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* Department of Pharmacology 1, Nagasaki University School of Medicine, Nagasaki, Japan;
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan; and
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, CA 90048
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Abstract |
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by splenocytes in response to TSHR Ag. Importantly, immune deviation toward Th2 was accompanied by significant inhibition of thyroid-stimulating Ab production and reduction in hyperthyroidism. However, in a therapeutic setting, injection of AdRGDCMVIL-4 alone or in combination with AdCMVTSHR into hyperthyroid mice had no beneficial effect. In contrast, coinjection of adenoviruses expressing IL-12 and the TSHR promoted the differentiation of Th1-type anti-TSHR immune responses as demonstrated by augmented Ag-specific IFN- secretion from splenocytes without changing disease incidence. Coinjection of adenoviral vectors expressing IL-4 or IL-12 had no effect on the titers of anti-TSHR Abs determined by ELISA or thyroid-stimulating hormone-binding inhibiting Ig assays, suggesting that Ab quality, not quantity, is responsible for disease induction. Our observations demonstrate the critical role of Th1 immune responses in a murine model of Graves’ hyperthyroidism. These data may raise a cautionary note for therapeutic strategies aimed at reversing Th2-mediated autoimmune responses in Graves’ disease in humans. |
Introduction |
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Th1/Th2-type responses analyzed using two murine models of Graves’ disease (8, 9) provide conflicting observations (10, 11, 12, 13, 14). The pioneering "Shimojo" model involves injecting fibroblasts coexpressing the human TSHR and MHC class II into syngeneic AKR/N mice (8). In this model, a Th1 adjuvant (CFA) delays and a Th2 adjuvant (alum) accelerates disease induction, suggesting the importance of Th2 immune responses (10). However, injection of the fibroblasts is associated with marked splenocyte production of the Th1 cytokine IFN- but not the Th2 cytokine IL-4 (11). In outbred NMRI mice vaccinated with TSHR-DNA, the presence of B cells, IL-4-producing T cells, and mast cells infiltrating the thyroid glands reflects Th2 responses (9). On the other hand, the same researchers have shown that anti-TSHR mAbs established from the spleens of TSHR DNA-vaccinated BALB/c mice are all IgG2a (12), a murine Th1 subclass. Consistent with the latter observation, splenocytes from TSHR-DNA-vaccinated BALB/c mice secrete IFN-, but not IL-4, in response to TSHR Ag (13) and T cell responses to TSHR-DNA vaccination are generated in IFN- KO BALB/c mice (14). The above discrepancies could be related to studies being performed in different laboratories, with different mouse strains and different cell types. Moreover, neither the Shimojo model nor naked DNA vaccination are suitable for definitive testing of the Th1/Th2 paradigm in Graves’ disease for the following reasons: 1) nonspecific immune activation induced by costimulatory molecules expressed on the injected fibroblasts prevents detailed Ab analysis or in vitro studies in the Shimojo model (11) and 2) induction of TSAb is very rare and hyperthyroidism fails to develop in inbred mice vaccinated with TSHR-DNA (13).
A novel murine model developed in our laboratory provides the opportunity for in vivo and in vitro analysis of the Th1/Th2 paradigm in Graves’ disease. Intramuscular injection of adenovirus coding the TSHR induces TSAb and hyperthyroidism in >50% of female BALB/c mice (15). In the present study, we determined the outcome of injecting adenoviruses expressing the TSHR in combination with adenovirus expressing IL-4 or IL-12. We found that coexpression of IL-4 significantly skewed the Ag-specific immune response to Th2 and inhibited the induction of Graves’ hyperthyroidism. In contrast, immune deviation to Th1 by coexpression of IL-12 had no effect on disease induction. Our findings reinforce the implication of the TSAb subclass data in humans, namely, that Th1-type responses mediate Graves’ disease.
