To reflect human autoimmune thyroiditis, thyroid peroxidase (not thyroglobulin) antibodies should be measured in female (not sex-independent) NOD.H2h4 mice.

NOD.H2h4 mice are the most commonly used model for human autoimmune thyroiditis. Because thyroid autoimmunity develops slowly (over months), NOD.H2h4 mice are usually exposed to excess dietary iodide to accelerate and amplify the process. However, unlike the female bias in human thyroid autoimmunity, autoantibodies to thyroglobulin (TgAb) are reported to be similar in male and female NOD.H2h4 . We sought evidence for sexual dimorphism in other parameters in this strain maintained on regular or iodized water. Without iodide, TgAb levels are higher in males than in females, the reverse of human disease. In humans, autoantibodies to thyroid peroxidase (TPOAb) are a better marker of disease than TgAb. In NOD.H2h4 mice TPOAb develop more slowly than TgAb, being detectable at 6 months of age versus 4 months for the latter. Remarkably, unlike TgAb, TPOAb levels are higher in female than male NOD.H2h4 mice on both regular and iodized water. As previously observed, serum T4 levels are similar in both sexes. However, thyroid-stimulating hormone (TSH) levels are significantly higher in males than females with or without iodide exposure. TSH levels correlate with TgAb levels in male NOD.H2h4 mice, suggesting a possible role for TSH in TgAb development. However, there is no correlation between TSH and TPOAb levels, the latter more important than TgAb in human disease. In conclusion, if the goal of an animal model is to closely reflect human disease, TPOAb rather than TgAb should be measured in older female NOD.H2h4 mice, an approach requiring patience and the use of mouse TPO protein.

High-level intrathymic thyrotrophin receptor expression in thyroiditis-prone mice protects against the spontaneous generation of pathogenic thyrotrophin receptor autoantibodies.

The thyrotrophin receptor (TSHR) A-subunit is the autoantigen targeted by pathogenic autoantibodies that cause Graves' hyperthyroidism, a common autoimmune disease in humans. Previously, we reported that pathogenic TSHR antibodies develop spontaneously in thyroiditis-susceptible non-obese diabetic (NOD).H2h4 mice bearing a human TSHR A-subunit transgene, which is expressed at low levels in both the thyroid and thymus (Lo-expressor transgene). The present study tested recent evidence that high intrathymic TSHR expression protects against the development of pathogenic TSHR antibodies in humans. By successive back-crossing, we transferred to the NOD.H2h4 background a human TSHR A-subunit transgene expressed at high levels in the thyroid and thymus (Hi-expressor transgene). In the sixth back-cross generation (> 98% NOD.H2h4 genome), only transgenic offspring produced spontaneously immunoglobulin (Ig)G class non-pathogenic human TSHR A-subunit antibodies. In contrast, both transgenic and non-transgenic offspring developed antibodies to thyroglobulin and thyroid peroxidase. However, non-pathogenic human TSHR antibody levels in Hi-expressor offspring were lower than in Lo-expressor transgenic mice. Moreover, pathogenic TSHR antibodies, detected by inhibition of TSH binding to the TSHR, only developed in back-cross offspring bearing the Lo-expressor, but not the Hi-expressor, transgene. High versus low expression human TSHR A-subunit in the NOD.H2h4 thymus was not explained by the transgene locations, namely chromosome 2 (127-147 Mb; Hi-expressor) and chromosome 1 (22.9-39.3 Mb; low expressor). Nevertheless, using thyroiditis-prone NOD.H2h4 mice and two transgenic lines, our data support the association from human studies that low intrathymic TSHR expression is associated with susceptibility to developing pathogenic TSHR antibodies, while high intrathymic TSHR expression is protective.

Evidence that MHC I-E dampens thyroid autoantibodies and prevents spreading to a second thyroid autoantigen in I-A(k) NOD mice.

