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Endocrinology Vol. 148, No. 5 2335-2344
Copyright © 2007 by The Endocrine Society

An Improved Graves’ Disease Model Established by Using in Vivo Electroporation Exhibited Long-Term Immunity to Hyperthyroidism in BALB/c Mice

Toshio Kaneda1, Asako Honda1, Atsushi Hakozaki, Tetsuya Fuse, Akihiro Muto and Tadashi Yoshida

Department of Pathophysiology, Faculty of Pharmaceutical Science, Hoshi University, Shinagawa, Tokyo 142-0581, Japan

Address all correspondence and requests for reprints to: Dr. Tadashi Yoshida, M.D./Ph.D., Department of Pathophysiology, Hoshi University, Shinagawa, Tokyo 142-8501, Japan. E-mail: tyoshida{at}hoshi.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In Graves’ disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using ß-galactosidase (ß-gal) expression vectors, ß-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0–31.8% BALB/c mice immunized with hTSHR and 79.2–95.7% immunized with hTSHR289His showed high total T4 levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN GRAVES’ DISEASE (GD), the generation of autoantibodies against the TSH receptor (TSHR) causes continuous stimulation of the thyroid gland and hyperthyroidism (1, 2). Animal models are very useful tools for studying the pathophysiology of autoimmune thyroid diseases. The model for Hashimoto’s disease, an autoimmune thyroiditis directed to thyroglobulin and thyroid peroxidase was reported to be inducible by classical immunization with a complete Freund’s adjuvant-containing antigen (3). However, Graves’ hyperthyroidism was not inducible using traditional immunization protocols with bacterial or insect recombinant human TSHR (hTSHR) protein (2). Therefore, GD models that demonstrate an autoimmune response to TSHR have not been reported until recent times.

In recent years, several animal models of GD have been developed. First, Shimojo et al. (4, 5) succeeded in inducing hyperthyroidism in AKR mice by repeatedly immunizing with hTSHR-transfected fibroblasts coexpressing the MHC class II antigen. After this breakthrough, Costagliola et al. (6, 7) reported that genetic immunization, i.e. im injection of an expression vector containing hTSHR cDNA, was an effective procedure for the induction of hyperthyroidism in outbred NMRI mice, but not BALB/c mice. These advancements revealed that the conformation of the expressed hTSHR, probably including sugar modifications, is crucial for the generation of the TSHR-stimulating antibody (TSAb) and for the induction of GD in mice. These models were pioneering and novel, but they have certain difficulties with regard to detailed analysis because of the low incidence of hyperthyroidism (~20%), time required for disease onset, and restricted availability of mouse strains. More recently, efficient murine models of GD generated by repeated im injection with an adenovirus vector expressing hTSHR, were reported by Nagayama et al. (8). By this protocol, approximately 30–50% of the immunized animals developed to hyperthyroidism. In further investigation, genetic immunization of the free A-subunit of hTSHR by using the adenovirus vector induced hyperthyroidism in a high proportion of BALB/c mice, i.e. approximately 65–80% (9). This exceptional method resulted in an outstandingly high incidence of hyperthyroidism involving TSAb, a high T4 value, and histological abnormalities of the thyroid gland. This model made it feasible to further develop the immunological investigation of GD (10, 11, 12, 13).

Among the nonviral techniques for in vivo gene transfer, direct gene transfer into the muscle is a simple and safe technique, although it is limited by the relatively low expression levels of the transferred gene. It was reported that over 80-fold protein was expressed by gene transfer into regenerating muscles pretreated with cardiotoxin or bupivacaine than that by mere im injection of the plasmid vector (14, 15, 16). The initial GD model developed by Costagliola et al. (6, 7) used cardiotoxin pretreatment before the injection of the hTSHR-expressing plasmid. However, the recent novel gene transfer technique, electroporation (EP), greatly improved the in vivo protein expression levels. Aihara and Miyazaki (17) reported that the serum IL-5 concentration in mice that were introduced with an IL-5-expressing plasmid by EP was more than 100-fold that in mice that were merely injected in the regenerating muscle; the former group of mice retained detectable levels of IL-5 for 3 wk. This procedure may be applicable to gene therapy because it does not involve unnecessary antigens like viral proteins.

In this study, by using in vivo EP, we developed a new animal model exhibiting GD-like hyperthyroidism. Repeated hTSHR cDNA transfer by EP could efficiently generate TSAb and induce hyperthyroidism in BALB/c mice. This prevalence of hyperthyroidism was approximately equal to that achieved by using the adenovirus vector. Moreover, surprisingly, the high T4 levels and autoantibody titers, including TSAb, persisted for over 8 months from the last immunization by using EP. Therefore, this model is useful not only for immunological analysis such as that of pathogenic epitopes in TSHR but also might be available for pharmacological analysis as a chronic hyperthyroidism model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell culture
ICR mice (male, 5–6 wk old) and BALB/c mice (female, 4–5 wk old) were purchased from SLC (Shizuoka Laboratories Animal Center, Shizuoka, Japan). CHO-K1 cells were provided by Riken Cell Bank (Tsukuba, Ibaraki, Japan) and were cultivated in F-12 (HAM) medium (Life Technologies, Gaithersburg, MD) containing 10% FBS (JRH Biosciences Inc., Lenexa, KS).

