help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0352
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Land, K. J.
Right arrow Articles by Seetharamaiah, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Land, K. J.
Right arrow Articles by Seetharamaiah, G. S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*LEVOTHYROXINE
Endocrinology Vol. 145, No. 8 3724-3730
Copyright © 2004 by The Endocrine Society

Signal Transducer and Activator of Transcription (Stat)-6-Dependent, But Not Stat4-Dependent, Immunity Is Required for the Development of Autoimmunity in Graves’ Hyperthyroidism

Kimberly J. Land, Jennifer S. Moll, Mark H. Kaplan and Gattadahalli S. Seetharamaiah

Department of Biochemistry and Molecular Biology (K.J.L., J.S.M., G.S.S.), Indiana University School of Medicine, Evansville, Indiana 47712; and Department of Microbiology and Immunology (M.H.K.), Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Gattadahalli S. Seetharamaiah, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 8600 University Boulevard, Evansville, Indiana 47712. E-mail: seethara{at}iupui.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of T helper (Th) cells in experimental models of Graves’ hyperthyroidism is still somewhat controversial. To further investigate the role of Th1- and Th2-dependent immunity during the development of Graves’ hyperthyroidism, we tested mice with targeted deletion of signal transducer and activator of transcription-4 (Stat4) or Stat6 genes that, respectively, have impaired Th1 and Th2 immunity. We immunized wild-type BALB/c, Stat4–/–, or Stat6–/– mice with human embryonic kidney cells (293 cells) expressing the extracellular domain of human TSH receptor (293-TBP cells). Fifty percent of wild-type BALB/c and Stat4–/– mice developed Graves’ hyperthyroidism with elevated serum T4 levels and thyroid stimulatory antibodies. In contrast, Stat6–/– mice resisted development of the disease. Stat4–/– mice exhibited a dominant Th2 immune response characterized by the production of IL-4 and IgG1 anti-TSH receptor antibodies. However, Stat6–/– mice displayed a strong Th1 immune response characterized by the production of interferon-{gamma} and IgG2a antibodies. Hyperthyroid mice showed enlargement of thyroid glands with hypertrophy and decreased amounts of colloid material, all characteristics of Graves’ disease. These data demonstrate that in this model, Stat6-dependent Th2 immunity is critical for the development of Graves’ hyperthyroidism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID-STIMULATORY ANTIBODIES (TSAbs) bind to the TSH receptor (TSHR), mimic the action of TSH, and cause hypersecretion of thyroid hormones, resulting in Graves’ disease (GD) (1, 2). Animal models are useful to study the role of TSHR-specific T cells and TSHR antibodies (Abs) during the development of GD. Recent studies have successfully established animal models of GD using different approaches (3, 4, 5, 6). Although GD is mediated by TSAbs, the generation of Abs requires TSHR-specific T cell help. CD4+ T cells functionally differentiate into two different phenotypes, T helper type 1 (Th1) and Th2 cells (7). The Th1 immune response is characterized by the production of interferon-{gamma} (IFN-{gamma}) and Abs of the IgG2a isotype, and the Th2 immune response is characterized by the production of IL-4 and IgG1 Abs (7, 8, 9, 10). Each subset mediates distinct types of inflammation; the Th1 cells mediate many autoimmune disorders, and Th2 cells regulate atopic disease.

Analysis of the involvement of Th1 and Th2 responses in murine models of Graves’ hyperthyroidism has provided conflicting results. Models using fibroblasts coexpressing TSHR and major histocompatibility complex (MHC) class II, along with a Th2 adjuvant, or TSHR DNA vaccination suggest that the Th2 response correlates with disease (5, 11, 12). In a study in which IFN-{gamma}–/– and IL-4–/– mice were immunized with M12 cells expressing TSHR, only IFN-{gamma}–/– mice, not IL-4–/– mice, developed Graves’ hyperthyroidism, demonstrating that the Th2 response is critical for the development of the disease in this model (13). In contrast, studies from others using fibroblasts coexpressing TSHR and MHC class II, TSHR DNA, and an adenovirus expressing TSHR suggest the dominance of Th1 responses in Graves’ hyperthyroidism (14, 15, 16).

