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Division of Medicine (P.V.R., J.P.B.), Guy’s, King’s and St. Thomas’ School of Medicine, London SE5 9PJ, United Kingdom; Clinical Sciences Centre (P.F.W., A.P.W.), University of Sheffield, Sheffield S5 7AU, United Kingdom; and Faculty of Medicine (G.C.), Memorial University of Newfoundland, St. John’s, Newfoundland, Canada A1B 3V6
Address all correspondence and requests for reprints to: Dr. J. P. Banga, Division of Medicine, Guy’s, King’s and St. Thomas’ School of Medicine, Bessemer Road, London SE5 9PJ, United Kingdom. E-mail: paul.banga{at}kcl.ac.uk.
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Introduction |
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Lately, different strategies have led to a breakthrough in the successful development of murine models of immune hyperthyroidism (10, 11, 12, 13). In the first model, repeated immunization with syngeneic fibroblasts expressing human TSHR and major histocompatibility complex (MHC) class II led to the induction of hyperthyroid disease and goiter in a proportion of H-2k female mice (10, 11, 12). In another study, the incidence of immune hyperthyroidism was increased by incorporating adjuvant in the injection schedule (14). In contrast, the second model relied on vaccination with TSHR plasmid DNA (13). Stable hyperthyroidism was only observed in a small number of female outbred mice (13), although plasmid DNA immunization of inbred strains such as BALB/c showed the presence of only a lymphocytic infiltrate (15). With regard to BALB/c mice, another group recently reported the poor anti-TSHR antibody responses and the lack of thyroiditis in the DNA vaccination model (16), but because their animals were housed under pathogen-free conditions than those in the original report (15), adds an extra complication to the model (16). In addition, other successful murine models described recently have relied on using transfected B cells (instead of fibroblasts) expressing TSHR, immunization with functional TSHR ectodomain protein in adjuvant or injections of recombinant adenovirus expressing TSHR to induce thyrotoxicosis (17, 18).
In this report, we describe our experience on the replication of the adjuvant modified, fibroblast injection model in AKR/N mice (14) and DNA vaccination in BALB/c mice (15), housed under conditions that were not pathogen free. We show the high incidence of the induction of TSAbs and large goiters in the AKR/N model, but the DNA vaccination model was characterized by a poor immune response to the TSHR, which was not accompanied by a lymphocytic infiltrate of the gland.
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Materials and Methods |
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Fibroblasts expressing functional TSHR
Mouse fibroblasts DAP.3 cells and RT4.15HP (expressing hybrid MHC class II) (20) (provided by Professor Robert Lechler) were stably transfected with pCI-neo-TSHR using Transfast reagent (Promega Corp., Southampton, UK) and selected with 50 µg/ml G418, which was gradually increased to 600 µg/ml for gene amplification. TSHR expression was determined by measurement of cAMP following stimulation with TSH. Positive lines were cloned by single cell cloning giving one stable clone, termed RT12, which showed the highest TSH-mediated cAMP enhancement.
Immunization of mice
Before injection, expanded cultures of TSHR expressing RT 12 fibroblasts or the RT4.15HP untransfected cells as controls were treated with mitomycin C (25 µg/ml) and dissociated from the plastic tissue culture flasks using cell dissociation medium (Sigma-Aldrich, Poole, UK). Female AKR/N (H-2k), 6-wk-old mice were purchased from Harlan UK Ltd. (Bicester, UK) and immunized with 2 x 107 RT12 (n = 10 mice) or RT4.15HP fibroblasts (n = 5 mice) in saline by ip injection in adjuvant (14). Briefly, all animals received Imject alum adjuvant (30 µl; Pierce \|[amp ]\| Warriner, Chester, UK) containing pertussis toxin (0.18 µg; Sigma-Aldrich) (14). Immunizations were repeated every 2 wk for a total of eight injections. Mice were bled 2 wk after the final immunization (wk 16 after first injection). All tests on immune sera were carried out on individual sera. All animals were housed in non-barrier-free conditions and fed on a high quality commercial soya bean concentrate pellet diet, with low protein levels ad libitum. All experiments were conducted and approved under United Kingdom Home Office regulations, with full animal veterinary welfare procedures on animal care followed and approved by the Institution.
