help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kikuoka, S.
Right arrow Articles by Niimi, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kikuoka, S.
Right arrow Articles by Niimi, H.
Endocrinology Vol. 139, No. 4 1891-1898
Copyright © 1998 by The Endocrine Society

ARTICLES

The Formation of Thyrotropin Receptor (TSHR) Antibodies in a Graves’ Animal Model Requires the N-Terminal Segment of the TSHR Extracellular Domain

Shuichi Kikuoka, Naoki Shimojo, Ken-Ichi Yamaguchi, Yukihiko Watanabe, Akira Hoshioka, Aizan Hirai, Yasushi Saito, Kazuo Tahara, Leonard D. Kohn, Naoki Maruyama, Yoichi Kohno and Hiroo Niimi

Department of Pediatrics (S.K., N.S., K.Y., Ak.H., Y.K., H.N.) and Second Department of Internal Medicine (Ai.H., Y.S., K.T.), Chiba University School of Medicine, Chiba 260, Japan; SRL, Inc. (Y.W.), Hachioji 192, Japan; Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (L.D.K.), Bethesda, Maryland 20892; and Department of Molecular Biology, Tokyo Metropolitan Institute of Gerontology (N.M.), Tokyo 173, Japan

Address all correspondence and requests for reprints to: Dr. Naoki Shimojo, Department of Pediatrics and Clinical Research, National Shimoshizu Hospital, Yotsukaido, Chiba 284, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves’, including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves’ patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90–165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves’ TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9–165 (Mc1+2), 90–165 (Mc2), or 261–370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves’ TSHRAbs, but it is important also in their formation in a mouse model of Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TSH RECEPTOR (TSHR) plays a critical role in both the function and growth of the thyroid and is one of the major thyroid autoantigens. Anti-TSHR antibodies (anti-TSHRAbs) of two types are the hallmark of Graves’ disease (GD): TSH-binding-inhibiting immunoglobulins (TBIIs) and stimulating TSHRAbs (reviewed in Refs. 1 and 2). Studies of monoclonal anti-TSHRAbs from a multiplicity of laboratories have concluded that the two types of antibodies are different (1, 2, 3, 4, 5, 6, 7, 8); nevertheless, separate evidence indicates that both types of TSHRAbs have their functional epitope on the N-terminus of the TSHR (1, 2, 8, 9, 10, 11, 12, 13, 14, 15, 16). Thus, site-directed mutagenesis has defined important amino acid residues for stimulating TSHRAbs between residues 25–61 (1, 2, 10, 15, 16). Studies with the Mc2 and Mc1+2 TSHR-lutropin/choriogonadotropin receptor (TSHR/LH-CGR) chimeras, with residues 90–165 and 8–165 of the TSHR, respectively, substituted by comparable LH-CGR residues, localized the functional epitope for stimulating TSHRAbs on residues 22–165 of the N-terminal segment of the TSHR extracellular domain and demonstrated the importance of residues 90–165, independent of residues 25–61, as a stimulating TSHRAb epitope (8, 11, 12, 13, 14). Thus, 95% of patients lost all or most of their stimulating TSHRAb activity when large cohorts of patients were evaluated with the Mc2 chimera (12, 14); it is very likely that residues 30–61 and 90–165 are conformationally linked and interdependent. Recent studies, using the TSHR/LH-CGR chimeras, additionally identified two types of Graves’ TBIIs, each with different functional properties, whose epitopes are on the N-terminus of the TSHR extracellular domain; one type of Graves’ TBII has its epitope within residues 90–165, and the other has its epitope on residues 25–90 (8, 13).

Although we have progressed in our knowledge of the epitopes of the TSHRAbs in Graves’ patients, we know little of the regions on the TSHR important in their formation. Recently, we developed a model of GD by immunizing AKR/N mice with murine fibroblasts transfected with the TSHR and a murine major histocompatibility complex (MHC) class II molecule, having the same H-2k haplotype, but not either alone (17). Contrary to studies that immunized mice with the extracellular domain of the TSHR alone (18, 19, 20, 21, 22, 23, 24, 25, 26, 27), we were able to induce immune thyroid disease with the humoral and histological features of human Graves’, including the presence of stimulating TSHRAbs and hyperthyroidism, in 17–25% of mice (17). We observed the formation of TBIIs in 90% of mice; the difference in stimulating TSHRAb and TBII formation supported conclusions that these were separate populations of antibodies.

The Graves’ model opens the door to studies of how TSHRAbs are formed. In this report, we used the Graves’ model to question whether the functional epitopes for stimulating TSHRAbs and TBIIs on the N-terminus of the extracellular domain were critical also in TSHRAb formation. We immunized the AKR/N mice with murine fibroblasts transfected with each of three human (h) TSHR/rat LH-CGR chimeras with residues 9–165 (Mc1+2), 90–165 (Mc2), or 261–370 (Mc4) substituted by equivalent residues of the rat LH-CGR. The first two chimeras are missing the N-terminal portion of the extracellular domain of the TSHR wherein most Graves’ stimulating TSHRAbs and TBIIs have their functional epitope (8, 11, 12, 13, 14). The Mc4 chimera is missing the C-terminal portion of the TSHR extracellular domain. We show that the N-terminal region of the TSHR, which is substituted in the Mc2 and Mc1+2 chimeras, is not only critical as an epitope for functional Graves’ TSHR autoantibodies, but also is critical for their formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental procedures were conducted in accordance with the policies of Chiba University’s Animal Care and Use Committee. All AKR/N (H-2k) mice were female and were used at 7 weeks of age in all experiments.