NOD.H2(k) and NOD.H2(h4) mice carry the major histocompatibility complex (MHC) class II molecule I-A(k) associated with susceptibility to experimentally induced thyroiditis. Dietary iodine-enhanced spontaneous thyroid autoimmunity, well known in NOD.H2(h4) mice, has not been investigated in NOD.H2(k) mice. We compared NOD.H2(h4) and NOD.H2(k) strains for thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) without or with dietary sodium iodide (NaI) for up to 32 weeks. TgAb levels were significantly higher in NOD.H2(h4) compared with NOD.H2(k) mice on NaI, and TPOAb developed in NOD.H2(h4) mice but not in NOD.H2(k) mice. DNA exome analysis revealed, in addition to the differences in the chromosome (Chr) 17 MHC regions, that NOD.H2(k) mice, and particularly NOD.H2(h4) mice, have substantial non-MHC parental DNA. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis highlighted thyroid autoimmunity and immune-response genes on Chr 17 but not on Chr 7, and 15 parental B10.A4R DNA. Studies of parental strains provided no evidence for non-MHC gene contributions. The exon 10 Tg haplotype, associated with experimentally induced thyroiditis, is absent in NOD.H2(h4) and NOD.H2(k) mice and is not a marker for spontaneous murine thyroid autoimmunity. In conclusion, the absence of I-E is a likely explanation for the difference between NOD.H2(h4) and NOD.H2(k) mice in TgAb levels and, as in humans, autoantibody spreading to TPO.

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.

Autoantibodies to the thyrotropin receptor.

This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion.

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (T(reg)) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A-Z). In transgenic and WT mice, regardless of T(reg) depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After T(reg) depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4(+) T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA-d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.

Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.

Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type.

IgG class thyroid peroxidase (TPO) autoantibodies with kappa light (L) chains predominate in serum and the genes for a large repertoire of such autoantibodies have been characterized. The present study was performed to clone and characterize TPO autoantibodies with lambda L chains which comprise approximately 20% of serum TPO autoantibodies. From a combinatorial IgG H/lambda L chain cDNA library in the phage display vector pComb3, 24 TPO-binding clones with lambda L chains were isolated, comprising three different heavy (H) and light (L) chain combinations. These combinations utilized two genes from the Vlambda II and IIIb families (closest germline genes DPL11 and hsigg11150) and three genes from the VH1, VH3 and VH4 families (VH26, 4.34 and hv1L1). The deduced amino acid sequences of these H chains were quite different from those of kappa F(ab) isolated using the same H chain library. We expressed the proteins for these three lambda F(ab), as well as for a lambda F(ab) (Humlv318 L chain/DP10-like H chain) previously isolated from another patient. The affinities for TPO of the lambda F(ab) (Kd 8 x 10(-10) M to 10(-7) M) were lower than those of the kappa F(ab) (Kd approximately 10(-10) M). For two lambda F(ab), both H and L chain genes were close to germline configuration, but there was no straightforward relationship between the extent of somatic mutation from germline configuration and affinity for TPO. All four lambda F(ab) bound less well to denatured TPO as to native TPO. The three F(ab) for which sufficient protein could be expressed for competition studies all recognized domain B within the immunodominant region on TPO previously identified using F(ab) with kappa L chains. Aside from these TPO-specific F(ab), only a few other human IgG class, organ-specific autoantibodies with lambda L chains have been characterized at the molecular level. Our study significantly augments the small database on this category of autoantibodies in general.

Profile of lambda light chain variable region genes in Graves' orbital tissue.

Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2/3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in both framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.

The quest for the autoantibody immunodominant region on thyroid peroxidase: guided mutagenesis based on a hypothetical three-dimensional model.

Thyroid peroxidase (TPO), a cell surface glycoprotein, is the major autoantigen in autoimmune thyroiditis in humans. The molecular cloning and expression of Ig genes from thyroid-infiltrating B cells has generated a large repertoire of human TPO Fab that have been used to map an immunodominant region on TPO. However, the topological site of this region, consisting of a cluster of highly conformational epitopes, remains unknown. Using the recently elucidated three-dimensional structure of myeloperoxidase as a model, we stably expressed on the surface of eukaryotic cells eight "guided" mutants of the TPO molecule. The sites of these mutations were strategically located to alter the surface contour of the molecule with minimal disruption to its core structure. Remarkably, in the present study (in contrast to previous unguided TPO mutagenesis studies), all eight TPO mutants retained recognition by the TPO Fab. These results support the validity of the model used for mutagenesis. Although not identifying the immunodominant region on TPO in thyroid autoimmunity, our data provide evidence against the involvement of certain topological segments and may help to narrow the search for this region. The most open region remaining as a candidate location is the antero-inferior portion of the molecule.

Evidence that cleavage of the thyrotropin receptor involves a "molecular ruler" mechanism: deletion of amino acid residues 305-320 causes a spatial shift in cleavage site 1 independent of amino acid motif.