Genetic immunization with TSHR plasmid in pcDNA3.1 vector was performed in saline or sucrose (13, 15); cardiotoxin could not be used as it was not permitted by the Home Office regulations in the United Kingdom. Female BALB/c mice of 6 wk age were purchased from Harlan UK Ltd., divided into groups of five animals and injected in the anterior tibialis muscle of each leg (25-µl vol per leg) on d 0 with a total of 100 µg 1) pcDNA3.1 plasmid (control group); 2) pcDNA3.1-TSHR in PBS; and 3) pcDNA3.1-TSHR in 25% sucrose. Injections were repeated 3 and 6 wk thereafter (13, 15). At 14 wk after initial immunization, the animals were killed by CO2 inhalation, blood collected by cardiac puncture, and the thyroid glands excised for histology.
cAMP assay for functional TSHR expression
The TSAb and TSBAb activity of sera was assayed in CHO cells expressing TSHR (JP09) and measuring the total intracellular cAMP using a commercial kit (cAMP EIA assay kit, Amersham Biosciences UK Ltd., Amersham, UK). Briefly, 30,000 cells (JP09 or untransfected CHO1 cells as controls) were added per well in duplicates to a flat bottomed 96-well plate and cultured overnight at 37 C. Before the assay, the medium was aspirated and 160 µl of fresh Ham’s F-12 medium (Invitrogen, Paisley, UK) containing 0.1% BSA (Sigma-Aldrich) and 0.2 mg/ml of 3-isobutyl-1-methyl-xanthine (Sigma-Aldrich) (termed F12 complete medium) added. A standard dose response of 3-fold dilutions of bovine TSH (bTSH) (National Institute for Medical Research, London, UK) starting at 10 mU/well diluted to 0.01 mU/well in 20 µl F-12 complete medium was used to determine the cAMP stimulatory response of JP09 cells; for control (zero concentration TSH), 20 µl of medium were added to the wells for basal cAMP levels. A dose-dependent increase in intracellular cAMP up to 1 mU/well TSH was obtained, with maximal cAMP response of 80–90 pmol/ml in JP09 cells in our laboratory. Mouse serum (3 µl diluted in a total volume of 180 µl F-12 complete medium) was used. For measurement of TSBAbs, a suboptimal stimulatory dose of 0.5 mU bTSH/well was added. After incubation for 2 h at 37 C, the intracellular cAMP was extracted with the cell lysis reagent from the kit and cAMP measured in 50-µl (TSH stimulated) or 100-µl (serum stimulated) lysates. The results are expressed as picomoles cAMP per milliliter.
Analysis of antibodies to native TSHR by flow cytometry
Antibodies recognizing the native TSHR were measured by flow cytometry using CHO cells expressing the extracellular domain of TSHR anchored via a glycophosphatidylinositol (GPI) link, expressing approximately 400,000 receptors have been described recently (21). For flow cytometry, 70–90% confluent monolayer cultures of GPI cells (or JP09 cells) were washed two times with PBS (Invitrogen), detached from the plastic surface with cell dissociation medium and transferred to Falcon tubes (200,000 cells/tube). After centrifugation at 500 x g for 3 min at 4 C, the cells were washed once in PBS containing 1% BSA and 0.1% sodium azide (PBS/BSA/azide). Aliquots of pelleted cells (100 µl) were incubated on ice with 1:5 diluted mouse serum in PBS/BSA/azide for 30 min on ice. As controls, culture supernatants containing monoclonal antibodies (mabs) to TSHR, A10 (22) or 2C11 (23) (from Dr. A. P. Johnstone) were used. Cells were washed twice with 5 ml of PBS/BSA/azide and incubated in 100 µl with 1:10 diluted fluorescein isothiocyanate antimouse IgG (Serotec, Oxford, UK) for 30 min on ice in dark. Cells were washed as described and resuspended in 0.5 ml cell fixative (CellFix, Becton Dickinson and Co. (Franklin Lakes, NJ). The fluorescence of 10,000 cells was measured using a FACSCalibur flow cytometer (Becton Dickinson and Co.). Results are expressed as mean fluorescence units, using CELLQuest software (Becton Dickinson and Co.).
TSH-binding inhibiting Ig (TBII) activity
TBII activity was determined using DYNOtest human TRAK II kits (BRAHMS AG, Hennigsdorf, Germany). Mouse sera (100 µl) were measured in single determinations.
Thyroid function tests and histology
Serum thyroid hormone total T3 was determined in single samples using 50 µl serum with a RIA kit (DYNOtest FT3, BRAHMS AG). Thyroid glands were fixed in formalin, embedded in methacrylate and 4-µm sections stained with hematoxylin and eosin (9).
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Results |
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). Furthermore, the GPI cells showed a log fold increase in fluorescence intensity in comparison to JP09 cells confirming the increased expression and sensitivity of staining with the GPI cells (Fig. 1) (21).
