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Regulation and Transfer of a Murine Model of Thyrotropin Receptor Antibody Mediated Graves’ Disease¹

Division of Endocrinology and Metabolism (M.K., L.A., R.C.M., H.V., P.N.G., T.F.D.), Departments of Medicine and Pathology (P.U.), Mount Sinai School of Medicine, New York, New York 10128

Address all correspondence and requests for reprints to: Dr. T. F. Davies, Mount Sinai Medical Center, Box 1055, 1 Gustave L. Levy Place, New York, New York 10128. E-mail: tdavies{at}smtplink.mssm.edu

	Abstract

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In order to replicate a recently described murine model of Graves’ disease, we immunized AKR/N (H-2^k) mice ip, every 2 weeks, with either a clone of fibroblasts expressing both the human TSH receptor (hTSHR) and murine major histocompatibility complex (MHC) class II molecules or with fibroblasts expressing the MHC class II molecules alone. Mice were bled, and their thyroid hormone levels measured, at 6, 12, and up to 18 weeks after the first immunization. Between 11–12 weeks after immunization, a significant number of mice began to die spontaneously and were found to have developed large goiters. Thirty to 40% of mice immunized with hTSHR transfected fibroblasts showed markedly increased serum T₃ and T₄ hormone levels by 12 weeks compared with controls, with the highest thyroid hormone levels being T₃: 420 ng/dl (normal < 70) and T₄: 16.5 µg/dl (normal < 5). The murine serum demonstrated the presence of antibodies to the TSHR, as evidenced by inhibition of labeled TSH binding to the hTSHR, and these sera had in vitro thyroid stimulating activity. Many of the hyperthyroid mouse exhibited weight loss and hyperactivity and, on examination, their thyroids had the histological features of thyroid hyperactivity including thyroid enlargement, thyroid cell hypertrophy, and colloid droplet formation - all consistent with Graves’ disease. In contrast, a small number of mice (< 5%) developed hypothyroidism with low serum T₄ levels and markedly increased TSH concentrations and evidence of thyroid hypoplasia. Both hyperthyroidism and hypothyroidism were successfully transferred to naive mice using ip cells of immunized mice. Surprisingly, hypothyroidism occurred in many recipient mice even after transfer from hyperthyroid donors.

These results confirmed that immunization with naturally expressed hTSHR in mammalian cells was able to induce functional TSHR autoantibodies that either stimulated or blocked the mouse thyroid gland and induced hyperthyroidism or thyroid failure. Furthermore, both blocking and stimulating antibodies coexisted in the same mice as evidenced so clearly by the transfer of hypothyroidism from hyperthyroid mice. The addition of a Th2 adjuvant (pertussis toxin) caused approximately 50% of the animals to become hyperthyroid beginning early at 9 weeks, whereas a Th1 adjuvant (CFA) delayed the disease onset such that only 10% were hyperthyroid by 12 weeks. As with human autoimmune thyroid disease, the T cell control of this murine model may be critical and requires more extensive investigation.

	Introduction

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ROBERT GRAVES’ thyroid disease is an autoimmune form of human hyperthyroidism often associated with an ophthalmopathy (orbitopathy) involving the extraocular connective tissues and muscles and more rarely with a dermopathy (1, 2). The disease has been defined, clinically, as hyperthyroidism associated with a generally soft, diffusely enlarged, thyroid gland in the presence of stimulating autoantibodies to the TSH receptor (TSHR-Abs) (3). This distinguishes the disorder from hCG-induced hyperthyroidism (4, 5) and the increasingly well recognized, but much rarer, forms of familial hyperthyroidism due to an activating mutation of the TSH receptor (6). The original self-infusion of plasma from patients with Graves’ disease by Adams and colleagues and the resulting thyroid stimulation (7) was the first example of the role of TSHR-Abs in the induction of human hyperthyroidism. Importantly, TSHR-Abs are disease specific, in great contrast to the high prevalence of autoantibodies to other thyroid antigens, such as thyroglobulin (Tg-Ab) and thyroid peroxidase (TPO-Ab), in the general population (8).

Recently, both Shimojo et al. (9) and Costagliola et al. (10) have reported new approaches to the induction of Graves’ disease in mice; the former using fibroblasts expressing the human TSHR (hTSHR) and the latter using hTSHR complementary DNA (cDNA) immunization. We report here our confirmation and extension of the work of Shimojo et al., who employed a murine L cell (fibroblast) line spontaneously expressing MHC class II antigen, and which was transfected with full-length hTSHR cDNA. The immunized mice became hyperthyroid and had many, but not all, of the features of Graves’ disease including thyroid-stimulating antibodies.