Some TSH receptors (TSHR) on the cell surface cleave into A and B subunits. Cleavage at upstream Site 1 is followed by the proteolytic excision of an intervening C peptide region terminating at a downstream Site 2. Although present evidence suggests that Site 1 lies between amino acid residues 303 and 317, the mechanism and exact amino acid(s) involved in cleavage are unknown. Previous amino acid substitutions at Site 1 failed to abrogate cleavage. We, therefore, performed deletion mutations within this region. Cleavage of cell surface TSHR, detected by 125I-TSH cross-linking to intact cells, was not prevented by deletion of four individual segments within the Site 1 cleavage region (delta305-308, delta309-312, delta313-316, delta317-320). However, deletion of the entire region (delta305-320) reduced the extent of cleavage and shifted the cleavage site upstream of the glycan at amino acid residue N302. Elimination of this glycan (N302Q substitution) reversed the effect of deleting amino acid residues 305-320 on TSHR cleavage, suggesting that reduced cleavage at the new, upstream cleavage site was caused by steric hindrance by the glycan at N302. In summary, deletion, as opposed to mutagenesis, of the TSHR cleavage Site 1 region produces a spatial shift in TSHR cleavage Site 1 from downstream to upstream of the glycan at N302. These observations provide strong evidence that TSHR cleavage at this site does not occur at a particular amino acid motif and suggests that cleavage involves a "molecular ruler" mechanism involving cleavage at a fixed distance from a protease attachment site.

Both the 5' and 3' noncoding regions of the thyrotropin receptor messenger ribonucleic acid influence the level of receptor protein expression in transfected mamalian cells.

The molecular basis for the difference in the bioresponsiveness of TSH receptor cell lines from two different laboratories has been investigated. We modified our 4-kb TSH receptor complementary DNA (cDNA) by deleting either the 5' untranslated region (UTR), the 3'UTR, or both UTRs. The 5'UTR contains two false AUG initiation codons followed by a stop codon. The cDNAs in the eukaryotic expression vector pSV2-NEO-ECE, as well as the 5'3'UTR-truncated cDNA in pSVL, were stably transfected into Chinese hamster ovary cells. Pools of more than 100 colonies were studied in order to minimize insertion site-dependent variation in the level of expression. Scatchard analysis of TSH binding indicated that the number of receptors on the surface of Chinese hamster ovary cells expressing the wild-type transcript (approximately 16,000/cell) increased approximately 2-fold with 5'UTR deletion, approximately 5-fold with 3'UTR deletion, and approximately 10-fold with both 5'UTR and 3'UTR deletion. TSH binding affinities of all constructs were in the range of 2-5 x 10(-10) M. No significant difference was evident between the 5'3'UTR truncated cDNAs in the two different vectors, pSV2-NEO-ECE and pSVL. The increase in the amplitude of the cAMP response to TSH stimulation was commensurate with the number of receptors expressed on the surface of the different cell lines. Truncation of the 5'UTR did not alter TSH receptor messenger RNA (mRNA) levels relative to the wild-type mRNA. In contrast, the level of the 3'UTR-truncated transcript, as well as the 5'3'UTR-deleted transcript, increased approximately 4-fold independent of the expression vector used. In summary, both the 5'UTR and 3'UTR of the human TSH receptor mRNA influence the level of receptor expression on transfected mammalian cells. In particular, the 3'UTR has a destabilizing influence on the MRNA. These data explain the greater level of TSH receptor expression in cell lines that are transfected with cDNA lacking these regions of the mRNA transcript.

The greater glycan content of recombinant human thyroid peroxidase of mammalian than of insect cell origin facilitates purification to homogeneity of enzymatically protein remaining soluble at high concentration.