Signal transducers and activator of transcription (Stat) are a class of molecules that mediate many cytokine-mediated responses (17, 18). Stat4 protein, which is critical for the Th1 immune response, is activated by IL-12 and induces the transcription of IFN-{gamma}. Stat4–/– mice lack IL-12-induced IFN-{gamma} production and Th1 differentiation (19, 20). In contrast, Stat6 protein is required for the development of the Th2 immune response and is activated by IL-4. Stat6–/– mice exhibit a reduction in Th2 cytokine production and decreased IL-4-induced B cell proliferation (21, 22). Stat4–/– mice are resistant to the development of T cell-mediated autoimmune diseases while becoming susceptible to infections with intracellular parasites, such as Toxoplasma gondii and Trypanosoma cruzi (23, 24, 25, 26). Stat6–/– mice have increased susceptibility to T cell-mediated autoimmune disease, whereas they are resistant to the development of allergic asthma (24, 27, 28). Thus, these mice provide an ideal model to establish the requirement for Th1 and Th2 responses in disease and immunity.

In this study we investigated the requirement for Th1 and Th2 responses in Graves’ hyperthyroidism using Stat4–/– or Stat6–/– mice. Our results show that Stat6–/–, but not Stat4–/–, mice are protected from the development of Graves’ hyperthyroidism. These data demonstrate that in this model, Th2 immunity is required for the development of Graves’ hyperthyroidism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and TSHR protein
Stat4–/– and Stat6–/– mice were generated as described previously (19, 21) and were backcrossed 10 generations to a BALB/c genetic background. BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Experiments were performed after receiving approval from the Indiana University animal care and use committee.

The glycosylated extracellular domain of human (h) TSHR (ET-gp) expressed in insect cells was purified as described previously (29, 30). Human embryonic kidney cells (293 cells) expressing the extracellular domain of hTSHR (293-TBP cells) were generated and maintained as described previously (4, 31). These cells were grown in DMEM/Ham’s F-12 with 10% fetal bovine serum and 200 µg/ml G-418 (Invitrogen, Carlsbad, CA). Soluble TSH-binding protein (TBP) was purified using a nickel affinity column as described previously (4, 31).

Immunization protocol
Six- to 8-wk-old female BALB/c, Stat4–/–, and Stat6–/– mice were immunized according to the following schedule. Experimental groups of mice (six mice per group) were injected ip with 100 µg ET-gp emulsified in Freund’s complete adjuvant (Sigma-Aldrich Corp., St. Louis, MO) on d 0 and with 100 µg ET-gp in Freund’s incomplete adjuvant (Sigma-Aldrich Corp.) on d 14 and 28. Subsequently, these mice were immunized six times at 2-wk intervals with 2 x 107 293-TBP cells along with cholera toxin B subunit (5 µg/mouse) (Sigma-Aldrich Corp.). Similarly, the control groups of mice (five mice per group) were immunized with PBS in Freund’s complete adjuvant, PBS in Freund’s incomplete adjuvant, and 293 cells in cholera toxin B subunit. The 293-TBP and 293 cells were pretreated with mitomycin C (50 µg/107 cells; Acros, Fairlawn, NJ). Blood was collected periodically through the tail vein and tested for anti-TSHR Abs and thyroid hormone levels. On d 300, all surviving mice were killed, and thyroid, spleen, and serum were collected from each mouse.

Measurement of TSH binding inhibitory Igs (TBII), T4, and thyroid-stimulating antibodies (TSAbs)
TBII values in sera were determined using a commercially available TRAb kit (Kronus, Boise, ID) as described previously (30, 32). This assay measures the ability of Abs in sera to inhibit the binding of 125I-labeled TSH to TSHR. Results are expressed as a percentage of the TBII values.

Total T4 in serum was measured with a commercially available RIA kit (Diagnostic Products Corp., Los Angeles, CA) as described previously (30, 33). This assay measures the ability of T4 in serum to compete with 125I-labeled T4 for binding to anti-T4 Ab-coated polypropylene tubes.

TSAbs in serum were measured with CHO cells expressing TSHR (29, 30). These cells were grown to confluence in 96-well plates in F-12 medium supplemented with 10% fetal bovine serum. Cells were incubated with serum diluted in hypotonic HBSS containing 0.5 mM 3-isobutylmethylxanthine for 2 h at 37 C. The cAMP released into the medium was measured with a cAMP RIA kit (PerkinElmer, Boston, MA).