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Materials and Methods |
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Adenovirus expressing the human TSHR (AdCMVTSHR) has previously been described (15). Adenoviruses expressing mouse IL-12 or mouse IL-4 were constructed according to a recently described method (16, 17). The plasmid pBSKS- IL-12BIA containing cDNAs for mouse IL-12 p35 and p40 linked with the internal ribosome entry site gene of encephalomyocarditis virus (18) (kindly provided by Dr. H. Yamamoto, Osaka University, Osaka, Japan) was digested with EcoRV and NotI. The plasmid pBSKS+ mIL4 containing cDNA for mouse IL-4 (RIKEN DNA Bank, Tsukuba, Japan) was digested with EcoRI, blunt-ended with DNA T4 polymerase, and digested with NotI. These fragments were ligated into pHMCMV6 (16) which had been digested with NheI, blunt-ended, and digested with XbaI. The resultant plasmids pHMCMVIL-12 and pHMCMVIL-4 were then digested with I-CeuI/PI-SceI, and ligated into I-CeuI/PI-SceI-digested pAdHM15-RGD (17). pAdHMRGDCMVIL-12 and pAdHMRGDCMVIL-4 were linearized with PacI and transfected into 293 human embryonal kidney cells with PolyFect (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. Recombinant adenoviruses expressing IL-12 or IL-4 (designated AdRGDCMVIL-12 and AdRGDCMVIL-4) were then plaque purified. Adenovirus was propagated in 293 human embryonal kidney cells and purified through two rounds of CsCl density gradient centrifugation. The viral particle concentration was determined as previously described (15). The multiplicity of infection (MOI) was defined as the ratio of total number of particles used in a particular infection divided by the number of cells.
Immunization protocol
Female BALB/c mice (6 wk old) were purchased from Charles River Breeding Laboratory (Tokyo, Japan). All experiments were conducted in accordance with the principles and procedures outlined in the Guidelines for the Care and Use of Laboratory Animals at the Nagasaki University (Nagasaki, Japan). Mice were kept in a specific pathogen-free condition through the experiments. For immunization, mice were injected i.m. with 50 µl of PBS containing 1011 particles of AdCMVTSHR alone or in combination with 5 x 1010 particles of AdRGDCMVIL-4 or AdRGDCMVIL-12. The same immunization schedule was repeated twice at 3-wk intervals. Blood was drawn from tails before the second and third immunizations and 3 wk after the third immunization and by cardiac puncture 8 wk after the third immunization. Thyroid glands were preserved for histology.
In a therapeutic setting, AdRGDCMVIL-4 (5 x 1010 particles/mouse) alone or in combination with AdCMVTSHR (1011 particles/mouse) was injected twice at a 2-wk interval into mice that were in a hyperthyroid state 8 wk after three injections of AdCMVTSHR.
Thyroxine (T4), TSAb, thyroid-stimulating hormone (TSH)-blocking Abs (TBAb), and TSH-binding inhibiting Ab (TBIAb) measurements
Total T4 in murine sera was measured with a commercially available RIA kit (RIA-gnost tT4; Nippon Schering, Osaka, Japan). The normal range was defined as the mean ± 3 SD of control mice.
TSAb activities in murine sera were measured with FRTL5 cells as previously described (15). The cells were seeded at 3 x 104 cells/well in a 96-well culture plate and incubated in 50 µl of hypotonic HBSS containing 0.5 mM isobutyl-methylxanthine, 20 mM HEPES, 0.25% BSA, and 5 µl of serum for 2 h at 37°C. cAMP released into the medium was measured with a cAMP RIA kit (Yamasa, Tokyo, Japan). A value over 150% of control mice was judged as positive. TBAb activities were measured in the same hypotonic buffer supplemented with 100 mU/ml bovine TSH (Sigma-Aldrich, St. Louis, MO) and were expressed as percent inhibition of TSH-induced cAMP generation by test sera.
TBIAb values were determined with a commercially available TRAb kit (BRAHMS Diagnostica, Berlin, Germany). Ten microliters of serum was used for each assay. A value over 15% inhibition of control binding was judged as positive.
ELISA for TSHR Abs
ELISA for detecting mouse IgG Abs against the TSHR was as previously reported (11, 13) with minor modifications. TSHR-289, a variant of the receptor expressed in eukaryotic cells that corresponds approximately to the extracellular A subunit (19), was affinity purified from culture medium (20). ELISA wells were coated overnight with 100 µl of TSHR-289 protein (1 µg/ml) and incubated with mouse sera (1/300 dilution). After incubation with HRP-conjugated anti-mouse IgG (diluted 3000 times, A3673; Sigma-Aldrich) or subclass-specific anti-mouse IgG1 and IgG2a (diluted 1000 and 1500 times, respectively; X56 and R19-15; BD PharMingen, San Diego, CA), color was developed using o-phenylenediamine and H2O2 as substrate and the OD read at 492 nm.
