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. 2005 Sep;141(3):405–411. doi: 10.1111/j.1365-2249.2005.02874.x

Adenovirus-mediated gene delivery of interleukin-10, but not transforming growth factor β, ameliorates the induction of Graves’ hyperthyroidism in BALB/c mice

O Saitoh *, Y Mizutori *,†, N Takamura ‡, H Yamasaki †, A Kita †, H Kuwahara †, Y Nagayama *
PMCID: PMC1809459  PMID: 16045729

Abstract

Interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) are well known anti-inflammatory cytokines. We have studied the effect of adenovirus-mediated IL-10 and TGF-β gene delivery on the induction of Graves’ hyperthyroidism in our mouse model that involves repeated injections of adenovirus expressing the thyrotropin receptor A subunit (AdTSHR). We first constructed adenoviruses encoding the two cytokines (AdIL10 and AdTGFβ) and confirmed expression by in vitro infection of COS cells. Susceptible BALB/c mice were injected twice with AdTSHR alone or together with AdIL10 or AdTGFβ, and bled two weeks after the second immunization. Significantly elevated serum thyroxine levels were seen in 26% of mice immunized with AdTSHR and AdIL10 versus 61% with AdTSHR alone. Levels of thyroid stimulating antibody, but not nonstimulating antibody, were also decreased, and TSHR-specific splenocyte secretion of interferon-γ in recall assays was impaired in mice treated with AdIL10. In contrast, AdTGFβ had little effect on hyperthyroidism. Overall, our findings demonstrate that gene delivery of IL-10, but not TGF-β, suppresses the induction of Graves’ hyperthyroidism in a mouse model. However, the effect of IL-10 is less powerful than we observed previously with T helper type 2-inducers including adenovirus expressing IL-4, Shistosoma mansoni infection or α-galactosylceramide.

Keywords: Graves’ disease, thyrotropin receptor, interleukin-10, transforming growth factor-β, adenovirus

Introduction

Graves’ disease is a thyroid-specific autoimmune disease characterized by the presence of agonistic antithyrotropin receptor (TSHR) autoantibodies (thyroid stimulating antibodies, TSAb) that lead to hyperthyroidism and diffuse hyperplasia of the thyroid gland [1]. The mechanism(s) for the loss of immunological tolerance to autoantigen, the TSHR, remain to be elucidated. Although there exist three forms of well-established treatments for Graves’ disease, antithyroid drugs, radioactive iodine and subtotal thyroidectomy, these are all palliative and the long-term prognosis is not satisfactory and disease recurrence or development of hypothyroidism are not uncommon [2]. We have therefore been focusing on the approaches for novel immuno-therapies for Graves’ disease using a mouse model that we established recently [3–5].

Interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) are well known as immunoregulatory or anti-inflammatory cytokines [6]. IL-10 was initially identified as a cytokine produced by T helper type 2 (Th2) cells in mice that inhibits interferon-γ (IFN-γ) synthesis from Th1 cells [7,8]. It is also produced by other cells including antigen-presenting cells (APCs) and monocytes, and suppresses production of other proinflammatory cytokines (tumour necrosis-α (TNF-α), IL-1 and IL-12), proliferation and cytokine response of T cells. Similarly, TGF-β suppresses the activities of different types of immune cells including T cells and APCs and inhibits the expression of proinflammatory cytokines [9]. On the other hand, these cytokines also have some stimulatory effects on cells of the immune system. Thus, IL-10 is reported to function as a B cell stimulator that enhances activation, proliferation, differentiation and antibody production of B cells [7,8]. Stimulatory effects of TGF-β on T cells and APCs have also been demonstrated in certain conditions [9]. Nevertheless, immunosuppressive properties appear to predominate for both cytokines because mice deficient in either IL-10 or TGF-β by gene disruption spontaneously develop autoimmune diseases [10–23]. We were therefore curious to determine the consequence of expressing IL-10 or TGF-β in the induction of Graves’ hyperthyroidism in our mouse model [3]. Of surprising, we found that adenovirus-mediated IL-10, but not TGF-β, gene delivery was associated with lower TSAb levels and a reduced incidence of Graves’ hyperthyroidism.

