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Absence of IL-4, and Not Suppression of the Th2 Response, Prevents Development of Experimental Autoimmune Graves’ Disease ¹

Rukiye-Nazan E. Dogan^*, Chenthamarakshan Vasu, Mark J. Holterman and Bellur S. Prabhakar^2,*

Departments of ^* Microbiology and Immunology and Surgery, University of Illinois, Chicago, IL 60612

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In autoimmune Graves’ disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) and cause hyperthyroidism. We studied the effects of fms-like tyrosine kinase receptor 3 ligand (Flt3-L) or GM-CSF treatment on the development of experimental autoimmune GD (EAGD) in mice, a slowly progressing Ab-mediated organ-specific autoimmune disease of the thyroid induced by immunization with syngeneic cells expressing TSHR. Flt3-L and GM-CSF treatment resulted in up-regulation of CD8a⁺ and CD8a^- dendritic cells, and skewing of cytokine and immune responses to TSHR in favor of Th1 and Th2, respectively. However, this skewing did not persist until the later stages, and thus failed to affect the course or severity of the disease. To determine whether the total absence of either IL-4 or IFN- could affect the development of EAGD, we immunized wild-type, IFN-^-/- and IL-4^-/- BALB/c mice with TSHR. Nearly 100% of the wild-type and IFN-^-/- mice developed EAGD with optimal TSHR-specific immune responses, while IL-4^-/- mice completely resisted disease and showed delayed and suboptimal pathogenic Ab response. These data demonstrated that skewing immune responses to TSHR, using either Flt3-L or GM-CSF, in favor of Th1 or Th2, respectively, may not be sufficient to alter the course of the disease, while the complete absence of IL-4, but not IFN-, can prevent the development of EAGD.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autoimmune Graves’ disease (GD)³ is the most common endocrine disorder and affects 1 in 10 women by 70 years of age. The disease is mediated by autoantibodies that bind to the thyrotropin receptor (TSHR) and stimulate thyroid hormone production. These stimulatory Abs belong predominantly to the IgG class and act as thyrotropin (TSH) agonists. Recently, we developed an animal model for GD (experimental autoimmune GD (EAGD)), in which BALB/c mice (H-2^d) are repeatedly immunized at 2-wk intervals with the M12 (H-2^d) B lymphoma cell line expressing recombinant mouse TSHR. By 4–5 wk after initiation of Ag inoculation, mice develop Abs that can bind to TSHR in an ELISA, but are unable to block TSH binding to the receptor. Upon continued immunization, Abs capable of blocking TSH binding to TSHR (TBII activity) appear in the circulation. Long after cessation of Ag inoculation, the Ab titer continues to increase, giving rise to stimulatory Abs, suggesting that the immunized mice have overcome tolerance to self-TSHR. The persistence of stimulatory Abs results in the development of hyperthyroidism with concomitant thyroid hypertrophy and loss of body weight, followed by lymphocytic infiltration of the thyroid (1).

An effective IgG Ab response against a protein Ag such as TSHR requires Ag-specific T cell help (2). It is thought that the subtype of Th cells that are activated in the early stages of an immune response will have a profound influence on the subsequent overall immune response to a given Ag. Th cells can be broadly divided into Th1 and Th2 types, and through the production of different cytokines they determine the type of immune response against an Ag (3). Th1 cells produce, among other cytokines, IFN-; facilitate delayed-type hypersensitivity responses, including macrophage activation; and play an important role in many destructive autoimmune disorders, such as experimental allergic encephalomyelitis, Hashimoto’s thyroiditis, and type 1 diabetes (4, 5). In contrast, cytokines such as IL-4, IL-5, and IL-13 produced by Th2 cells dominate in immune responses against certain parasites and provide help for B cells in the generation of Abs, including IgE and IgA (6). Cytokines produced by Th1 cells inhibit the development of Th2 responses, while cytokines produced by Th2 cells cause reciprocal inhibition of Th1 responses (7, 8). Differences in the relative amounts of Th1/Th2 cytokines can affect the qualitative nature of immune responses against bacterial, viral, and parasitic infections (9).

