Contrasting Activities of Thyrotropin Receptor Antibodies in Experimental Models of Graves’ Disease Induced by Injection of Transfected Fibroblasts o
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 3V6i^uk, 百拇医药
Abstracti^uk, 百拇医药
The development of experimental models of autoimmune hyperthyroid Graves’ disease has proved a difficult challenge, but recently two novel methods have led to their successful development in mice. We describe our studies on replicating the adjuvant modified, human TSH receptor (TSHR) and major histocompatibility complex class II transfected fibroblast injection system, and the plasmid DNA vaccination method as models resembling the human disorder. The fibroblast injection model in female AKR/N (H-2k) mice led to 70% of the animals developing thyroid-stimulating antibodies and their thyroid glands showed large goiters with histological features of thyroid cell activation characteristic of Graves’ glands. Consistent with the clinical homolog, there was no inflammatory cell infiltrate of the thyroid gland. Detailed studies on the anti-TSHR antibodies such as thyroid-stimulating blocking antibody, antibodies to the native TSHR by flow cytometry, and TSH-binding inhibiting Ig showed that they were heterogeneous and did not correlate with disease activity, thus resembling those present in patients with Graves’ disease. In contrast, the plasmid DNA vaccination model in female BALB/c (H-2d) mice led to the generation of low levels of anti-TSHR antibodies by flow cytometry, which were undetectable for thyroid-stimulating antibodies, TSH-stimulating blocking antibodies, and TSH-binding inhibiting Ig activity. Moreover, this model too was not accompanied by lymphocytic cell infiltration. The data demonstrate the high incidence of hyperthyroid disease induced in the adjuvant modified, transfected fibroblast model in AKR/N mice to allow pathological mechanisms of disease to be studied.
Introductionrd{s, 百拇医药
GRAVES’ DISEASE IS an antibody-mediated autoimmune disorder in which autoantibodies specific for the TSH receptor (TSHR) mimic the hormone, TSH, leading to the uncontrolled stimulation of the thyroid gland with consequent hyperthyroidism (1, 2). An added complication of Graves’ disease is thyroid eye disease in a subset of patients, but whether the TSHR is also the target antigen has remained controversial (3). The autoantibody response to the TSHR is heterogeneous in nature, comprising thyroid-stimulating antibodies (TSAbs), thyroid-stimulating blocking antibodies (TSBAbs), and neutral antibodies (4). It is the sum of these serum antibodies that determines the final clinical outcome of disease in patients. The autoantibodies can also fluctuate during the course of disease, thereby leading to alterations in the clinical presentation of disease (5). There are no spontaneous models of Graves’ disease, and thus induced animal models have been sought for some time. Whereas numerous studies by injection of different strains of mice with purified recombinant TSHR or its extracellular domain region in adjuvant have been reported, none have led to the generation of pathogenic TSAbs, although high titers of anti-TSHR antibodies are easily generated (6, 7). Additionally, only some preparations of recombinant TSHR ectodomain lead to induction of a lymphocytic infiltrate of the gland (8, 9).