Materials and methods

Construction, amplification and purification of recombinant adenoviruses

Adenovirus expressing TSHR289 (AdTSHR), a variant of the TSHR corresponding approximately to the A-subunit cleaved from the full-length receptor, was constructed previously [24] and kindly provided by Drs S. M. McLachlan and B. Rapoport (Autoimmune Disease Unit, Cedars-Sinai Medical Center, Los Angeles, CA, USA).

For the construction of adenovirus expressing mouse IL-10, pBluescript-IL-10 (from RIKEN DNA bank, Tukuba, Japan) was digested with Sac II, blunt ended with T4 DNA polymerase and digested with Apa I. The IL-10 cDNA fragment was then ligated into the shuttle vector pHMCMV6 [3] that had been digested with Kpn I, blunt-ended and digested with Apa I to yield pHMCMVIL10. For adenovirus expressing mouse TGF-β, TGF-β cDNA was obtained with a reverse transcription-polymerase chain reaction method by mRNA extraction from Con A-stimulated BLAB/c splenocytes using ISOGEN (Nippon Gene, Tokyo, Japan), followed by reverse-transcription with random primers and amplification of TGF-β cDNA with two specific primers (5′-tcg cta gcg aag tgc cgt ggg gcg c-3′ and 5′-agt cta gac ttc agc tgc act tgc ag-3′; the underlines indicate Nhe I and Xba I sites) (TAKARA RNAPCR kit Ver 2·1, TAKARA, Tokyo, Japan). The PCR product obtained was digested with Nhe I and Xba I and ligated into the compatible restriction sites of pHMCMV6 (pHMCMVTGFβ). The sequence was confirmed with ABI PRISM 310 automated capillary DNA sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). pHMCMVIL10 and pHMCMVTGFβ were digested with I-Ceu I and PI-Sce I and ligated into pAdHM15-RGD digested with the same enzymes [4]. The resultant plasmids pAdHMCMVIL10 and pAdHMCMVTGFβ were linealized with Pac I and transfected into 293 human embryonal kidney cells with SuperFect (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. Recombinant adenoviruses expressing IL-10 or TGF-β (designated AdIL10 and AdTGFβ) were then plaque-purified. Amplification and purification of adenovirus, and the determination of viral particle concentrations have previously been described [3–5]. The multiplicity of infection (MOI) was defined as the ratio of total number of particles used in a particular infection per number of cells.

Cytokine assay

COS cells seeded at 1 × 105 cells per well in a 24-well culture plate were infected with AdIL10 or AdTGFβ at a MOI of 10 000 particles per cell. The next day, the cells were washed with PBS and the culture was continued with fresh medium for two days. The concentrations of each cytokines in the culture supernatants were determined with an ELISA kit (IL-10, BioSource, Camarillo, CA, USA; TGF-β, R & D systems; Minneapolis, MN, USA) according to the manufacturer's protocols.

Splenocytes were cultured (triplicate aliquots) at 5 × 105 cells per well in a 96-well round-bottomed plate in the presence or absence of 5 µg/ml TSHR289 protein (from Drs S. M. McLachlan and B. Rapoport). TSHR protein was affinity purified from culture medium as described [25] and dialysed against 10 mM Tris-HCl, pH 7·4, 50 mM NaCl. Four days later, the concentrations of IFN-γ and IL-10 in the culture medium were determined with ELISA kits (Pharmingen, San Diego, CA, USA & BioSource) according to the manufacturer's protocols. Serum TGF-β concentrations were also measured with an ELISA kit (R & D systems).

Immunization protocol

Female BALB/c (six weeks old) were purchased from Charles River Japan Laboratory Inc. (Tokyo, Japan). All experiments were conducted in accordance with the principles and procedures outlined in the Guideline for the Care and Use of Laboratory Animals in Nagasaki University. Mice were kept in a specific pathogen free condition through the experiments. For immunization with adenovirus, mice were intramuscularly (i.m.) injected with 100 µl PBS containing 1 × 1011 particles of AdTSHR alone or in combination with 5 × 1010 particles of AdIL10 or AdTGFβ. This immunization schedule was performed twice at three-week intervals.

Thyroid function test

Total thyroxine (T4) concentrations in mouse sera were measured with a commercially available radioimmunoassay kit (SPAC T4 RIA kit; TFB, Tokyo, Japan). The normal range was defined as the mean ± 3 SD. of control mice.