Polarization of Th cell responses can be influenced by several factors, including dendritic cells (DCs). Through their cytokine production, DCs are capable of influencing the differentiation of Th cells into either Th1 or Th2 cell types (10, 11). There are at least two subtypes of DCs, namely CD8a⁺ and CD8a^-, and they exert their influence through the production of distinct cytokines and skew the differentiation of T cells toward Th1 or Th2, respectively (12, 13, 14). fms-like tyrosine kinase receptor 3 ligand (Flt3-L) and GM-CSF can mobilize different DC subsets or their precursors in vivo and modulate immune responses in favor of either a Th1 or Th2 response, respectively (15, 16, 17).

Flt3-L can induce the expansion of both CD8a⁺ and CD8a^- DC subsets, but its administration along with a protein immunogen favors the development of a Th1-type immune response characterized by the production of IFN- and IgG2a Abs (15, 18, 19). However, GM-CSF is a potent growth factor that induces CD8a^- DCs in the spleen, which, in turn, favors Th2-type immune responses characterized by IL-4 production and a predominant IgG1 Ab response (14, 16, 20).

Subsequent to DC-mediated, Ag-specific T cell activation, cytokines released by activated T cells can help maintain the skewing in favor of either the Th1 or Th2 type throughout the immune response (17). These principles have been exploited to alter the Th1/Th2 balance in a number of experimental T cell-mediated autoimmune diseases (21, 22). In these animal models, Ag-specific T cell response results in acute clinical onset of the disease, usually within 14–21 days after immunization. These diseases are characterized by predominantly Th1-type, cell-mediated immune responses that cause target tissue damage, resulting in the clinical disease. Earlier studies have shown that skewing immune responses away from the Th1 toward a Th2 response can prevent the development of T cell-mediated autoimmune diseases (4, 5).

These observations show the promise of developing effective immunotherapies against T cell-mediated autoimmune diseases with an acute onset; however, the implications of these findings for the development of therapy for slowly progressing Ab-mediated autoimmune diseases are not as clear. Although it would seem that skewing immune responses toward a Th2 type might be beneficial in T cell-mediated autoimmune diseases, it is unlikely to be effective against very slowly progressing Ab-mediated EAGD that requires repeated immunizations with TSHR and Th2-promoting adjuvant. On the contrary, either the suppression of a Th2-type response or eliciting a predominantly Th1-type response might prevent the development of Ab-mediated autoimmune diseases, such as EAGD.

In this study we investigated the effects of treatment with either Flt3-L or GM-CSF, and the absence of IL-4 or IFN-, on the development of EAGD. Our results showed that skewing the immune response to TSHR in favor of either Th1 or Th2 at earlier stages is not sufficient to affect the disease development. However, the complete absence of IL-4, but not IFN-, can prevent the development of EAGD. These data also suggest that continued suppression of IL-4 might be of therapeutic value in GD.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals and cell lines

Six- to 8-wk-old female wild-type (WT), IL4^-/- and IFN-^-/- BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). This study was approved by the institutional animal care and use committee of University of Illinois (Chicago, IL) and was performed according to an approved protocol. The mouse B cell line (M12) expressing full-length mouse TSHR (mM12 cells) and the human fibroblast cell line expressing mouse TSHR ectodomain were prepared and maintained as described previously (1).

Cytokine modulators

GM-CSF and Flt3-L were provided by Dr. C. R. Maliszewski (Immunex, Seattle, WA). GM-CSF was polyethylene glycol-modified to prevent rapid degradation and clearance from the animal. Recombinant mouse IL-12 was obtained from R&D Systems (Minneapolis, MN).