Structural studies on thyroid peroxidase (TPO), a major thyroid autoantigen, require milligram amounts of pure protein. We found that the human TPO ectodomain (amino acid residues 1-848) generated in insect cells did not remain in solution at high concentrations after affinity purification. In contrast, the TPO ectodomain secreted by mammalian (Chinese hamster ovary) cells, although generated to a lesser extent (1 vs. 8 mg/liter), remained in solution at high concentration (10 mg/ml) after purification to homogeneity. This purified material was well recognized by TPO autoantibodies, but lacked enzymatic activity. We attempted to restore activity by culturing the Chinese hamster ovary cells in the presence of added heme. TPO enzymatic activity was clearly detected in conditioned medium from cells cultured in hematin and hemin, but not in protoporphyrin IX (all at 1 mg/liter). Heme prosthetic group incorporation into affinity-purified TPO was highest for hematin and hemin, but unchanged for protoporphyrin IX (OD 410/280 nm ratios of 0.25, 0.23, and 0.14, respectively). Enzymatic activity was now evident with hemin (mean +/- SE, 27.2 +/- 2.6; n = 3; guaiacol units/mg protein), hematin (24.1 +/- 1.6), and, to a lesser extent, protoporphyrin IX (3.6 +/- 0.2). Culturing cells in 20 mg/liter hematin, the maximum concentration tolerated, increased enzymatic activity even further (45.6 +/- 0.6 guaiacol units/mg protein). All purified TPO preparations were homogeneous on PAGE and of similar size (105 kDa). Enzymatic deglycosylation showed a complex carbohydrate contribution of 13 kDa (unlike the 2.3 kDa in insect cell TPO). In conclusion, this is the first report on the purification to homogeneity of recombinant human TPO of mammalian cell origin. Unlike TPO generated in insect cells, mammalian TPO remains soluble at high concentration, possibly because of its greater carbohydrate content. This enzymatically active, recombinant human TPO may be useful for future structural studies.

On the functional importance of thyrotropin receptor intramolecular cleavage.

We examined the relationship between TSH receptor (TSHR) cleavage into two subunits and ligand-independent, constitutive activity characteristic of this receptor. Because of homology to the thrombin receptor-tethered ligand, we focused initially on a region in the vicinity of the second, downstream cleavage site of the TSHR ectodomain. We introduced into the wild-type TSHR three mutations. One mutation, TSHR(GQE(367-369)NET) prevents cleavage at site 2. The other two mutations, ELK(369-371)T-Y (TSHR-E1a2) and NPQE(372-375)SAIF (TSHR-E1b), introduce major changes into the potential tethered ligand. Basal, steady state intracellular cAMP levels in cloned, stably transfected Chinese hamster ovary cells were expressed as a function of the number of receptors (cAMP/receptor). None of these three mutations decreased ligand-independent constitutive activity, thereby excluding the tethered ligand hypothesis as well as a requirement for cleavage at site 2 in this process. Turning to the more upstream site 1 in the TSHR ectodomain, we examined a receptor (TSHR-delta50AA) with deletion of a unique 50-amino acid insertion (residues 317-366) that appears to be involved in cleavage at this site. Constitutive cAMP production was similar to that of the wild-type TSHR. Finally, we studied a TSHR mutant that cleaves at neither site 1 (deletion of residues 317-366) nor site 2 (GQE(367-369)NET substitution) and, therefore, does not cleave into A and B subunits. Again, the basal, constitutive level of cAMP production was similar to that of the wild-type TSHR. In summary, contrary to the prevailing hypothesis based on several lines of evidence, TSHR cleavage into subunits is not associated with constitutive, ligand-independent activity.

Recombinant thyroid peroxidase-specific autoantibodies. I. How diverse is the pool of heavy and light chains in immunoglobulin gene libraries constructed from thyroid tissue-infiltrating plasma cells.

Thyroid peroxidase (TPO) autoantibodies are a distinguishing feature of autoimmune thyroid disease. We have previously constructed immunoglobulin G heavy (H) and light (L) chain cDNA libraries from intrathyroidal B-cells. TPO-selected autoantibodies expressed by combined H and L chain libraries (combinatorial libraries) recognized a limited number of epitopes on TPO and used only a few of the many H and L chain variable region genes present in the genome (germline genes). One possible explanation for this restriction is a lack of diversity in the parental H and L chain gene libraries used to construct the combinatorial library. To address this issue, we determined the nucleotide sequences of randomly selected H and kappa L chain variable region genes from a pair of H and L chain libraries. The 12 H chain gene sequences analyzed were highly diverse, and none resembled the genes of TPO-selected autoantibodies. The sequences of 14 randomly selected kappa L chain genes were less diverse; 12 of 14 were closely related to the same germline gene (KL012) used by TPO-specific autoantibodies. However, we observed previously that only about 1 in 500 of the L chains in this library can pair with an H chain and bind TPO. We now find that, with 1 exception, the randomly selected KL012-like genes in the L chain library differ significantly from the antigen-specific KL012-like genes, particularly in the antigen-binding regions. In summary, the present data indicate that 1) the restricted number of H chain genes used by TPO-specific autoantibodies cannot be ascribed to limited H chain gene diversity in the parent library; and 2) L chains from combinatorial libraries (even when related to the same germline gene) cannot simply be regarded as plastic, or promiscuous, partners for high affinity antigen binding by a particular H chain.