TSAb, thyroid blocking antibodies (TBAb) and TSH binding inhibiting antibodies (TBIAb) measurements

TSAb and TBAb activities in mouse sera were measured with FRTL5 cells as previously described [3–5]. Briefly, for TSAb assay the cells were seeded at 5 × 104 cells/well in a 96-well culture plate and incubated in 50 µl hypotonic HBSS containing 0·5 mM isobutyl-methylxanthine, 20 mM HEPES, 0·25% BSA and 5 µl serum for 2 h at 37 C. cAMP released into the medium was measured with a cAMP radioimmunoassay kit (Yamasa, Choshi, Japan). A value over 150% of control mice was judged as positive. TBAb activities were measured in the same buffer supplemented with 100 µU/ml bovine TSH (Sigma, St. Louis, MO, USA), 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 GmbH, Berlin, Germany). Ten µl of serum was used for each assay. A value over 15% inhibition of control binding was judged as positive.

ELISA for anti-TSHR antibodies

ELISA wells were coated overnight with 100 µl TSHR289 protein (see above; 1 µg/ml) and incubated with mouse sera (1 : 100 dilution). After incubation with horseradish peroxidase conjugated antimouse IgG (diluted 3000×, A3673, Sigma), or subclass-specific antimouse IgG1 and IgG2a (diluted 1000× and 1500×; X56 and R19-15, BD PharMingen), colour was developed using orthophenylene diamine and H2O2 as substrate and optical density read at 492 nm.

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 haematoxylin and eosin.

Data analysis

Data were analysed by unpaired Students’t-test or by the chi-square test using a StatView 4·02 software (Abacus Concept Inc., Berkeley, CA, USA). Probability values less than 0·05 were considered statistically significant.

Results

The functional integrity of AdIL10 and AdTGFβ constructed for this study were first evaluated by infecting COS cells. As shown in Fig. 1a,b, adenovirus-infected COS cells produced significant amounts of each cytokine compared with uninfected cells.

Fig. 1.

Fig. 1

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In vitro production of IL-10 and TGF-β from control and AdIL10- or AdTGFβ-infected COS cells. The concentration of each cytokine released into the culture supernatants from control and adenovirus-infected COS cells (MOI of 10 000 particles/cell) after two days was measured by ELISA. Data are shown as the mean ± range (n = 2). N.D., not detectable.

To evaluate the effect of IL-10, BALB/c mice were untreated or injected i.m. with AdTSHR alone (1010 particles/mouse) or in combination with AdIL10 (5 × 109 particles/mouse) on two occasions three weeks apart. Blood and thyroid tissues were obtained two weeks after the last immunization. In mice immunized with AdTSHR alone (TSHR group) or with AdTSHR and AdIL10 (TSHR/IL-10 group), serum T4 levels were significantly increased compared with those in control mice (Fig. 2a). The incidence of Graves’ hyperthyroidism was defined as T4 levels exceeding the mean +3 SD. of control mice. On this basis, 61% (14 of 23) in the TSHR group were hyperthyroid versus 26% (6 of 23) in the TSHR/IL-10 group. Thus, induction of hyperthyroidism was significantly reduced in the TSHR/IL-10 group compared with the TSHR group (P = 0·016, χ2 test).

Fig. 2.

Fig. 2

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T4, TSAb, TBAb and TBIAb in BALB/c mice immunized with AdTSHR alone or in combination with AdIL10. Mice were immunized twice with AdTSHR alone or together with AdIL10 with a three weekly interval and bled two weeks after the second injection. Each value represents the mean of duplicate determinations. Horizontal lines, the upper normal limits in control (naïve) mice. ○ hyperthyroid; • euthyroid.

Anti-TSHR antibodies were measured in four different ways. The first and second methods were the TSAb and TBAb assays that detect stimulating and blocking antibodies capable of causing hyperthyroidism and hypothyroidism, respectively. TSAb activities were significantly higher in the TSHR group than the TSHR/IL-10 group (P = 0·029). Moreover, TSAb activities were positive in the majority of hyperthyroid mice and some euthyroid mice in the TSHR group but in only one of the TSHR/IL-10 group (Fig. 2b). However, no significant difference was observed between two groups in TBAb assay (Fig. 2c). The third method was the TBIAb assay, which measures the ability of anti-TSHR antibodies to displace 125I-TSH binding to the TSHR and cannot discriminate between stimulatory and inhibitory antibodies. TBIAb activities were strongly positive in both TSHR and TSHR/IL-10 groups (Fig. 2d). Fourthly, IgG class anti-TSHR antibodies measured by ELISA revealed significantly higher levels in the TSHR group than the TSHR/IL-10 group (Fig. 3a). TSHR antibody IgG subclasses, also determined by ELISA, showed a slight elevation of IgG1 to IgG2a (Th2 to Th1) ratios in the TSHR/IL-10 group, although the difference was not statistically significant because of variability between individual mice (Fig. 3b).