ELISA to measure anti-TSHR Abs
Anti-TSHR Abs were measured by ELISA as described previously (33, 34). Briefly, wells were coated with 100 ng ET-gp protein in 100 µl carbonate-bicarbonate buffer, pH 9.6, and incubated overnight at 4 C. The following reagents were added in succession after incubation for 1 h at 37 C, followed by washing the plate between each step: 1% BSA (to reduce nonspecific binding), serially diluted mouse serum, and horseradish peroxidase-conjugated goat antimouse IgG, IgG1, IgG2a, or IgG2b (Southern Biotechnology Associates, Birmingham, AL). Color was developed using 2,2'-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (Roche, Indianapolis, IN) and H2O2 as substrate in citrate buffer, pH 4.9, and absorbance was measured at 405 nm.

T cell proliferation in response to TBP
Splenocytes were cultured in triplicate (5 x 105 cells) in 96-well, flat-bottom plates in RPMI 1640 medium containing 2% normal mouse serum in the presence or absence of purified TBP protein (10 µg/ml) or 1 µg/ml Con A as a control. After 48 h of incubation at 37 C and 6% CO2, 1 µCi [3H]thymidine (PerkinElmer) was added to each well. Cultures were harvested after 18 h and were counted in a scintillation counter.

Cytokine production in response to TBP
Splenocytes were cultured in triplicate (2.5 x 106 cells) in 24-well, flat-bottom plates in RPMI 1640 medium containing 2% normal mouse serum in the presence or absence of purified TBP protein (10 µg/ml). After 48 h of incubation at 37 C and 6% CO2, supernatants were collected, and concentrations of IFN-{gamma} and IL-4 were determined using ELISA kits (BD Pharmingen, San Diego, CA). The amount of cytokines produced was determined using standard curves corresponding to recombinant murine cytokines and is expressed as picograms per milliliter.

Thyroid histology
Thyroid tissues were removed and fixed with 10% formalin. Tissues were embedded in paraffin, and 5-µm thick sections were prepared and stained with hematoxylin and eosin.

Statistical analysis
Statistical significance was calculated using a t test. A value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To investigate the requirement for Th1 or Th2 immunity during the development of Graves’ hyperthyroidism, we immunized wild-type BALB/c, Stat4–/–, and Stat6–/– mice with 293-TBP. First we monitored the progression of development of Graves’ hyperthyroidism. Blood samples were collected periodically and tested for TBII, T4, and TSAbs. As shown in Table 1AGo, all mice immunized with 293-TBP developed significant TBII levels, compared with control mice, by d 150 and levels continued to increase until d 300. This showed that they all generated anti-TSHR Abs that bind to TSHR and inhibit the binding of 125I-labeled TSH to TSHR.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Kinetics of TBII, T4, and TSAb of mice during the development of Graves’ hyperthyroidism1

 
Next, to investigate any perturbation in thyroid hormone levels, the sera from these mice were tested for T4 levels. Two each of BALB/c and Stat4–/– mice immunized with 293-TBP showed elevated T4 levels by d 210 compared with control mice, indicating that they had developed hyperthyroidism (Table 1BGo). In addition, a third mouse in both of these groups developed elevated T4 levels by d 240. All of these mice remained hyperthyroid until the end of the experiment. However, Stat6–/– mice immunized with 293-TBP showed normal levels of T4, comparable to those in the control group of mice immunized with 293 cells. One hyperthyroid BALB/c mouse with a T4 of 11.1 µg/dl and one Stat4–/– mouse with a T4 of 9.9 µg/dl died before d 250, and each was found to have large diffuse goiter. The differences in T4 levels between TSHR and control groups of both BALB/c and Stat4–/– mice were significant (P < 0.05; Table 1BGo). However, these differences were not as highly significant as TBII levels between TSHR and control groups of mice (P < 0.001; Table 1AGo). This is because only 50% of both BALB/c and Stat4–/– mice showed elevated T4 levels, whereas 100% of mice immunized with TSHR developed elevated TBII levels. We repeated this experiment with an additional six mice per group; similar to the data shown in Table 1Go, 50% of BALB/c and Stat4–/– mice developed hyperthyroidism (not shown).

To determine whether the elevated T4 is due to the activity of TSAbs, sera were tested for their ability to activate TSHR and produce cAMP. Sera from BALB/c and Stat4–/– mice immunized with TSHR generated higher amounts of cAMP compared with control mice at all time points tested (Table 1CGo). Stat6–/– mice, which did not develop hyperthyroidism, had no significant TSAb activity.