Immunization of mice

Following treatment with GM-CSF and Flt3-L, mice were immunized with mM12 cells as described previously (1). Briefly, female BALB/c mice (12 mice/group) were pretreated i.p. with GM-CSF (2 µg/mouse/day for 5 days), Flt3-L (10 µg/mouse/day for 9 days), or Flt3-L for 6 days, followed by Flt3 and IL-12 for 3 days (10 and 0.5 µg of each/mouse/day). We included IL-12 along with Flt3-L because previous studies have shown that IL-12 appears to be a dominant cytokine driving the differentiation of Th1 lymphocytes in vitro and in vivo (23). Therefore, we used the combined treatment to induce a more potent Th1 response that we believed might be required to skew the response toward Th1 in BALB/c mice with a propensity to mount a Th2 response. On day 0 two mice were sacrificed for DC enumeration. The remaining 10 mice in each group received their first i.p. immunization with the syngeneic B lymphoma cell line, M12 or M12 cells expressing mouse TSHR (mM12; 1 x 10⁷ cells/mouse) pretreated with mitomycin C (50 µg/10⁷ cells; Sigma-Aldrich, St. Louis, MO), along with cholera toxin B subunit (CTB; 5 µg/mouse) (1). Starting on day 10 mice were left untreated or were treated with GM-CSF or Flt3-L for an additional 5 days. Starting on day 15 mice were immunized six more times at 2-wk intervals. Mice untreated but immunized with mM12 cells served as positive controls, while mice immunized with M12 cells and CTB served as negative controls. Blood samples were collected from all surviving mice periodically. On day 280 all animals were sacrificed. Our earlier studies showed that CTB is the most effective adjuvant of choice in inducing EAGD (1, 24); hence, it was used in this study.

Enumeration of DC by flow cytometry

Splenocytes were obtained from unimmunized mice that were treated with GM-CSF, Flt3-L, or Flt3-L and IL-12 (two mice per group) on day 0 and stained with FITC-labeled anti-mouse CD11c Ab and with either PE-labeled anti-mouse MHC class II or CD8a Ab (Caltag, San Francisco, CA). Propidium iodide uptake (10 µg/ml) was used to detect dead cells. Samples were analyzed (at least 10,000 cells from each mouse) by flow cytometry on a FACScan, and data were acquired using CellQuest software (BD Biosciences, San Jose, CA). These experiments were repeated independently with very similar results.

Purification of mETSHR

293 cells expressing the extracellular domain of mouse TSHR (mETSHR) as a fusion protein were used to obtain the soluble protein using a protocol identical with that described previously (25). The fusion protein was treated with thrombin to release the ectodomain, which was subsequently purified using an anti-TSHR mAb affinity column. This purified protein was used in all in vitro experiments that required TSHR.

Detection of Abs to mETSHR

The Ab response to mouse TSHR was determined by ELISA. Plates were coated with 100 ng/well (100 µl) of mETSHR in 0.01 M carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. To reduce nonspecific binding, wells were treated with 250 µl of PBS containing 1% BSA for 1 h at room temperature. After washing, serially diluted mouse serum was added into wells in triplicate and incubated for 1 h at 37°C. Abs bound to mETSHR were detected by adding HRP-labeled goat anti-mouse IgG, IgG1, or IgG2a (Caltag) for 1 h at room temperature, followed by the addition of tetramethylbenzidine-H₂O₂ substrate. The OD₄₅₀ was determined using an ELISA plate reader (model 550; Bio-Rad, Hercules, CA).

Measurement of TSH binding inhibitory index (TBII) values of sera

The ability of Abs to inhibit the binding of a ¹²⁵I-labeled TSH to porcine TSHR was measured using an assay kit that has been approved for clinical use (Kronus, Dana Point, CA). The TBII activity in the sera was determined following the manufacturer’s protocol. Briefly, duplicate samples of each serum (50 µl/tube) were incubated with detergent-solubilized porcine thyroid membrane and bovine ¹²⁵I-labeled TSH for 30 min. The TSHR Ab complexes were precipitated using polyethylene glycol, and the radioactivity was determined using a gamma counter. Results are expressed as the percent TBII activity using the formula: 1 - ([cpm sample - cpm NSB]/[cpm control - cpm NSB]). Since we used the sera from M12-immunized mice in lieu of human normal serum, which was provided in the kit, the mean TBII value for control mice was subtracted from the test values. Therefore, the values for control mice are 0 in Tables I and II. The TBII values in sera from normal mice range from 8–10%.

Thyroid-stimulating Abs in the sera of mice were detected using a cAMP generation assay. CHO cells expressing hTSHR (26, 27) were grown to confluence in 96-well plates in F-12 medium supplemented with 10% FBS. Before the assay, spent medium was removed, and cells were washed with fresh medium. Mouse sera diluted in hypotonic HBSS without NaCl, but containing 0.5 mM 3-isobutyl-ethyl-xanthine, were added and incubated for 3 h in a CO₂ incubator at 37°C. The cAMP released into the medium was measured employing an RIA kit (NEN, Boston, MA). Background cAMP production measured using this kit is 2–3%.