Cytokine assays
COS cells seeded at 1 x 105 cells/well in a 24-well culture plate were infected with adenovirus encoding IL-12 or IL-4 at the indicated MOIs. The next day, the cells were washed three times with PBS and the culture was continued in fresh medium for 2 days. The concentrations of IL-12 or IL-4 in culture supernatants were determined with ELISA kits (BioSource International, Camarillo, CA) according to the manufacturer’s protocols. Serum levels of IL-4 and IL-12 were also measured with ELISA kits (BioSource International).
Splenocytes were cultured (triplicate aliquots) at 4 x 105 cells/well in a 96-well round-bottom plate in the presence or absence of TSHR-289 protein (5 µg/ml). Five days later, the concentrations of IFN- and IL-4 in the medium were determined with ELISA kits (BioSource International). Cytokine production was expressed as picograms per milliliter using standard curves of recombinant murine IL-12 and IL-4 or as a fold increase relative to cultures without TSHR Ag.
Thyroid histology
Thyroid tissues were removed and fixed with 10% Formalin in PBS. Tissues were embedded in paraffin and 5-µm thick sections were prepared and stained with H&E.
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Results |
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v
3- and v5-integrin, on the HI loop of the fiber knob, thus facilitating RGD/integrin-dependent viral infection (17). Although this mutant virus retains the binding activity with its cognate receptor, it exhibits higher infectivity to a variety of cells than wild-type adenovirus (17). COS cells infected with AdRGDCMVIL-4 and AdRGDCMVIL-12 produced significant amounts of IL-4 and IL-12, respectively, in a MOI-dependent manner (Fig. 1). Furthermore, i.m. injection of AdRGDCMVIL-4 and AdRGDCMVIL-12 (5 x 1010 particles/mouse) to mice also increased serum concentrations of IL-4 and IL-12, respectively, although the expression was transient (Fig. 2). In vitro infection (MOI of 3000 particles/cell) or in vivo injection (1011 particles/mouse) of AdCMVTSHR did not induce detectable IL-4 or IL-12 (data not shown).
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2 test).
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Th1 vs Th2 balance was examined in two ways: 1) in terms of the IgG subclass-specific ELISA (Fig. 3C) and 2) cytokines secreted by splenocytes in response to TSHR Ag (Fig. 5). The IgG2a:IgG1 (Th1:Th2) ratio of anti-TSHR Abs was significantly lower in the TSHR plus IL-4 group 3 wk after the second and third immunizations as compared with TSHR and TSHR plus IL-12 groups (p = 0.021 and 0.009, respectively, Student’s t test). However, this difference was not observed before the second immunization or 8 wk after the third immunization (p = 0.173 and 0.065). Although the mean IgG2a:IgG1 ratios in the TSHR/IL-12 group were slightly elevated compared with the mean values in the TSHR group throughout the experiment, the differences were not statistically significant.
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in response to TSHR Ag (Fig. 5, A and B). When expressed as fold increase, IFN- secretion was significantly lower in the TSHR plus IL-4 group vs the TSHR group (
6-fold vs 2.5-fold, p = 0.011) and significantly higher in the TSHR plus IL-12 group as compared with the TSHR group (36-fold, p = 0.0038; Fig. 5B). However, this difference was no longer observed in splenocytes isolated from mice at the end of the experiments (Fig. 5, C and D). These data clearly demonstrate the shifting of immune response, albeit transiently, to Th1 and Th2 by IL-12 and IL-4, respectively. No IL-4 secretion was observed in mice of any group (data not shown). Incidentally, the histology of the thyroid glands from hyperthyroid mice was essentially same among the three groups. In particular, the hyperthyroid mice had diffuse goiters with hypertrophy and hypercellularity of thyroid epithelial cells as previously reported (15), findings consistent with those in overstimulated thyroid glands. No lymphocytic infiltration was observed.