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). As a percentage of stimulatory cAMP levels with control serum, the sera from RT12-injected animals enhanced cAMP responses (above mean + 3 SD of control serum) ranging from 110% (mouse no. 8) to 280% (mouse no. 9). However, thyroid function tests showed only one animal (no. 10) with significant increase in serum T3 (over mean ± 3 SD) in the RT12-injected group (Table 1). Examination of TSBAbs in the immune sera with JP09 cells in the presence of 0.5 mU bTSH/well showed that they were not detectable in any of the RT12 injected group nor in the control, RT4.15HP injected mice (Table 1). Eight sera from RT12 injected immune animals were positive for TBII activity (nos. 1, 3, 4, 5, 7, 8, 9, 10), exhibiting 125I-TSH displacement ranging from 50–93% (Table 1). None of the sera from control RT4.15HP-injected animals were positive for TBII activity (Table 1).
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). Serum from six animals injected with RT12 cells recognized the native receptor (nos. 1, 3, 5, 7, 9, 10) by showing a significant shift in the fluorescence signal in comparison to that with control serum (Fig. 2A and Table 1
). To ensure that the binding of the serum Igs by flow cytometry was specific for the TSHR moiety on GPI cells rather than to other cell surface proteins, we examined serum reactivity to the parent, empty plasmid transfected CHO 1 cells (which do not express TSHR). Five of the six positive sera on GPI cells (nos. 1, 5, 7, 9, 10) continued to show specific binding to GPI cells, with no significant binding to CHO1 cells (Fig. 2B), confirming that these five animals produced high levels of anti-TSHR antibodies. Interestingly, despite the inability to measure antibodies to the native TSHR by flow cytometry in serum no. 3 (due to high backgrounds), the serum was positive for TSAbs and TBII activity, indicating the presence of anti-TSHR antibodies. Two animals (nos. 2 and 6) were negative for anti-TSHR antibodies in all the different assays used to monitor the antibodies, although mouse no. 2 was endowed with a enlarged gland (Table 1). Thus, only one animal from the group of ten (no. 6) failed to respond in terms of anti-TSHR antibodies and thyroid gland enlargement (Table 1).
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) were positive for TSHR specific antibodies assessed in different systems with TSAbs, TBII, and positivity on GPI cells by flow cytometry. Interestingly, one animal (no. 2) remained negative for anti-TSHR antibodies, but developed a goiter, whereas another animal (no. 6) did not respond to the immunization (Table 1).
Thyroid gland histology
The thyroid glands from 6 of the 10 immune animals immunized with RT 12 cells were enlarged and goitrous (nos. 2, 3, 5, 7, 9, 10; Table 1). A representative goitrous gland from the hyperthyroid mouse (no. 10) is shown in Fig. 3A. None of the thyroid glands from the control animals injected with RT4.15 cells showed the presence of any goiter (Table 1). Upon histological examination, there was no lymphocytic infiltration in any of the thyroid glands. The goitrous glands showed tall columnar follicular epithelium and irregular follicles with reduced colloid material within the follicles (Fig. 3B), highlighting the intense functional activity of the thyroid follicles in the goitrous glands.
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Discussion |
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The antibodies induced to TSHR in the adjuvant injection-modified, fibroblast injection model were heterogeneous, when measured in different assays. Thus, examination of anti-TSHR antibodies present in individual sera from mice showed some sera to be strongly positive for all types of anti-TSHR antibodies, such as TSAbs, TBII activity, and by flow cytometry with GPI cells. In contrast, some immune animals were positive for some types of anti-TSHR antibodies, but there was no correlation to the presence of goiter to a specific type of anti-TSHR response. This was supported by the fact that one animal was negative for all types of anti-TSHR antibodies, but with a goiter (animal no. 2, Table 1), indicating the lack of correlation anti-TSHR antibodies and thyroid enlargement. It is possible that growth-stimulating immunoglobulins are responsible for the large goiter in this animal, and their measurement would be interesting (24). Thus the heterogeneity of anti-TSHR antibodies in the adjuvant modified, transfected fibroblast immunization model mirrors the pattern observed for receptor antibodies in patients with Graves’ disease, which show no correlation to disease activity (4). Although we have not assessed the epitope specificity of the anti-TSHR antibodies present in different animals in the RT12 immunized group, it is notable that in another recent study, the TSAbs induced in BALB/c with recombinant adenovirus were shown to have a similar epitope specificity to those present in patients with Graves’ disease (18). Taken together, our data with this model demonstrate that despite using an inbred strain of mice, the induced anti-TSHR antibodies are heterogeneous and resemble those present in patients with Graves’ disease.