Measurement of total thyroxine (T₄) and 3,5,3-triiodothyroinine (T₃)

Commercially available RIA kits (Diagnostic Products, Los Angeles, CA) were used to detect mouse thyroid hormones, T₄ and T₃. In these assays the thyroid hormones in the sera compete with ¹²⁵I-labeled T₄ or T₃ for binding to specific Ab-coated polypropylene tubes. Briefly, assay tubes containing sera and 1 ml of ¹²⁵I-labeled T₄ or T₃ were incubated for 1 h at 37°C. The contents of the tubes were decanted, and the bound radioactivity was measured in a gamma counter. The amount of T₄ or T₃ was calculated from a standard curve generated using reference standards provided by the manufacturer. Normal ranges for T₄ and T₃ in mouse sera when measured using these kits are 4–5.5 µg/dl and 30–45 ng/dl, respectively.

T cell proliferation in response to mETSHR

Splenocytes were plated in 96-well, flat-bottom tissue culture plates at 5 x 10⁵ cells/well in RPMI 1640 medium containing 2% normal mouse serum, 1 mM glutamine, 1 mM sodium pyruvate, and 20 µg/ml gentamicin. The plates were incubated with medium alone or medium containing either an optimal amount of Ag (10 µg/ml recombinant mETSHR) or 2 µg/ml Con A for 48 h at 37°C in the presence of 5% CO₂. At the end of the incubation period, 1 µCi of ³H-labeled TdR (NEN) was added to each well and let to stand for 18 h. Cultures were harvested on glass-fiber filter paper using a 96-well cell harvester (MachIII-M; Tomtec, Hamden, CT). The incorporated ³H-labeled TdR was quantified using a Microbeta counter (PerkinElmer, Fremont, CA).

Cytokine production ex vivo

Spleen cells (5 x 10⁶/well) were cultured in 1.5 ml of RPMI 1640 medium, with or without 10 µg/ml recombinant mETSHR containing 2% normal mouse serum, 1 mM glutamine, 1 mM sodium pyruvate, and 20 µg/ml gentamicin for 48 h in the presence of 5% CO₂ at 37°C. Supernatants collected from both stimulated and unstimulated cultures were analyzed in triplicate for IFN-, IL-4, or IL-2 production in an ELISA using the manufacturer’s protocols. Cytokine detection kits were obtained from BD PharMingen (San Diego, CA) or Caltag. The amount of cytokine produced was determined by extrapolating values from standard curves obtained for the corresponding recombinant cytokine. The lower sensitivity limits for these assays, as suggested by manufacturers, are 3 pg/ml for IL-2, 8 pg/ml for IL-4, and 31 pg/ml for both IL-12 and IFN-.

Statistical analysis

Means, statistical deviations, and statistical significance were calculated using Microsoft excel application (Redmond, WA). Statistical significance (p value) was calculated using Student’s t test. A p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Treatment with Flt3-L or GM-CSF affects DC phenotype and cytokine production

Mice from each group were sacrificed on day 0, and their DCs were analyzed for number and phenotype to determine the efficacy of pretreatment with Flt3-L or GM-CSF. Earlier studies have shown that a majority of both CD8a⁺ and CD8a^- DC subsets express CD11c, MHC-II, CD40, CD80, and CD86 (15, 18, 19). Therefore, we tested cells for the expression of CD11c and MHC class II. As shown in Fig. 1A, mice treated with GM-CSF, Flt3-L, or Flt3-L and IL-12 had a significantly higher percentage of DCs (10.54, 6.61, and 8.28%, respectively) relative to the untreated control group (2.9%). In addition, the total number of cells in the spleen increased from 67 x 10⁶ in controls to 296 x 10⁶, 132 x 10⁶, and 128 x 10⁶ in GM-CSF, Flt3-L, and Flt3-L plus IL-12 groups, respectively. The percentage of the CD8a⁺ population was 5–6 times higher in mice receiving either Flt3-L alone or Flt3-L plus IL12 relative to GM-CSF-treated or untreated mice (Fig. 1B). Thus, treatment with FLt3-L or GM-CSF not only increased the total number of cells in the spleen and the percentage of DCs, but also differentially affected the development of different DC subsets. Next, we assessed the in vivo effects on cytokine production by splenocytes from treated and untreated mice that were cultured for 36 h without any in vitro antigenic stimulation. All treated mice, relative to controls, showed a modest increase in the levels of IL-12 and IL-2. Mice treated with GM-CSF showed a modest increase in IL-4 and IL-10, while mice treated with Flt3-L showed an increase in IFN-, but these increases were not significant (data not shown).

View larger version (54K): [in this window] [in a new window]   FIGURE 1. Effects of treatment with cytokine modulators. Mice were treated with GM-CSF, Flt3-L, or Flt3-L plus IL-12 as described in Materials and Methods. Mice were sacrificed on day 0, and spleen cells were analyzed for the expression of either MHC-II or CD11c (A) or for CD8a and MHC-II (B) by cell surface double staining with FITC-labeled anti-mouse CD11c and PE-labeled MHC-II or CD8a mAb conjugates. Quadrants were set for each Ab using an isotype-matched control.

It is generally accepted that the cytokines produced by DCs early after Ag exposure determine whether a subsequent immune response will be skewed toward the Th1 or Th2 type. Therefore, to test the effects of cytokine modulation, we immunized mice that were left untreated or were treated with Flt3-L or GM-CSF, with M12 cells expressing mouse TSHR (mM12 cells). To ensure immunization with an optimal amount of Ag, these cells were monitored for TSHR expression before each immunization by flow cytometry. As shown in Fig. 2A, mM12 cells expressed relatively high levels of TSHR protein, and cells showing similar levels of expression were used for immunizations.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Expression and purification of mouse ETSHR. A, Before each immunization, mouse TSHR expression on stably transfected mM12 cells was confirmed by flow cytometry using a TSHR-specific Ab. Propidium iodide was used to exclude dead cells. The figure shows a typical FACS profile of cells used for immunizations. B, SDS-PAGE analysis of affinity-purified mETSHR. Lane 1, Molecular mass markers of 106, 81, and 47.5 kDa. Lane 2, Purified mETSHR protein. This purified protein was used in all ex vivo assays that required TSHR.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Early response after immunization with mM12 cells. Proliferative and cytokine responses of spleen cells from different groups of mice against mETSHR. Mice were immunized following treatment with GM-CSF, Flt3-L, or IL-12 and Flt3-L as described in Materials and Methods. Control mice received M12 cells along with CTB. Mice were sacrificed on day 28, and spleen cells were collected. A, Proliferative response was determined in the presence (stimulated) or the absence (unstimulated) of mETSHR. B–D, Ex vivo cytokine production in the presence (stimulated) or the absence (unstimulated) of mETSHR for 36 h. Results are expressed as the mean ± SD of triplicate values for two mice tested individually. This experiment was repeated separately with very similar results. *, p < 0.05 vs immunized control mice.

Influence of Flt3-L or GM-CSF treatment on disease progression

We monitored mice receiving different treatments for their susceptibility to the development of EAGD. Periodic blood samples were collected and tested for TSHR-specific Ab responses and thyroid hormone levels. Initially, the sera were tested for TBII activity, which detects Abs capable of blocking TSH binding to TSHR. Immunized mice showed some TBII activity by day 110, which continued to increase until day 250 (Table IA). Next, we tested these sera for their ability to activate cAMP production as an indicator of their TSHR stimulatory activity. These results (Table IB) showed that all immunized groups had developed reasonable levels of stimulatory Abs compared with the control group beginning on day 180, and this activity continued to increase until day 250. Next, sera were tested for free T₄ levels, as an indicator of hyperthyroidism. Compared with the control group, T₄ levels were only marginally higher on days 110, 150, and 180, but were much higher by day 250 in all mM12-immunized groups with a rare exception (Table IC). Since deiodination of T₄ results in the formation of T₃, we tested for T₃ levels on day 250. Regardless of the treatment, all groups showed elevated levels of T₃ (210–229 ng/ml) compared with the control group (39 ng/ml). It is interesting to note that the increases in T₄ and T₃ levels were concomitant with increases in stimulatory Abs.