Susceptibility rather than resistance to hyperthyroidism is dominant in a thyrotropin receptor adenovirus-induced animal model of Graves' disease as revealed by BALB/c-C57BL/6 hybrid mice.

We investigated why TSH receptor (TSHR) adenovirus immunization induces hyperthyroidism more commonly in BALB/c than in C57BL/6 mice. Recent modifications of the adenovirus model suggested that using adenovirus expressing the TSHR A subunit (A-subunit-Ad), rather than the full-length TSHR, and injecting fewer viral particles would increase the frequency of hyperthyroidism in C57BL/6 mice. This hypothesis was not fulfilled; 65% of BALB/c but only 5% of C57BL/6 mice developed hyperthyroidism. TSH binding inhibitory antibody titers were similar in each strain. Functional TSHR antibody measurements provided a better indication for this strain difference. Whereas thyroid-stimulating antibody activity was higher in C57BL/6 than BALB/c mice, TSH blocking antibody activity was more potent in hyperthyroid-resistant C57BL/6 mice. F(1) hybrids (BALB/c x C57BL/6) responded to A-subunit-Ad immunization with hyperthyroidism and TSHR antibody profiles similar to those of the hyperthyroid-susceptible parental BALB/c strain. In contrast, ELISA of TSHR antibodies revealed that the IgG subclass distribution in the F(1) mice resembled the disease-resistant C57BL/6 parental strain. Because the IgG subclass distribution is dependent on the T helper 1/T helper 2 cytokine balance, this paradigm can likely be excluded as an explanation for susceptibility to hyperthyroidism. In summary, our data for BALB/c, C57BL/6, and F(1) strains suggest that BALB/c mice carry a dominant gene(s) for susceptibility to induction of a thyroid-stimulating antibody/TSH blocking antibody balance that results in hyperthyroidism. Study of this genetic influence will provide useful information on potential candidate genes in human Graves' disease.

Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells.

The complementary DNA for the human TSH receptor (TSHR) translated region was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprecipitation of TSHR in whole cells precursor-labeled with [35S]methionine and [35S]cysteine revealed an approximately 10-fold increase in TSHR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,000 cells) compared to cells with the same gene not subjected to amplification (TSHR-0 cells). Similarly, [125I]TSH cross-linking to the surface of intact CHO cells revealed a progressive increase in TSH-binding sites with dihydrofolate reductase minigene amplification, with a 12.8-fold increase in TSHR in TSHR-10,000 vs. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express approximately 1.9 x 10(6) TSHR on their surface. Two ligand-TSHR complexes were evident under reducing conditions, representing the single chain holoreceptor of about 115 kDa and a dissociated A subunit of about 60 kDa. In the absence of TSH, basal cAMP levels in TSHR-10,000 cells were greater than those in TSHR-0 cells (6-fold in isotonic medium and 18.5-fold in hypotonic medium), indicating that the unliganded TSHR has significant constitutive activity. We assessed the kinetics of TSH binding to CHO cells overexpressing the TSHR using [125I]TSH in the presence of increasing concentrations of unlabeled TSH as well as by attempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (Kd = approximately 5 x 10(-10) M), we observed progressively lower affinities for TSH binding by TSHR-800 cells (Kd = approximately 10(-9) M) and TSHR-10,000 cells (Kd = approximately 2 x 10(-9) M). In summary, we report a high level of expression of TSHR in CHO cells and confirm the high constitutive activity of the TSHR in the absence of ligand as well as the binding of TSH to the single subunit, uncleaved TSHR. Moreover, we found that a high level of expression is associated with apparent negative cooperativity among the TSHR in terms of their affinity for ligand.

The influence of propranolol on the thyrotropin receptor.