	Materials and Methods

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Antigenic cells
The protocol described by Shimojo et al. (9) was established and used. A murine fibroblast L cell line (RT4.15HP), expressing a hybrid gene containing A_ß^k and A_ß ^d of murine MHC class II, kindly provided by Dr. R. N. Germain (National Institute of Allergy and Infectious Diseases, National Institutes of Health). The A_ß^d region present in the transfected MHC class II construct apparently has no role to play in antigen binding (11). RT4.15HP cells were transfected with the pSVL vector subcloned with the full-length hTSHR gene, kindly provided by Dr. G. Vassart (Free University of Bruxelles, Belgium) (12), together with pRC/CMV (Invitrogen, Carlsbad, CA) using Lipofectamine (Gibco BRL, Gaithersburg, MD). Cells were selected for neomycin resistance using 500 µg of G418 per ml (Gibco BRL) and stable transfectants were selected by cAMP generation in the presence of bovine TSH (13). Highly positive cells were cloned by limiting dilution and clone mk.C.6 cells were used throughout the subsequent studies after release from their plastic support using EGTA.

cAMP generation assay
The RT4.15 HP cell line, the RT4.15HP cell clone mk.C.6. transfected with hTSHR, or CHO cells expressing hTSHR (JP09) were plated in 24-well plates (4 x 10⁴ cells/well), fed fresh medium 24 h later, and used at confluence. Medium in the wells was removed, cells were washed, and 300 µl bTSH in serial concentrations or control hypotonic assay medium were added to the cells as described previously (14). Cells were incubated for 2 h at 37 C. Medium was collected and the cAMP released into the medium was measured by RIA after diluting with sodium acetate buffer (Diagnostic Products Corp., Los Angeles, CA).

Immunization of mice with transfectants
Six-week-old female AKR/N (H-2^k) mice (unless stated otherwise) (Jackson Laboratory, Bar Harbor, ME) were ip immunized eight times every 2 weeks with 2 x 10⁷ hTSHR-transfected fibroblasts (mk.C.6.) after treatment with mitomycin C (Gibco BRL). Different adjuvant regimes were employed: in Group 1, mice were immunized without adjuvant; in Group 2, they received Imject Alum adjuvant (30 µl/IP; Pierce Chemical Co., Rockford, IL) containing pertussis toxin (0.18 µg, Sigma Chemical Co., St. Louis, MO) in addition to the ip cells and in Group 3 they received Complete Freund’s adjuvant (CFA; 50 µl IP and 100 µl into hind footpads; Gibco BRL). Control mice were injected ip with 2 x 10⁷ vector-only transfected fibroblasts in the same protocol. Mice were bled at 6, 12, and up to 18 weeks after the first immunization and usually killed two weeks after a final immunization.

Histology
Mouse thyroids were removed and examined histologically after fixing in 10% formalin. Multiple sections were examined at 3–5 µ intervals after staining with H & E. We also used the immunoperoxidase method with anti-Leukocyte Common Antigen (LCA; PharMingen) to detect lymphocytic infiltrates (15).

Thyroid function tests
Total thyroxine (T₄) was measured by blood spot assay (Diagnostic Products Corp.) and serum triiodothyronine (T₃) was measured by a fluorescence polarization immunoassay (FPIA), (Axsym; Abbot Laboratories, Abbot Park, IL). Murine TSH was measured in selected animals using mTSH-specific antibody and mouse TSH/LH NIDDKD standard provided by Dr. A. F. Parlow as described elsewhere (16) (Drs. Angel Campos Barros and Douglas Forrest, Mount Sinai School of Medicine, New York, NY). Antibodies to mouse Tg were assessed by ELISA using a mouse Tg preparation (17).

Detection of antibodies to the TSHR
Thyroid stimulating antibodies in the serum of hyperthyroid mice were detected by stimulation of cAMP generation in CHO-TSHR (JP09) cells (kindly provided by Dr. Gilbert Vassart, Brussels, Belgium) as described above. Attempts were also made to utilize an ELISA system for their detection. Here, 96-well plates (Immulon 2, Dynatech Laboratories, Chantilly, VA) were coated overnight with 100 ng/well of purified and refolded mTSHR-ecd in carbonate-bicarbonate buffer (15 mM Na₂CO₃ and 35 mM NaHCO₃, pH 9.6) (18). After washing and blocking, wells were incubated with individual mouse antisera (preimmune, immune and control; 1:1000 dilution). Bound antibodies were detected using alkaline phosphatase-labeled sheep antimouse IgG (Sigma Chemical Co.; 1:500 dilution) and developed with p-nitrophenyl phosphate substrate. Using a similar ELISA method, epitope mapping of the mTSHR-Abs was performed. Instead of mTSHR-ecd, 96-well plates were coated with a set of 26 overlapping hTSHR peptides, 20 residues each, spanning the hTSHR ectodomain and control peptides spanning the hTSHR transmembrane region (kindly provided by Dr. J. Morris, Mayo Medical School, Rochester, MN) (19).

Flow cytometry and direct immunofluorescence analysis
Fibroblasts (10⁶ cells) were blocked with 1% ovalbumin (Sigma Chemical Co.) in PBS at 4 C for 30 min and then incubated with 1 µg of monoclonal FITC conjugated antimouse H-2D^k antibody (PharMingen, San Diego, CA) or biotin-conjugated mouse antimouse I-A^k (A_ß^k) monoclonal antibody (PharMingen) for 30 min on ice. Antimouse I-A^k stained cells were further incubated with 1 µg of Streptavidin-R-Phycoerythrin (PE) conjugate (Sigma Chemical Co.) for 30 min on ice. Cells were washed and analyzed by flow cytometry on a FACScan Cytometer (Becton Dickinson and Co., Mountain View, CA).

	Results

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Characterization of clone mk.C.6.
A clone of L cells highly responsive to bTSH stimulation, as assessed by cAMP generation, was selected from the RT4.15HP transfected cells (Fig. 1). The mk.C.6. clone appeared to have similar TSH responsiveness to the CHO-TSHR cells when measured under the conditions detailed in Materials and Methods. Analysis of MHC class I & II expression in this transfected clone showed the maintenance of a high degree of spontaneous expression as observed by fluorescence with specific antibodies (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 1. cAMP generation assay. CHO cells expressing hTSHR (JPO 9, closed triangle), RT 4.15HP clone mk.C.6 (filled circle) and RT 4.15 HP (open circle) were stimulated with increasing doses of bTSH, and the extracellular accumulation of cAMP in the culture media was measured. 10-9 M bTSH was taken as equal to 103 µU/ml.

View larger version (68K): [in this window] [in a new window]   Figure 2. Thyroid glands from hyperthyroid mice. Left, A thyroid gland from a control mouse immunized with RT 4.15HP cells. Right, An enlarged thyroid from a thyrotoxic mouse immunized with mk.C.6. cells.

View larger version (133K): [in this window] [in a new window]   Figure 3. Thyroid histology from hyperthyroid mice (H&E, X100). Left, Control mouse thyroid tissue. Right, Thyroid tissue from a thyrotoxic mouse. Cells were hypertrophic and colloid droplet formation was observed. There was no lymphocytic infiltration.

Group 2. Mice immunized using the same protocol as Group 1 but with the addition of a Th2 adjuvant (Group 2, alum and pertussis toxin IP; n = 24) developed hyperthyroidism earlier than expected with > 25% of the mice hyperthyroid by 9 weeks (Fig. 4). In all, 9 of 19 (47%) immunized animals had increased thyroid hormone levels and goiters (Table 1). Moreover, 4 of the 9 mice were fragile animals and either died spontaneously or at the time of bleeding. Two mice developed hyothyroidism as shown by their increased TSH levels (Table 1).

View larger version (20K): [in this window] [in a new window]   Figure 4. The influence of adjuvants on the onset of hyperthyroidism. The % of mice becoming hyperthyroid is shown in relation to time after first immunization. The pertussis adjuvant regime (Group 2, open circle) and the CFA adjuvant regime (Group 3, closed circle) results are contrasted.

Male susceptibility to thyroid dysfunction
Although female mice were used in most of the studies described, 11 male AKR/N mice were immunized with 10 female mice as part of Group 3 and were found to be indistinguishable with the same prevalence of hyperthyroidism by 16 weeks (4/11 males, 4/10 females). They were, therefore, incorporated into the overall analysis of Group 3 (Table 1).

Serum antibody response to immunization
Sera from the mk.C.6. immunized mice inhibited the binding of labeled TSH to CHO-TSHR cells (for examples, see Table 2a). This indicated the presence of TSHR-Abs. In addition, each of the hyperthyroid mouse sera examined (at a dilution of 1:70) enhanced the generation of cAMP release from CHO-TSHR cells consistent with the presence of thyroid stimulating antibodies (for examples, see Table 2b). Sera from hypothyroid mice inhibited the TSH stimulation of CHO-TSHR cells, consistent with the presence of TSHR blocking antibodies (not shown). Serum anti-Tg was not detected in any of the mouse sera by ELISA (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 5. Serum T4 levels of recipient mice after transfer of ip cells. These data show the serum T4 levels of donor and recipient mice which received ip cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A useful model of human Graves’ disease should meet several criteria. These include: 1) hyperthyroidism; 2) goiter formation; 3) an intrathyroidal lymphocytic infiltration but an absence of thyroidal destruction; 4) retroorbital involvement in the autoimmune process; 5) the presence of serum thyroid-stimulating antibodies; and 6) the ability to transfer the disease. The mouse Graves’ model analyzed here fits many of these criteria with the notable exception of no lymphocytic infiltration in the thyroid (or retroorbital tissues which have remained normal to date). Although a mild lymphocytic infiltration was reported by Shimojo et al. (9) and also by others using recombinant TSHR ectodomain (21), no infiltrates were seen in our hyperthyroid or hypothyroid mice. Furthermore, an adjuvant favoring a Th1 response, where cytotoxic T cells should flourish, also failed to induce thyroiditis. Recent observations using recombinant TSHR ectodomain have demonstrated that unique regions of the ectodomain may be responsible for the development of thyroiditis (22). It is, however, difficult to understand how these epitopes may not have been well represented in the functional, natural receptor used in the present immunization series.

The immunization protocol described by Shimojo et al. (9) and used here was the first to predictably develop thyroid-stimulating antibodies in a mouse model. Previous attempts at immunization with the TSHR (21) used a prokaryotic fusion protein of the hTSHR ectodomain as antigen. Responding Balb/c mice remained euthyroid but apparently developed a mild intrathyroidal lymphocytic infiltration; further, the choice of mouse strain appeared to be important (23). The mouse serum in these earlier studies contained high titers of TSHR-Abs; they did not act as TSH agonists but, rather, blocked the TSH receptor. Using insect cell expressed, either nonglycosylated (without leader sequence) or glycosylated, hTSHR-ecd, we and other investigators were also able to induce hTSHR-ecd antibodies (19, 21, 24, 25). However, such antibodies also did not have any thyroid stimulating activity. We concluded that when the TSHR ectodomain was used as an immunogen, polyclonal and monoclonal antibodies were obtained which were able to compete for TSH binding (19, 26) but were blocking or neutral in their activity rather than stimulating. However, binding to TSHR peptides revealed recognition of multiple epitopes biased toward hydrophilic sequences near the N- and C-termini of the ectodomain (27).

Our peptide-binding data examined the potential epitopes recognized by TSHR antibodies. Widely recognized was the region incorporating the 50 residue insert in the ectodomain between residues 317–366 (Table 3) and a single N-terminal peptide that represented residues 97–116. However, there were no distinct differences in the epitope mapping using sera from hyperthyroid mice. This suggested that all the TSHR antibodies recognized the same peptides and that the unique stimulating antibodies were not reflected in the binding to linear peptides and may have required complex conformational epitopes. Hence, it was likely that the blocking TSHR antibodies were those recognizing the C-terminal insert region. This conclusion would be consistent with that of Kikuoka et al. (28) using the same mouse model, who found that the N-terminal region, when expressed on the immunizing fibroblasts, was essential for the formation of stimulating antibodies. A number of studies using folded and unfolded recombinant human and murine TSHR-ecds have previously shown that folding of the TSHR was critical for high affinity binding of hTSHR-Abs, underlining the importance of conformational epitopes (18, 29). Furthermore, these complex conformational receptor epitopes are likely to be generated by posttranslational events involving the entire TSH holoreceptor including holoreceptor cleavage and associated conformational changes that we have described in detail elsewhere (29). Hence, the normal conformation(s) of the TSHR target must be required for the hyperthyroid autoimmune response, and this cannot be deciphered using peptide epitope mapping.

While our data confirm and extend the previous examination of this Graves’ disease model (9, 28, 30), it does not yet explain the role of MHC antigen expression on the immunizing fibroblast. The fibroblast cells employed in this technique were hybrid H-2^k and H-2^d, but the H-2^d antigen sequence is unlikely to have acted as an immune stimulant in the disease induction because it does not interact with the T cell receptor (11). The spontaneous expression of the MHC class II antigens may, however, be critical for effective presentation of TSHR antigenic peptides for which there is much evidence (31, 32). We need to learn a great deal more about this new animal model, as it provides an attractive basis for developing a true form of Graves’ hyperthyroidism in mice.

	Acknowledgments

 
We thank Dr. Edward Diamond and his staff in the Mount Sinai Endocrine Laboratory for help with T₄ and T₃ RIAs.

	Footnotes

 
¹ Supported in part by: NIH Grants DK-52464, DK-35764 and DK-45011 (to T.F.D.), Cellular and Molecular Endocrinology Training Grant DK-07645 (to L.A.), and the David Owen Segal Endowment (to M.K.).

Received September 10, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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