Construction of expression vector for gene transfer
The pC1-IRES-{Delta}CD4 expression vector contains an internal ribosomal entry site (IRES) and human truncated CD4 receptor ({Delta}CD4) cDNA as a selection marker. This vector was constructed from a part of pMACS4-IRES II (18) (Miltenyi Biotec, Bergisch Gladbach, Germany) and pEGFPc1 (19) (accession no. U55763; BD Biosciences Clontech, Palo Alto, CA). IRES-{Delta}CD4 from pMACS4-IRES II was digested with EcoRI and XbaI and inserted into pEGFPc1. The complete pC1-IRES-{Delta}CD4 vector was formed by ligation after the elimination of the EGFP sequence by digestion with AgeI and BspE1. The hTSHR or hTSHR289His insert was amplified by PCR (KOD-plus; Toyobo, Nagoya, Japan) from the hTSHR gene in pGEM-7Z. Mouse TSHR (mTSHR) cDNA was cloned from the BALB/c thyroid gland by RT-PCR. The hTSHR, mTSHR, or hTSHR289His cDNA fragment was inserted into pC1-IRES-{Delta}CD4 at an EcoRI site.

The pBacMam-2 vector (Novagen, Inc., Madison, WI) contains a hybrid promoter with a cytomegalovirus enhancer, chicken ß-actin promoter, and rabbit ß-globin terminator for high expression in mammalian cells and in vivo (20). The amplified hTSHR or hTSHR289His cDNA was inserted at a KpnI site in pBacMam-2. The reverse primer for the first PCR contained the DNA sequence ranging to 289aa of hTSHR and a part of His-tag. For the second PCR, the reverse primer was designed to include the His-tag, a stop codon, and a KpnI site. The primers used were as follows: first and second PCRs, forward primer, 5'-AGG GTA CCG AGC TGA GAA TGA GGC GAT-3'; first PCR, reverse primer, 5'-ATG GTG GTG GTG ATG TTG ATT CTT AAA AGC ACA GC-3'; second PCR, reverse primer, 5'-TTG GTA CCT TAG TGA TGG TGG TGG TGA TGT TG-3'.

Gene transfer into the muscle and genetic immunization with hTSHR or hTSHR289His
Mice were anesthetized with pentobarbital (50 mg/kg ip) and were injected with plasmid DNA (50 µg/site, 1 mg/ml 0.9% NaCl) divided into four times shot in the middle of biceps femoris muscle of both legs. A pair of electrode needles (CUY560-5, 5-mm gap; Nepagene, Tokyo, Japan) were inserted into the muscle across the site at which the plasmid DNA was injected, and electric pulses were delivered using an electroporator (CUY21 EDIT; Nepagene). Three pulses (50 V) and three additional pulses of the opposite polarity were administered to each injection site at a rate of 1 pulse per 200 ms; the duration of each pulse was 50 ms (21).

The control group of the classical genetic immunization was treated with cardiotoxin (10 µM, 50 µl/site, Naja nigricollis; Latoxan, Valence, France) (6), 7 d before plasmid DNA injection (50 µg/site, im).

Female BALB/c mice (6 wk old) were injected im with the plasmid vectors (50 µg/50 µl/site), pC1-hTSHR-IRES-{Delta}CD4, pBacMam-2-hTSHR, or empty vectors, followed immediately by in vivo EP. The mice were treated four times at 3-wk intervals, and blood was drawn 1 wk after each treatment. Representative mice were euthanized 3 wk after the fourth immunization, which was 12 wk after the first immunization, to obtain their thyroid glands.

All animal studies were approved by the Institutional Animal Care and Use Committee and performed with the highest standards of animal care in a pathogen-free facility.

Assay for ß-gal activity in muscle tissues
Muscle tissues were excised and homogenized in lysis buffer [15 mM Tris-HCl (pH 8.0), 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.4 mM aminoethyl benzenesulfonyl fluoride]. After sonication and centrifugation (12,000 x g, 4 C, 10 min), supernatants were collected. ß-D-Galactosidase (Wako, Osaka, Japan) derived from Escherichia coli was used as the standard enzyme (0.97–500.0 µIU/ml) for the verification of quantitative assessment. The supernatants were diluted with lysis buffer, mixed with 2x assay buffer [200 mM sodium phosphate buffer (pH 7.3), 2 mM MgCl2, 100 mM 2-mercaptoethanol, 1.33 mg/ml, o-nitrophenyl-ß-D-galactopyranoside (Wako)], and incubated for 3 h at 37 C. ß-Gal activity was monitored based on the absorbance of the generated o-nitrophenol at 415 nm. Subsequently, the ß-gal activity was corrected by the protein concentration in the homogenates that was measured using Coomassie Protein Assay Reagent (Pierce Biotechnology Inc., Rockford, IL).