In addition to the comparison between TSHR and control groups of mice, we compared the differences among TSHR groups between BALB/c and Stat4–/– mice, BALB/c and Stat6–/– mice, and Stat4–/– and Stat6–/– mice. Stat4–/– mice immunized with TSHR had marginally higher levels of TBII than BALB/c and Stat6–/– mice, and these differences were not significant (Fig. 1AGo). However, as shown in Fig. 1BGo, BALB/c and Stat4–/– mice immunized with TSHR developed significantly elevated T4 levels compared with similarly immunized Stat6–/– mice (P < 0.05). Similarly, sera from hyperthyroid BALB/c and Stat4–/– mice exhibited significantly increased cAMP levels, compared with Stat6–/– mice, indicating the increased TSAb activity (P < 0.05; Fig. 1CGo).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. TBII (A), T4 (B), and TSAb (C) levels in serum obtained on d 300 from mice immunized with control 293 cells ({square}) or 293-TBP cells ({blacksquare}). TBII and T4 were determined using commercially available kits. TSAb activity was measured using CHO cells expressing TSHR as described in Materials and Methods. Results are expressed as the mean ± SD (n = 5 mice in the control group; n = 6 mice in the TSHR group). *, P < 0.05. The results shown are representative of two independent experiments using a total of 10–12 mice in each group.

 
We next tested the levels of total anti-TSHR IgG and IgG isotypes by ELISA. As shown in Fig. 2AGo, all mice immunized with 293-TBP cells developed a high titer IgG response to TSHR. Stat4–/– mice, which developed hyperthyroidism, showed a significantly lower IgG response to TSHR than Stat6–/– mice, which did not develop the disease. In addition, there were significant differences between different groups in the subclass specificity of anti-TSHR Abs. The IgG1 response was significantly higher in BALB/c and Stat4–/– mice compared with Stat6–/– mice (Fig. 2BGo). In contrast, IgG2a was the predominant subclass in Stat6–/– mice and was significantly higher than levels in BALB/c and Stat4–/– mice (Fig. 2CGo).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Anti-TSHR Ab responses determined by ELISA. Sera obtained on d 300 from mice immunized with 293-TBP cells were tested at different dilutions for IgG (A), IgG1 (B), and IgG2a (C) against ET-gp protein as described in Materials and Methods. Results are expressed as the mean ± SD (n = 5 mice in the control group; n = 6 mice in the TSHR group). A, *, P < 0.05 comparing Stat4–/– vs. Stat6–/– at the same point. B and C, *, P < 0.05 comparing BALB/c vs. Stat6–/– or Stat4–/– vs. Stat6–/– at the same point. The results shown are representative of two independent experiments using a total of 10–12 mice in each group.

 
Splenocytes obtained from different groups of mice were tested for T cell proliferation in response to TSHR antigen. Spleen cells from BALB/c, Stat4–/–, and Stat6–/– mice immunized with 293-TBP cells showed good T cell proliferation in response to TSHR (Fig. 3AGo), indicating that the cells were similarly primed. The Th1 and Th2 cytokine production in culture supernatant was examined by quantifying the secretion of IFN-{gamma} and IL-4 in response to TSHR antigen by ELISA. The IFN-{gamma} production in culture supernatants of splenocytes of Stat4–/– mice was significantly lower than that in Stat6–/– and BALB/c mice after stimulation with TSHR protein (Fig. 3BGo). IL-4 production in culture supernatants from Stat4–/– mice was significantly higher than that in BALB/c and Stat6–/– mice (Fig. 3CGo).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 3. Proliferation and cytokine production of splenocytes from BALB/c, Stat4–/–, and Stat6–/– mice in response to purified TBP protein in vitro. The proliferation (A) and production of IFN-{gamma} (B) and IL-4 (C) from mice immunized with control 293 cells or 293-TBP cells were determined in the absence ({square}) or the presence ({blacksquare}) of purified TBP protein (10 µg/ml). Results are expressed as the mean ± SD of triplicate values obtained using spleen cells from five or six mice tested individually. *, P < 0.05; **, P < 0.001.

 
Three each of the hyperthyroid BALB/c and Stat4–/– mice showed enlargement of thyroid glands compared with thyroids from control mice (Fig. 4AGo). Hematoxylin- and eosin-stained sections of thyroids from hyperthyroid mice showed hypertrophy, with occasional protrusion into the follicular lumen and decreased amounts of colloid material, indicating thyroid hyperactivity (Fig. 4BGo). The thyroid from control mice showed normal histology (Fig. 4CGo). Histological examination of thyroid glands from all other experimental mice immunized with TSHR that did not develop hyperthyroidism (i.e. BALB/c, Stat4–/–, and Stat6–/– mice) showed no significant hypertrophy or hyperactivity compared with thyroids from control mice (not shown).



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 4. A, Enlarged thyroid from a hyperthyroid mouse (left) compared with normal thyroid from a control mouse (right). The ruler shows 1-mm divisions. Shown is the histology of thyroid glands (hematoxylin- and eosin-stained paraffin sections) from a hyperthyroid mouse (B; x200 magnification) and a control mouse (C; x200 magnification).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we investigated the roles of Stat4 and Stat6, which are critical for the Th1 and Th2 immunity, respectively, in the development of Graves’ hyperthyroidism. The use of Stat4- and Stat6-deficient mice showed that Stat6–/– mice were protected from Graves’ hyperthyroidism, whereas Stat4–/– mice developed Graves’ hyperthyroidism similar to wild-type BALB/c mice. BALB/c and Stat4–/– mice developed the disease with similar TBII and TSAb responses, accompanied by elevated thyroid hormone levels. In contrast, Stat6–/–- mice, which failed to develop the disease, had normal thyroid hormone levels and no TSAb activity.

The Th1 vs. Th2 balance in these mice was examined by comparing the TSHR-specific IgG subclass as well as the cytokines produced by splenocytes in response to TSHR between different groups. BALB/c and Stat4–/– mice, which developed the disease, had predominantly an IgG1 (Th2) anti-TSHR Ab response. In addition, as expected, splenocytes from Stat4–/– mice secreted significantly higher amounts of Th2 cytokine IL-4, compared with Stat6–/– mice, in response to TSHR. In contrast, Stat6–/–mice, which did not develop the disease, had predominantly an IgG2a (Th1) anti-TSHR Ab response. This was accompanied by secretion of significantly higher quantities of a Th1 cytokine IFN-{gamma} by Stat6–/– splenocytes in response to TSHR. It is of interest to note that T cells from wild-type, Stat4–/–, and Stat6–/– mice proliferated similarly in response to TSHR protein, indicating that they were primed. In addition, levels of anti-TSHR Abs measured by TBII assay and anti-TSHR Abs, belonging to IgG, determined by ELISA were similar in Stat4–/– and Stat6–/– mice. In fact, Stat4–/– mice had lower Ab titers than Stat6–/– mice. However, Stat4–/– and Stat6–/– mice significantly differed in the subclass distribution of Abs and secretion of cytokines. These results suggest that the expression of different cytokines influences the quality, not the quantity, of disease-inducing Abs. These studies as well as those by Dogan et al. (13) demonstrate the importance of the IgG1 isotype in disease development and highlight that the production of a vigorous IgG2a anti-TSHR Ab response is not sufficient to cause disease.

Stat4–/– or Stat6–/– mice have been used to investigate the role of Th1/Th2 immune responses during the development of different autoimmune diseases. Stat6–/– mice are susceptible, and Stat4–/– mice are resistant, to the induction of experimental autoimmune encephalomyelitis, which is mediated by CD4+T cells (24). Similarly, disruption of the Stat4 signaling pathway protects against the development of CD4+T cell-dependent autoimmune diabetes (23). In addition, a more recent study showed that Stat6–/– mice develop more frequent and more severe myasthenia gravis than Stat4–/– mice (35). These results demonstrate that the Stat4 gene and the Th1 immune response are required for the development of T cell-mediated autoimmune diseases. However, in Ab-mediated autoimmune diseases, such as lupus, Stat4–/– mice develop accelerated disease, and Stat6–/– mice display a significant reduction in the development of disease (36, 37). These studies along with current data indicate that the Stat6 signaling pathway and the Th2-type immune response are critical for the development of Ab-mediated autoimmune diseases.

The analysis of the Th1/Th2 immune response in four different animal models of Graves’ hyperthyroidism has provided conflicting observations. Studies from Rapoport’s group (14, 15, 16), using three different animal models, have shown that splenocytes secrete the Th1 cytokine IFN-{gamma}, but not the Th2 cytokine IL-4, in response to TSHR. These studies correlate a Th1 response with developing Graves’ hyperthyroidism. However, our current data show that a Th2-type response is critical for the development of Graves’ hyperthyroidism in the 293-TBP animal model. Furthermore, our results are consistent with an earlier study in which wild-type BALB/c, IL-4–/–, or IFN-{gamma}–/– mice were immunized with M12 cells expressing mouse TSHR. Dogan et al. (13) reported that IFN-{gamma}–/– mice developed Graves’ hyperthyroidism accompanied by TSAbs and production of a Th2-dominant cytokine IL-4 and anti-TSHR Abs belonging to IgG1 subclass. They also reported that IL-4–/– mice failed to develop Graves’ hyperthyroidism and exhibited a strong Th1 response, characterized by the production IFN-{gamma} and anti-TSHR Abs belonging to IgG2a. Similar to that report, our study shows that IL-4 is critical for the development of Graves’ hyperthyroidism. Other studies using different animal models of Graves’ hyperthyroidism also suggest that a Th2-type response is required for disease (5, 11, 12). It was shown that the incidence of hyperthyroidism or TSAbs was higher in mice immunized with fibroblasts coexpressing TSHR and MHC class II along with a Th2 adjuvant than in mice immunized without Th2 adjuvant (11, 12). There are several potential explanations for the apparent differences in the results reported by Rapoport’s group and others, including our current study. Differences may be due to the utilization of different strategies to analyze Th1/Th2 responses. Rapoport’s group has used in vivo treatment of mice to study the Th1/Th2 immune response. This approach may restrict the availability of cytokines to only certain times during the genesis of disease. In contrast, we and Dogan et al. (13) used cytokine-deficient mice (IL-4–/– and IFN-{gamma}–/–) or mice that were deficient in genes that control Th1/Th2 responses (Stat4–/– and Stat6–/–) that would eliminate the presence of single or subsets of cytokines throughout the course of disease development. These distinct models may have a dramatic effect on the level of immune deviation occurring in these models. For example, although Stat6–/– mice have a drastic shift in anti-TSHR Ab isotypes, the shift from in vivo treatment may not be as severe. Alternatively, differences could be due to the variations in TSHR preparations, routes of immunizations, adjuvants, or strains of mice used in different studies. Together, these studies suggest that both Th1 and Th2 immunities play important roles in the development of Graves’ hyperthyroidism according to the animal model used to develop the disease (DNA/adenovirus TSHR vs. 293-TSHR). It is relatively easy to analyze the Th1/Th2 response in murine models of GD. However, it is more complex to analyze the Th1/Th2 immunity in human GD, because most of these patients also exhibit autoantibodies against other thyroid antigens, such as thyroglobulin and thyroid peroxidase, in addition to TSHR, suggesting the importance of both Th1 and Th2 immunities in human GD.

In summary, our data show that disruption of the Stat6 gene protects mice from the development of Graves’ hyperthyroidism. These data also demonstrate that in this model, the Th2-type immune response and the Stat6 signaling pathway are critical for the development of Graves’ hyperthyroidism. Finally, our data are consistent with an earlier study that showed that the extracellular domain of TSHR is sufficient for the induction of Graves’ hyperthyroidism (4).


   Acknowledgments
 
We thank Dr. Aaron J. W. Hsueh (Stanford University School of Medicine, Stanford, CA) for providing the 293-TBP cells, and Drs. Leonard D. Kohn and Kazuo Tahara (National Institutes of Health, Bethesda, MD) for providing us the CHO cells expressing hTSHR. We thank Dr. Dale W. Saxon for taking photographs of the thyroid glands, and Dr. Godfrey Tunnicliff for critical review of the manuscript. We also thank Dr. T. G. Niranjan, Mr. Gary W. White, and Ms. Emily Judy for technical help.


   Footnotes
 
This work was supported by a research enhancement grant from Indiana University School of Medicine (to G.S.S.) and National Institutes of Health Grant AI-45515 (to M.H.K.).

Abbreviations: Ab, Antibody; ET-gp, glycosylated extracellular domain of human TSH receptor; GD, Graves’ disease; h, human; IFN-{gamma}, interferon-{gamma}; MHC, major histocompatibility complex; Stat, signal transducer and activator of transcription; TBII, TSH binding inhibitory index; 293-TBP cells, human embryonic kidney cells expressing the extracellular domain of human TSH receptor; TBP, TSH-binding protein; Th, T helper; TSAb, thyroid stimulatory antibodies; TSHR, TSH receptor.

Received March 18, 2004.

Accepted for publication April 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rees Smith B, McLachlan SM, Furmaniak L 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev 9:106–121[Abstract/Free Full Text]
  2. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  3. Shimojo N, Kohno Y, Yamaguchi K, Kikuoka S, Hoshioka A, Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K 1996 Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA 93:11074–11079[Abstract/Free Full Text]
  4. Kaithamana S, Fan J, Osuga Y, Liang SG, Prabhakar BS 1999 Induction of experimental autoimmune Graves’ disease in BALB/c mice. J Immunol 163:5157–5164[Abstract/Free Full Text]
  5. Costagliola S, Many MC, Denef JF, Pohlenz J, Refetoff S, Vassart G 2000 Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves’ disease. J Clin Invest 105:803–811[Medline]
  6. Nagayama Y, Kita-Furuyama M, Ando T, Nakao K, Mizuguchi H, Hayakawa T, Eguchi K, Niwa M 2002 A novel murine model of Graves’ hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor. J Immunol 168:2789–2794[Abstract/Free Full Text]
  7. Mosmann TR, Coffman RL 1989 TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145–173[CrossRef][Medline]
  8. Stevens TL, Bossie A, Sanders VM, Fernandez-Botran R, Coffman RL, Mosmann TR, Vitetta ES 1988 Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334:255–258[CrossRef][Medline]
  9. Paul WE, Seder RA 1994 Lymphocyte responses and cytokines. Cell 76:241–251[CrossRef][Medline]
  10. Liew FY 2002 TH1 and TH2 cells: a historical perspective. Nat Rev Immunol 2:55–60[CrossRef][Medline]
  11. Kita M, Ahmad L, Marians RC, Vlase H, Unger P, Graves PN, Davies TF 1999 Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves’ disease. Endocrinology 140:1392–1398[Abstract/Free Full Text]
  12. Rao PV, Watson PF, Weetman AP, Carayanniotis G, Banga JP 2003 Contrasting activities of thyrotropin receptor antibodies in experimental models of Graves’ disease induced by injection of transfected fibroblasts or deoxyribonucleic acid vaccination. Endocrinology 144:260–266[Abstract/Free Full Text]
  13. Dogan RN, Vasu C, Holterman MJ, Prabhakar BS 2003 Absence of IL-4, and not suppression of the Th2 response, prevents development of experimental autoimmune Graves’ disease. J Immunol 170:2195–2204[Abstract/Free Full Text]
  14. Yan XM, Guo J, Pichurin P, Tanaka K, Jaume JC, Rapoport B, McLachlan SM 2000 Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens. Clin Exp Immunol 122:170–179[CrossRef][Medline]
  15. Pichurin P, Yan XM, Farilla l, Guo J, Chazenbalk GD, Rapoport B, McLachlan SM 2001 Naked TSH receptor DNA vaccination: a TH1 T cell response in which interferon-{gamma} production, rather than antibody, dominates the immune response in mice. Endocrinology 142:3530–3536[Abstract/Free Full Text]
  16. Nagayama Y, Mizuguchi H, Hayakawa T, Niwa M, McLachlan SM, Rapoport B 2003 Prevention of autoantibody-mediated Graves’-like hyperthyroidism in mice with IL-4, a Th2 cytokine. J Immunol 170:3522–3527[Abstract/Free Full Text]
  17. Hoey T, Grusby MJ 1999 STATs as mediators of cytokine-induced responses. Adv Immunol 71:145–162[Medline]
  18. Kaplan MH, Grusby MJ 1998 Regulation of T helper cell differentiation by STAT molecules. J Leukoc Biol 64:2–5[Abstract]
  19. Kaplan MH, Sun YL, Hoey T, Grusby MJ 1996 Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382:174–177[CrossRef][Medline]
  20. Thierfelder WE, van Deursen JM, Yamamoto K, Tripp RA, Sarawar SR, Carson RT, Sangster MY, Vignali DA, Doherty PC, Grosveld GC, Ihle JN 1996 Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. Nature 382:171–174[CrossRef][Medline]
  21. Kaplan MH, Schindler U, Smiley ST, Grusby MJ 1996 Stat 6 is required for mediating responses to IL-4 and for the development of Th2 cells. Immunity 4:313–319[CrossRef][Medline]
  22. Takeda K, Tanaka T, Shi W, Matsumoto M, Minami K, Kashiwamura S, Nakanishi K, Yoshida N, Kishimoto T 1996 Essential role of Stat6 in IL-4 signaling. Nature 380:627–630[CrossRef][Medline]
  23. Holz A, Bot A, Coon B, Wolfe T, Grusby MJ, von Herrath MG 1999 Disruption of the STAT4 signaling pathway protects from autoimmune diabetes while retaining antiviral immune competence. J Immunol 163:5374–5382[Abstract/Free Full Text]
  24. Chitnis T, Najafian N, Benou C, Salama AD, Grusby MJ, Sayegh MH, Khoury SJ 2001 Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis. J Clin Invest 108:739–747[CrossRef][Medline]
  25. Cai G, Radzanowski T, Villegas EN, Kastelein R, Hunter CA 2000 Identification of STAT4-dependent and independent mechanisms of resistance to Toxoplasma gondii. J Immunol 165:2619–2627[Abstract/Free Full Text]
  26. Tarleton RL, Grusby MJ, Zhang L 2000 Increased susceptibility of Stat4-deficient and enhanced resistance in Stat6-deficient mice to infection with Trypanosoma cruzi. J Immunol 165:1520–1525[Abstract/Free Full Text]
  27. Akimoto T, Numata F, Tamura M, Takata Y, Higashida N, Takashi T, Takeda K, Akira S 1998 Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice. J Exp Med 187:1537–1542[Abstract/Free Full Text]
  28. Tekkanat KK, Maassab HF, Cho DS, Lai JJ, John A, Berlin A, Kaplan MH, Lukacs NW 2001 IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent. J Immunol 166:3542–3548[Abstract/Free Full Text]
  29. Seetharamaiah GS, Dallas JS, Patibandla SA, Thotakura NR, Prabhakar BS 1997 Requirement of glycosylation of the human thyrotropin receptor ectodomain for its reactivity with autoantibodies in patients’ sera. J Immunol 158:2798–2804[Abstract]
  30. Seetharamaiah GS, Dallas JS, Prabhakar BS 1999 Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes. Autoimmunity 29:21–31[Medline]
  31. Osuga Y, Liang SG, Dallas JS, Wang C, Hsueh AJ 1998 Soluble ecto-domain mutant of thyrotropin (TSH) receptor incapable of binding TSH neutralizes the action of thyroid-stimulating antibodies from Graves’ patients. Endocrinology 139:671–676[Abstract/Free Full Text]
  32. Seetharamaiah GS, Kaithamana S, Desai RK, Prabhakar BS 1999 Regulation of thyrotropin receptor expression in insect cells. J Mol Endocrinol 23:315–323[Abstract]
  33. Patibandla SA, Fan JL, Prabhakar BS, Seetharamaiah GS 1999 Comparison of immune responses to extracellular domains of mouse and human thyrotropin receptor. J Autoimmun 13:205–213[CrossRef][Medline]
  34. Seetharamaiah GS, Wagle NM, Morris JC, Prabhakar BS 1995 Generation and characterization of monoclonal antibodies to the human thyrotropin receptor: antibodies can bind to discrete conformational or linear epitopes and block TSH binding. Endocrinology 136:2817–2824[Abstract]
  35. Wang W, Ostlie NS, Conti-Fine BM, Milani M 2004 The susceptibility to experimental myasthenia gravis of STAT6–/– and STAT4–/– BALB/c mice suggests a pathogenic role of Th1 cells. J Immunol 172:97–103[Abstract/Free Full Text]
  36. Singh RR, Saxena V, Zang S, Li L, Finkelman FD, Witte DP, Jacob CO 2003 Differential contribution of IL-4 and Stat 6 vs Stat4 to the development of lupus nephritis. J Immunol 170:4818–4825[Abstract/Free Full Text]
  37. Jacob CO, Zang S, Li L, Ciobanu V, Quismorio F, Mizutani A, Satoh M, Koss M 2003 Pivotal role of Stat4 and Stat6 in the pathogenesis of the lupus-like disease in the New Zealand mixed 2328 mice. J Immunol 171:1564–1571[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
K. J. Land, P. Gudapati, M. H. Kaplan, and G. S. Seetharamaiah
Differential Requirement of Signal Transducer and Activator of Transcription-4 (Stat4) and Stat6 in a Thyrotropin Receptor-289-Adenovirus-Induced Model of Graves' Hyperthyroidism
Endocrinology, January 1, 2006; 147(1): 111 - 119.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
S. M. McLachlan, Y. Nagayama, and B. Rapoport
Insight into Graves' Hyperthyroidism from Animal Models
Endocr. Rev., October 1, 2005; 26(6): 800 - 832.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Land, K. J.
Right arrow Articles by Seetharamaiah, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Land, K. J.
Right arrow Articles by Seetharamaiah, G. S.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*LEVOTHYROXINE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals