Differential Requirement of Signal Transducer and Activator of Transcription-4 (Stat4) and Stat6 in a Thyrotropin Receptor-289-Ade
http://www.100md.com
《内分泌学杂志》
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine (K.J.L., P.G., G.S.S.), Evansville, Indiana 47712
Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine (M.H.K.), Indianapolis, Indiana 46202
Abstract
T helper type 1 (Th1) and Th2 cells have critical roles in the development of cell-mediated and humoral immune responses, respectively. This division of function predicts that Th1 cells mediate inflammatory diseases and Th2 cells promote antibody (Ab)-mediated autoimmunity. Our previous studies using HEK-293 cells expressing the extracellular domain of the TSH receptor (TSHR) showed that Stat4–/– mice, which lack Th1 cells, are susceptible, whereas Stat6–/– mice, which lack Th2 cells, are resistant to the induction of Graves’ hyperthyroidism. To investigate the role of Stat4 and Stat6 genes in other murine models of hyperthyroidism, we injected wild-type BALB/c, Stat4–/–, and Stat6–/– mice with an adenovirus expressing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad). The viral system induces a much stronger immune response with much more rapid onset of disease. Our results showed that 56% of wild-type, 75% of Stat4–/–, and 39% of Stat6–/– mice developed hyperthyroidism. Hyperthyroid mice exhibited thyroid stimulatory Abs. The Stat4–/– mice developed a higher incidence and greater severity of hyperthyroidism compared with wild-type and Stat6–/– mice. BALB/c and Stat4–/– mice showed significantly higher TSHR Abs of the IgG1 subclass and IL-4 compared with Stat6–/– mice. In contrast, Stat6–/– mice had predominantly the IgG2a subclass of TSHR Ab and produced significantly higher amounts of IFN- than BALB/c and Stat4–/– mice. All hyperthyroid mice showed enlarged thyroid glands with hyperactivity. These results suggest that in the TSHR-289-ad model, the Th2 cells are more efficient in mediating disease, but in the absence of Th2 cells, Th1 cells may still initiate a reduced incidence of Graves’ hyperthyroidism.
Introduction
GRAVES’ DISEASE (GD) is an autoimmune disorder characterized by hyperthyroidism. The thyroid stimulatory Abs (TSAbs) stimulate the TSH receptor (TSHR) and cause elevation of thyroid hormone levels (1, 2, 3). Although GD is mediated by TSAbs, the generation of high-affinity Abs requires B cell interaction with antigen-specific T cells. The immune response to protein antigens, such as TSHR, is determined by the cytokines produced by CD4+ T helper (Th) cells. Naive CD4+ T cells, when stimulated with an antigen, functionally differentiate into Th1 or Th2 cells depending on the type of pathogen (4, 5). In general, Th1 cells produce cytokines such as IFN- and mainly promote cell-mediated immunity, whereas Th2 cells secrete cytokines such as IL-4 and IL-5 and enhance antibody (Ab)-mediated immunity. Cytokines are the dominant factors guiding the development of Th1 and Th2 cells. The Th1 and Th2 cells mutually regulate the function of each other and determine the outcome of immune responses. The differentiation of Th cells into Th1 and Th2 cells is controlled by a number of factors, including intracellular signaling molecules.
Signal transducer and activator of transcription (Stat) proteins are intracellular signaling molecules that mediate many cytokine-mediated responses (6, 7, 8, 9, 10). Stat4 is required for the development of fully functional Th1 cells, is activated by IL-12, and induces the transcription of IFN- (6, 7, 8). In contrast, Stat6 is essential for the development of Th2 cells and is activated by IL-4 (9, 10). Stat4–/– mice develop Th2-like immune responses in vivo and are resistant to the development of T cell-mediated autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) and type I diabetes in nonobese diabetic mice (11, 12). However, Stat4–/– mice develop accelerated Ab-mediated autoimmune disease, lupus (13, 14). In contrast, Stat6–/– mice develop a Th1-like immune response, are susceptible to T cell-mediated autoimmune diseases such as EAE (11), and display a significant reduction in the incidence of lupus (13, 14).
In GD patients, Th2 immune response was suggested to be dominant because GD is caused by Abs. This concept is supported by a study which showed that GD patients predominantly produce the Th2 cytokines IL-4 and IL-10 (15). In contrast, another study reported that TSAbs in humans are almost exclusively of IgG1 (16), which is a Th1 type in humans. In addition, other studies show a mixed Th1 and Th2 immune response (17, 18). The analysis of the Th1/Th2 response in animal models of GD has also provided conflicting results. In the TSHR-ad model, both IFN-- and IL-4-deficient BALB/c mice were resistant to the induction of Graves’ hyperthyroidism (19), suggesting that both Th1 and Th2 responses are required in this model. In contrast, in the TSHR-M12 model, hyperthyroidism was induced in BALB/c mice deficient in IFN-, but not in IL-4 (20). Recently, we showed that BALB/c mice deficient in Stat4, but not Stat6, injected with TSHR-HEK-293 cells developed hyperthyroidism (21). These studies indicate a critical role for Th2 cells in this model of GD and a limited role for Th1 cells. To investigate the role of Stat4 and Stat6 genes in other murine models of Graves’ hyperthyroidism, we injected wild-type BALB/c, Stat4–/–, and Stat6–/– mice with an adenovirus containing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad). Our results showed that 56% of wild-type (WT), 75% of Stat4–/–, and 39% of Stat6–/– mice developed hyperthyroidism, suggesting that even in the absence of Th2 cells, Ab-mediated autoimmune disease can develop.
Materials and Methods
Immunization of mice with TSHR-289-ad
Construction and purification of adenovirus containing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad) and control adenovirus (con-ad) containing -galactosidase have been described previously (22, 23). These adenoviruses were provided by Drs. B. Rapoport and S. M. McLachlan (Cedars-Sinai Medical Center, University of California, Los Angeles, CA). Adenoviruses (289-ad and con-ad) were propagated in HEK-293 cells and purified by CsCl density gradient centrifugation. The viral particle concentration was determined by measuring the absorbance at 260 nm (24), and it was stored in aliquots at –80 C.
Stat4–/– and Stat6–/– mice were generated as described previously (6, 9) 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 following approval from the Indiana University animal care and use committee. Mice were maintained in conventional housing facility. Six- to 8-wk-old female BALB/c, Stat4–/–, and Stat6–/– mice were injected im in the thigh muscle with 1010 particles of 289-ad or con-ad in 50 μl PBS. All mice in this study were immunized simultaneously using the same batch of adenovirus. Mice were injected three times at 3-wk intervals (d 0, 21, and 42), and blood was drawn 2 wk after the third injection (d 56). All surviving mice were killed 4 wk after the third injection (d 70) to obtain blood, spleen cells, and thyroid glands.
Serum TSH binding inhibitory Igs (TBII) and T4
TBII in sera were determined using a commercially available TRAb kit (Kronus, Boise, ID) according to the manufacturer’s protocol and as described previously (25, 26). This assay measures the ability of Abs in sera to inhibit the binding of 125I-labeled TSH to TSHR. Results are expressed as percent TBII values.
Total T4 in sera was measured with a commercially available RIA kit (Diagnostic Products Corp., Los Angeles, CA) according to the manufacturer’s protocol and as described previously (25, 27). This assay measures the ability of T4 in sera to compete with 125I-labeled T4 for binding to anti-T4 Ab-coated polypropylene tubes.