RT-PCR
Total RNA was extracted from the muscle tissues after gene transfer of pBacMam-2-hTSHR and was used as a template for cDNA synthesis. cDNA was prepared using Transcriptor Reverse Transcriptase (Roche Diagnostics, Basel, Switzerland). Primers were synthesized on the basis of the hTSHR and GAPDH sequences. The sequences of the primers used for PCR were as follows: hTSHR forward, 5'-GTGCCATCAGGAGGAGGACTT-3'; hTSHR reverse, 5'-CATCCAGCTTTGTCCCATTGA-3'; GAPDH forward, 5'-TCATCATCTCCGCCCCTTC-3'; GAPDH reverse, 5'-TGCCTGCTTCACCACCTTCT-3'.

Establishment of stable mTSHR-expressing clone FMA5
The pC1-mTSHR-IRES-{Delta}CD4 vector, expressing mTSHR and {Delta}CD4 as a selection marker, was transfected in CHO-K1 cells by using FuGENE6 (Roche Diagnostics). Every week, the transfected cells were enriched using a MACS separation unit (Miltenyi Biotec) after labeling them with anti-hCD4 microbeads or anti-TSHR-Ab (clone 2C11; Serotec Co., Kidlington, UK) (22) and antimouse-IgG microbeads. After the fifth MACS separation, the {Delta}CD4-positive cells simultaneously expressing mTSHR were seeded into 96-well plates for cloning by limiting dilution. After cloning the mTSHR-transfected CHO-K1 cells, the mTSHR expression was monitored by flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA). The obtained clones were suspended in ice-cold PBS containing 0.5% BSA, 0.1% sodium azide, and 1 mM glucose. The clones were stained for 30 min with FITC-labeled antihuman CD4 (Beckman Coulter, Inc., Fullerton, CA) or with unlabeled anti-TSHR antibody and FITC-labeled antimouse Ig (Dako Cytomation, Glostrup, Denmark). The clone that was selected based on mTSHR expression and cAMP productivity in response to bovine TSH (bTSH) stimulation, was designated FMA5.

TSAb measurements
For detecting TSAb activity, FMA5 cells were incubated for 30 min at 37 C with 5% mouse serum in Tyrode-HEPES buffer [20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4·12H2O, 1.5 mM KH2PO4, 1 mM MgCl2, 0.3% BSA (pH 7.4)] containing 0.5 mM 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO). The cAMP accumulated in the cells and supernatant was measured using an RIA kit (Yamasa Corp., Choshi, Japan).

Total T4 measurement
The total T4 in the serum was measured using a commercially available RIA kit (DiaSorin Inc., Stillwater, MN). Hyperthyroidic mice were assessed based on the normal T4 range, which was defined as the mean + 3 SD of the values obtained for 11 or 13 mice immunized with the control vector.

Histological analysis
Thyroid glands or muscle tissues injected with ß-gal-expressing vectors were excised and fixed with 10% formalin in 0.9% NaCl. The tissues embedded in Tissue Mount (Chiba Medical, Chiba, Japan) were rapidly frozen in acetone/dry-ice to prepare cryostat sections (Leica Microsystems, Nussloch, Germany). For the detection of ß-gal activity, the muscle sections (10 µm thick) were soaked in substrate solution [0.2% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, 2 mM MgCl2, 5 mM K4Fe(CN)6·3H2O, 5 mM K3Fe(CN)6, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4·12H2O, 1.5 mM KH2PO4] for 1 h. Thyroid sections (5-µm thick) were stained with hematoxylin and eosin.