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.pi$!, http://www.100md.com
Materials and Methodspi$!, http://www.100md.com
Human TSHR cDNApi$!, http://www.100md.com
Human TSHR cDNA in pCDM8 (19) was excised using XhoI and NotI and subcloned into pCI-neo for stable transfections in fibroblast cells. For DNA vaccination, the TSHR cDNA was subcloned into pcDNA 3.1. Plasmid DNA was prepared using QIAfilter Plasmid Giga kit (QIAGEN, Crawley, UK).pi$!, http://www.100md.com
Fibroblasts expressing functional TSHRpi$!, http://www.100md.com
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 mice2}cfw{[, http://www.100md.com
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./6, 百拇医药
cAMP assay for functional TSHR expression/6, 百拇医药
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 cytometry7', 百拇医药
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^-tlu, http://www.100md.com
TBII activity was determined using DYNOtest human TRAK II kits (BRAHMS AG, Hennigsdorf, Germany). Mouse sera (100 µl) were measured in single determinations.^-tlu, http://www.100md.com
Thyroid function tests and histology^-tlu, http://www.100md.com
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).^-tlu, http://www.100md.com
Results^-tlu, http://www.100md.com
Adjuvant fibroblast injection model TSHR expression in transfected fibroblasts and GPI cells^-tlu, http://www.100md.com
One clone of TSHR-transfected fibroblasts, termed RT12 cells, was strongly positive by bTSH stimulation of cAMP and was selected for immunizations (not shown). We also confirmed that the RT12 cells continued to express MHC class II (H-2k) by flow cytometry using anti-I-Ak specific mab, 10.2.16 (not shown). However, examination of the TSHR expression in the transfected, RT12 fibroblasts by flow cytometry with TSHR-specific mabs, A10 (22) or 2C11 (23) did not allow the specific detection of receptor expression, due to the low receptor expression (not shown). In contrast, TSHR expression by flow cytometry was easily detectable in GPI and JP09 cells, whereas the control CHO1 cells gave negative background staining (Fig. 1). 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).
fig.ommitteed@^(, 百拇医药
Figure 1. Flow cytometric analysis to show increased sensitivity of staining for TSHR antibodies recognizing the native receptor in GPI cells compared with JP09 cells. The staining with the control CHO 1 cells for background staining is also shown. The experiment shown was performed with anti-TSHR mab, 2C11, although other mabs to TSHR such as A10 also gave similar staining profiles (not shown).@^(, 百拇医药
Antibodies to TSHR and hyperthyroid disease@^(, 百拇医药
Serum from individual immune animals were assessed for induced anti-TSHR antibodies by a variety of assays, including TSAbs, TSBAbs, flow cytometry (for neutral antibodies recognizing the native receptor), and TBII. Analysis of TSAbs in the RT12-injected immune animals showed 7 sera (nos. 3, 4, 5, 7, 8, 9, 10) significantly enhanced intracellular cAMP stimulation in JP09 cells, compared with the activity of the control serum (Table 1). 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).
fig.ommitteedd.y, 百拇医药
Table 1. Induced anti-TSHR antibody production (assessed in different assays), thyroid function (T3), and thyroid status in AKR/N mice following injection of transfected fibroblasts expressing MHC class II (RT4.15 HP) or MHC class II and human TSHR (RT12) cellsd.y, 百拇医药
Immune sera were also tested for antibodies to the native TSHR by flow cytometry on GPI cells. For background staining, individual serum from control group of mice immunized with MHC class II-expressing RT4.15HP cells were used. None of the sera from the control group of mice showed any specific binding to GPI cells by flow cytometry (Fig. 2A). 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.
fig.ommitteed#.6u, 百拇医药
Figure 2. A, Analysis by flow cytometry on GPI cells of induced anti-TSHR antibodies in individual mice sera (1:5 dilution) injected with TSHR and MHC class II-expressing fibroblasts (RT12 cells). For controls, individual serum from the five mice injected with MHC class II expressing fibroblasts (RT4.15HP cells) were tested (1:5 dilution); for simplicity, serum from only one control animal giving the highest background binding is shown as a broken line trace in the histograms. Histogram nos. 1–10 refer to immune sera from RT12-injected mice, in solid line trace and the mean fluorescence unit (MFU) for each histogram is shown; values above mean ± 3 SD obtained with the RT4.15 HP-injected mice (MFU 34.25) were considered to be positive and indicated in bold (Table 1). B, Sera with positive staining by flow cytometry on GPI cells from RT12 injected mice (from above) are specific for cell surface TSHR. Mice nos. 1, 3, 5, 7, 9, and 10, which were positive by flow cytometry on GPI cells, were tested for binding to the control CHO 1 cells by flow cytometry (1:5 dilution). Except for mouse no. 3, serum for the other five mice (nos. 1, 5, 7, 9, and 10) show statisticallly significant levels of antibodies for TSHR in GPI cells in comparison to CHO 1 cells (P = 0.007). The MFU values in this experiment differ from those in Table 1 because serum from wk 16, after the first injection (when the animals were killed), was used and the experiment was conducted on a different day.