Fig. 3.

Fig. 3

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Anti-TSHR antibodies analysed by ELISA in mice immunized with AdTSHR and/or AdTSHR + AdIL10. Values for IgG class antibodies and the IgG1 to IgG2a ratios of TSHR antibodies are presented as the mean + S.E. *P < 0·01; NS, not significant.

As another marker of the effect of AdIL10, cytokine secretion was evaluated by splenocytes from immunized mice challenged in vitro with TSHR antigen. IFN-γ production increased approximately 9-fold in splenocytes from AdTSHR-immunized mice compared with a 1·2-fold in the AdTSHR/AdIL10 immunized mice (Fig. 4). Of interest, the basal level of IFN-γ was increased in splenocytes from the latter group of mice, reminiscent of the data we obtained using AdIL4 or Schistosoma mansoni[4,5]. Unlike the clear-cut IFN-γ response, there was no difference in IL-10 production by splenocytes from mice immunized with AdTSHR versus AdTSHR/AdIL10 (data not shown).

Fig. 4.

Fig. 4

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IFN-γ production by splenocytes from BALB/c mice immunized with AdTSHR alone or together with AdIL10. Splenocytes were prepared 10 days after 1 × immunization and were stimulated with TSHR289 protein (5 µg/ml) for four days. IFN-γ levels in the culture supernatants were measured by ELISA. Data are the mean + S.D. (n = 4).

The outcome of TGF-β expression on immunization with AdTSHR was evaluated using the same approach. Unlike the findings for AdIL10, coinjection of AdTGFβ had no effect on the incidence of hyperthyroidism (66·7% in the TSHR group versus 73·3% in the TSHR/TGF-β group), T4 levels, TSAb, or TBIAb (Figs 5a). Nor was there a change in the IgG1/IgG2a ratios for TSHR antibodies (Fig. 6b). However, IgG class antibody levels were lower in the TSHR/TGF-β group than in TSHR group (Figs 6a, P < 0·01), and antigen-specific splenocyte secretion of IFN-γ was also suppressed (Fig. 7). Serum TGF-β concentrations were determined after AdTGFβ injection to confirm its in vivo expression, showing a clear increase form 30·9 ± 5·1 pg/ml (mean ± S.E., n = 3) before injection (day 0) to 51·0 ± 2·6 and 73·7 ± 29·2 pg/ml on days 2 and 4, respectively.

Fig. 5.

Fig. 5

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T4, TSAb and TBIAb in BALB/c mice immunized with AdTSHR alone or in combination with AdTGFβ. Mice were immunized twice with AdTSHR alone or together with AdTGFβ with a three weekly interval, and blood was obtained two weeks after the second injection. Data are the means of duplicate determinations. Horizontal lines, the upper normal limits in control (naïve) mice. ○ hyperthyroid; • euthyroid.

Fig. 6.

Fig. 6

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Anti-TSHR antibodies analysed by ELISA in mice immunized with AdTSHR and/or AdTSHR + AdTGFβ. Values for IgG class antibodies and the IgG1 to IgG2a ratios are presented as the mean ± S.E.

Fig. 7.

Fig. 7

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IFN-γ production by splenocytes from BALB/c mice immunized with AdTSHR alone or together with AdTGFβ. Splenocytes were prepared 10 days after 1 × immunization and were stimulated with TSHR289 protein (5 µg/ml) for four days. IFN-γ levels in the culture supernatants were measured by ELISA. Data are the mean ± S.D. (n = 4).

Finally, it was of interest to determine whether the expression of IL-10 or TGF-β had an effect on lymphocytic infiltration of the thyroid glands. In NOD.H-2h4 mice, for example, spontaneous thyroiditis was inhibited by treatment with an antibody to TGF-β[26]. However, as previously reported [3,24], hyperthyroid mice had diffuse goitres with hypertrophy and hypercellularity of thyroid epithelial cells but no lymphocytic infiltration (data not shown).