Production of the rhTSHR A-subunit, rhTSHR289His, and antibody detection by ELISA
The pBacMam-2-TSHR289His vector was transfected into HEK293 cells cultivated in DMEM. After 1 d, the culture medium was changed to Opti-MEM (Invitrogen Corp., Carlsbad, CA) without FBS. Recombinant hTSHR289His (rhTSHR289His) that was secreted in the medium was collected using Ni-NTA-agarose (Qiagen, Inc., Valencia, CA). Purified rhTSHR289His protein was analyzed by digestion with N-glycosidase F (Endo-F; Roche Diagnostics) [10 IU/ml Endo-F, 50 mM Tris-HCl (pH 8.4), 1% Triton X-100, 0.1% SDS]. The obtained rhTSHR289His or the rhTSHR289His digested by Endo-F was detected by Western blot analysis using anti-His antibody (sc-7816; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The amount of antibody bound to rhTSHR289His was measured by ELISA. rhTSHR289His (20 µg/ml) was add to the wells of the ELISA plate (Maxisorp; Nalge Nunc International, Rochester, NY) and incubated overnight. Mouse serum that was diluted to 0.25% with 50% BlockAce (Snow Brand Milk Products Co. Ltd., Tokyo, Japan) was incubated for 1 h in the ELISA wells coated with purified rhTSHR289His protein. For the detection of the bound antibody, HRP-conjugated anti-mouse IgG (Amersham Bioscience Corp., Piscataway, NJ) and 3,3',5,5'-tetramethyl benzidine (1-Step Ultra TMB-ELISA; Pierce Biotechnology Inc.) were used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preliminary study of in vivo EP: superiority of EP for gene transfer and in vivo hTSHR expression
Preliminarily, we investigated the applicability of in vivo EP for gene transfer into the muscle by using the LacZ expression vector pBacMam-2-LacZ. Histological analysis revealed that ß-gal was strongly expressed in the muscle fibers (Fig. 1AGo). In a time-dependent analysis, the activity of the ß-gal introduced via EP peaked at d 5, and was detectable up to 2 wk (Fig. 1BGo). Five days after EP treatment, the ß-gal activity increased to 60-fold of that in the group in which cardiotoxin-pretreated muscles were injected with naked DNA. The ß-gal introduced via pBacMam-2-LacZ showed more than 20-fold the activity of that introduced via pC1-LacZ-IRES-{Delta}CD4 (Fig. 1CGo). These results suggested that the pBacMam-2 vector would satisfy our purposes, i.e. high in vivo expression of hTSHR and establishment of a GD model. Then we investigated the expression of hTSHR mRNA in the muscle tissue after transferring pBacMam-2-hTSHR by EP (Fig. 1DGo). Although hTSHR mRNA was expressed in EP-treated muscle tissue within 2 wk, the mRNA that was introduced by the injection of pBacMam-2-hTSHR into the cardiotoxin-pretreated muscle was hardly detectable. These results demonstrated the superiority of EP as a procedure for in vivo gene transfer and induction of GD.


Figure 1
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  		FIG. 1. Preliminary study of in vivo introduction by EP. A, Histochemical staining for ß-gal activity. Male ICR mice were injected with 50 µg of pBacMam-2-LacZ in saline at the biceps femoris muscle. Cross-section of muscle tissue at a thickness of 10 µm after gene introduction by EP. Muscle tissue was excised 5 d after EP and stained for ß-gal activity. Magnification, x100. B, Time course of ß-gal activity expressed in muscle tissues. Male ICR mice were injected with pBacMam-2-LacZ (closed circle) or an empty vector (open circle) as a control, immediately followed by EP. For comparison with a previous DNA transfer procedure, mice were pretreated with (open triangle) or without (open square) cardiotoxin in the muscle. After 1 wk, pBacMam-2-LacZ was injected in the same cardiotoxin-pretreated sites. ß-Gal activities were assayed based on the enzymatic activity in homogenates of the excised muscle tissues by using o-nitrophenyl-ß-D-galactopyranoside as the substrate. The absorbance of the generated o-nitrophenol was measured at 415 nm. Values are means ± SD for six muscle tissues of three mice. C, Comparison of expression vectors based on ß-gal activity. Lane 1, Control (saline, nontreated); lane 2, pC1-LacZ-IRES-{Delta}CD4 (direct DNA injection); lane 3, pBacMam-2-LacZ (direct DNA injection); lane 4, pC1-LacZ-IRES-{Delta}CD4 (EP); lane 5, pBacMam-2-LacZ (EP). Values are means ± SD for six muscle tissues of three mice. Muscles of the mice in the direct DNA injection group were pretreated with cardiotoxin. D, Difference in hTSHR mRNA expression between the EP and direct in vivo injection methods of DNA. After genetic introduction in ICR mice, mRNA expression of hTSHR was detected by RT-PCR using hTSHR primers, and that of GAPDH was detected as a control. Numbers within parentheses indicate the number of PCR cycles.

 
Establishment of the mTSHR-expressing cell line FMA5 and its characterization for analysis of TSAb
The established cell line FMA5 was analyzed for mTSHR expression by using flow cytometry. The expression of mTSHR or {Delta}CD4 was confirmed using the anti-TSHR-Ab 2C11 or FITC-labeled anti-hCD4 (Fig. 2AGo and B). bTSH stimulation dose-dependently increased the cAMP level in FMA5 cells; the level reached approximately 80-fold at a bTSH concentration of 1 mIU (Fig. 2CGo). These results demonstrated that FMA5 was useful for the detection of TSAb in GD model mice.


Figure 2
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  		FIG. 2. Establishment of the TSHR-expressing cell line FMA5. A and B, Detection of mTSHR expression in an established cell line by using flow cytometry. FMA5 cells were stained with the anti-TSHR monoclonal antibody 2C11 (A) or with anti-hCD4 antibody (B). The histogram of the control CHO-K1 cells that were used for background staining is shown as an open histogram. C, FMA5 (open circle) and control CHO-K1 cells (closed circle) were incubated with bTSH for 30 min at 37 C (0.1–1000 µIU/ml). cAMP that was accumulated in the cells and supernatant was measured by RIA. Values are the means from three assays in a representative experiment.