Propranolol, a beta adrenergic blocking drug, is known to inhibit the thyrotropin (TSH) stimulation of adenosine-3',5'-monophosphate (cyclic AMP production in thyroid membranes but the mechansim of this inhibitory action is known. We have therefore investigated the influence of propranolol on the binding of 125I-labelled TSH to human thyroid membranes. Both d- and l-propranolol were found to enhance the binding of 125I-labelled TSH to thyroid membranes. The amount of label bound increased from about 30% in the absence of propranolol to about 60% in the presence of 3.3 x 10(-3)M propranolol. Scatchard analysis of the binding data indicated that propranolol increased the association constant of the thyrotropin-thyrotropin receptor interaction. Practolol, lithium carbonate, methimazole, and somatostatin had no effect on thyrotropin binding. This effect of propranolol appeared to be due to a direct reversible action of propranolol on the thyroid membranes and could be attributed to the membrane-disrupting properties of the drug rather than its beta-blocking activity. The increased TSH receptor occupancy which resulted from the increased association constant of the TSH-thyroid membrane interaction corresponded with a decrease in TSH-stimulated cyclic AMP formation. These data could indicate that propranolol reduced the efficiency of the receptor-adenylyl cyclase coupling system.

Evidence that the thyrotropin receptor ectodomain contains not one, but two, cleavage sites.

TSH receptor (TSHR) cleavage into two subunits (A and B) was explored using two new mammalian cell lines expressing the recombinant receptor; 1) TSHR-10,000 CHO cells overexpressing the TSHR; 2) TSHRmyc cells with a c-myc epitope inserted at residues 338-349. Immunoprecipitation or immunoblotting of TSHR-10,000 cells with mAb to either the A subunit or the B subunit revealed multiple forms of the TSHR: 1) uncleaved receptors of approximately 115 kDa and approximately 100 kDa with complex carbohydrate and high mannose carbohydrate, respectively; 2) two subunit TSHR with an approximately 62 kDa A subunit containing complex carbohydrate. The A subunit was approximately 35 kDa after enzymatic deglycosylation (predicted C-terminus near residue 330). The nonglycosylated B subunit was evident primarily as an approximately 42 kDa band (predicted N terminus near residue 380). The sum of the A and B subunit polypeptide backbones was smaller than the predicted size of the TSHR, a polypeptide backbone (84.5 kDa), raising the possibility that an approximately 5-kDa polypeptide fragment was excised during intramolecular cleavage. This hypothesis was supported by data obtained with the TSHRmyc cells. Thus, mAb to the c-myc epitope and to amino acid residues 22-35 (mAb A10) were equally effective in detecting the single chain forms of the TSHR in these cells. However, the 35 kDa, deglycosylated A subunit was clearly visible on immunoprecipitation with mAb A10 to the TSHR amino terminus, but not with the anti-myc mAb, indicating loss of the c-myc epitope at residues 338-349. Further, even though the A subunit was not detected in TSHRmyc cells with anti-myc mAb, 125I-TSH cross-linking to the cell surface showed similar A subunit expression in TSHRmyc and wild-type TSHR expressing cells. In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.

Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition.

Most thyroid peroxidase (TPO) autoantibodies in man recognize closely associated epitopes in two domains (A and B) on TPO. These epitopes were defined by recombinant monoclonal human autoantibodies expressed as antigen-binding fragments [F(ab)]. Only five heavy (H) and light (L) chain gene combinations encoded 34 F(ab), all of which have high affinity (Kd, approximately 10(-10) M) for TPO. We, therefore, investigated the roles of H and L chain genes in TPO domain recognition in two ways. First, we created hybrid F(ab) by forced recombination of H and L chain genes from 4 F(ab) recognizing the A or B domains. These hybrid F(ab) proteins, expressed in bacteria, bound extremely poorly (or not at all) to TPO, even at concentrations more than 100-fold higher than those required for detection of TPO binding by the original F(ab). Nucleotide sequencing of the cDNA as well as gel electrophoresis of the expressed proteins confirmed that poor hybrid F(ab) binding to TPO was not the result of cloning artifacts. Therefore, contrary to prevailing views on combinatorial libraries, we found no tolerance for H and L chain cross-combinations in high affinity TPO binding. These observations strengthen the likelihood that the H and L chain combinations from combinatorial libraries reflect those of TPO autoantibodies in vivo. In a second approach to examine the roles of H and L chains in TPO binding, we focused on three original F(ab) with similar L chains (encoded by KL012-like germline genes) and similar H chains (encoded by V1-3B-like germline genes), but different diversity (D) regions. All F(ab) bound predominantly to TPO domain A, as observed previously for a F(ab) with a KL012 L chain and a different H chain. Conversely, a F(ab) with a V1-3B-like H chain but a different L chain (A') bound to TPO domain B. These data indicate that the L chain plays a major role in defining TPO epitope recognition.