Fibroblasts
The murine L cell fibroblast line (28), which expresses a hybrid gene containing Aßk and Aßd of murine MHC class II (RT4.15HP) and was kindly provided by Dr. R. N. Germain (NIAID, NIH), is the same as used in our previous study (17). The Aßd determinant is membrane proximal and was shown not to be associated with antigen presentation (28); thus, this shuffled I-Ak molecule is not different from I-Ak in antigen- presenting activity, and there are no qualitative differences in either T cell or antibody-recognition of I-Ak molecules containing either hybrid or wild-type Aßd (28). The control class II-untransfected murine L cell fibroblasts (DAP.3) also were provided by Drs. R. N. Germain and Prof. T. Saito (NIH and Chiba University School of Medicine).

TSHR gene transfection
The cloning and characterization of the hTSHR, amplification of rat LH-CGR complementary DNA (cDNA) fragments by PCR, and creation of the TSHR/LH-CGR chimeras were reported previously (11). The chimeric receptors used in this report are designated to indicate the substituted segment of the hTSHR, numbered from the methionine start site (Fig. 1Go). In Mc1+2, residues 8–165 of the hTSHR were replaced by residues 10–166 of the LH-CGR; in Mc2, residues 90–165 of the TSHR were replaced by residues 91–166; in Mc4, TSHR residues 261–370 were replaced by residues 261–329. Chimeric receptor cDNA, as well as the hTSHR and rat LH-CGR cDNAs, were subcloned into pSG5 expression vectors, and amplified DNA was purified by CsCl gradient centrifugation (11, 29).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Structure of the Mc1+2, Mc2, and Mc4 hTSHR/rat LH-CGR chimeras. The wild-type hTSHR is diagrammatically represented on top; the restriction sites used for chimera construction are noted. Open bars indicate the sequence of the extracellular domain of the TSHR; cross-hatched bars denote rat LH-CGR sequence. Numbers indicate the amino acid residue starting each segment, as numbered from the methionine start site. Chimera receptors are named according to the segment substituted by LH-CGR sequence.

 
After subcloning into a pSG5 vector (Stratagene, La Jolla, CA), the hTSHR or the chimeras were transfected into RT4.15HP cells, together with pMAMneo (Clontech, Palo Alto, CA), using lipofectin (GIBCO BRL, Gaithersburg, MD), as described (17). Cells were selected for neomycin resistance using 500 µg/ml G418 (GIBCO BRL); stable transfectants were selected by their ability to increase cAMP levels in the presence of TSH (17). Positive cells were cloned by limiting dilution. Control RT4.15HP cells, transfected with the pSG5 vector alone, also were established.

Immunization of mice with transfectants
Immunization of mice with the transfectants was as described (17). Briefly, mice were ip immunized every 2 weeks with 107 fibroblasts that had been pretreated with mitomycin C (Kyowa Hakko Kogyo, Tokyo). Two weeks after the sixth and final immunization, mice were killed and bled. These mice were chosen because they have the same class I and a homologous class II I-A molecule to that of the fibroblasts containing the transfected class II and TSHR cDNAs (17). The immunization protocol was chosen because mice first developed hyperthyroidism in significant numbers after the sixth immunization (17).

TSHRAb and thyroid function assays
Commercial RIA kits (RSR Limited, Cardiff, UK) were used for the ability of antibodies in the serum to inhibit [125I]TSH binding (TBII activity) and to measure serum T4 levels (Dai-ichi Radioisotopes, Tokyo). Stimulating TSHRAb activity was measured using hTSHR-transfected Chinese hamster ovary (CHO) cells (13). In brief, 4,000 hTSHR-transfected CHO cells were plated in 96-well flat-bottom plates and cultured for 48 h in growth medium. Cells were washed with HBSS and incubated with 25 µl protein A-purified IgG (2 mg/ml) and 175 µl assay buffer (8 mM Na2PO4, 1.5 mM KH2PO4, 0.9 mM CaCl2, 2.7 mM KCl, 0.5 mM MgCl2, 222 mM sucrose, 0.5 mM 3-isobutyl-1-methylxanthine, 0.1% glucose, and 1% BSA). After a 3-h incubation at 37 C, supernatants were collected, and cAMP was measured with a commercial RIA kit (Yamasa Co. Ltd., Chiba, Japan). The ability of the IgG to inhibit TSH activity was measured using the same incubation conditions and hTSHR-transfected CHO cells, except that 10 µU/ml bovine TSH was present with each IgG. IgG was obtained from the sera of pairs of animals within each experimental group to overcome technical problems related to obtaining sufficient serum for all measurements in this report.

In all cases, incubations were in duplicate, and all wells were assayed in duplicate. All experiments included normal mouse IgG as a negative control and both a known Graves’ stimulating TSHRAb and purified bTSH as positive controls.