 
Level of TSAb activity in serum of mice genetically immunized by EP
The TSAb activity in the serum was measured on a basis of the cAMP accumulation in FMA5 cells. In the mice that were genetically immunized with the pC1-IRES-{Delta}CD4 vector, the rate of mice having significant TSAb activity induced by hTSHR or hTSHR289His increased gradually with the round of immunization by EP. After the fourth EP, two of the 25 mice (8.0%) immunized with hTSHR and all the 23 mice (100.0%) immunized with hTSHR289His showed TSAb activity (Fig. 3AGo).


Figure 3
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  		FIG. 3. TSHR-stimulating activity in serum of genetically immunized mice. TSAb measurement in FMA5 cells. FMA5 cells were incubated for 30 min at 37 C with 5% test serum in Tyrode-HEPES buffer containing 0.5 mM 3-isobutyl-1-methylxanthine and 0.3% BSA. cAMP accumulation was measured by RIA. BALB/c mice were immunized by in vivo EP with an hTSHR-expressing (shaded circle), hTSHR289His-expressing (closed circle), or empty vector (open circle). A, pC1-IRES-{Delta}CD4 vector; B, pBacMam-2 vector. Results are expressed as the percentage of basal cAMP level released in the presence of control serum from the mice immunized with the empty vector. TSAb activity was measured after the second, third, and fourth EP. The shaded area indicates the mean ± 3 SD of values of 13 or 11 control vector-immunized mice.

 
With regard to the pBacMam-2 vector, consequently, 12 of the 22 mice (54.5%) immunized with hTSHR and 17 of the 24 mice (70.8%) immunized with hTSHR289His showed positive TSAb activity after the fourth EP (Fig. 3BGo).

Elevation of total serum T4 level in mice genetically immunized by EP
The total serum T4 level in mice was measured 1 wk after the second, third, and fourth EP. We assessed the hyperthyroid mice on a basis of the serum T4 levels above the mean + 3 SD of the values of 11 or 13 control vector-immunized mice. With regard to the pC1-IRES-{Delta}CD4 vector, three of the 25 mice (12.0%) immunized with hTSHR and 22 of the 23 mice (95.7%) immunized with hTSHR289His showed high T4 levels (Fig. 4AGo) after the fourth EP. With regard to the pBacMam-2 vector, seven of the 22 mice (31.8%) immunized with hTSHR showed high T4 levels after the fourth EP, whereas 19 of the 24 mice (79.2%) immunized with hTSHR289His showed high T4 levels after the third EP (Fig. 4BGo). The correlation coefficient between the TSAb activity and serum T4 level was 0.64 (Fig. 5AGo) or 0.62 (Fig. 5BGo) in the corresponding experiments after the fourth EP. These data suggested that the hyperthyroidism induced in the mice depended on the TSAb activity, and demonstrated that a novel animal model for hyperthyroidism had been established using EP.


Figure 4
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  		FIG. 4. Elevation of total serum T4 level in genetically immunized mice. BALB/c mice were immunized by EP with an hTSHR-expressing (shaded circle), hTSHR289His-expressing (closed circle), or empty vector (open circle). A, pC1-IRES-{Delta}CD4 vector; B, pBacMam-2 vector. Total serum T4 levels in mice were measured 1 wk after the second, third, and fourth EP. The shaded area indicates the mean ± 3 SD of values of 13 or 11 empty vector-immunized mice.

 

Figure 5
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  		FIG. 5. Correlation between TSAb and total T4 value. Correlation between TSAb and the total T4 level after the fourth EP with the pC1-IRES-{Delta}CD4 vector (A, n = 61) or the pBacMam-2 vector (B, n = 57). The dashed lines indicate each mean + 3 SD of values of 13 or 11 control vector-immunized mice.

 
Pathological feature of thyroid glands in the genetically immunized mice by EP
Four weeks after the fourth EP, representative normal and hyperthyroid mice were anatomized. The thyroid glands of the hyperthyroid mice were enlarged unlike the normal thyroid gland (Fig. 6Go, A–C). Frozen sections of the thyroid glands of the representative normal and hyperthyroid mice were stained with hematoxylin and eosin. The diseased glands showed hyperplastic and highly irregular follicles with considerable marginal scalloping and vacuolization, indicating the hyperactivity of the gland (Fig. 6Go, E, F, G, and I). However, we could have not observed inflammation or lymphocyte infiltration in thyroid glands at 4 wk after the fourth immunization.