To summarize the antibody and thyroid function status in the transfected fibroblast injection model, seven sera from RT12 immunized group were positive for TSAbs, and their thyroid glands were enlarged; thus, these animals were hyperthyroid. The anti-TSHR antibodies were also detectable by flow cytometry in six sera, but this was compounded by nonspecific antibodies to other cell surface antigens in 1 sera. Overall, 5 sera (nos. 1, 5, 7, 9, 10; Table 1) 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).+d, http://www.100md.com
Thyroid gland histology+d, http://www.100md.com
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.
fig.ommitteed&qv, http://www.100md.com
Figure 3. A, Enlarged thyroid gland from hyperthyroid mouse (no. 10), in comparison to a normal size gland from an animal injected with control, RT4.15 HP fibroblasts. Magnification, x100. The ruler shows 1-mm divisions. B, Histology of a normal thyroid gland compared with that from a hyperthyroid mouse (no. 10). The diseased gland shows highly irregular follicles with extensively reduced colloidal space, indicative for hyperstimulation of the gland. Magnification, x100.&qv, http://www.100md.com
Plasmid DNA immunization model&qv, http://www.100md.com
BALB/c mice were injected with pcDNA3.1-TSHR plasmid in saline or in 25% sucrose (15); control animals were injected with the empty pcDNA3.1 plasmid in sucrose. Upon killing on wk 14, the serum from all the immune and control animals were analyzed for anti-TSHR antibodies and thyroid function tests. None of the animals showed the presence of TSAbs, TSBAbs, nor TBII activity and the serum T3 levels were not significantly different from the control plasmid injected animals (not shown). However, testing of the sera (at 1:5 dilution) by flow cytometry on GPI cells showed two animals in the sucrose group injected with TSHR plasmid to be weakly positive for anti-TSHR antibodies, when compared with sera from the control group of empty plasmid injected mice by flow cytometry on GPI cells, whereas sera from all the animals in the saline group injected with TSHR plasmid were negative by this assay (not shown). Histological examination of the excised thyroid glands showed normal histology, with no lymphocytic infiltrate, comparable to that present in the control empty plasmid vaccinated mice (not shown).
Discussion@[.l-f, http://www.100md.com
We report our experience on the two recently introduced murine models of hyperthyroid Graves’ disease, the adjuvant injection modified, fibroblast model in AKR/N (H-2k) mice (14) and the plasmid DNA vaccination model in BALB/c (H-2d) mice (15). Immunization of AKR/N mice with transfected fibroblasts expressing MHC class II and human TSHR in adjuvant resulted in 70% of the animals with TSAbs, and their thyroid glands showed large goiters. On this basis, 70% of the animals displayed immune hyperthyroidism, and corroborates the data of Kita et al. (14) on the high incidence of hyperthyroid disease using adjuvant supplementation with transfected fibroblasts. In terms of serum thyroid hormones, surprisingly only one animal showed stability in the elevated serum T3 levels, despite a larger number of the animals with TSAbs and large goiter. At the same time, histological analysis of the glands showed the presence of tall, columnar thyroid follicular cells, with empty colloid indicating the activated status of the follicular cells, which is typical in Graves’ disease. However, the plasmid DNA immunization model in BALB/c mice (15) proved difficult to replicate because low titer antibodies were induced, but more importantly there was no infiltration of the gland.
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.w, 百拇医药
Acknowledgmentsw, 百拇医药
We wish to thank Professor R. Lechler for the provision of RT4.15HP fibroblasts and the anti I-Ak mab; Professor G. Vassart for JP09 cells; Dr. A. Diamond for the human TSHR cDNA; and Dr. Alan Johnstone for the anti-TSHR mab, 2C11. We also wish to acknowledge BRAHMS AG for the generous gifts of TRAK II and DYNOtest kits.