Discussion

IL-10 and TGF-β are well known anti-inflammatory cytokines, although they have dual and sometimes nonoverlapping effects on the immune system. The effects of these cytokines have been studied in a variety of autoimmune diseases. With regard to IL-10, in organ-restricted cell-mediated autoimmune diseases, administration of IL-10 protein suppressed EAE, diabetes and collagen-induced arthritis [12–14]. However, conflicting observations have been described for the outcome of IL-10 gene delivery. For example, adenovirus-mediated, but not herpes simplex virus-mediated, IL-10 gene delivery inhibited EAE [15,16]. Systemic expression of IL-10 showed a beneficial effect on diabetes but its local expression exacerbated the same disease [17,18]. These variable effects may depend on dosage, timing, site of IL-10 expression. In addition, IL-10 enhances some autoantibody-mediated diseases including lupus and myasthenia gravis [27,28], presumably due to the ability of IL-10 to stimulate B cells. However, the same effect was not observed in autoimmune haemolytic anaemia [29]. Because Graves’ disease is an autoantibody-mediated autoimmune disease, it seemed possible that IL-10 administration might have the desired effect in our Graves’ model, namely reduction of TSHR antibody production and suppression of disease, or an undesirable outcome, namely enhance TSHR antibody production and exacerbation of disease.

Turning to TGF-β, protective effects on other autoimmune diseases (diabetes, EAE, experimental colitis, SLE) have been described using this cytokine both as a protein [19,20] and by gene delivery [21–23]. Conversely, however, local expression of TGF-β in pancreas together with TNF-α induced diabetes [30].

Against this background, we investigated the ability of adenovirus-mediated IL-10 and TGF-β gene delivery to change the induction of Graves’ hyperthyroidism. Although these cytokines can be expressed systemically by i.m. injection of adenovirus vectors, the local concentration of these cytokines is also likely to be extremely high at the injection sites. Consequently, using this approach, both systemic and local effects of these cytokines could be anticipated.

Our findings demonstrate that adenovirus-mediated IL-10, but not TGF-β, gene delivery has a beneficial effect and reduced the induction of Graves’ hyperthyroidism. As for Th2-inducers we have used recently [4,5], IL-10 suppressed TSAb production and hyperthyroidism, but did not change TSHR antibody levels as shown in TBIAb and TBAb assays. These observations suggest that IL-10 does not inhibit the initiation of anti-TSHR autoimmunity, but does prevent the progression from autoimmunity to overt autoimmune disease.

We have recently shown that in our model, Graves’ hyperthyroidism is a Th1-dominant autoimmune disease [4,5]. Thus, coinjection of adenovirus expressing the Th2 cytokine IL-4, prior infection of S. mansoni or simultaneous administration of α-galactosylceramide all polarized anti-TSHR immune responses away from a Th1 phenotype and protected susceptible BALB/c mice from developing Graves’ hyperthyroidism. In contrast, the Th1 cytokine IL-12 was associated with deviation of TSHR-specific immune response to Th1 without changing disease incidence. The similar results were also demonstrated in another study with the combined intradermal injection of TSHR-DNA and cytokine-DNAs (31). Because IL-10 is also a Th2 cytokine in mice [32], its ability to suppress the induction of TSAb and hyperthyroidism is likely to be attributable, at least in part, to immune deviation to a Th2 phenotype. Consistent with these previous findings, IL-10 suppressed TSHR-specific splenocyte production of a Th1 cytokine IFN-γ, although the increase in the IgG1/IgG2a (Th2/Th1) ratios of anti-TSHR antibodies did not reach statistical significance. It should be noted here that Th1-immune response is not necessarily equal to cell-mediated immune response because antibodies are also produced in Th1-immune response. It is indeed reported that TSAbs in human Graves’ disease belong to Th1 type of IgG subclass (33).

It should be emphasized that the suppressive effect of IL-10 (present study) was not as impressive as with other Th2-inducers we have employed recently [4,5]. Thus, disease incidence declined from 60 to 26% by AdIL10 in the present study versus from 60 to 70-7-12% using AdIL4, S. mansoni and α-galactosylceramide. A possible explanation for these observations is that the suppressive effect of IL-10-mediated Th2-immune deviation on hyperthyroidism is masked by the enhancing effect of IL-10 on antibody production.