Figure 6
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  		FIG. 6. Thyroid gland appearance and its histology in mice genetically immunized by EP. Appearance of the thyroid gland in mice immunized with an empty pBacMam-2 (A; control), hTSHR-expressing (B), and hTSHR289His-expressing vectors (C). Thyroid glands from hyperthyroidic mice were enlarged unlike the normal thyroid gland. Frozen sections of the thyroid glands from normal and hyperthyroidic mice were stained with hematoxylin and eosin. D, pBacMam-2 control vector; E, pBacMam-2-TSHR (hyperthyroidic); F, pBacMam-2-hTSHR289His (hyperthyroidic); G, pC1-hTSHR289His-IRES-{Delta}CD4 (hyperthyroidic); H, pBacMam-2 control vector; I, pBacMam-2-hTSHR289His (hyperthyroidic). Magnification, D–G, x75; H and I, x300.

 
Detection of antibodies against rhTSH289His in serum of genetically immunized mice by using ELISA
The serum of mice immunized with the vectors expressing hTSHR or hTSHR289His or the control vector was collected 1 wk after the second, third, and fourth EP. The titers of the corresponding antibodies were measured using ELISA wells coated with rhTSHR289His expressed in HEK293 cells that corresponds to the extracellular A-subunit of hTSHR. We verified that rhTSHR289His possessed an N-linked sugar modification that was digested with Endo-F (Fig. 7AGo). In the experiments using pC1-IRES-{Delta}CD4 (Fig. 7BGo), even after the fourth immunization, antibodies to hTSHR289His were detectable in only one of the 25 mice (4.0%) immunized with hTSHR and in two of the 23 mice (8.7%) immunized with hTSHR289His. In contrast, antibodies to rhTSHR289His, after the second immunization with pBacMam-2 by EP, were detected in 20 of the 22 mice (90.9%) immunized with hTSHR, and in 22 of the 24 mice (91.7%) immunized with hTSHR289His (Fig. 7CGo). The antibody titers were positive in all mice (100%) immunized with hTSHR or hTSHR289His after the fourth EP (Fig. 7CGo). However, the titers detected by ELISA for rhTSHR289His were not related to the TSAb activity or total T4 values. These data suggested that pBacMam-2 induced higher protein expression than that induced by pC1-IRES-{Delta}CD4, and that TSAb induction was not regularly dependant on the quantity of the antigen (Figs. 3Go and 7Go, B and C).


Figure 7
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  		FIG. 7. Detection of antibodies in serum by ELISA using rhTSHR289His. BALB/c mice were immunized by EP with the hTSHR-expressing (shaded circle), hTSHR289His-expressing (closed circle), and empty vectors (open circle). Serum was collected 1 wk after the second, third, and fourth in vivo EP. Antibody binding, in 0.25% serum, was measured by ELISA wells coated with rhTSHR289His protein. Western blotting of rhTSHR289His detected by anti-His-Ab, digested without/with Endo-F (A), pC1-IRES-{Delta}CD4 vector (B), pBacMam-2 vector (C). Data shown are the OD values. The shaded area indicates the mean ± 3 SD of values of 13 or 11 control vector-immunized mice.

 
Long-term persistence of TSAb, i.e. acquired immunity, in genetically immunized mice
The hyperthyroid mice started dying approximately 4 months after the last immunization using EP, and they were confirmed to have hypertrophic thyroid glands at autopsy. Eight months after the last EP treatment, the autoantibodies including TSAb and total T4 in the mice were assayed. Surprisingly, 50–52.9% surviving mice immunized with hTSHR289His retained to positive TSAb or T4 level in both the pC1-IRES-{Delta}CD4 and pBacMam-2 groups (Fig. 8Go, A and B). Six of the 18 mice immunized with hTSHR via the pBacMam-2 vector were hyperthyroid. In the pBacMam-2 group, the antibodies evaluated by ELISA maintained the same level as that of 1 wk after the fourth EP, although this was not observed in the pC1-IRES-{Delta}CD4 group (Fig. 8CGo). However, the titers of the antibodies detected by ELISA did not correlate to the TSAb or T4 levels even 8 months after the last immunization (data not shown).


Figure 8
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  		FIG. 8. Hyperthyroidism caused by long-term immunity to TSHR. TSAb (A), total T4 (B), and the titer of the autoantibodies (C) in surviving mice were measured 8 months after the last immunization with the pC1-IRES-{Delta}CD4 (A-1, B-1, C-1) and pBacMam-2 (A-2, B-2, C-2) vectors. hTSHR-expressing (shaded circle), hTSHR289His-expressing (closed circle), and control vectors (open circle). The shaded area indicates the mean ± 3 SD of values of 12 or 10 control vector-immunized mice.

 
In the anatomic observation, hypertrophy of the thyroid glands was observed in all mice with high T4 levels (Fig. 9Go, A and B). However, in histological analysis, we could not observe lymphocyte infiltration in thyroid glands even at 8 months after last immunization same as at 3 wk after the fourth immunization (data not shown). In a correlation analysis between the total T4 levels and TSAb, the correlation coefficient in the pC1-IRES-{Delta}CD4 group was 0.55 (Fig. 10AGo), and that in the pBacMam-2 group was 0.70 (Fig. 10BGo). These results suggested that the sustained TSAb activity caused hyperthyroidism in the mice; this hyperthyroidism was not transient and progressed to the chronic state after the mice were genetically immunized four times by EP.