In the adjuvant modified, fibroblast immunization procedure, the injection of viable cells can lead to the induction of antibodies to a variety of cell surface proteins, including the transfected protein of interest, the TSHR. In the flow cytometry assay for assessment of anti-TSHR antibodies to the native receptor, the presence of nonspecific antibodies to other cell surface proteins can lead to high nonspecific backgrounds. In a recent study, the presence of nonspecific antibodies induced in the fibroblast injection model prevented the analysis of the anti-TSHR response by ELISA and flow cytometry (16). In our study, from the six mice that scored positive in the flow cytometry assay on GPI cells, it was apparent that only one serum gave strong signals with the control, empty vector transfected CHO cells, indicating the presence of antibodies to other surface proteins. Thus, the fibroblast injection method can lead to the induction of antibodies to the protein of interest, such as the TSHR, as well as antibodies to other cell surface antigens. The low incidence of nonspecific antibodies induced in this study by the adjuvant modified, fibroblast injection method compared with those in other studies (16) may be related to the adjuvant modification procedure for immunization or to levels of TSHR protein expressed in the transfected cells because the latter study relied on 125I-TSH binding to monitor TSH expression, whereas in this study, like the other reports (10, 11, 12, 14) monitored functional receptor expression by TSH mediated cAMP stimulation of the fibroblasts.
Although we have been able to successfully replicate the adjuvant fibroblast injection model of Graves’ disease and report new knowledge on the heterogeneity of the induced anti-TSHR antibodies, our attempts to replicate the DNA vaccination model proved more difficult. Although restricted to plasmid vaccinations in saline or sucrose, our data parallel the recent report of Pinchurin and colleagues (16) who, using the more potent cardiotoxin system for injections also found low levels of induced anti-TSHR antibodies in their BALB/c mice. Furthermore, like the preceeding study, there was a lack of inflammatory infiltrate in the thyroid gland in the immune animals, in contrast to the thyroiditis reported in the original study (15). Although the animals in the Pinchurin study were kept in germ-free conditions (16), the animals in this and other studies, were not kept under pathogen-free conditions (15). It is possible that differences in the source of animals may be responsible for the lack of thyroiditis, but it is interesting that immunization of recombinant adenovirus expressing TSHR also led to a lack of infiltration in the thyroid gland, although in this instance the animals were also housed in pathogen-free conditions (18). Another difference in the plasmid DNA vaccination study reported here resides in the volume of the im injection, as our study was restricted to injecting 25-µl vol into the anterior tibialis muscle in each leg, in comparison to the single 100-µl plasmid injections in the other studies (15, 25). Detailed studies on plasmid vaccination into the anterior tibialis muscle in mice have highlighted the importance of such diverse factors as the injection volume, rate of delivery, and injection depth to be critical factors in determining the expression of the transgene in the muscle cells and the subsequent immune response (26). Most importantly, exceeding a volume of 50 µl was critical for increasing the hydrostatic pressure within the anterior tibialis tissue in mice for optimal DNA uptake into the muscle cells (26). This raises the possibility that subtle differences in the plasmid vaccination procedure into the anterior tibialis muscle, including the smaller inoculation volumes used in this study, may be contributory factors for the diverse results on the induced anti-TSHR antibodies and thyroid pathology observed in different studies (13, 15, 16).
In summary, the current studies show that the adjuvant modified, fibroblast model leads to a high incidence and induction of anti-TSHR antibodies that may resemble those present in human Graves’ disease patients. Our studies, like those reported recently (16), show that plasmid DNA vaccination by the im route may require far more stringent conditions in terms of animal source, husbandry, and injection reproducibility to induce anti-TSHR antibodies and thyroid gland pathology. We anticipate that the adjuvant fibroblast injection model will provide information on the anti-TSHR antibodies and the development of monoclonal antibodies with disease-modifying activities will assist in understanding disease mechanisms in this common disorder.
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Acknowledgments |
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Footnotes |
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Abbreviations: bTSH, Bovine TSH; GPI, glycophosphatidylinositol; mab, monoclonal antibody; MFU, mean fluorescence unit; MHC, major histocompatibility complex; TBII, TSH-binding inhibiting Ig; TSHR, TSH receptor; TSAbs, thyroid-stimulating antibodies; TSBAbs, thyroid-stimulating blocking antibodies.
Received July 8, 2002.
Accepted for publication October 8, 2002.
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production rather than antibody dominates the immune response in mice. Endocrinology 142:3530–3536 knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination. Endocrinology 143:1182–1189This article has been cited by other articles:
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