Why TGF-β is unable to influence the induction of Graves’ hyperthyroidism is unknown at present. Some reports have demonstrated different outcomes using cytokines with similar properties to treat autoimmune diseases. Broberg et al. [15] recently reported the efficacy of IL-4, but not IL-10, to treat EAE in their experimental setting using replicative herpes simplex virus as a gene delivery vehicle. Conversely, in experimental autoimmune thyroiditis, a model for Hashimoto thyroiditis in humans, IL-10, but not IL-4, had a suppressive effect on thyroglobulin (Tg)-specific cytotoxic T cells [34]. Moreover, direct injection of plasmid coding IL-10 into the thyroid gland effectively cured Tg-induced thyroiditis and also reduced IFN-γ levels and anti-Tg antibody responses [35]. Although the reason(s) for the discrepancies remain to be elucidated, these reports together with our current data suggest that different Th2 or regulatory cytokines play different roles in the pathogenesis of individual autoimmune diseases. Of interest, TGF-β also suppressed antigen-specific splenocyte secretion of IFN-γ, suggesting suppression of IFN-γ is not sufficient for protection from development of hyperthyroidism.

Our model may not precisely mimic human disease. Nevertheless, the model permits testing the effects of potential novel therapies. Future studies will thus be necessary to evaluate the effect of these anti-inflammatory cytokines in the different approaches. For example, use of regulatory (or tolerogenic) APCs induced by in vitro treatment with regulatory cytokines [36], or APCs or autoantigen-reactive T lymphocytes genetically engineered to secrete regulatory cytokines [37] will be of interest. These approaches will also avoid another disadvantage of systemic administration of regulatory cytokines, namely indiscriminate suppression of immune responses in general.

Acknowledgments

We thank RIKEN DNA bank (Tsukuba, Japan) for mouse IL-10 cDNA and Drs S. M. McLachlan and B. Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA, USA) for the AdTSHR and protein, helpful discussions, and also comments regarding this manuscript.

References

  • 1.Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM. The thyrotropin receptor: interaction with thyrotropin and autoantibodies. Endocr Rev. 1998;19:673–716. doi: 10.1210/edrv.19.6.0352. [DOI] [PubMed] [Google Scholar]
  • 2.Cooper DS. Treatment of thyrotoxicosis. In: Braverman LE, Utiger RD, editors. The Thyroid. 8th edn. Philadelphia: Lippincott. Williams & Wilkins; 2000. pp. 691–715. [Google Scholar]
  • 3.Nagayama Y, Kita-Furuyama M, Nakao K, Ando T, Mizuguchi H, Hayakawa T, Eguchi K, Niwa M. A novel murine model of Gravesí hyperthyroidism with intramuscular injection of adenovirus expressing thyrotropin receptor. J Immunol. 2002;168:2789–94. doi: 10.4049/jimmunol.168.6.2789. [DOI] [PubMed] [Google Scholar]
  • 4.Nagayama Y, Mizuguchi H, Hayakawa T, Niwa M, McLachlan SM, Rapoport B. Prevention of autoantibody-mediated Gravesí-like hyperthyroidism in mice by IL-4, a Th2 cytokine. J Immunol. 2003;170:3522–7. doi: 10.4049/jimmunol.170.7.3522. [DOI] [PubMed] [Google Scholar]
  • 5.Nagayama Y, Niwa M, McLachlan SM, Rapoport B. Schistosoma mansoni and α-galactosylceramide: prophylactic effect of Th1 immune suppression in a mouse model of Gravesí hyperthyroidism. J Immunol. 2004;173:2167–73. doi: 10.4049/jimmunol.173.3.2167. [DOI] [PubMed] [Google Scholar]
  • 6.Roncarolo MG, Bacchetta R, Bordignon C, Narula S, Livins MK. Type 1 T regulatory cells. Immunol Rev. 2001;182:68–79. doi: 10.1034/j.1600-065x.2001.1820105.x. [DOI] [PubMed] [Google Scholar]
  • 7.Asadullah K, Sterry W, Volk AHD. Interleukin-10 therapy — review of a new approach. Pharmacol Rev. 2003;55:241–69. doi: 10.1124/pr.55.2.4. [DOI] [PubMed] [Google Scholar]
  • 8.Moore KW, de Waal Malefyt R, Coffman RL, OíGarra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683–765. doi: 10.1146/annurev.immunol.19.1.683. [DOI] [PubMed] [Google Scholar]
  • 9.Letterio JJ, Roberts AB. Regulation of immune responses by TGF-β. Annu Rev Immunol. 1998;16:37–161. doi: 10.1146/annurev.immunol.16.1.137. [DOI] [PubMed] [Google Scholar]
  • 10.Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W. Interleukin-10 — deficient mice develop chronic enterocolitis. Cell. 1993;75:263–74. doi: 10.1016/0092-8674(93)80068-p. [DOI] [PubMed] [Google Scholar]
  • 11.Shull MM, Ormsby I, Kier AB, et al. Targeted disruption of the mouse transforming growth factor-b1 gene results in multifocal inflammatory disease. Nature. 1992;359:693–9. doi: 10.1038/359693a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Rott O, Fleischer B, Cash E. Interleukin-10 prevents experimental allergic encephalomyelitis in rats. Eur J Immunol. 1994;24:1434–40. doi: 10.1002/eji.1830240629. [DOI] [PubMed] [Google Scholar]
  • 13.Pennline KJ, Roque-Gaffney E, Monahan M. Recombinant human interleukin-10 prevents the onset of diabetes in the nonobese diabetic (NOD) mouse. Clin Immunol Immunopathol. 1994;71:169–75. doi: 10.1006/clin.1994.1068. [DOI] [PubMed] [Google Scholar]
  • 14.Persson S, Mikulowska A, Narula S, OíGarra A, Holmdahl R. Interleukin-10 suppresses the development of collagen type II arthritis in mice and ameliorates sustained arthritis in rats. Scand J Immunol. 1996;44:607–14. doi: 10.1046/j.1365-3083.1996.d01-355.x. [DOI] [PubMed] [Google Scholar]
  • 15.Broberg E, Setala N, Roytta M, Salmi A, Eralinna J-P, He B, Roizman B. Expression of interleukin-4 but not interleukin-10 from a replicative herpes simplex virus type I viral vector precludes experimental allergic encephalitis. Gene Ther. 2001;8:769–77. doi: 10.1038/sj.gt.3301465. [DOI] [PubMed] [Google Scholar]
  • 16.Cua DJ, Hutchins B, Laface DM, Stohlman SA, Coffmann RL. Central nervous system expression of IL-10 inhibits autoimmune encephalomyelitis. J Immunol. 2001;166:602–8. doi: 10.4049/jimmunol.166.1.602. [DOI] [PubMed] [Google Scholar]
  • 17.Goudy KS, Song S, Wasserfall C, et al. Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice. Proc Natl Acad Sci USA. 2001;98:13913–8. doi: 10.1073/pnas.251532298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Wogensen L, Lee MS, Sarvetnick N. 1994. Production of interleukin 10 by islet cells accelerates immune-mediated destruction of beta cells in nonobese diabetic mice. J Immunol. 2001;166:4124–30. doi: 10.1084/jem.179.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lennart DJ, Flanders KC, Rangers GE, Sriram S. Successful treatment of experimental allergic encephalomyelitis with transforming growth factor-β1. J Immunol. 1991;147:1792–6. [PubMed] [Google Scholar]
  • 20.Kuruvilla AP, Shah PR, Hochwald GM, Liggitt HD, Palladino MA, Thorbecke GJ. Protective effect of transforming growth factor-β1 on experimental autoimmune diabetes in mice. Proc Natl Acad Sci USA. 1991;88:2918–21. doi: 10.1073/pnas.88.7.2918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Giladi E, Raz E, Karmeli F, Okon E, Rachmilewitz D. Transforming growth factor-β gene therapy ameliorates experimental colitis in rats. Eur J Gastroent Hepatol. 1995;7:341–7. [PubMed] [Google Scholar]
  • 22.Raz E, Lotz M, Baris SM, Berry CC, Eisenberg RA, Carson D. Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene therapy. Lupus. 1995;4:286–92. doi: 10.1177/096120339500400409. [DOI] [PubMed] [Google Scholar]
  • 23.Picchirillo CA, Chang Y, Prudíhomme GJ. TGF-β1 somatic gene therapy prevents autoimmune disease in no obese diabetes mice. J Immunol. 1998;161:3950–6. [PubMed] [Google Scholar]
  • 24.Chen C-R, Pichurin P, Nagayama Y, Latrofa F, Rapoport B, McLachlan SM. The thyrotropin receptor autoantigen in Gravesí disease is the culprit as well as the victim. J Clin Invest. 2003;111:1897–904. doi: 10.1172/JCI17069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chazenbalk GD, McLachlan SM, Pichurin P, Yan X-M, Rapoport B. A ‘prion-like’ shift between two conformational forms of a recombinant thyrotropin receptor A subunit module: Purification and stabilization using chemical chaperones of the form reactive with Graves’ autoantibodies. J Clin Endocrinol Metab. 2001;86:1287–93. doi: 10.1210/jcem.86.3.7363. [DOI] [PubMed] [Google Scholar]
  • 26.Braley-Mullen H, Chen K, Wei Y, Yu S. Role of TGF-β in development of spontaneous autoimmune thyroiditis in NOD.H-2h4 mice. J Immunol. 2001;167:7111–8. doi: 10.4049/jimmunol.167.12.7111. [DOI] [PubMed] [Google Scholar]
  • 27.Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S, Howard M. Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J Exp Med. 1994;179:305–10. doi: 10.1084/jem.179.1.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zhang G-X, Xiao B-G, Yu L-Y, van der Meide PH, Link H. Interleukin-10 aggravates experimental autoimmune myasthenia gravis through inducing Th2 and B cell responses to AChR. J Neuroimmunol. 2001;113:10–8. doi: 10.1016/s0165-5728(00)00411-2. [DOI] [PubMed] [Google Scholar]
  • 29.Youssef AR, Shen CR, Lin CL, Barker RN, Elson CJ. IL-4 and IL-10 modulate autoimmune haemolytic anaemia in NZB mice. Clin Exp Immunol. 2005;139:84–9. doi: 10.1111/j.1365-2249.2005.02663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Sanvito F, Nichols A, Herrera PL, Wohlwend A, Vassali JD, Orci L. TGF-β overexpression in murine pancreas induces chronic pancreatitis and together with TNF-α, triggers insulin-dependent diabetes. Biochem Biophys Res Commun. 1995;217:1279–86. doi: 10.1006/bbrc.1995.2906. [DOI] [PubMed] [Google Scholar]
  • 31.Barrett K, Liakata E, Rao PV, Watson PF, Weetman AP, Lymberi P, Banga JP. Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor. Clin Exp Immunol. 2004;136:413–22. doi: 10.1111/j.1365-2249.2004.02483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Mestas J, Hughes CCW. of mice and not men: differences between mouse and human immunology. J Immunol. 2004;172:2731–8. doi: 10.4049/jimmunol.172.5.2731. [DOI] [PubMed] [Google Scholar]
  • 33.Weetman AP, Yateman ME, Ealey PA, Black CM, Reimer CB, Williams Jr RC, Shine B, Marshall NJ. Thyroid-stimulating antibody activity between different immunoglobulin G subclasses. J Clin Invest. 1990;86:723–7. doi: 10.1172/JCI114768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Mignon-Godefroy K, Brazillet MP, Rott O, Charreire J. Distinctive modulation by IL-4 and IL-10 of the effector function of murine thyroglobulin-primed cells in ìtransfer-experimental autoimmune thyroiditisî. Cell Immunol. 1995;162:171–7. doi: 10.1006/cimm.1995.1066. [DOI] [PubMed] [Google Scholar]
  • 35.Batteux F, Trebeden H, Charreire J, Chiocchia G. Curative effect of experimental autoimmune thyroiditis by in vivo administration of plasmid coding for interleukin-10. Eur J Immunol. 1999;29:958–63. doi: 10.1002/(SICI)1521-4141(199903)29:03<958::AID-IMMU958>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  • 36.Rutella S, Lemoli RM. Regulatory T cells and tolerogenic dendritic cells: from basic biology to clinical applications. Immunol Lett. 2004;94:11–26. doi: 10.1016/j.imlet.2004.04.015. [DOI] [PubMed] [Google Scholar]
  • 37.Tarner IH, Fathman CG. Gene therapy in autoimmune disease. Cur Opin Immunol. 2001;13:676–82. doi: 10.1016/s0952-7915(01)00278-3. [DOI] [PubMed] [Google Scholar]

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