Flow cytometry analysis of transfectants
Fibroblasts (106 cells) were incubated with 1 µg monoclonal anti-I-Ak (MHC class II-specific) or anti-Dk (MHC class I-specific) antibodies obtained from the American Tissue Culture Collection, 10.2.16 or 15–5-S, respectively, or an isotype-specific control monoclonal antibody (Becton Dickinson, Mountain View, CA). After 30 min on ice, cells were washed with PBS at pH 7.4 and incubated for 30 min with fluorescein-isothiocyanate-conjugated goat antimouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), then analyzed by flow cytometry on a FACScan Cytometer using CellQuest software (Becton Dickinson).

Proliferation assay
The spleens from the mice immunized with hTSHR-transfected RT4.15HP cells were excised, and a cell suspension was prepared. Thirty million cells were incubated with 106 mitomycin C-treated hTSHR-transfected RT4.15HP cells in a 25-cm2 culture flask, at 37 C, in 7% CO2, and in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 50 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin. After 10 days, cells were harvested and assayed for proliferative response to Mc2 or Mc1+2 chimera-transfected fibroblasts. In brief, 2 x 104 cells were incubated with 105 mitomycin C-treated fibroblasts in triplicate and in flat-bottom microplates. After a 72 h incubation, the cultures were pulsed with [3H]thymidine (0.5 µCi/well) for 16 h. Cells were collected with a harvester, and [3H]thymidine incorporation was measured by liquid scintillation spectrometry.

Other assays and statistics
Protein concentration was determined by Bradford’s method (Bio-Rad, Richmond, CA); recrystallized BSA was the standard. Statistical analysis was performed by one-way ANOVA to determine the P value between different groups and by Spearman’s rank correlation coefficient to validate the correlation between two series of data.

Materials
Purified bovine TSH preparation was the kind gift of the NIH hormone distribution program (NIDDK-bTSH, 30 U/mg).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the TSHR- and TSHR/LH-CGR-transfected fibroblasts
When the murine MHC class II-transfected fibroblast cell line, RT4.15HP, was transfected with the hTSHR or the TSHR/LH-CGR chimeras, all expressed the receptor in a functional array, exhibiting TSH-increased stimulation of the cAMP signal system (Fig. 2Go). The ability of the Mc2 or Mc1+2 chimeras to exhibit higher activity than the wild-type TSHR has been observed in CHO cells transfected with these chimeras (12).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. Bovine TSH-induced cAMP response of hTSHR-transfected fibroblasts. RT4.15HP cells transfected with the hTSHR (open squares), the Mc2 chimera (dark squares) Mc1+2 (dark diamonds), Mc4 (open circles), or vector alone (pSG5; closed circles) were stimulated with the indicated concentrations of bovine TSH for 3 h. Then the supernatants were collected, and cAMP in the supernatant was measured using a commercial RIA kit.

 
The transfection procedures did not result in different levels of class I expression, which were similar in all cells. Thus, flow cytometry analysis showed that hTSHR- and TSHR/LH-CGR chimera-transfected RT4.15HP cells expressed comparable levels of class I molecules on their cell surface, both with respect to each other and to the DAP.3 controls (Fig. 3Go, column 2). The hTSHR- and TSHR/LH-CGR chimera-transfected RT4.15HP cells all, however, exhibited increased surface expression of class II antigen, by comparison with the DAP.3 control cells, which exhibited no class II surface expression (Fig. 3Go, column 3).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 3. Surface expression of MHC class I (column 2) and class II (column 3) molecules on the surface of murine fibroblasts used for immunization. Procedures were performed as described in Materials and Methods; experiments in column 1 were performed using a control antibody.

 
Mc1+2- or Mc4-transfected fibroblasts elicited a proliferative response when incubated with spleen cells from mice immunized with the TSHR, who do develop TBIIs and stimulating TSHRAbs (Fig. 4Go), indicating that Mc1+2- and Mc4-transfected cells have T cell epitope(s). The hTSHR- and TSHR/LH-CGR chimera-transfected RT4.15HP and DAP.3 cells, or vector-transfected RT4.15HP cells, were used to immunize AKR/N mice.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Proliferative responses of spleen cells from mice immunized with TSHR-transfected fibroblasts to chimera receptor-transfected fibroblasts. Spleen cells from mice immunized with RT4.15HP cells transfected with wild-type TSHR were stimulated with 106 mitomycin C-treated fibroblasts transfected with wild-type TSHR for 10 days, then tested for their responses to chimera receptor-transfected fibroblasts in a 3-day proliferation assay. Data are presented as mean ± SD.

 
TSHRAbs produced by TSHR/LH-CGR chimeras
As previously reported (17), measurements of TBII activity showed that most mice immunized with hTSHR-transfected RT4.15HP cells developed serum TBII activity (Fig. 5Go). This was not true of the mice in the same experiment which were immunized with vector-transfected RT4.15HP cells (Fig. 5Go) or with DAP.3 cells expressing hTSHR alone (data not shown; see Ref.17). Three independent experiments yielded the same results: most mice immunized with hTSHR-transfected RT4.15HP cells, but not with control cells, developed serum TBII activity. A reasonably constant proportion of mice, 17 to 25%, developed elevated T4 values and stimulating TSHRAbs (17). This was again not true of mice immunized with vector-transfected RT4.15HP cells or with DAP.3 cells expressing hTSHR alone (17).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 5. TBII levels in the sera of mice immunized with fibroblasts transfected with hTSHR/rat LH-CGR chimeras. Each dot indicates the TBII titer of an individual mouse. Inhibition activity exceeding 20% (+2 SD > mean of controls) was considered positive, based on experiments in which mice were immunized with vector-transfected RT4.15HP (21).