Finally, the feasibility of IL-4 for treatment of Graves’ disease was evaluated. Groups of hyperthyroid mice (n = 6–7 in each group) were left untreated or injected i.m. with AdRGDCMVIL-4 (5 x 1010 particles/mouse) alone or in combination with AdCMVTSHR (1011 particles/mouse) twice at a 2-wk interval. Serum T4 was measured 2 wk after the second immunization. Although the disease remitted spontaneously in some mice, the incidence of disease and mean T4 levels were not significantly different after treatment in three groups (Fig. 6).
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Discussion |
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, not IL-4, from splenocytes in response to TSHR Ag in TSHR-immunized mice reflect the induction of both Th2 and, particularly, Th1 immune responses in this model.
Coinjection of adenovirus expressing IL-4 skewed the development of the immune response against the TSHR toward the Th2 phenotype, as reflected in the decreased ratio of IgG2a:IgG1 TSHR Abs (Th1:Th2) as well as Ag-specific secretion by splenocytes of a Th1 cytokine IFN-. This deviation of anti-TSHR immune response away from Th1 domination was accompanied by significant suppression of stimulating TSHR Abs and hyperthyroidism. In contrast, coinjection of adenovirus expressing IL-12 enhanced IFN- secretion from splenocytes, that biasing toward Th1 did not affect induction of stimulating Abs or disease incidence. It is worth noting that Ab titers determined with ELISA and TBIAb assays were not affected quantitatively by expression of either cytokine, suggesting that Ab quality, not quantity, is responsible for disease induction. Similar results have recently been observed in our adenovirus model with different mouse strains (21) as well as for DNA vaccination of outbred mice (9). The significantly lower levels of TSAb and comparable levels of TBAb in the TSHR plus IL-4 group vs the TSHR group indicate that the protective effect of IL-4 is not attributed to the increased proportion of blocking to stimulating Abs. On the other hand, the significantly lower TBAb activities in the TSHR plus IL-12 group than in the other two groups suggest that the Th1/Th2 balance influences the ratio of stimulating/blocking Abs. Overall, these data strongly indicate the crucial role of the Th1 immune response in the pathogenesis of Graves’ disease in our model. Incidentally, it is unlikely that the different abilities of IgG1 and IgG2a to bind distinct FcRs and to activate complement (22) contribute to disease pathogenesis.
In this context, it is of interest that the Th2 adjuvant alum, but not the Th1 adjuvant polyriboinosinic polyribocytidylic acid, completely suppresses Ab production and disease development in another murine Graves’ model we have recently established. This model involves injecting female BALB/c mice with bone marrow-derived dendritic cells infected with adenovirus expressing TSHR (23). These data suggesting the importance of Th1 immune responses in Graves’ models utilizing TSHR-expressing adenovirus are consistent with our present observations, but are in sharp contrast to the previous studies with the Shimojo model of Graves’ disease (10).
These apparently opposing effects of alum in different animal models, along with controversial data regarding the Th1/Th2 paradigm in experimental murine and human disease (reviewed in the Introduction), indicate that it is unwise to conclude that Graves’ manifestations are the outcome of Th1-type immune responses. Moreover, the view that Th2 cytokines play a primary role in humoral autoimmune diseases may be unwarranted. Indeed, the importance of Th1 immune responses has been demonstrated in other Ab-mediated organ-specific autoimmune diseases such as autoimmune hemolytic anemia (AIHA), myasthenia gravis, and Goodpasture’s disease (24). For example, preferential secretion of Th1 cytokines in response to autoantigens has been demonstrated in AIHA and myasthenia gravis (24, 25). Moreover, amelioration in AIHA was observed when mice were injected with DNA encoding IL-4 or with anti-IL-12 Ab (24). On the other hand, in murine lupus, an Ab-mediated systemic autoimmune disease (26), disease activity was suppressed in IFN- and IL-4 KO mice as well as in IL-4-transgenic mice (27, 28). These divergent results are not easy to reconcile and may point to the complexity of cytokine interactions.