Thyroid-stimulating Abs (TSAbs) and TSH-blocking Abs (TBAbs)
TSAbs and TBAbs in sera were measured in a bioassay using Chinese hamster ovary (CHO) cells expressing TSHR as described previously (25, 28). In brief, these cells were grown to confluence in 96-well plates in F-12 medium supplemented with 10% fetal bovine serum (FBS). Cells were incubated in triplicate with mouse serum diluted 1:30 in hypotonic HBSS containing 0.5 mM 3-isobutylmethylxanthine for 2 h at 37 C. The cAMP released into the buffer was measured with a cAMP RIA kit (PerkinElmer, Boston, MA). TSAb was expressed as a percentage of the basal cAMP produced in the presence of buffer alone.
To measure TBAbs, cells were incubated in triplicate with mouse serum diluted 1:25 in hypotonic HBSS containing 0.5 mM 3-isobutylmethylxanthine. After 30 min at 37 C, 5 x 10–10 M bovine TSH (Sigma-Aldrich Corp., St. Louis, MO) was added, and incubation was continued for 2 h. The cAMP released into the buffer was measured with a cAMP RIA kit (PerkinElmer). The TBAb was calculated as: 100 x (1 – [cAMP of test serum in presence of bTSH ÷ cAMP of control serum in presence of bTSH]).
ELISA to measure IgG subclass of anti-TSHR Abs
Anti-TSHR Ab were measured by ELISA using purified TSHR-289 protein. This protein corresponds approximately to the TSHR-A subunit and was provided by Drs. B. Rapoport and S. M. McLachlan. TSHR-289 protein secreted into the culture medium by CHO cells was purified by affinity chromatography as described previously (29, 30). Wells of ELISA plates (Immulon2, Thermo Electron, Franklin, MA) were coated with 100 ng TSHR-289 protein in 100 μl carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 C. After washing the plate, 200 μl 0.2% FBS in PBS containing 0.05% Tween 20 was added and incubated for 1.5 h at room temperature to reduce nonspecific binding. The following reagents (100 μl) were added in succession after incubation at room temperature, followed by washing the plate between each step: serially diluted mouse serum in duplicate for 1.5 h, biotinylated goat antimouse IgG (Sigma-Aldrich Corp.) for 1 h, and avidin-horseradish peroxidase conjugate (Sigma-Aldrich Corp.) for 30 min. Color was developed in dark using 3,3',5,5'-tetramethylbenzidine (BD Pharmingen, San Diego, CA). The reaction was stopped using 50 μl 2 N sulfuric acid, and absorbance was measured at 450 nm. The subclass specificity of anti-TSHR Abs was determined using biotinylated goat antimouse IgG1 or IgG2a (Caltag Laboratories, Burlingame, CA).
Splenocyte proliferative response to purified extracellular domain of TSHR (ETSHR) protein
HEK-293 cells expressing the ETSHR were provided by Dr. Aaron J. W. Hsueh (Stanford University School of Medicine, Stanford, CA) and were maintained as described previously (31, 32). Soluble ETSHR protein was purified using a nickel affinity column as described previously (31, 32). Splenocytes of individual mice were cultured in triplicate (5 x 105 cells) in 96-well, round-bottom plates in RPMI 1640 medium containing 5% FBS in the presence or absence of purified ETSHR protein (10 μg/ml) or 5 μg/ml Con A as a control. After 72 h of incubation at 37 C in 6% CO2, 1 μCi [3H]thymidine (PerkinElmer) was added to each well. Cultures were harvested after 18 h and counted in a scintillation counter. Proliferation is expressed as the stimulation index and is calculated as the ratio between the average counts per minute of cultures in the presence of ETSHR protein and the average counts per minute of cultures in the presence of medium. The use of the stimulation index normalizes results and allows comparison of experiments performed at different times.
Cytokine production in response to purified ETSHR protein
Splenocytes of individual mice were cultured in triplicate (1 x 106 cells) in 96-well, round-bottom plates in RPMI 1640 medium containing 5% FBS in the presence or absence of purified ETSHR protein (10 μg/ml) or 5 μg/ml Con A as a control. After 48 h of incubation at 37 C in 6% CO2, supernatants were collected, and concentrations of IFN- and IL-4 were determined using ELISA kits (BD Pharmingen). The amount of cytokines produced was determined using standard curves corresponding to recombinant murine cytokines and expressed as picograms per milliliter.
Thyroid weight and histology
Thyroid tissues were removed, weighed, and fixed with 10% formalin. Tissues were embedded in paraffin, and 5-μm-thick sections were prepared and stained with hematoxylin-eosin.
Statistical analysis
Statistical significance was calculated using Student’s t test. P < 0.05 was considered significant.
Results
Our earlier studies using HEK-293 cells expressing ETSHR showed that 50% of WT BALB/c and Stat4–/– mice developed hyperthyroidism. However, none of the Stat6–/– mice developed hyperthyroidism (21). To investigate the role of Stat4 and Stat6 genes in other murine models of Graves’ hyperthyroidism, we immunized wild-type BALB/c, Stat4–/–, and Stat6–/– mice with an adenovirus expressing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad). We conducted two independent experiments, and data from these two experiments are shown in this report. Blood samples collected on d 56 were tested for TBII, T4, TSAbs, and TBAbs. We first measured the ability of Abs to bind to TSHR and inhibit the binding of [125I]TSH (TBII). In both experiments, all groups of mice (BALB/c, Stat4–/– and Stat6–/–) injected with 289-ad developed significant TBII levels compared with the corresponding con-ad-injected mice (Fig. 1). This showed that all groups of mice injected with 289-ad generated anti-TSHR Abs.
Next, to investigate any perturbation in thyroid hormone levels, the sera from these mice were tested for T4 levels. In both experiments, BALB/c and Stat4–/– mice injected with 289-ad developed significantly elevated T4 compared with the corresponding control mice (Fig. 2). However, T4 levels in Stat6–/– mice injected with 289-ad were not significantly different from those in the corresponding control mice (P = 0.112 in experiment 1; P = 0.126 in experiment 2). In the first experiment, 50% of BALB/c mice, 80% of Stat4–/– mice, and 37% of Stat6–/– mice injected with 289-ad developed hyperthyroidism (Fig. 2A). In the second experiment, 62% of BALB/c mice, 70% of Stat4–/– mice, and 40% of Stat6–/– mice injected with 289-ad developed hyperthyroidism (Fig. 2B).
We also compared differences in TBII and T4 among the experimental groups. The data combined from both experiments showed that experimental BALB/c and Stat4–/– mice developed significantly higher TBII than Stat6–/– mice (P < 0.01; not shown). Stat4–/– mice injected with 289-ad developed significantly higher T4 compared with similarly immunized Stat6–/– mice (P < 0.01; not shown). Serum T4 levels were elevated in nine of 16 (56%) WT, 15 of 20 (75%) Stat4–/–, and seven of 18 (39%) Stat6–/– mice. The Stat4-deficient mice developed a higher incidence and greater severity of hyperthyroidism than WT and Stat6-deficient mice. The majority of mice with high TBII (70% or more) also had elevated T4 levels. In general, serum TBII levels correlated well with serum T4 levels.
Patients with GD often show loss of body weight. Therefore, body weight might serve as a clinical parameter in these mice. Mice were weighed once a week to assess any weight loss. The differences in weight of the mice were not statistically significant between control group and experimental groups (not shown). However, some of the mice with very high levels of T4 lost 10% or more of their weight suddenly toward the end of the experiment. Of 54 experimental mice, four mice were either killed or died before d 70 because they exhibited significant weight loss and exhaustion. Of these mice, one was BALB/c, and the remaining three were Stat4–/–. All had very high T4 levels (>15 μg/dl).