 
In neither case did immunization of mice with fibroblasts transfected with the Mc2 or Mc1+2 TSHR/LH-CGR chimeras result in the formation of TBII antibodies (Fig. 5Go). This was true in 3 separate experiments, with a total of 20 mice, and this was statistically significant (P < 0.01). No mouse immunized with fibroblasts containing the Mc2 or Mc1+2 chimera developed increased thyroid hormone levels (Fig. 6Go), nor did they develop IgGs with stimulating TSHRAb activity (Fig. 7AGo) or IgGs with the ability to inhibit TSH-increased cAMP levels in CHO cells transfected with the wild-type TSHR (Fig. 7BGo), as illustrated for the Mc1+2-immunized mice. As previously reported (17), the ability to inhibit TSH-increased cAMP levels is an activity associated with Graves’ TBII activity (8). The absence of hyperthyroidism and stimulating TSHRAb activity relative to mice immunized with wild-type TSHR was statistically significant (P < 0.05). Mice immunized with the Mc4 chimera developed TBIIs with the same frequency as mice immunized with the wild-type TSHR (Fig. 5Go).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Serum T4 levels in mice immunized with fibroblasts transfected with Mc1+2 and Mc2 hTSHR/rat LH-CGR chimeras. Each dot indicates serum T4 of an individual mouse. T4 levels higher than 4 µg/dl were considered significantly elevated, based on the T4 levels in the control mice immunized with vector-transfected RT4.15HP cells (mean + 3 SD). Data are from a representative of 3 independently performed experiments. Whereas in all experiments, 24% of mice immunized with hTSHR-transfected RT4.15HP cells had elevated T4 values, no mice immunized with Mc1+2- or Mc2-transfected RT4.15HP cells (n = 40 total) had elevated T4s. Serum T4 in mice immunized with Mc4-transfected RT4.15HP cells are not shown because of an insufficient amount of sera to be measured.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Ability of IgG from mice immunized with fibroblasts transfected with hTSHR or TSHR/LH-CGR chimeras to increase cAMP levels (A) or to inhibit TSH-increased cAMP levels, an activity associated with TBII activity (B). Because only small amounts of serum could be obtained from individual mice, the sera from 2 mice were pooled, and the IgG was purified on a protein A-Sepharose column. The data presented were obtained from vector-transfected (pSG5) RT4.15HP-immunized mice whose TBII and T4 levels were within normal limits (Fig. 4Go), from mice immunized with hTSHR-transfected RT4.15HP cells who had high serum T4 levels with positive TBII activity, or from mice immunized with the Mc1+2 chimera. Data are shown as the mean of duplicate determinations. These data were duplicated by multiple different pools (>6) of IgG from comparable animals in each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GD is an autoimmune thyroid disease characterized by the presence of antibodies against the TSHR that stimulate the thyroid to cause hyperthyroidism and/or goiter. By immunizing mice with fibroblasts transfected with the hTSHR and a MHC class II molecule, but not by either alone, we have induced immune hyperthyroidism that has the major humoral and histological features of GD in AKR/N mice: stimulating TSHRAbs, TBIIs which are different from the stimulating TSHRAbs, increased thyroid hormone levels, thyroid enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen (17).

The development of a GD model in mice enabled us to examine questions concerning the mechanism by which TSHR autoantibodies might form. Evidence had accumulated that the N-terminus of the extracellular domain of the TSHR, residues 25–165, contained critical functional epitopes for both stimulating TSHRAbs and TBIIs (8, 9, 10, 11, 12, 13, 14, 15, 16), both of which develop in the AKR/N mice immunized with murine fibroblasts transfected with the TSHR and murine MHC class II cDNAs (17). Though the importance of the N-terminal region as epitope for the stimulating TSHRAbs to exert their function is clear, its role in induction of TSHRAb in GD, so far, has not been studied. In this report, to see whether the functional epitope was related to the formation of the TSHRAbs, we immunized mice with the Mc2 and Mc1+2 TSHR/LH-CGR chimeras that substitute TSHR residues 90–165 and 8–165 with comparable LH-CGR residues, the same chimeras that were used to identify these critical functional epitopes (8, 11, 12, 13, 14). We anticipated that N-terminal substitution by LH-CGR might lead to inability to generate stimulating TSHRAb and, in fact, we found no such activity in the IgG fraction from mice immunized with Mc1+2- or Mc2-transfected fibroblasts. These data are in accord with, and support, previous results that indicate that the N-terminal region is important for stimulating TSHRAb recognition (8, 9, 10, 11, 12, 13, 14, 15, 16).

Of special interest was the fact that mice immunized with the N-terminal region-substituted chimeric receptors did not, at all, form TSHRAb detected by the TBII assay. Mc1+2 or Mc2 has no substitution at the C-terminal portion of the extracellular domain of the TSHR including immunodominant epitope, residues 357–372. This last point is of interest for the following reasons. Several groups reported that immunization of animals with the immunodominant peptide led to the induction of TSHRAb (30, 31, 32, 33). Furthermore, the immunodominant peptide could adsorb TSHRAb in Graves’ patients (34, 35, 36), suggesting that this region might be quite important for the formation of the TSHRAb. If this assumption is true, immunization with Mc1+2 or Mc2 should lead to induction of TSHRAb; however, this was not the case in our system. The discrepancy in results, i.e. TSHRAb production by peptide immunization vs. our present result, may be caused by the difference in systems employed; however, TSHR peptide immunization also gave different results, compared with immunization with the soluble TSHR extracellular domain. Thus, immunization with peptides at the N-terminal region of the TSHR extracellular domain induced stimulating TSHRAb without any TBII activity (37), whereas immunization of the entire extracellular domain of the TSHR largely resulted in production of TBII but not stimulating TSHRAb (18, 19, 20, 21, 22, 23, 24). These data suggest that synthetic peptides are not necessarily antigens presented to the immune system in vivo and that production of TSHRAb by immunization with the TSHR peptides may not reflect the mechanism of TSHRAb formation in Graves’, nor may they represent functional antibodies, because they are not associated with significant hypo- or hyperthyroidism (37). Because our system to immunize mice with TSHR-transfected fibroblasts induces not only stimulating TSHRAb and TBII, both found in Graves’, but also hyperthyroidism and morphological changes in the thyroid consistent with Graves’ (17), in vivo mechanisms for generation of TSHRAb, may strictly require the N-terminal region of the TSHR. Immunization with the Mc4 chimera, which retains the N-terminal sequence of the extracellular domain but substitutes the C-terminus, resulted in the formation of TBIIs, in contrast to immunization with Mc2 and Mc1+2 chimeras. The importance of the N-terminus of the extracellular domain of the TSHR, and not the C-terminus, in the formation of TBIIs, therefore, was reinforced.

There are several possible explanations for the inability of fibroblasts expressing N-terminal region-substituted chimeric receptor to raise TSHRAb. First, a different primary and/or tertiary structure in the N-terminal region-substituted chimeric receptors might not allow the generation of antibodies with substantial binding affinity for conformational epitope(s) in native TSHR, because some Graves’ IgGs span the N- and C-terminus and recognize determinants on both termini (2, 9, 34, 35, 36). It would be possible that immunization with N-terminal region-substituted chimeric receptors raise antibodies to N-terminus of the LH-CGR and/or those to TSHR amino acids 166–415. We tried to detect antibodies to chimera-receptor by flow cytometry analysis; however, this procedure did not work, perhaps because of low expression levels of transfected receptor or because this procedure is not very sensitive, as recently reported (38, 39). Thus, less than 50% of Graves’ patients have antibodies detectable by this procedure, and there is no correlation with either TBII or stimulating TSHRAb. Detection of antibodies to the immunogenic peptide, aa 352–366, by Western blotting, also failed; but again, this could reflect poor assay sensitivity. A second possibility is that the N-terminal region contains B cell epitope(s) initially recognized by the immune system. In contrast to epitope spreading from C- to N-terminus of the extracellular domain suggested by some reports (14, 34, 35, 40), the Davies group has suggested, by studying the binding ability of TSHRAb to a panel of TSHR peptides, that TSHRAb epitopes in CBA/J mice spread from N- to C-terminus during serial immunization with the extracellular domain of TSHR (27). Because AKR/N mice have the same H-2 haplotype (H-2k) with CBA/J, the N-terminal region may be a primary B cell epitope for AKR/N mice, the strain used in these experiments. Deletion of a primary B cell epitope on the TSHR may be the cause for inability in epitope spreading, which subsequently leads to high affinity TSHRAb that inhibit TSH binding to the TSHR. This hypothesis and the first possibility are not mutually exclusive. Finally, a role of the N-terminal region as T cell epitopes may exist. Although we showed Mc1+2 contained epitopes recognized by T cells from mice immunized with native TSHR, suggesting that T cell epitope(s) exist on the portion other than segment 1+2, T cells that recognize the N-terminal region may be critical in generation of TSHRAb, in terms of help for B cells. There have been no data reported on the relationship between T cell epitope specificity and their functions in terms of help for TBII vs. stimulating TSHRAb. Because our murine model induces both types of antibodies, isolation and characterization of TSHR-specific T cells will give important information, to understand the mechanisms for generation of TSHRAbs in GD.

In summary, our present report clearly shows that the same N-terminal region of the extracellular domain that is important as epitope for TBIIs and stimulating TSHRAbs, but not C-terminal region, is important in their formation in the mouse model of GD.


   Acknowledgments
 
We thank Dr. R. N. Germain and Prof. T. Saito for providing RT4.15HP and DAP.3 cells; Ms. S. Uchiyama for help in measuring TBII and thyroid hormone levels; Dr. T. Ban for helpful discussions; and Ms. Y. Tsuchikawa and Ms. Y. Okuda for excellent technical assistance.

Received July 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M 1995 The thyrotropin receptor. Vitam Horm 50:287–384[Medline]
  2. Kohn LD, Giuliani C, Montani V, Napolitano G, Ohmori M, Ohta M, Saji M, Schuppert F, Shong M-H, Suzuki K, Taniguchi S-I, Yano K, Singer DS 1995 Antireceptor immunity. In: Rayner D, Champion B (eds) Thyroid Immunity. R. G. Landes Biomedical Publishers, Austin/Georgetown, pp 115–170
  3. Valente WA, Vitti P, Yavin Z, Grollman EF, Schneider M, Rotella CM, Zonefrati R, Toccafondi RS, Kohn LD 1982 Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves’ disease. Proc Natl Acad Sci USA 79:6680–6684[Abstract/Free Full Text]
  4. Ealey PA, Kohn LD, Ekins RP, Marshall NJ 1984 Characterization of monoclonal antibodies derived from lymphocytes from Graves’ disease patients in a cytochemical bioassay for thyroid stimulators. J Clin Endocrinol Metab 58:909–914[Abstract/Free Full Text]
  5. Yokoyama N, Izumi M, Katamine S, Nagataki S 1987 Heterogeneity of Graves’ immunoglobulin G: comparison of thyrotropin receptor and antibodies in serum and in culture supernatants of lymphocytes transformed by Epstein-Barr virus infection. J Clin Endocrinol Metab 64:215–218[Abstract/Free Full Text]
  6. Li H, Akamizu T, Okuda J, Sugawa H, Matsuda F, Tsubata T, Mori T 1995 Isolation of Epstein-Barr virus transformed lymphocytes producing IgG class monoclonal antibodies using a magnetic cell separator (MACS): preparation of thyroid stimulating IgG antibodies from patients with Graves’ disease. Biochem Biophys Res Commun 207:985–993[CrossRef][Medline]
  7. Morgenthaler NG, Kim MR, Tremble J, Huang GC, Richter W, Gupta M, Scherbaum WA, McGregor AM, Banga JP 1996 Human immunoglobulin G autoantibodies to the thyrotropin receptor from Epstein-Barr Virus transformed B lymphocytes: characterization by immunoprecipitation with recombinant antigen and biological activity. J Clin Endocrinol Metab 81:3155–3161[Abstract]
  8. Kohn LD, Suzuki K, Hoffman WH, Tombaccini D, Marcocci C, Shimojo N, Watanabe Y, Amino N, Cho BY, Kohno Y, Hirai A, Tahara K 1997 Characterization of monoclonal thyroid stimulating and thyrotropin binding inhibiting autoantibodies from a Hashimoto’s patient whose children had intrauterine and neonatal thyroid disease. J Clin Endocrinol Metab 82:3998–4009[Abstract/Free Full Text]
  9. Nagayama Y, Wadsworth HL, Russo D, Chazenbalk GD, Rapoport B 1991 Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors. J Clin Invest 88:336–340
  10. Nagayama Y, Rapoport B 1992 Thyroid stimulatory autoantibodies in different patients with autoimmune thyroid disease do not all recognize the same components of the human thyrotropin receptor: selective role of receptor amino acids Ser25-Glu30. J Clin Endocrinol Metab 75:1425–1430[Abstract]
  11. Tahara K, Ban T, Minegishi T, Kohn LD 1991 Immunoglobulins from Graves’ disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or Immunoglobulins from idiopathic myxedema patients. Biochem Biophys Res Commun 179:70–77[CrossRef][Medline]
  12. Kim WB, Cho BY, Park, HY, Lee HK, Kohn LD, Tahara K, Koh CS 1996 Epitopes for thyroid stimulating antibodies in Graves’ sera: a possible link of heterogeneity to differences in response to antithyroid drug treatment. J Clin Endocrinol Metab 81:1758–1767[Abstract]
  13. Watanabe Y, Tahara K, Hirai A, Tada H, Kohn LD, Amino N 1997 Subtypes of anti-TSH receptor antibodies classified by bio- and conversion assays using CHO cells expressing wild type human TSH receptor or TSH receptor-LH/CG receptor chimera. Thyroid 7:13–20[Medline]
  14. Kim WB, Chung HK, Lee HK, Kohn LD, Tahara K, Cho BY 1997 Changes in epitopes for thyroid stimulating antibodies in Graves’ sera during treatment of hyperthyroidism: therapeutic implications. J Clin Endocrinol Metab 82:1953–1988[Abstract/Free Full Text]
  15. Kosugi S, Ban T, Akamizu T, Kohn LD 1992 Identification of separate determinants on the thyrotropin receptor reactive with Graves’ thyroid stimulating antibodies and with thyroid stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors. Mol Endocrinol 6:168–180[Abstract/Free Full Text]
  16. Kosugi S, Ban T, Kohn LD 1993 Identification of thyroid-stimulating antibody-specific interaction sites in the N-terminal region of the thyrotropin receptor. Mol Endocrinol 7:114–130[Abstract/Free Full Text]
  17. Shimojo N, Kohno Y, Yamaguchi K-I, Kikuoka S-I, Hoshioka A, Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K 1996 Induction of Graves’-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA 93:11074–11079[Abstract/Free Full Text]
  18. Seetharamaiah GS, Desai RK, Dallas JS, Tahara K, Kohn LD, Prabhakar BS 1993 Induction of TSH binding inhibitory immunoglobulins with the extracellular domain of human thyrotropin receptor produced using a baculovirus expression system. Autoimmunity 14:315–320[Medline]