Received July 8, 2002.##{, http://www.100md.com
Accepted for publication October 8, 2002.##{, http://www.100md.com
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Abstracti^uk, 百拇医药
The development of experimental models of autoimmune hyperthyroid Graves’ disease has proved a difficult challenge, but recently two novel methods have led to their successful development in mice. We describe our studies on replicating the adjuvant modified, human TSH receptor (TSHR) and major histocompatibility complex class II transfected fibroblast injection system, and the plasmid DNA vaccination method as models resembling the human disorder. The fibroblast injection model in female AKR/N (H-2k) mice led to 70% of the animals developing thyroid-stimulating antibodies and their thyroid glands showed large goiters with histological features of thyroid cell activation characteristic of Graves’ glands. Consistent with the clinical homolog, there was no inflammatory cell infiltrate of the thyroid gland. Detailed studies on the anti-TSHR antibodies such as thyroid-stimulating blocking antibody, antibodies to the native TSHR by flow cytometry, and TSH-binding inhibiting Ig showed that they were heterogeneous and did not correlate with disease activity, thus resembling those present in patients with Graves’ disease. In contrast, the plasmid DNA vaccination model in female BALB/c (H-2d) mice led to the generation of low levels of anti-TSHR antibodies by flow cytometry, which were undetectable for thyroid-stimulating antibodies, TSH-stimulating blocking antibodies, and TSH-binding inhibiting Ig activity. Moreover, this model too was not accompanied by lymphocytic cell infiltration. The data demonstrate the high incidence of hyperthyroid disease induced in the adjuvant modified, transfected fibroblast model in AKR/N mice to allow pathological mechanisms of disease to be studied.
Introductionrd{s, 百拇医药
GRAVES’ DISEASE IS an antibody-mediated autoimmune disorder in which autoantibodies specific for the TSH receptor (TSHR) mimic the hormone, TSH, leading to the uncontrolled stimulation of the thyroid gland with consequent hyperthyroidism (1, 2). An added complication of Graves’ disease is thyroid eye disease in a subset of patients, but whether the TSHR is also the target antigen has remained controversial (3). The autoantibody response to the TSHR is heterogeneous in nature, comprising thyroid-stimulating antibodies (TSAbs), thyroid-stimulating blocking antibodies (TSBAbs), and neutral antibodies (4). It is the sum of these serum antibodies that determines the final clinical outcome of disease in patients. The autoantibodies can also fluctuate during the course of disease, thereby leading to alterations in the clinical presentation of disease (5). There are no spontaneous models of Graves’ disease, and thus induced animal models have been sought for some time. Whereas numerous studies by injection of different strains of mice with purified recombinant TSHR or its extracellular domain region in adjuvant have been reported, none have led to the generation of pathogenic TSAbs, although high titers of anti-TSHR antibodies are easily generated (6, 7). Additionally, only some preparations of recombinant TSHR ectodomain lead to induction of a lymphocytic infiltrate of the gland (8, 9).
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.pi$!, http://www.100md.com
Materials and Methodspi$!, http://www.100md.com
Human TSHR cDNApi$!, http://www.100md.com
Human TSHR cDNA in pCDM8 (19) was excised using XhoI and NotI and subcloned into pCI-neo for stable transfections in fibroblast cells. For DNA vaccination, the TSHR cDNA was subcloned into pcDNA 3.1. Plasmid DNA was prepared using QIAfilter Plasmid Giga kit (QIAGEN, Crawley, UK).pi$!, http://www.100md.com
Fibroblasts expressing functional TSHRpi$!, http://www.100md.com
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 mice2}cfw{[, http://www.100md.com
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./6, 百拇医药
cAMP assay for functional TSHR expression/6, 百拇医药
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 cytometry7', 百拇医药
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^-tlu, http://www.100md.com
TBII activity was determined using DYNOtest human TRAK II kits (BRAHMS AG, Hennigsdorf, Germany). Mouse sera (100 µl) were measured in single determinations.^-tlu, http://www.100md.com
Thyroid function tests and histology^-tlu, http://www.100md.com
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).^-tlu, http://www.100md.com
Results^-tlu, http://www.100md.com
Adjuvant fibroblast injection model TSHR expression in transfected fibroblasts and GPI cells^-tlu, http://www.100md.com
One clone of TSHR-transfected fibroblasts, termed RT12 cells, was strongly positive by bTSH stimulation of cAMP and was selected for immunizations (not shown). We also confirmed that the RT12 cells continued to express MHC class II (H-2k) by flow cytometry using anti-I-Ak specific mab, 10.2.16 (not shown). However, examination of the TSHR expression in the transfected, RT12 fibroblasts by flow cytometry with TSHR-specific mabs, A10 (22) or 2C11 (23) did not allow the specific detection of receptor expression, due to the low receptor expression (not shown). In contrast, TSHR expression by flow cytometry was easily detectable in GPI and JP09 cells, whereas the control CHO1 cells gave negative background staining (Fig. 1). 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).
fig.ommitteed@^(, 百拇医药
Figure 1. Flow cytometric analysis to show increased sensitivity of staining for TSHR antibodies recognizing the native receptor in GPI cells compared with JP09 cells. The staining with the control CHO 1 cells for background staining is also shown. The experiment shown was performed with anti-TSHR mab, 2C11, although other mabs to TSHR such as A10 also gave similar staining profiles (not shown).@^(, 百拇医药
Antibodies to TSHR and hyperthyroid disease@^(, 百拇医药
Serum from individual immune animals were assessed for induced anti-TSHR antibodies by a variety of assays, including TSAbs, TSBAbs, flow cytometry (for neutral antibodies recognizing the native receptor), and TBII. Analysis of TSAbs in the RT12-injected immune animals showed 7 sera (nos. 3, 4, 5, 7, 8, 9, 10) significantly enhanced intracellular cAMP stimulation in JP09 cells, compared with the activity of the control serum (Table 1). 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).
fig.ommitteedd.y, 百拇医药
Table 1. Induced anti-TSHR antibody production (assessed in different assays), thyroid function (T3), and thyroid status in AKR/N mice following injection of transfected fibroblasts expressing MHC class II (RT4.15 HP) or MHC class II and human TSHR (RT12) cellsd.y, 百拇医药
Immune sera were also tested for antibodies to the native TSHR by flow cytometry on GPI cells. For background staining, individual serum from control group of mice immunized with MHC class II-expressing RT4.15HP cells were used. None of the sera from the control group of mice showed any specific binding to GPI cells by flow cytometry (Fig. 2A). 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.
fig.ommitteed#.6u, 百拇医药
Figure 2. A, Analysis by flow cytometry on GPI cells of induced anti-TSHR antibodies in individual mice sera (1:5 dilution) injected with TSHR and MHC class II-expressing fibroblasts (RT12 cells). For controls, individual serum from the five mice injected with MHC class II expressing fibroblasts (RT4.15HP cells) were tested (1:5 dilution); for simplicity, serum from only one control animal giving the highest background binding is shown as a broken line trace in the histograms. Histogram nos. 1–10 refer to immune sera from RT12-injected mice, in solid line trace and the mean fluorescence unit (MFU) for each histogram is shown; values above mean ± 3 SD obtained with the RT4.15 HP-injected mice (MFU 34.25) were considered to be positive and indicated in bold (Table 1). B, Sera with positive staining by flow cytometry on GPI cells from RT12 injected mice (from above) are specific for cell surface TSHR. Mice nos. 1, 3, 5, 7, 9, and 10, which were positive by flow cytometry on GPI cells, were tested for binding to the control CHO 1 cells by flow cytometry (1:5 dilution). Except for mouse no. 3, serum for the other five mice (nos. 1, 5, 7, 9, and 10) show statisticallly significant levels of antibodies for TSHR in GPI cells in comparison to CHO 1 cells (P = 0.007). The MFU values in this experiment differ from those in Table 1 because serum from wk 16, after the first injection (when the animals were killed), was used and the experiment was conducted on a different day.
To summarize the antibody and thyroid function status in the transfected fibroblast injection model, seven sera from RT12 immunized group were positive for TSAbs, and their thyroid glands were enlarged; thus, these animals were hyperthyroid. The anti-TSHR antibodies were also detectable by flow cytometry in six sera, but this was compounded by nonspecific antibodies to other cell surface antigens in 1 sera. Overall, 5 sera (nos. 1, 5, 7, 9, 10; Table 1) 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).+d, http://www.100md.com
Thyroid gland histology+d, http://www.100md.com
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.
fig.ommitteed&qv, http://www.100md.com
Figure 3. A, Enlarged thyroid gland from hyperthyroid mouse (no. 10), in comparison to a normal size gland from an animal injected with control, RT4.15 HP fibroblasts. Magnification, x100. The ruler shows 1-mm divisions. B, Histology of a normal thyroid gland compared with that from a hyperthyroid mouse (no. 10). The diseased gland shows highly irregular follicles with extensively reduced colloidal space, indicative for hyperstimulation of the gland. Magnification, x100.&qv, http://www.100md.com
Plasmid DNA immunization model&qv, http://www.100md.com
BALB/c mice were injected with pcDNA3.1-TSHR plasmid in saline or in 25% sucrose (15); control animals were injected with the empty pcDNA3.1 plasmid in sucrose. Upon killing on wk 14, the serum from all the immune and control animals were analyzed for anti-TSHR antibodies and thyroid function tests. None of the animals showed the presence of TSAbs, TSBAbs, nor TBII activity and the serum T3 levels were not significantly different from the control plasmid injected animals (not shown). However, testing of the sera (at 1:5 dilution) by flow cytometry on GPI cells showed two animals in the sucrose group injected with TSHR plasmid to be weakly positive for anti-TSHR antibodies, when compared with sera from the control group of empty plasmid injected mice by flow cytometry on GPI cells, whereas sera from all the animals in the saline group injected with TSHR plasmid were negative by this assay (not shown). Histological examination of the excised thyroid glands showed normal histology, with no lymphocytic infiltrate, comparable to that present in the control empty plasmid vaccinated mice (not shown).
Discussion@[.l-f, http://www.100md.com
We report our experience on the two recently introduced murine models of hyperthyroid Graves’ disease, the adjuvant injection modified, fibroblast model in AKR/N (H-2k) mice (14) and the plasmid DNA vaccination model in BALB/c (H-2d) mice (15). Immunization of AKR/N mice with transfected fibroblasts expressing MHC class II and human TSHR in adjuvant resulted in 70% of the animals with TSAbs, and their thyroid glands showed large goiters. On this basis, 70% of the animals displayed immune hyperthyroidism, and corroborates the data of Kita et al. (14) on the high incidence of hyperthyroid disease using adjuvant supplementation with transfected fibroblasts. In terms of serum thyroid hormones, surprisingly only one animal showed stability in the elevated serum T3 levels, despite a larger number of the animals with TSAbs and large goiter. At the same time, histological analysis of the glands showed the presence of tall, columnar thyroid follicular cells, with empty colloid indicating the activated status of the follicular cells, which is typical in Graves’ disease. However, the plasmid DNA immunization model in BALB/c mice (15) proved difficult to replicate because low titer antibodies were induced, but more importantly there was no infiltration of the gland.
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.w, 百拇医药
Acknowledgmentsw, 百拇医药
We wish to thank Professor R. Lechler for the provision of RT4.15HP fibroblasts and the anti I-Ak mab; Professor G. Vassart for JP09 cells; Dr. A. Diamond for the human TSHR cDNA; and Dr. Alan Johnstone for the anti-TSHR mab, 2C11. We also wish to acknowledge BRAHMS AG for the generous gifts of TRAK II and DYNOtest kits.
Received July 8, 2002.##{, http://www.100md.com
Accepted for publication October 8, 2002.##{, http://www.100md.com
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