Cytokines exert variable actions on the induction/suppression of immune response, depending on the level and location of production or the route and timing of administration. For example, IL-4, which is generally believed to stimulate humoral immunity and inhibit a Th1 immune response (29), is also shown to have the indispensable role in establishment of an early phase of Th1 immune response (30, 31). Divergent effects of certain cytokines have been reported in Th1-dominated, T cell-mediated autoimmune diseases. Thus, the therapeutic efficacy of adenovirus-mediated transiently expressed IL-4 (32) and a pathogenic role of sustained administration of rIL-4 (33) have both been demonstrated in inflammatory bowel disease. Prevention of autoimmune diabetes in nonobese diabetic mice has been achieved with systemic injection of adenovirus-expressing IL-4 (34) and also in IL-4R KO mice (35).
In conclusion, Graves’ disease induced by injecting TSHR-expressing adenovirus is characterized by Th1 and to a lesser extent Th2 responses. Despite its protective effect on induction of murine Graves’ disease, IL-4 had no beneficial effect in a therapeutic setting. Skewing an established immune response might be more difficult than inducing a polarized response in naive mice. Indeed, it is reported that IL-4 fails to convert an already established Th1 response into a Th2 response (36). Nevertheless, the pathogenic potential of the Th1-type immune response in Graves’ hyperthyroidism suggests the need for caution in developing therapeutic strategies aimed at deviating a presumed Th2-mediated autoimmune response to Th1-dominant immunity. Furthermore, unlike murine Graves’ models, Th1-dominated subclinical chronic thyroiditis likely coexists with Graves’ disease in humans, as indicated by the presence of autoantibodies against thyroglobulin and thyroid peroxidase in most Graves’ sera, which may also worsen with such treatment. Further studies will be required to explore the therapeutic potential of cytokine therapy in humans with Graves’ disease.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TSHR, thyrotropin receptor; TSAb, thyroid-stimulating Ab; TSH, thyroid-stimulating hormone; TBIAb, TSH-binding inhibiting Ab; TBAb, TSH-blocking Ab; T4, thyroxine; MOI, multiplicity of infection; AIHA, autoimmune hemolytic anemia.
Received for publication October 21, 2002. Accepted for publication January 23, 2003.
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References |
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production, rather than antibody, dominates the immune response in mice. Endocrinology 142:3530. knock-out mice does not enhance TSH receptor antibody production following naked DNA vaccination. Endocrinology 143:1182.R). Ann. Hematol. 76:231.[Medline]
subunit presented by the susceptibility allele HLA-DR52a. Ann. Neurol. 45:224.[Medline]
and interleukin-4 in murine lupus. J. Clin. Invest. 99:1936.[Medline]
gene protects mice against autoimmune diabetes. Proc. Natl. Acad. Sci. USA 97:12700.This article has been cited by other articles:
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T. Kaneda, A. Honda, A. Hakozaki, T. Fuse, A. Muto, and T. Yoshida An Improved Graves' Disease Model Established by Using in Vivo Electroporation Exhibited Long-Term Immunity to Hyperthyroidism in BALB/c Mice Endocrinology, May 1, 2007; 148(5): 2335 - 2344. [Abstract] [Full Text] [PDF]
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J. A. Gilbert, S. L. Kalled, J. Moorhead, D. M. Hess, P. Rennert, Z. Li, M. Z. Khan, and J. P. Banga Treatment of Autoimmune Hyperthyroidism in a Murine Model of Graves' Disease with Tumor Necrosis Factor-Family Ligand Inhibitors Suggests a Key Role for B Cell Activating Factor in Disease Pathology Endocrinology, October 1, 2006; 147(10): 4561 - 4568. [Abstract] [Full Text] [PDF]
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C.-R. Chen, P. Pichurin, G. D. Chazenbalk, H. Aliesky, Y. Nagayama, S. M. McLachlan, and B. Rapoport Low-Dose Immunization with Adenovirus Expressing the Thyroid-Stimulating Hormone Receptor A-Subunit Deviates the Antibody Response toward That of Autoantibodies in Human Graves' Disease Endocrinology, January 1, 2004; 145(1): 228 - 233. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Mice immunized with AdCMVTSHR alone; •, those with AdCMVTSHR and AdGRDCMVIL-12; 
, those with AdCMVTSHR and AdRGDCMVIL-4; 
, naive mice. Data are means ± SD in B and D and means ± SE in C, each determined in duplicates. Arrows indicate adenovirus injections. * and **, p = 0.021 and 0.009 for the TSHR plus IL-4 group vs the TSHR group, respectively (Student’s t test).