  19. Costagliola S, Alcalde L, Ruf J, Vassart G, Ludgate M 1994 Overexpression of the extracellular domain of the thyrotropin receptor in bacteria; production of thyrotropin binding inhibiting immunoglobulins. J Mol Endocrinol 13:11–21[Abstract/Free Full Text]
  20. Costagliola S, Alcade L, Tonacchera M, Ruf J, Vassart G, Ludgate M 1994 Induction of thyrotropin receptor (TSH-R) autoantibodies and thyroiditis in mice immunized with the recombinant TSH-R. Biochem Biophys Res Commun 199:1027–1034[CrossRef][Medline]
  21. Costagliola S, Many MC, Stalmans-Falys M, Tonacchera M, Vassart G, Ludgate M 1994 Recombinant thyrotropin receptor and the induction of autoimmune thyroid disease in BALB/c mice: a new animal model. Endocrinology 135:2150–2159[Abstract]
  22. Marion S, Braun JM, Ropars A, Kohn LD, Charreire J 1994 Induction of autoimmunity by immunization of mice with human thyrotropin receptor. Cell Immunol 158:329–341[CrossRef][Medline]
  23. Wagle NM, Dallas JS, Seetharamaiah GS, Fan JL, Desai RK, Memar O, Rajaraman S, Prabhakar BS 1994 Induction of hyperthyroxinemia in BALB/c but not in several other strains of mice. Autoimmunity 18:103–112[Medline]
  24. Carayanniotis G, Huang GC, Nicholson LB, Scott T, Allain P, McGregor AM, Banga JP 1995 Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves’ disease. Clin Exp Immunol 99:294–302[Medline]
  25. Seetharamaiah GS, Wagle NM, Morris JC, Prabhakar BS 1995 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. Endocrinology 136:2817–2824[Abstract]
  26. Wagle NM, Patibandla SA, Dallas JS, Morris JC, Prabhakar BS 1995 Thyrotropin receptor-specific antibodies in BALB/cJ mice with experimental hyperthyroxinemia show a restricted binding specificity and belong to the immunoglobulin G1 subclass. Endocrinology 136:3461–3469[Abstract]
  27. Vlase H, Nakashima M, Graves PN, Tomer Y, Morris JC, Davies TF 1995 Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading. Endocrinology 136:4415–4423[Abstract]
  28. Germain RN, Ashwell JD, Lechler RI, Margulies DH, Nickerson KM, Suzuki G, Tou JY 1985 "Exon-shuffling" maps control of antibody-and T-cell-recognition sites to the NH2-terminal domain of the class II major histocompatibility polypeptide A. Proc Natl Acad Sci USA 82:2940–2944[Abstract/Free Full Text]
  29. Davis LG, Dibner MD, Battey JF 1986 Basic Methods in Molecular Biology. Elsevier, New York, pp 93–98
  30. Desai RK, Dallas JS, Gupta MK, Seetharamaiah GS, Fan JL, Tahara K, Kohn LD, Prabhakar BS 1993 Dual mechanism of perturbation of thyrotropin mediated activation of thyroid cells by antibodies to the thyrotropin receptor (TSHR) and TSHR derived peptides. J Clin Endocrinol Metab 77:658–663[Abstract]
  31. Sakata S, Ogawa T, Matsui I, Manshouri T, Atassi MZ 1992 Biological activities of rabbit antibodies against synthetic human thyrotropin receptor peptides representing thyrotropin binding regions. Biochem Biophys Res Commun 182:1369–1375[CrossRef][Medline]
  32. Ohmori M, Endo T, Onaya T 1991 Development of chicken antibodies toward the human thyrotropin receptor peptides and their bioactivities. Biochem Biophys Res Commun 174:399–403[CrossRef][Medline]
  33. Dallas JS, Cunningham SJ, Patibandla SA, Seetharamaiah GS, Morris JC, Tahara K, Kohn LD, Prabhakar BS 1996 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. Endocrinology 137:3329–3339[Abstract]
  34. Mori T, Sugawa H, Piraphatdist T, Inoue D, Enomoto T, Imura H 1991 A synthetic oligopeptide derived from the human thyrotropin receptor sequence binds to Graves’ immunoglobulin and inhibits thyroid stimulating antibody activity but lacks interactions with TSH. Biochem Biophys Res Commun 178:165–172[CrossRef][Medline]
  35. Ueda Y, Sugawa H, Akamizu T, Okuda J, Ueda M, Kosugi S, Ohta C, Kihou Y, Mori T 1995 Thyroid stimulating antibodies in sera from patients with Graves’ disease are heterogeneous in epitope recognition. Eur J Endocrinol 132:62–68[Abstract/Free Full Text]
  36. Morris JC, Gibson JL, Haas EJ, Bergert ER, Dallas JS, Prabhakar BS 1994 Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSHR peptides. Autoimmunity 17:287–299[Medline]
  37. Ohmori M, Endo T, Ikeda M, Onaya T 1991 Role of N-terminal region of the thyrotropin (TSH) receptor in signal transduction for TSH or thyroid stimulating antibody. Biochem Biophys Res Commun 178:733–738[CrossRef][Medline]
  38. Jaume JC, Kakinuma A, Chazenbalk GD, Rapoport B, McLachlan SM 1997 Thyrotropin receptor autoantibodies in serum are present at much lower levels than thyroid peroxidase autoantibodies: analysis by flow cytometry. J Clin Endocrinol Metab 82:1885–1893[Abstract/Free Full Text]
  39. Patibandla SA, Dallas JS, Seetharamaiah GS, Tahara K, Kohn LD, Prabhakar BS 1997 Flow cytometric analyses of antibody binding to Chinese hamster ovary cells expressing human thyrotropin receptor. J Clin Endocrinol Metab 82:500–507[Abstract/Free Full Text]
  40. Sugawa H, Ueda Y, Zhang M, Kosugi S, Ueda M, Mori T 1995 Determinant spreading shown by monoclonal thyroid stimulating antibody produced against a TSH receptor peptide. Thyroid [Suppl 1]5:S5