To determine whether elevated T4 is due to the stimulatory Ab activity, sera were tested for their ability to activate TSHR and produce cAMP. TSAbs in sera were measured in a bioassay using CHO cells expressing TSHR and were expressed as a percentage of basal cAMP produced in the presence of buffer alone. The data combined from both experiments are shown in Fig. 3A. Sera from Stat4–/– mice injected with TSHR-289-ad, which developed the highest T4 levels, generated significantly higher levels of TSAbs compared with Stat6–/– mice (P < 0.01). In addition, BALB/c mice injected with 289-ad developed significant TSAbs compared with the corresponding con-ad-injected mice (P < 0.05). Similarly, Stat4–/– and Stat6–/– mice injected with 289-ad also developed significant TSAbs compared with the corresponding con-ad-injected mice (P < 0.05, not shown). Sera from hyperthyroid mice stimulated TSHR, leading to increased cAMP production, confirming the presence of TSAbs. Sera from mice that did not develop hyperthyroidism had no significant TSAbs. There was good agreement between T4 and TSAbs. These data confirm that the hyperthyroidism is due to the activity of TSAbs.
Next, we determined the ability of Abs to block TSH-mediated cAMP production. The majority of sera from mice immunized with TSHR-289-ad showed TBAb activity compared with con-ad-injected mice (Fig. 3B). TBAb activity was not significantly different among WT, Stat4–/–, and Stat6–/– mice injected with TSHR-289-ad. However, mice with elevated TBII and normal T4 levels had significant TBAb activity (Fig. 3B). This showed that TBAbs in these mice account for the elevated TBII.
We next measured serum anti-TSHR Abs belonging to IgG and different subclasses by ELISA against purified TSHR-289 protein. Mice injected with con-ad showed background OD values (<0.1) at 1:100 dilutions of sera. In contrast, mice injected with TSHR-289-ad developed TSHR Abs detectable even at 1:800 (Fig. 4A). The differences in IgG TSHR Abs among BALB/c, Stat4–/–, and Stat6–/– mice were not statistically significant (Fig. 4A). However, there were significant differences among the groups in the IgG1 subclass distribution of anti-TSHR Abs. WT BALB/c and Stat4–/– mice showed significantly higher levels of TSHR Abs of the IgG1 subclass compared with Stat6–/– mice (Fig. 4B). In contrast, Stat6–/– mice had predominantly IgG2a, with little of the IgG1 subclass of Ab (Fig. 4C). These data suggest that the IgG1 subclass of Ab is critical for the development of Graves’ hyperthyroidism.
Splenocytes from different groups of mice were tested for proliferation in response to purified ETSHR protein. As shown in Fig. 5A, splenocytes from all groups of TSHR-289-ad-injected mice showed significantly higher proliferative responses to ETSHR compared with con-ad mice (P < 0.001). Significant proliferative responses from all experimental groups showed that cells were similarly primed. We next analyzed the Th1 and Th2 cytokines secreted into the culture supernatants by splenocytes in response to ETSHR protein. As shown in Fig. 5B, splenocytes from BALB/c and Stat6–/– mice produced significantly higher IFN- than those from Stat4–/– mice in response to ETSHR protein (P < 0.001). In contrast, splenocytes from BALB/c and Stat4–/– mice produced significantly higher IL-4 than those from Stat6–/– mice (Fig. 5C; P < 0.05). All TSHR-289-ad injected mice produced IFN- in response to ETSHR protein (Fig. 5B). However, IL-4 was detectable in only 40% of BALB/c and Stat4–/– mice and in none of the Stat6–/– mice in response to ETSHR protein (Fig. 5C). We also tested for the other Th2 cytokine, IL-5. Similar to IL-4, IL-5 was detectable in only 30–40% of BALB/c and Stat4–/– mice and in none of the Stat6–/– mice (not shown). It is relatively difficult to detect Th2 cytokines compared with Th1 cytokines due to low levels of Th2 cells. Additionally, the variation in the secretion of IL-4 and IL-5 among individual BALB/c and Stat4–/– mice could be due to the variation in the dose of TSHR antigen presented to naive T cells after adenoviral infection. It is also possible that immunization with TSHR-ad leads to the production of lower quantities of IL-4 compared with immunization with M12 or HEK-293 cells expressing TSHR (20, 21). It is of interest to note that others have reported that IL-4 was not detected in BALB/c mice immunized with TSHR-289-ad (19).
Hyperthyroidism is usually associated with goiter. We therefore determined the size and weight of thyroid from each mouse. All hyperthyroid mice exhibited enlarged thyroid glands. A representative thyroid from each group is shown in Fig. 6. The thyroids of the largest size were seen in Stat4–/– mice injected with TSHR-289-ad. Thyroid sizes of WT BALB/c mice were intermediate, and those from Stat6–/– mice were the smallest compared with the other two experimental groups. In general, thyroid sizes correlated well with serum T4. The weights of thyroids of Stat4–/– mice were significantly higher compared with those in BALB/c and Stat6–/– mice (Fig. 7). The weights of the thyroids of all groups of mice immunized with TSHR-289-ad were significantly higher compared with those from con-ad mice (P < 0.001). The sizes and weights of thyroids from con-ad-injected BALB/c mice were normal (Figs. 6 and 7). Similarly, the sizes and weights of thyroids of con-ad-injected Stat4–/– and Stat6–/– mice were also normal (not shown).
The thyroids from con-ad-injected mice showed normal histology (Fig. 8, A and B). Histological examination of thyroids from hyperthyroid mice showed hypertrophy and hypercellularity of follicular epithelia with extensive protrusion into follicular lumen and a decreased amount of colloid material, indicating thyroid hyperactivity (Fig. 8, C–H). There were no intrathyroidal lymphocytic infiltrations. Histological examination of thyroids of other experimental mice that did not develop hyperthyroidism showed no significant hypertrophy or hyperactivity compared with those from control mice (Fig. 8, A and B).
Discussion
The factors that influence the development of Graves’ hyperthyroidism are not completely understood. Studies to define the role of Th1/Th2 immune responses in different animal models of GD have yielded different results. IFN- and IL-4 are the prototypic cytokines secreted by Th1 and Th2 cells, respectively. Studies using IFN-–/– or IL-4–/– BALB/c mice have reported different outcomes in two different models of GD. In the TSHR-M12 model, IFN-–/–, but not IL-4–/–, mice developed hyperthyroidism, demonstrating the importance of Th2 signaling in this model (20). In contrast, in the TSHR-ad model, Graves’ hyperthyroidism was attenuated in IFN-–/– and IL-4–/– BALB/c mice, demonstrating the importance of both Th1 and Th2 signaling in this model (19).
Stat4 and Stat6 are signaling molecules required for the development of fully functional Th1 and Th2 cells, respectively (6, 7, 8, 9, 10). Stat4–/– and Stat6–/– mice are similar to, but not identical with, IFN-–/– and IL-4–/– mice. Stat4–/– and Stat6–/– mice produce significantly lower amounts of IFN- and IL-4, respectively, compared with WT mice. Stat4–/– mice lack IL-12-activated IFN- production, but still secrete small quantities of IFN- induced by IL-18 or antigen receptor (6, 7, 8). Similarly, Stat6–/– mice lack IL-4-activated IL-4 production and are able to secrete IL-4 by cells such as natural killer T cells, mast cells, etc. (33, 34). However, IFN-–/– and IL-4–/– mice completely lack the secretion of IFN- and IL-4, respectively. Therefore, to investigate the role of Th1/Th2 immune responses in Graves’ hyperthyroidism, we recently immunized Stat4–/– or Stat6–/– BALB/c mice with TSHR-HEK-293 cells (21). The results from this earlier study showed that Stat4–/–, and not Stat6–/–, mice develop hyperthyroidism. These data are consistent with an earlier study, using TSHR-M12 cells and IFN-–/– and IL-4–/– mice, which showed that a Th2-type immune response is critical for the development of Graves’ hyperthyroidism in this model (20).
To investigate the roles of Stat4 and Stat6 genes in other models of Graves’ hyperthyroidism, we injected BALB/c, Stat4–/–, and Stat6–/– mice with the TSHR-289-ad. Similar to previous studies (22, 35), our data confirm the induction of Graves’ hyperthyroidism in BALB/c mice and extend this model to Stat4–/– and Stat6–/– BALB/c mice. Our present results showed that Stat4–/– mice developed significantly higher TBII, T4, and TSAbs compared with Stat6–/– mice. Stat4–/– mice developed a Th2-type immune response accompanied by significantly higher levels of TSHR Abs of the IgG1 subclass and IL-4 compared with Stat6–/– mice. In contrast, Stat6–/– mice developed a Th1-type immune response, showed predominantly the IgG2a subclass of TSHR Abs, and produced a significantly greater amount of IFN- than Stat4–/– mice. Although all hyperthyroid mice showed enlarged thyroid glands, Stat4–/– mice exhibited the largest thyroids. In addition to the higher incidence of disease (75% vs. 39%), the Stat4–/– mice exhibited a greater severity of hyperthyroidism (average T4, 11.8 vs. 6.5 μg/dl) compared with Stat6–/– mice.
The incidence (75% vs. 56%) and severity (average T4, 11.8 vs. 9.2 μg/dl) of hyperthyroidism were higher in Stat4–/– mice compared with WT BALB/c mice, although the reason for this is still unclear. There were no significant differences in their TBII, TSAbs, anti-TSHR Abs, or secretion of IL-4. However, BALB/c mice produced significantly higher levels of IFN- than Stat4–/– mice. This alone may not explain the differences between Stat4–/– and BALB/c mice. There may be differences in the affinity and fine specificity of anti-TSHR Abs between the groups; this will be examined in future studies. It is also possible that Stat4 regulates genes distinct from IFN- that reduce Ab-mediated autoimmune disease.
Although all experimental conditions, such as immunization procedure and housing, were identical, 100% of BALB/c and Stat4–/– mice and only 72% (13 of 18) of Stat6–/– mice developed higher TBII levels. We observed this variation in immune response in Stat6–/– mice in both experiments. This could be due to variation in the amount of T cell help that can be provided in the absence of fully functional Th2 cells in Stat6–/– mice.
There are some interesting differences between the present study and those using IFN-- and IL-4-deficient mice (19). Importantly, Stat4- and Stat6-deficient mice do recapitulate the phenotype of cytokine-deficient mice. There are several reasons for this. First, Stat4-deficient T cells can make some IFN-, albeit at reduced levels. Additionally, some cells, such as CD8 cells, can make normal antigen receptor-induced IFN- in the absence of Stat4. Similarly, natural killer T cells and mast cells can make IL-4 in the absence of Stat6. Thus, STAT-deficient mice are not devoid of cytokine production, and this accounts for the normal IgG2a anti-TSHR level in Stat4-deficient mice. However, Stat4- and Stat6-deficient mice represent a more comprehensive deficiency of Th1 and Th2 functions, respectively, than the restricted elimination of a single cytokine. The distinctions between these models may elucidate important components of the pathogenesis of GD.
There were other differences between the previous and the present study. In the previous study, IFN-- and IL-4-deficient mice were immunized twice with TSHR-289-ad (19). However, in the present study, Stat4- and Stat6-deficient mice were immunized three times with TSHR-289-ad. To determine the possible reasons for the differences in the incidence of disease, we repeated the experiments with Stat4- and Stat6-deficient mice involving two immunizations. There were no significant differences between two and three immunizations in the incidence of hyperthyroidism (not shown). However, the severity of hyperthyroidism was marginally higher after three immunizations than after two.
There are several advantages of the TSHR-289-ad model compared with the TSHR-HEK-293 model (21). First, the duration required for the development of disease is short (70 vs. 300 d). Second, the incidence of disease is higher (56% vs. 50% in BALB/c, 75% vs. 50% in Stat4–/–, and 39% vs. 0% in Stat6–/– mice). Third, the severity of disease is greater (average T4, 9.2 vs. 7.2 μg/dl in BALB/c, 11.8 vs. 5.9 μg/dl in Stat4–/–, and 6.5 vs. 2.9 μg/dl in Stat6–/– mice). These data demonstrate that TSHR-289-ad generates a stronger immune response than TSHR-HEK-293 cells and agrees with previous data showing that adenovirus infection induces strong immune responses (22). This could be due the expression of TSHR protein with proper conformation by adenovirus infection. It also suggests that a strong immune stimulation is required to generate Ab-mediated autoimmune disease in the absence of Th2 cells.
Stat4–/– and Stat6–/– mice have been used to study the role of Th1/Th2 immune responses in different autoimmune diseases. Stat4–/– mice were protected from the development of lymphocytic choriomeningitis virus-induced CD4+ T cell-dependent autoimmune diabetes (12). A more recent study reported that nonobese diabetic Stat4–/– mice were protected from the development of diabetes and EAE (36). Similarly, Stat4–/–, but not Stat6–/–, mice exhibited delayed onset and reduced severity of disease compared with WT mice in diabetes induced by multiple low doses of streptozotocin (37). Stat4–/– mice are resistant and Stat6–/– are susceptible to the induction of EAE mediated by CD4+ T cells (11). In addition, Stat6–/– mice develop more frequent and more severe myasthenia gravis than Stat4–/– mice (38). 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 (13, 14). These studies along with our current data indicate that the Stat6 signaling pathway and the Th2-type immune response are critical for the development of Ab-mediated autoimmune diseases.
Similar to the TSHR-HEK-293 model (21), the TSHR-289-ad model also demonstrated that the incidence of Graves’ hyperthyroidism was much higher in Stat4–/– mice compared with Stat6–/– mice. However, in contrast to the TSHR-HEK-293 model, the TSHR-ad model showed that Stat6–/– mice were not completely protected from the development of Graves’ hyperthyroidism. These results suggest that in the TSHR-289-ad model, Th2 cells are more efficient in mediating disease, but in the absence of Th2 cells, Th1 cells may still initiate a reduced incidence of Graves’ hyperthyroidism.
Acknowledgments
We thank Drs. B. Rapoport and S. M. McLachlan (Cedars-Sinai Medical Center, University of California, Los Angeles, CA) for providing us with TSHR-289 adenovirus and TSHR-289 protein, and Dr. Aaron J. W. Hsueh (Stanford University School of Medicine, Stanford, CA) for providing us with HEK-293 cells expressing ETSHR protein.
Footnotes
This work was supported by a Research Enhancement grant from the Indiana University School of Medicine (to G.S.S.) and National Institutes of Health Grant AI-45515 (to M.H.K.).
The authors K.J.L., P.G., M.H.K., and G.S.S. have nothing to declare related to the material being published.
First Published Online September 29, 2005
Abbreviations: Ab, Antibody; ad, adenovirus; CHO, Chinese hamster ovary; con, control; EAE, experimental autoimmune encephalomyelitis; ETSHR, extracellular domain of TSH receptor; FBS, fetal bovine serum; GD, Graves’ disease; Stat, signal transducer and activator of transcription; TBAb, TSH-blocking antibody; TBII, TSH binding inhibitory Ig; Th1, T helper type 1; TSAb, thyroid stimulatory antibody; TSHR, thyrotropin receptor; TSHR-289-ad, adenovirus expressing amino acid residues 1–289 of TSH receptor; WT, wild type.
Accepted for publication September 21, 2005.
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Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine (M.H.K.), Indianapolis, Indiana 46202
Abstract
T helper type 1 (Th1) and Th2 cells have critical roles in the development of cell-mediated and humoral immune responses, respectively. This division of function predicts that Th1 cells mediate inflammatory diseases and Th2 cells promote antibody (Ab)-mediated autoimmunity. Our previous studies using HEK-293 cells expressing the extracellular domain of the TSH receptor (TSHR) showed that Stat4–/– mice, which lack Th1 cells, are susceptible, whereas Stat6–/– mice, which lack Th2 cells, are resistant to the induction of Graves’ hyperthyroidism. To investigate the role of Stat4 and Stat6 genes in other murine models of hyperthyroidism, we injected wild-type BALB/c, Stat4–/–, and Stat6–/– mice with an adenovirus expressing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad). The viral system induces a much stronger immune response with much more rapid onset of disease. Our results showed that 56% of wild-type, 75% of Stat4–/–, and 39% of Stat6–/– mice developed hyperthyroidism. Hyperthyroid mice exhibited thyroid stimulatory Abs. The Stat4–/– mice developed a higher incidence and greater severity of hyperthyroidism compared with wild-type and Stat6–/– mice. BALB/c and Stat4–/– mice showed significantly higher TSHR Abs of the IgG1 subclass and IL-4 compared with Stat6–/– mice. In contrast, Stat6–/– mice had predominantly the IgG2a subclass of TSHR Ab and produced significantly higher amounts of IFN- than BALB/c and Stat4–/– mice. All hyperthyroid mice showed enlarged thyroid glands with hyperactivity. These results suggest that in the TSHR-289-ad model, the Th2 cells are more efficient in mediating disease, but in the absence of Th2 cells, Th1 cells may still initiate a reduced incidence of Graves’ hyperthyroidism.
Introduction
GRAVES’ DISEASE (GD) is an autoimmune disorder characterized by hyperthyroidism. The thyroid stimulatory Abs (TSAbs) stimulate the TSH receptor (TSHR) and cause elevation of thyroid hormone levels (1, 2, 3). Although GD is mediated by TSAbs, the generation of high-affinity Abs requires B cell interaction with antigen-specific T cells. The immune response to protein antigens, such as TSHR, is determined by the cytokines produced by CD4+ T helper (Th) cells. Naive CD4+ T cells, when stimulated with an antigen, functionally differentiate into Th1 or Th2 cells depending on the type of pathogen (4, 5). In general, Th1 cells produce cytokines such as IFN- and mainly promote cell-mediated immunity, whereas Th2 cells secrete cytokines such as IL-4 and IL-5 and enhance antibody (Ab)-mediated immunity. Cytokines are the dominant factors guiding the development of Th1 and Th2 cells. The Th1 and Th2 cells mutually regulate the function of each other and determine the outcome of immune responses. The differentiation of Th cells into Th1 and Th2 cells is controlled by a number of factors, including intracellular signaling molecules.
Signal transducer and activator of transcription (Stat) proteins are intracellular signaling molecules that mediate many cytokine-mediated responses (6, 7, 8, 9, 10). Stat4 is required for the development of fully functional Th1 cells, is activated by IL-12, and induces the transcription of IFN- (6, 7, 8). In contrast, Stat6 is essential for the development of Th2 cells and is activated by IL-4 (9, 10). Stat4–/– mice develop Th2-like immune responses in vivo and are resistant to the development of T cell-mediated autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) and type I diabetes in nonobese diabetic mice (11, 12). However, Stat4–/– mice develop accelerated Ab-mediated autoimmune disease, lupus (13, 14). In contrast, Stat6–/– mice develop a Th1-like immune response, are susceptible to T cell-mediated autoimmune diseases such as EAE (11), and display a significant reduction in the incidence of lupus (13, 14).
In GD patients, Th2 immune response was suggested to be dominant because GD is caused by Abs. This concept is supported by a study which showed that GD patients predominantly produce the Th2 cytokines IL-4 and IL-10 (15). In contrast, another study reported that TSAbs in humans are almost exclusively of IgG1 (16), which is a Th1 type in humans. In addition, other studies show a mixed Th1 and Th2 immune response (17, 18). The analysis of the Th1/Th2 response in animal models of GD has also provided conflicting results. In the TSHR-ad model, both IFN-- and IL-4-deficient BALB/c mice were resistant to the induction of Graves’ hyperthyroidism (19), suggesting that both Th1 and Th2 responses are required in this model. In contrast, in the TSHR-M12 model, hyperthyroidism was induced in BALB/c mice deficient in IFN-, but not in IL-4 (20). Recently, we showed that BALB/c mice deficient in Stat4, but not Stat6, injected with TSHR-HEK-293 cells developed hyperthyroidism (21). These studies indicate a critical role for Th2 cells in this model of GD and a limited role for Th1 cells. To investigate the role of Stat4 and Stat6 genes in other murine models of Graves’ hyperthyroidism, we injected wild-type BALB/c, Stat4–/–, and Stat6–/– mice with an adenovirus containing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad). Our results showed that 56% of wild-type (WT), 75% of Stat4–/–, and 39% of Stat6–/– mice developed hyperthyroidism, suggesting that even in the absence of Th2 cells, Ab-mediated autoimmune disease can develop.
Materials and Methods
Immunization of mice with TSHR-289-ad
Construction and purification of adenovirus containing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad) and control adenovirus (con-ad) containing -galactosidase have been described previously (22, 23). These adenoviruses were provided by Drs. B. Rapoport and S. M. McLachlan (Cedars-Sinai Medical Center, University of California, Los Angeles, CA). Adenoviruses (289-ad and con-ad) were propagated in HEK-293 cells and purified by CsCl density gradient centrifugation. The viral particle concentration was determined by measuring the absorbance at 260 nm (24), and it was stored in aliquots at –80 C.
Stat4–/– and Stat6–/– mice were generated as described previously (6, 9) 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 following approval from the Indiana University animal care and use committee. Mice were maintained in conventional housing facility. Six- to 8-wk-old female BALB/c, Stat4–/–, and Stat6–/– mice were injected im in the thigh muscle with 1010 particles of 289-ad or con-ad in 50 μl PBS. All mice in this study were immunized simultaneously using the same batch of adenovirus. Mice were injected three times at 3-wk intervals (d 0, 21, and 42), and blood was drawn 2 wk after the third injection (d 56). All surviving mice were killed 4 wk after the third injection (d 70) to obtain blood, spleen cells, and thyroid glands.
Serum TSH binding inhibitory Igs (TBII) and T4
TBII in sera were determined using a commercially available TRAb kit (Kronus, Boise, ID) according to the manufacturer’s protocol and as described previously (25, 26). This assay measures the ability of Abs in sera to inhibit the binding of 125I-labeled TSH to TSHR. Results are expressed as percent TBII values.
Total T4 in sera was measured with a commercially available RIA kit (Diagnostic Products Corp., Los Angeles, CA) according to the manufacturer’s protocol and as described previously (25, 27). This assay measures the ability of T4 in sera to compete with 125I-labeled T4 for binding to anti-T4 Ab-coated polypropylene tubes.
Thyroid-stimulating Abs (TSAbs) and TSH-blocking Abs (TBAbs)
TSAbs and TBAbs in sera were measured in a bioassay using Chinese hamster ovary (CHO) cells expressing TSHR as described previously (25, 28). In brief, these cells were grown to confluence in 96-well plates in F-12 medium supplemented with 10% fetal bovine serum (FBS). Cells were incubated in triplicate with mouse serum diluted 1:30 in hypotonic HBSS containing 0.5 mM 3-isobutylmethylxanthine for 2 h at 37 C. The cAMP released into the buffer was measured with a cAMP RIA kit (PerkinElmer, Boston, MA). TSAb was expressed as a percentage of the basal cAMP produced in the presence of buffer alone.
To measure TBAbs, cells were incubated in triplicate with mouse serum diluted 1:25 in hypotonic HBSS containing 0.5 mM 3-isobutylmethylxanthine. After 30 min at 37 C, 5 x 10–10 M bovine TSH (Sigma-Aldrich Corp., St. Louis, MO) was added, and incubation was continued for 2 h. The cAMP released into the buffer was measured with a cAMP RIA kit (PerkinElmer). The TBAb was calculated as: 100 x (1 – [cAMP of test serum in presence of bTSH ÷ cAMP of control serum in presence of bTSH]).
ELISA to measure IgG subclass of anti-TSHR Abs
Anti-TSHR Ab were measured by ELISA using purified TSHR-289 protein. This protein corresponds approximately to the TSHR-A subunit and was provided by Drs. B. Rapoport and S. M. McLachlan. TSHR-289 protein secreted into the culture medium by CHO cells was purified by affinity chromatography as described previously (29, 30). Wells of ELISA plates (Immulon2, Thermo Electron, Franklin, MA) were coated with 100 ng TSHR-289 protein in 100 μl carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 C. After washing the plate, 200 μl 0.2% FBS in PBS containing 0.05% Tween 20 was added and incubated for 1.5 h at room temperature to reduce nonspecific binding. The following reagents (100 μl) were added in succession after incubation at room temperature, followed by washing the plate between each step: serially diluted mouse serum in duplicate for 1.5 h, biotinylated goat antimouse IgG (Sigma-Aldrich Corp.) for 1 h, and avidin-horseradish peroxidase conjugate (Sigma-Aldrich Corp.) for 30 min. Color was developed in dark using 3,3',5,5'-tetramethylbenzidine (BD Pharmingen, San Diego, CA). The reaction was stopped using 50 μl 2 N sulfuric acid, and absorbance was measured at 450 nm. The subclass specificity of anti-TSHR Abs was determined using biotinylated goat antimouse IgG1 or IgG2a (Caltag Laboratories, Burlingame, CA).
Splenocyte proliferative response to purified extracellular domain of TSHR (ETSHR) protein
HEK-293 cells expressing the ETSHR were provided by Dr. Aaron J. W. Hsueh (Stanford University School of Medicine, Stanford, CA) and were maintained as described previously (31, 32). Soluble ETSHR protein was purified using a nickel affinity column as described previously (31, 32). Splenocytes of individual mice were cultured in triplicate (5 x 105 cells) in 96-well, round-bottom plates in RPMI 1640 medium containing 5% FBS in the presence or absence of purified ETSHR protein (10 μg/ml) or 5 μg/ml Con A as a control. After 72 h of incubation at 37 C in 6% CO2, 1 μCi [3H]thymidine (PerkinElmer) was added to each well. Cultures were harvested after 18 h and counted in a scintillation counter. Proliferation is expressed as the stimulation index and is calculated as the ratio between the average counts per minute of cultures in the presence of ETSHR protein and the average counts per minute of cultures in the presence of medium. The use of the stimulation index normalizes results and allows comparison of experiments performed at different times.
Cytokine production in response to purified ETSHR protein
Splenocytes of individual mice were cultured in triplicate (1 x 106 cells) in 96-well, round-bottom plates in RPMI 1640 medium containing 5% FBS in the presence or absence of purified ETSHR protein (10 μg/ml) or 5 μg/ml Con A as a control. After 48 h of incubation at 37 C in 6% CO2, supernatants were collected, and concentrations of IFN- and IL-4 were determined using ELISA kits (BD Pharmingen). The amount of cytokines produced was determined using standard curves corresponding to recombinant murine cytokines and expressed as picograms per milliliter.
Thyroid weight and histology
Thyroid tissues were removed, weighed, and fixed with 10% formalin. Tissues were embedded in paraffin, and 5-μm-thick sections were prepared and stained with hematoxylin-eosin.
Statistical analysis
Statistical significance was calculated using Student’s t test. P < 0.05 was considered significant.
Results
Our earlier studies using HEK-293 cells expressing ETSHR showed that 50% of WT BALB/c and Stat4–/– mice developed hyperthyroidism. However, none of the Stat6–/– mice developed hyperthyroidism (21). To investigate the role of Stat4 and Stat6 genes in other murine models of Graves’ hyperthyroidism, we immunized wild-type BALB/c, Stat4–/–, and Stat6–/– mice with an adenovirus expressing amino acid residues 1–289 of TSHR (TSHR-289-ad or 289-ad). We conducted two independent experiments, and data from these two experiments are shown in this report. Blood samples collected on d 56 were tested for TBII, T4, TSAbs, and TBAbs. We first measured the ability of Abs to bind to TSHR and inhibit the binding of [125I]TSH (TBII). In both experiments, all groups of mice (BALB/c, Stat4–/– and Stat6–/–) injected with 289-ad developed significant TBII levels compared with the corresponding con-ad-injected mice (Fig. 1). This showed that all groups of mice injected with 289-ad generated anti-TSHR Abs.
Next, to investigate any perturbation in thyroid hormone levels, the sera from these mice were tested for T4 levels. In both experiments, BALB/c and Stat4–/– mice injected with 289-ad developed significantly elevated T4 compared with the corresponding control mice (Fig. 2). However, T4 levels in Stat6–/– mice injected with 289-ad were not significantly different from those in the corresponding control mice (P = 0.112 in experiment 1; P = 0.126 in experiment 2). In the first experiment, 50% of BALB/c mice, 80% of Stat4–/– mice, and 37% of Stat6–/– mice injected with 289-ad developed hyperthyroidism (Fig. 2A). In the second experiment, 62% of BALB/c mice, 70% of Stat4–/– mice, and 40% of Stat6–/– mice injected with 289-ad developed hyperthyroidism (Fig. 2B).
We also compared differences in TBII and T4 among the experimental groups. The data combined from both experiments showed that experimental BALB/c and Stat4–/– mice developed significantly higher TBII than Stat6–/– mice (P < 0.01; not shown). Stat4–/– mice injected with 289-ad developed significantly higher T4 compared with similarly immunized Stat6–/– mice (P < 0.01; not shown). Serum T4 levels were elevated in nine of 16 (56%) WT, 15 of 20 (75%) Stat4–/–, and seven of 18 (39%) Stat6–/– mice. The Stat4-deficient mice developed a higher incidence and greater severity of hyperthyroidism than WT and Stat6-deficient mice. The majority of mice with high TBII (70% or more) also had elevated T4 levels. In general, serum TBII levels correlated well with serum T4 levels.
Patients with GD often show loss of body weight. Therefore, body weight might serve as a clinical parameter in these mice. Mice were weighed once a week to assess any weight loss. The differences in weight of the mice were not statistically significant between control group and experimental groups (not shown). However, some of the mice with very high levels of T4 lost 10% or more of their weight suddenly toward the end of the experiment. Of 54 experimental mice, four mice were either killed or died before d 70 because they exhibited significant weight loss and exhaustion. Of these mice, one was BALB/c, and the remaining three were Stat4–/–. All had very high T4 levels (>15 μg/dl).
To determine whether elevated T4 is due to the stimulatory Ab activity, sera were tested for their ability to activate TSHR and produce cAMP. TSAbs in sera were measured in a bioassay using CHO cells expressing TSHR and were expressed as a percentage of basal cAMP produced in the presence of buffer alone. The data combined from both experiments are shown in Fig. 3A. Sera from Stat4–/– mice injected with TSHR-289-ad, which developed the highest T4 levels, generated significantly higher levels of TSAbs compared with Stat6–/– mice (P < 0.01). In addition, BALB/c mice injected with 289-ad developed significant TSAbs compared with the corresponding con-ad-injected mice (P < 0.05). Similarly, Stat4–/– and Stat6–/– mice injected with 289-ad also developed significant TSAbs compared with the corresponding con-ad-injected mice (P < 0.05, not shown). Sera from hyperthyroid mice stimulated TSHR, leading to increased cAMP production, confirming the presence of TSAbs. Sera from mice that did not develop hyperthyroidism had no significant TSAbs. There was good agreement between T4 and TSAbs. These data confirm that the hyperthyroidism is due to the activity of TSAbs.
Next, we determined the ability of Abs to block TSH-mediated cAMP production. The majority of sera from mice immunized with TSHR-289-ad showed TBAb activity compared with con-ad-injected mice (Fig. 3B). TBAb activity was not significantly different among WT, Stat4–/–, and Stat6–/– mice injected with TSHR-289-ad. However, mice with elevated TBII and normal T4 levels had significant TBAb activity (Fig. 3B). This showed that TBAbs in these mice account for the elevated TBII.
We next measured serum anti-TSHR Abs belonging to IgG and different subclasses by ELISA against purified TSHR-289 protein. Mice injected with con-ad showed background OD values (<0.1) at 1:100 dilutions of sera. In contrast, mice injected with TSHR-289-ad developed TSHR Abs detectable even at 1:800 (Fig. 4A). The differences in IgG TSHR Abs among BALB/c, Stat4–/–, and Stat6–/– mice were not statistically significant (Fig. 4A). However, there were significant differences among the groups in the IgG1 subclass distribution of anti-TSHR Abs. WT BALB/c and Stat4–/– mice showed significantly higher levels of TSHR Abs of the IgG1 subclass compared with Stat6–/– mice (Fig. 4B). In contrast, Stat6–/– mice had predominantly IgG2a, with little of the IgG1 subclass of Ab (Fig. 4C). These data suggest that the IgG1 subclass of Ab is critical for the development of Graves’ hyperthyroidism.
Splenocytes from different groups of mice were tested for proliferation in response to purified ETSHR protein. As shown in Fig. 5A, splenocytes from all groups of TSHR-289-ad-injected mice showed significantly higher proliferative responses to ETSHR compared with con-ad mice (P < 0.001). Significant proliferative responses from all experimental groups showed that cells were similarly primed. We next analyzed the Th1 and Th2 cytokines secreted into the culture supernatants by splenocytes in response to ETSHR protein. As shown in Fig. 5B, splenocytes from BALB/c and Stat6–/– mice produced significantly higher IFN- than those from Stat4–/– mice in response to ETSHR protein (P < 0.001). In contrast, splenocytes from BALB/c and Stat4–/– mice produced significantly higher IL-4 than those from Stat6–/– mice (Fig. 5C; P < 0.05). All TSHR-289-ad injected mice produced IFN- in response to ETSHR protein (Fig. 5B). However, IL-4 was detectable in only 40% of BALB/c and Stat4–/– mice and in none of the Stat6–/– mice in response to ETSHR protein (Fig. 5C). We also tested for the other Th2 cytokine, IL-5. Similar to IL-4, IL-5 was detectable in only 30–40% of BALB/c and Stat4–/– mice and in none of the Stat6–/– mice (not shown). It is relatively difficult to detect Th2 cytokines compared with Th1 cytokines due to low levels of Th2 cells. Additionally, the variation in the secretion of IL-4 and IL-5 among individual BALB/c and Stat4–/– mice could be due to the variation in the dose of TSHR antigen presented to naive T cells after adenoviral infection. It is also possible that immunization with TSHR-ad leads to the production of lower quantities of IL-4 compared with immunization with M12 or HEK-293 cells expressing TSHR (20, 21). It is of interest to note that others have reported that IL-4 was not detected in BALB/c mice immunized with TSHR-289-ad (19).
Hyperthyroidism is usually associated with goiter. We therefore determined the size and weight of thyroid from each mouse. All hyperthyroid mice exhibited enlarged thyroid glands. A representative thyroid from each group is shown in Fig. 6. The thyroids of the largest size were seen in Stat4–/– mice injected with TSHR-289-ad. Thyroid sizes of WT BALB/c mice were intermediate, and those from Stat6–/– mice were the smallest compared with the other two experimental groups. In general, thyroid sizes correlated well with serum T4. The weights of thyroids of Stat4–/– mice were significantly higher compared with those in BALB/c and Stat6–/– mice (Fig. 7). The weights of the thyroids of all groups of mice immunized with TSHR-289-ad were significantly higher compared with those from con-ad mice (P < 0.001). The sizes and weights of thyroids from con-ad-injected BALB/c mice were normal (Figs. 6 and 7). Similarly, the sizes and weights of thyroids of con-ad-injected Stat4–/– and Stat6–/– mice were also normal (not shown).
The thyroids from con-ad-injected mice showed normal histology (Fig. 8, A and B). Histological examination of thyroids from hyperthyroid mice showed hypertrophy and hypercellularity of follicular epithelia with extensive protrusion into follicular lumen and a decreased amount of colloid material, indicating thyroid hyperactivity (Fig. 8, C–H). There were no intrathyroidal lymphocytic infiltrations. Histological examination of thyroids of other experimental mice that did not develop hyperthyroidism showed no significant hypertrophy or hyperactivity compared with those from control mice (Fig. 8, A and B).
Discussion
The factors that influence the development of Graves’ hyperthyroidism are not completely understood. Studies to define the role of Th1/Th2 immune responses in different animal models of GD have yielded different results. IFN- and IL-4 are the prototypic cytokines secreted by Th1 and Th2 cells, respectively. Studies using IFN-–/– or IL-4–/– BALB/c mice have reported different outcomes in two different models of GD. In the TSHR-M12 model, IFN-–/–, but not IL-4–/–, mice developed hyperthyroidism, demonstrating the importance of Th2 signaling in this model (20). In contrast, in the TSHR-ad model, Graves’ hyperthyroidism was attenuated in IFN-–/– and IL-4–/– BALB/c mice, demonstrating the importance of both Th1 and Th2 signaling in this model (19).
Stat4 and Stat6 are signaling molecules required for the development of fully functional Th1 and Th2 cells, respectively (6, 7, 8, 9, 10). Stat4–/– and Stat6–/– mice are similar to, but not identical with, IFN-–/– and IL-4–/– mice. Stat4–/– and Stat6–/– mice produce significantly lower amounts of IFN- and IL-4, respectively, compared with WT mice. Stat4–/– mice lack IL-12-activated IFN- production, but still secrete small quantities of IFN- induced by IL-18 or antigen receptor (6, 7, 8). Similarly, Stat6–/– mice lack IL-4-activated IL-4 production and are able to secrete IL-4 by cells such as natural killer T cells, mast cells, etc. (33, 34). However, IFN-–/– and IL-4–/– mice completely lack the secretion of IFN- and IL-4, respectively. Therefore, to investigate the role of Th1/Th2 immune responses in Graves’ hyperthyroidism, we recently immunized Stat4–/– or Stat6–/– BALB/c mice with TSHR-HEK-293 cells (21). The results from this earlier study showed that Stat4–/–, and not Stat6–/–, mice develop hyperthyroidism. These data are consistent with an earlier study, using TSHR-M12 cells and IFN-–/– and IL-4–/– mice, which showed that a Th2-type immune response is critical for the development of Graves’ hyperthyroidism in this model (20).
To investigate the roles of Stat4 and Stat6 genes in other models of Graves’ hyperthyroidism, we injected BALB/c, Stat4–/–, and Stat6–/– mice with the TSHR-289-ad. Similar to previous studies (22, 35), our data confirm the induction of Graves’ hyperthyroidism in BALB/c mice and extend this model to Stat4–/– and Stat6–/– BALB/c mice. Our present results showed that Stat4–/– mice developed significantly higher TBII, T4, and TSAbs compared with Stat6–/– mice. Stat4–/– mice developed a Th2-type immune response accompanied by significantly higher levels of TSHR Abs of the IgG1 subclass and IL-4 compared with Stat6–/– mice. In contrast, Stat6–/– mice developed a Th1-type immune response, showed predominantly the IgG2a subclass of TSHR Abs, and produced a significantly greater amount of IFN- than Stat4–/– mice. Although all hyperthyroid mice showed enlarged thyroid glands, Stat4–/– mice exhibited the largest thyroids. In addition to the higher incidence of disease (75% vs. 39%), the Stat4–/– mice exhibited a greater severity of hyperthyroidism (average T4, 11.8 vs. 6.5 μg/dl) compared with Stat6–/– mice.
The incidence (75% vs. 56%) and severity (average T4, 11.8 vs. 9.2 μg/dl) of hyperthyroidism were higher in Stat4–/– mice compared with WT BALB/c mice, although the reason for this is still unclear. There were no significant differences in their TBII, TSAbs, anti-TSHR Abs, or secretion of IL-4. However, BALB/c mice produced significantly higher levels of IFN- than Stat4–/– mice. This alone may not explain the differences between Stat4–/– and BALB/c mice. There may be differences in the affinity and fine specificity of anti-TSHR Abs between the groups; this will be examined in future studies. It is also possible that Stat4 regulates genes distinct from IFN- that reduce Ab-mediated autoimmune disease.
Although all experimental conditions, such as immunization procedure and housing, were identical, 100% of BALB/c and Stat4–/– mice and only 72% (13 of 18) of Stat6–/– mice developed higher TBII levels. We observed this variation in immune response in Stat6–/– mice in both experiments. This could be due to variation in the amount of T cell help that can be provided in the absence of fully functional Th2 cells in Stat6–/– mice.
There are some interesting differences between the present study and those using IFN-- and IL-4-deficient mice (19). Importantly, Stat4- and Stat6-deficient mice do recapitulate the phenotype of cytokine-deficient mice. There are several reasons for this. First, Stat4-deficient T cells can make some IFN-, albeit at reduced levels. Additionally, some cells, such as CD8 cells, can make normal antigen receptor-induced IFN- in the absence of Stat4. Similarly, natural killer T cells and mast cells can make IL-4 in the absence of Stat6. Thus, STAT-deficient mice are not devoid of cytokine production, and this accounts for the normal IgG2a anti-TSHR level in Stat4-deficient mice. However, Stat4- and Stat6-deficient mice represent a more comprehensive deficiency of Th1 and Th2 functions, respectively, than the restricted elimination of a single cytokine. The distinctions between these models may elucidate important components of the pathogenesis of GD.
There were other differences between the previous and the present study. In the previous study, IFN-- and IL-4-deficient mice were immunized twice with TSHR-289-ad (19). However, in the present study, Stat4- and Stat6-deficient mice were immunized three times with TSHR-289-ad. To determine the possible reasons for the differences in the incidence of disease, we repeated the experiments with Stat4- and Stat6-deficient mice involving two immunizations. There were no significant differences between two and three immunizations in the incidence of hyperthyroidism (not shown). However, the severity of hyperthyroidism was marginally higher after three immunizations than after two.
There are several advantages of the TSHR-289-ad model compared with the TSHR-HEK-293 model (21). First, the duration required for the development of disease is short (70 vs. 300 d). Second, the incidence of disease is higher (56% vs. 50% in BALB/c, 75% vs. 50% in Stat4–/–, and 39% vs. 0% in Stat6–/– mice). Third, the severity of disease is greater (average T4, 9.2 vs. 7.2 μg/dl in BALB/c, 11.8 vs. 5.9 μg/dl in Stat4–/–, and 6.5 vs. 2.9 μg/dl in Stat6–/– mice). These data demonstrate that TSHR-289-ad generates a stronger immune response than TSHR-HEK-293 cells and agrees with previous data showing that adenovirus infection induces strong immune responses (22). This could be due the expression of TSHR protein with proper conformation by adenovirus infection. It also suggests that a strong immune stimulation is required to generate Ab-mediated autoimmune disease in the absence of Th2 cells.
Stat4–/– and Stat6–/– mice have been used to study the role of Th1/Th2 immune responses in different autoimmune diseases. Stat4–/– mice were protected from the development of lymphocytic choriomeningitis virus-induced CD4+ T cell-dependent autoimmune diabetes (12). A more recent study reported that nonobese diabetic Stat4–/– mice were protected from the development of diabetes and EAE (36). Similarly, Stat4–/–, but not Stat6–/–, mice exhibited delayed onset and reduced severity of disease compared with WT mice in diabetes induced by multiple low doses of streptozotocin (37). Stat4–/– mice are resistant and Stat6–/– are susceptible to the induction of EAE mediated by CD4+ T cells (11). In addition, Stat6–/– mice develop more frequent and more severe myasthenia gravis than Stat4–/– mice (38). 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 (13, 14). These studies along with our current data indicate that the Stat6 signaling pathway and the Th2-type immune response are critical for the development of Ab-mediated autoimmune diseases.
Similar to the TSHR-HEK-293 model (21), the TSHR-289-ad model also demonstrated that the incidence of Graves’ hyperthyroidism was much higher in Stat4–/– mice compared with Stat6–/– mice. However, in contrast to the TSHR-HEK-293 model, the TSHR-ad model showed that Stat6–/– mice were not completely protected from the development of Graves’ hyperthyroidism. These results suggest that in the TSHR-289-ad model, Th2 cells are more efficient in mediating disease, but in the absence of Th2 cells, Th1 cells may still initiate a reduced incidence of Graves’ hyperthyroidism.
Acknowledgments
We thank Drs. B. Rapoport and S. M. McLachlan (Cedars-Sinai Medical Center, University of California, Los Angeles, CA) for providing us with TSHR-289 adenovirus and TSHR-289 protein, and Dr. Aaron J. W. Hsueh (Stanford University School of Medicine, Stanford, CA) for providing us with HEK-293 cells expressing ETSHR protein.
Footnotes
This work was supported by a Research Enhancement grant from the Indiana University School of Medicine (to G.S.S.) and National Institutes of Health Grant AI-45515 (to M.H.K.).
The authors K.J.L., P.G., M.H.K., and G.S.S. have nothing to declare related to the material being published.
First Published Online September 29, 2005
Abbreviations: Ab, Antibody; ad, adenovirus; CHO, Chinese hamster ovary; con, control; EAE, experimental autoimmune encephalomyelitis; ETSHR, extracellular domain of TSH receptor; FBS, fetal bovine serum; GD, Graves’ disease; Stat, signal transducer and activator of transcription; TBAb, TSH-blocking antibody; TBII, TSH binding inhibitory Ig; Th1, T helper type 1; TSAb, thyroid stimulatory antibody; TSHR, thyrotropin receptor; TSHR-289-ad, adenovirus expressing amino acid residues 1–289 of TSH receptor; WT, wild type.
Accepted for publication September 21, 2005.
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