Figure 9
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  		FIG. 9. Thyroid gland appearance in chronic hyperthyroidic mice. Thyroid glands were excised 8 months after the last immunization with the pC1-IRES-{Delta}CD4 (A) or pBacMam-2 vector (B). A-1, Control (normal); A-2, hTSHR (euthyroidic); A-3, A-4, and A-5, hTSHR289His (hyperthyroidic); B-1, control (normal); B-2, hTSHR (hyperthyroidic); B-3 and B-4, hTSHR289His (hyperthyroidic).

 

Figure 10
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  		FIG. 10. Correlation between TSAb and total T4 values in chronic hyperthyroidic mice. Correlation between TSAb and total T4 levels after the fourth EP with the pC1-IRES-{Delta}CD4 vector (A, n = 52) or the pBacMam-2 vector (B, n = 46). The dashed lines indicate each mean + 3 SD of values of 12 or 10 control vector-immunized mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We could induce hyperthyroidism in BALB/c mice and establish an efficient murine model that manifested GD-like hyperthyroidism; this was done by repeated im genetic immunization with hTSHR-expressing vectors by using the EP technique. Previous methodology for the establishment of GD models was broadly classified into two categories (23): one involves immunization by transferring a TSHR-expressing cell line, and the other, genetic immunization by the injection of TSHR-expressing vectors. Kaithamana et al. (24) reported that hyperthyroidism was inducible in nearly 100% BALB/c mice by repeated injection of M12 cells expressing mTSHR or hTSHR; however, it took as long as 6–8 months for the induction (24, 25). A recent procedure established by Nagayama et al. (8) used hTSHR-expressing adenovirus vector and yielded notable achievements among previously reported GD models. This model facilitated the investigation of longstanding problems concerning the in vivo GD pathology (10, 11, 12, 13). The most striking verification in these works was that hTSHR289, which corresponds to the free A-subunit of hTSHR, effectively induced hyperthyroidism without the generation of a TSHR-inhibitory antibody (9, 26).

In this study, we could verify that hTSHR289His corresponding A-subunit of hTSHR, which have sugar modification digestible with Endo-F (Fig. 7AGo), effectively induced hyperthyroidism in BALB/c mice. These results showed that 289aa of hTSHR with N-linked glycosylation fulfill the requirements for the antigen recognition and for the induction of TSAb in BALB/c mice. However, lymphocyte infiltration in thyroid gland was not observed in this model, the same results as reported in the model induced by adenovirus system (8). The A-subunit of hTSHR is cleaved at membrane by protease (27). Chen et al. (9) reported that the inducibility of hyperthyroidism by noncleavage TSHR mutant, which was not shed from membrane, was very weak comparing to wild-type hTSHR in BALB/c mice using adenovirus system. Therefore, the induction of TSAb by hTSHR might require the generation of shed A-subunit. Furthermore, speculating from the results of in vitro analysis in transfected HEK293, mature TSHR289His should be readily released out from muscular tissues (Fig. 7AGo). Thus, the shed A-subunit or TSHR289His might be recognized in lymphoid organ, but not be in thyroid gland. The onset of hyperthyroidism might be caused by only TSAb, and not necessarily need involvement of cytotoxic T cell, unlike insulin-dependent diabetes mellitus. The hyperthyroidism without lymphocyte infiltration might be interpreted by the place outside thyroid gland for antigen recognition, and by the Th2-dominant strain, BALB/c mice.

The superiority of EP among the gene transfer methods was confirmed by our preliminary study using the ß-gal- and the TSHR-expressing vectors (Fig. 1Go, B–D). Although a maximum of 31.8% mice displayed hyperthyroidism induced by genetic immunization with hTSHR using EP (Fig. 4BGo), this immunization method could possibly be used to induce hyperthyroidism in inbred BALB/c mice. Furthermore, 95.7% mice that were immunized with the pC1-hTSHR289His-IRES-{Delta}CD4 vector by using EP developed hyperthyroidism and showed positive TSAb activity (Figs. 3AGo and 4AGo). This remarkable prevalence was higher than that observed in most previous models including the adenovirus-inducing system. Therefore, our model using EP has advantages over previous models with regard to the prevalence of hyperthyroidism and to the time required for disease onset.

A preliminary study using the LacZ gene (Fig. 1CGo) revealed that the potential of protein expression via the pC1-IRES-{Delta}CD4 vector was relatively low. The low hTSHR expression level was also estimated by detecting the generated anti-hTSHR289His antibodies by using ELISA (Fig. 7Go, B and C). These results suggested that the induction of hTSHR expression by the pC1-IRES-{Delta}CD4 vector is insufficient to trigger immune reactions and the subsequent hyperthyroidism. However, mild immunization with the pC1-hTSHR289His-IRES-{Delta}CD4 vector effectively induced TSAb and elevated T4 levels that progressively increased with the rounds of immunization, and finally, the prevalence of hyperthyroidism rose to 95.7% (Fig. 4AGo). The pC1-IRES-{Delta}CD4 vector coexpresses human {Delta}CD4 as an unnecessary antigen that possesses the ability to facilitate the immunological reaction; however, the influence of {Delta}CD4 expression in the immune reaction and/or the prevalence of GD is currently unknown. Initially, we speculated that the high expression of hTSHR leads to the efficient induction of hyperthyroidism, and therefore, we selected the pBacMam-2 vector. In keeping with our speculation, pBacMam-2 induced a high titer of antibodies to rhTSHR289His that progressively increased with the rounds of immunization by EP (Fig. 7CGo). However, 79.2% mice immunized with pBacMam-2-hTSHR289His developed hyperthyroidism unexpectedly (Fig. 4BGo).

As mentioned above, the A-subunit of hTSHR is shed at membrane by protease. The induction of TSAb by wild-type hTSHR might require much protein expression in muscle for generation of shed A-subunit from hTSHR. Hence, pBacMam-2 vector that express much protein than pC1-IRES-{Delta}CD4, might effectively induce hyperthyroidism. However, although these speculations might be relevant to the group immunized with hTSHR, the prevalence of the hyperthyroidism induced via hTSHR289His might not depend on the quantity of the antigen. These data suggested that continuous low expression of hTSHR289His, rather than high protein expression, is critical for the dominant generation of TSAb without nonsignificant antibodies against hTSHR289His (Fig. 7Go, B and C); these findings also supported the hypothesis discussed by Chen et al. (26) based on the results obtained by using a low-dose adenovirus system.

Besides those mentioned above, the EP method has other merits over previous methods.

The second advantage is that our method does not involve unnecessary antigens, such as components and proteins derived from the cell line or virus, in the induction of GD. This would be a critical advantage of our model in immunological investigations in TSHR for pathogenic epitopes or antigenic conformation. In genetic immunization, CpG included in the injecting DNA sequences was reported to have immunomodulating effects. CpG motifs have adjuvant effects that could enhance immunity and preferentially induce Th1 responses involving IL-2, IFN-{gamma}, and IL-12 production (28, 29). Although the induction of GD via various expression vectors by using EP also has these problems, it would be the best technique for eliminating the influence of unnecessary antigens at this time.

Third, the antibody titers, including TSAb, surprisingly persisted for over 8 months after the fourth EP, the final immunization. Most previous reports have not indicated acquired immunity against TSHR. In the procedure reported by Kaithamana et al. (24), the levels of T4 and TSAb increased gradually up to 90–150 d after the last immunization with M12 cells expressing recombinant mTSHR or hTSHR. Maruyama et al. (30) reported that im introduction of the erythropoietin gene via a single EP resulted in the persistent protein expression for at least 11 or 15 wk (30, 31). However, the immunity against TSHR for over 8 months, observed in our results, was not likely to be caused by the persistent expression of the introduced hTSHR gene. Our results and the procedures reported by Kaithamana et al. suggested that the antibodies to TSHR, i.e. acquired immunity, might be a consequence of continuous in vivo stimulation by endogenous mTSHR. One week after the fourth EP, over 90% mice immunized with pC1-hTSHR289His-IRES-{Delta}CD4 showed hyperthyroidism (Fig. 4AGo). Therefore, at 8 months after the last EP, some hyperthyroid mice reverted to euthyroidism irrespective of the positive TSAb titer (Fig. 8Go, A and B). At present, the details of the mice in remission are under investigation.

Fourth, the construction of expression vectors for EP is easier and faster than that of viral vectors, and it does not require time for establishment of a cell line expressing hTSHR or mutant hTSHR. Furthermore, in principle, the cotransfer of multiple genes by EP would be possible. The application of various truncated or mutant receptors in this model will enable us to analyze the autoantibody epitopes in GD, and to investigate, in particular, the pathophysiology of GD.

In summary, we could efficiently induce GD-like hyperthyroidism in BALB/c mice by repeated im genetic immunization with hTSHR-expressing vectors by using the EP technique. The methodology in this report is comprehensively the most practical procedure for the induction of hyperthyroidism, although there is scope for improvement. It would be useful not only for immunological analysis but might also be available for pharmacological analysis and drug evaluation.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 25, 2007

1 T.K. and A.H. contributed equally to this work. Back

Abbreviations: bTSH, Bovine TSH; {Delta}CD4, truncated CD4 receptor; EP, electroporation; Endo-F, N-glycosidase F; ß-gal, ß-galactosidase; GD, Graves’ disease; hTSHR, human TSH receptor; IRES, internal ribosomal entry site; mTSHR, mouse TSH receptor; TSHR, TSH receptor; TSAb, TSHR-stimulating antibody.

Received August 8, 2006.

Accepted for publication January 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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