This article has been cited by other articles:


Home page
 EndocrinologyHome page
O. Martinez, E. Gangi, D. Mordi, S. Gupta, S. Dorevitch, M.-P. Lefranc, and B. S. Prabhakar
Diversity in the Complementarity-Determining Region 3 (CDR3) of Antibodies from Mice with Evolving Anti-Thyroid-Stimulating Hormone Receptor Antibody Responses
Endocrinology, February 1, 2007; 148(2): 752 - 761.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
S. M. McLachlan, Y. Nagayama, and B. Rapoport
Insight into Graves' Hyperthyroidism from Animal Models
Endocr. Rev., October 1, 2005; 26(6): 800 - 832.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Muehlberg, J. A. Gilbert, P. V. Rao, A. M. McGregor, and J. P. Banga
Dynamics of Thyroid-Stimulating and -Blocking Antibodies to the Thyrotropin Receptor in a Murine Model of Graves' Disease
Endocrinology, April 1, 2004; 145(4): 1539 - 1545.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
B. S. Prabhakar, R. S. Bahn, and T. J. Smith
Current Perspective on the Pathogenesis of Graves' Disease and Ophthalmopathy
Endocr. Rev., December 1, 2003; 24(6): 802 - 835.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. Schwarz-Lauer, P. N. Pichurin, C.-R. Chen, Y. Nagayama, C. Paras, J. C. Morris, B. Rapoport, and S. M. McLachlan
The Cysteine-Rich Amino Terminus of the Thyrotropin Receptor Is the Immunodominant Linear Antibody Epitope in Mice Immunized Using Naked Deoxyribonucleic Acid or Adenovirus Vectors
Endocrinology, May 1, 2003; 144(5): 1718 - 1725.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. V. Rao, P. F. Watson, A. P. Weetman, G. Carayanniotis, and J. P. Banga
Contrasting Activities of Thyrotropin Receptor Antibodies in Experimental Models of Graves' Disease Induced by Injection of Transfected Fibroblasts or Deoxyribonucleic Acid Vaccination
Endocrinology, January 1, 2003; 144(1): 260 - 266.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Nagayama, M. Kita-Furuyama, T. Ando, K. Nakao, H. Mizuguchi, T. Hayakawa, K. Eguchi, and M. Niwa
A Novel Murine Model of Graves' Hyperthyroidism with Intramuscular Injection of Adenovirus Expressing the Thyrotropin Receptor
J. Immunol., March 15, 2002; 168(6): 2789 - 2794.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
E. S. Park, H. Kim, J. M. Suh, S. J. Park, S. H. You, H. K. Chung, K. W. Lee, O-Y. Kwon, B. Y. Cho, Y. K. Kim, et al.
Involvement of JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) in the Thyrotropin Signaling Pathway
Mol. Endocrinol., May 1, 2000; 14(5): 662 - 670.
[Abstract] [Full Text]

Home page
 J. Immunol.Home page
S. Kaithamana, J. Fan, Y. Osuga, S.-G. Liang, and B. S. Prabhakar
Induction of Experimental Autoimmune Graves' Disease in BALB/c Mice
J. Immunol., November 1, 1999; 163(9): 5157 - 5164.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Kita, L. Ahmad, R. C. Marians, H. Vlase, P. Unger, P. N. Graves, and T. F. Davies
Regulation and Transfer of a Murine Model of Thyrotropin Receptor Antibody Mediated Graves' Disease
Endocrinology, March 1, 1999; 140(3): 1392 - 1398.
[Abstract] [Full Text]

Home page
 J. Clin. Endocrinol. Metab.Home page
J. Wortsman, P. McConnachie, K. Tahara, and L. D. Kohn
Thyrotropin Receptor Epitopes Recognized by Graves' Autoantibodies Developing under Immunosuppressive Therapy
J. Clin. Endocrinol. Metab., July 1, 1998; 83(7): 2302 - 2308.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kikuoka, S.
Right arrow Articles by Niimi, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kikuoka, S.
Right arrow Articles by Niimi, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals