understand the role of glycosylation on autoantibody reactivity, we expressed cDNA encoding amino acid residues 22 to 416 of the human thyrotropin receptor (TSHR), along with the baculovirus-encoded glycoprotein 67 signal sequence (ETSHR-gp) in insect cells. N-terminal sequence analysis revealed that the signal peptide was cleaved and confirmed the identity of ETSHR-gp protein. The molecular mass of the ETSHR-gp protein was 63 kDa and was higher than the expected molecular mass of 45 kDa, suggesting that the protein was glycosylated.

Lipoprotein from Yersinia enterocolitica contains epitopes that cross-react with the human thyrotropin receptor.

Yersinia enterocolitica has recently been shown to produce a low molecular mass envelope protein that contains an epitope(s) that is cross-reactive with the extracellular domain of the human thyrotropin receptor (ETSHR). In this study, we have generated mAb to this cross-reactive protein and have obtained amino acid sequences for peptide fragments obtained from Lys-c digestion of the protein. The amino acid sequences of these peptides were identical to sequences present in bacterial lipoprotein (LP).

Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells.

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells.

Yersinia enterocolitica envelope proteins that are crossreactive with the thyrotropin receptor (TSHR) also have B-cell mitogenic activity.

Autoantibodies to the thyrotropin receptor (TSHR) have been shown to mediate the hyperthyroidism associated with Graves' disease (GD). A number of hypotheses have been proposed which link an infectious agent to the mechanism(s) involved in the induction of GD. Several studies have suggested that the development of GD may be linked to infection with the enteric pathogen Yersinia enterocolitica.

Thyrotropin (TSH) receptor antibodies (TSHrAb) can inhibit TSH-mediated cyclic adenosine 3',5'- monophosphate production in thyroid cells by either blocking TSH binding or affecting a step subsequent to TSH binding.

In the present study, rabbit antibodies that possess thyroid stimulation-blocking activity were used to investigate potential mechanisms by which TSH receptor antibodies can inhibit thyroid cell function. The antibodies were produced against two synthetic peptides corresponding to amino acids 357-372 (p357) and 367-386 (p367) of the human TSHr (hTSHr). By enzyme-linked immunosorbent assay, both antisera (alpha 357 and alpha 367) had high titers ( > 1:100,000) of IgG against their respective peptides and recombinant extracellular TSHr protein (ETSHr); alpha 357 had a low IgG titer to p367 (1:800), and alpha 367 had a low IgG titer to p357 ( < 1:200).

Recombinant desmoglein 3 has the necessary epitopes to adsorb and induce blister-causing antibodies.

The development of an animal model for studying the pathogenesis of pemphigus vulgaris (PV) has been hampered by the unavailability of the purified full-length autoantigen desmoglein 3 (Dsg 3).Therefore, we expressed Dsg 3 using a baculovirus expressed system. The expressed protein was identified as Dgs 3 by its reactivity with a pan-cadherin anti-serum, an anti-serum to a Dsg 3 synthetic peptide, or patient serum, and by amino-terminal sequencing.

Influence of adjuvants on the induction of autoantibodies to the thyrotropin receptor.

To determine the influence of adjuvant on the induction of antibodies to thyrotropin receptor (TSHR), we immunized BALB/c mice with a extracellular domain of the TSHR (ETSHR) protein in complete Freund's adjuvant (CFA), Titer Max (TM) and Gerbu. Similarly, control groups of mice were immunized with bovine serum albumin (BSA) in each of the different adjuvants. As determined by ELISA, ETSHR given along with CFA elicited high titers of antibodies to ETSHR which were mainly restricted to the IgG1 subclass.

Experimental autoimmunity to thyrotropin receptor.

Since the cloning of a full length cDNA encoding the thyrotropin receptor (TSHr), several laboratories have been actively trying to develop an optimal animal model to understand the pathogenesis of TSHr mediated autoimmune diseases and have made considerable progress. To date, results from our laboratory have indicated that the nature of the antigen, and the adjuvant used for immunization, immunogenetic background of the animal and fine specificities of antibodies elicited might play an important role in determining the qualitative nature of the antibody response. Although an ideal animal model for either Graves' disease or primary myxedema is not yet available, ongoing studies in our laboratory and elsewhere hold promise for establishing animal models for various TSHr mediated autoimmune diseases in the near future.

Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: antibodies can bind to discrete conformational or linear epitopes and block TSH binding.

Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG2a, and IgG2b. The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others.

A region on the human thyrotropin receptor which can induce antibodies that inhibit thyrotropin-mediated activation of in vitro thyroid cell function also contains a highly immunogenic epitope.

Autoantibodies to the thyrotropin receptor (TSHr) bind to the extracellular domain of the TSHr (ETSHr) and either stimulate or inhibit thyroid cell function and/or growth. In order to investigate the regulation and the specificity of the immune response to the TSHr, our laboratory recently produced recombinant human ETSHr protein by using the baculovirus expression system. In the present study, we used the recombinant ETSHr protein, a panel of overlapping synthetic peptides derived from the TSHr, and polyclonal rabbit antibodies produced against recombinant ETSHr and synthetic peptides to define a highly immunogenic region (aa 352-388) of the TSHr.

High frequency of B cells capable of producing anti-thyrotropin receptor antibodies in patients with Graves' disease.

Hyperthyroidism in Graves' disease (GD) is mediated by antibodies to the thyrotropin receptor (TSHr). Patients that go into remission show a decline in antibody titer. However, upon cessation of treatment with anti-thyroid drugs a significant proportion of patients relapse and TSHr antibodies (TSHrAb) are present in their circulation.