Influence of Flt3-L or GMCSF treatment on late immune responses to TSHR

Regardless of the treatment, by day 110 Ab responses reached comparable levels in all groups of mice immunized with mM12 cells (Fig. 4). We tested lymphocytes obtained on day 250 for their ability to proliferate (Fig. 5A) and produce cytokines (Fig. 5, B–D) in response to TSHR and found that the responses in various treatment groups were comparable to those of the untreated, but TSHR-immunized, control mice. Collectively, our studies showed that the TSHR Ab responses at early stages of the disease (i.e., days 28–56) were skewed more in favor of Th1 or Th2 responses in mice treated with Flt3-L, with or without IL-12, and GM-CSF, respectively (Figs. 3 and 4). However, this skewing was less dramatic later in the course of the disease, with little or no apparent differences in various treatment groups in Ab production, disease frequency, or time of clinical onset (Table I and Fig. 4). These results indicated that GM-CSF or Flt3-L treatment did not have a lasting effect on TSHR-specific immune responses.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Ab response against mETSHR. Sera were tested using serial 2-fold dilutions for detecting IgG, IgG1, and IgG2a Ab responses against mETSHR using an ELISA. Results are expressed as the mean Ab titer values of sera collected from 7 to 10 mice/group analyzed individually.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. TSHR induced responses at a later stage of the disease. A, Spleen cell proliferation in the presence (stimulated) or the absence (unstimulated) of mETSHR. B–D, Spleen cells were tested for ex vivo cytokine production in the presence (stimulated) or the absence (unstimulated) of mTSHR. Results are expressed as the mean ± SD of triplicate assays using spleen cells from 7–10 mice, tested individually. The p values were calculated by comparing each value with the corresponding value for TSHR group (*, statistically significant).

To determine whether, unlike cytokine skewing induced by GM-CSF or Flt3-L, which failed to affect disease outcome, the complete absence of IL-4 or IFN- would have any effect on the development of GD, we immunized WT, IL-4^-/-, and IFN-^-/- BALB/c mice with mM12 cells. The results showed that WT and IFN-^-/- mice had developed significant TBII activity by day 180 and continued to increase until day 250. IL-4^-/- mice showed lower levels of TBII activity than the other two TSHR-immunized groups (Table IIA). Sera from immunized WT and IFN-^-/- mice showed increased stimulatory Ab activity by day 250. Although the IL-4^-/- mice showed an increase in stimulatory Abs compared with the control group, the levels of these Abs were significantly lower than those in the other two immunized groups (Table IIB). As expected, mM12-immunized WT mice showed a significant elevation in T₄ levels by day 250 (12.8 ± 0.4 µg/ml). IFN-^-/- mice immunized with TSHR also showed increases in T₄ levels comparable to those noted in WT mice (12.9 ± 0.9 µg/ml). However, T₄ levels in immunized IL-4^-/- mice were within the normal range (5.9 ± 3.6 µg/ml) and were comparable to the levels seen in negative control mice immunized with M12 cells (5.0 ± 0.3 µg/ml; Table IIC). Similarly, the mean T₃ level in IL-4^-/- mice (81 ng/ml) was significantly lower than that in TSHR-immunized IFN-^-/- (185.4 ng/ml) and WT (210 ng/ml) mice.

Since IL-4^-/- and IFN-^-/- mice showed profound differences in their susceptibility to the disease, we compared both the early and late immune responses of these mice to mTSHR. On day 28 IL-4^-/- mice had a slightly lower IgG response to TSHR relative to IFN-^-/- and WT mice (Fig. 6A). However, the IgG1 response was higher in WT and IFN-^-/- mice, with IL-4^-/- mice showing a minimal response (Fig. 6B). In contrast, relative to WT mice, IL-4^-/- and IFN-^-/- mice showed higher and lower IgG2a responses, respectively (Fig. 6C). Although the Ab levels were higher, the relative amounts of different isotypes remained essentially the same within each group until day 250. This is in contrast to the loss of early isotype skewing (day 56) seen in GM-CSF- and Flt3-L-treated mice at a later stage of the disease (day 110 and beyond).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Anti-ETSHR Ab responses. Mice were immunized on days 0 and 15 with mM12 cells and CTB as described in Materials and Methods. Sera were collected on days 28 and 250 and were tested at different dilutions for IgG (A), IgG1 (B), and IgG2a (C) Ab responses against mETSHR using ELISA. Results are expressed as the mean ± SD of triplicate assays using sera from 9–10 mice, tested individually. The p values were determined by comparing each value with the corresponding value for WT mice (*, statistically significant). Histograms show OD values at a dilution of 1/400. Mean Ab titer values are overlaid on the individual histogram bars.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. TSHR induced responses at a later stage of the disease. Mice were sacrificed on day 280, and spleen cells were collected. A, Cell proliferation was determined in the presence (stimulated) or the absence (unstimulated) of mETSHR. B–D, Ex vivo cytokine production. Results are expressed as the mean ± SD of triplicate values obtained using spleen cells from 9–10 mice tested individually. The p values were calculated by comparing each value with the corresponding value for the wild-type group (*, statistically significant).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In general, Th1 responses are characterized by the production of IFN-, whereas Th2 responses are characterized by the production of IL-4, IL-5, and IL-13. Since Th1 cytokines have suppressive effects on Th2 responses and vice versa (7), one way to skew the immune response in favor of either Th1 or Th2 is to use appropriate cytokine treatment at an early stage of immune response induction. Alternatively, Abs against cytokines, including anti-IL-4 and anti-IL-12, which favor either Th2 or Th1 responses, respectively, are used to alter the cytokine balance (28, 29). For example, anti-IL-4 Abs can neutralize IL-4 and result in the suppression of Th2 responses and can enhance Th1 responses. However, treatment with cytokines or anti-cytokine Abs has limitations due to their systemic effects. Another approach to skew the immune response is to treat mice, before Ag inoculation, with cytokine modulators such as Flt3-L or GM-CSF (14, 16, 18, 20).

Treatment with Flt3-L leads to the expansion of CD11c⁺ CD8a⁺ and CD11c⁺ CD8a^- DCs, while GM-CSF causes preferential expansion of CD11c⁺, CD8a^- DCs. These treatments have been used to expand particular subsets of DCs with consequent effects on the ensuing immune responses (17, 30). Although the mechanism by which DC subsets influence selective expansion of Th1 or Th2 T cells is not fully understood, it is generally believed to be due to the production of certain cytokines that aid in the selective differentiation of T cell subsets (31, 32). It is thought that the cytokine milieu early after Ag inoculation not only influences the differentiation of Th1- or Th2-type T cells, but also sustains those cells due to appropriate cytokine production by the activated T cells. Based on these assumptions, we treated mice with Flt3-L or GM-CSF and then challenged them with THSR.

Treatment with GM-CSF before TSHR inoculation resulted in a predominant Th2 response, while treatment with Flt3-L alone or in combination with IL-12 resulted in a Th1 response against the TSHR at least until days 56–110. GM-CSF-treated mice showed higher IgG1 Ab (Fig. 4) and IL-4 (Fig. 3) production. In contrast, Flt3-L-treated mice showed higher IgG2a (Fig. 4) and IFN- (Fig. 3) production. However, these treatments had no apparent effect on the frequency or the time of onset of the disease. Analyses of immune responses against TSHR showed that all mice regardless of treatment status showed very similar responses beginning on day 110 until day 250. Lack of significant differences in Ab isotype on day 110 indicated that by this time the effects of treatment with Flt3-L or GM-CSF had begun to wane. Furthermore, it suggested that because of delayed appearance of pathogenic Abs required for induction of the disease, the effects of treatment with Flt3-L or GM-CSF might have to persist until the appearance of stimulatory Abs.

Although anti-TSHR Ab responses can be detected shortly after the second or third immunization, these Abs are of little, if any, functional consequence. However, upon repeated antigenic inoculation along with the adjuvant, mice develop Abs that can initially compete for TSH binding to the TSHR (Table IA) and eventually stimulate TSHR-mediated function (i.e., induction of cAMP; Table IB). Slow development of functionally relevant Abs might occur through epitope spreading (e.g., from immunodominant to cryptic epitopes), leading to Ab responses against the pathogenic cryptic epitopes, and/or somatic hypermutations required for the development of high affinity Abs that can act as TSH agonists. This particular requirement distinguishes stimulatory Abs against the TSHR from other autoantibodies that block the action of hormones, as seen in primary myxedema, myasthenia gravis, and insulin resistance, or that form immune complexes, as in systemic lupus erythematosus.

How does one reconcile these results with those from earlier studies, which had indicated that the cytokines produced early after Ag exposure could influence the overall immune response to the Ag (30, 31, 32, 33). Not many studies have evaluated the long term (i.e., for 6–8 mo) effects of treatment with either the cytokines or the cytokine modulators. Recent studies evaluated the effects of nasal or oral administration of acetylcholine receptor and glutamine acid decarboxylase peptides and found that these peptides can skew the immune response against them in favor of the Th2 type and suppress experimental myasthenia gravis and diabetes, respectively, for longer observation periods (34, 35, 36). We also noted that the effects of cytokine modulators can be seen weeks (i.e., until day 110) after cessation of treatment (i.e., on day 15), but the effects were lost much before the appearance of functional Abs (after day 180) and development of hyperthyroidism. Although the reason for the lack of persistence of the effects of cytokine modulators for a longer period of time is not known, we speculate that it could be because a majority of initially activated TSHR-specific T cells eventually undergo activation-induced death due to repeated exposure to the Ag. Alternatively, since an ongoing Ab response is less dependent on T cells, there could be recruitment of insufficient number of new T lymphocytes required to sustain a particular skewing. It is also possible that repeated immunization with TSHR and CTB eventually induced Th2 cells in mice treated with Flt-3L and rendered them susceptible. This might be particularly relevant because unlike in earlier studies in which the Ag was given once or twice (37, 38, 39), in this study the Ag was inoculated repeatedly and with a potent adjuvant well after the cessation of treatment. This may have contributed to the failure of Flt3-L and IL-12 to have longer term downstream effects. In fact, other studies underway in our laboratory strongly indicate a shift in T cell repertoire, as evidenced by a change in the TSHR peptide specificity and the TCR V usage of T cells (not shown) during the course of the disease. These results indicate that treatment of mice with Flt3-L or GM-CSF at later stages of the disease development might be more effective. Alternatively, mere skewing of the response against TSHR may not be sufficient to alter the course of the disease, but might require total suppression of the predominant cytokines.

To determine whether the absence of IL-4 or IFN- could affect the disease outcome, we conducted additional studies using WT, IL-4^-/-, and IFN-^-/- mice. Since the cytokine profiles of these mice have been extensively characterized, in this study we did not analyze early cytokine production. Both WT and IFN-^-/- mice developed disease with very similar TBII and stimulatory Ab responses and with thyroid hormone perturbation consistent with hyperthyroidism. In contrast, IL-4^-/- mice failed to develop the disease, as indicated by normal levels of thyroid hormones. These mice showed both delayed appearance and lower TBII and stimulatory Ab responses relative to the other two groups. The WT mice showed both IgG1 and IgG2a Ab responses, while the IL-4^-/- and IFN-^-/- mice produced mainly IgG2a and IgG1 Ab responses, respectively. As expected, upon stimulation with mETSHR, lymphocytes from IL-4^-/- and IFN-^-/- mice produced IFN- and IL-4, respectively. The levels of these cytokines were comparable to that seen in WT mice. Development of disease in Flt3-L-treated, but not in IL-4^-/-, mice indicates that although an IL-4-dominant response (i.e., Th2 cytokine) might be less critical in the early stages of disease induction, it is required for disease progression.

We examined the thyroids of all sacrificed mice when the experiment was terminated. We expected more thyroid infiltration in Flt3-L- and Flt3-L plus IL-12-treated and IL-4^-/- mice because of expected Th1 skewing. In contrast, we expected to see little or no infiltration in GM-CSF-treated and IFN-^-/- mice because of suboptimal T cell responses and Th2 skewing. In general, as expected, all hyperthyroid mice showed enlargement of the thyroid follicles with little or no lymphocytic infiltration, relative to unaffected mice, regardless of the treatment. There was no significant difference in thyroid histopathology among affected mice in different groups. These findings are not entirely surprising, because our earlier study (1) showed that thyroid infiltration was not apparent until 60 days after the development of hyperthyroidism. Since we diagnosed the disease based on elevations in the levels of stimulatory Abs, T₄, and T₃, these mice were not followed for the duration required to evaluate thyroid pathology accurately.

In summary, our data showed that the absence of IL-4 can confer resistance against the development of EAGD, while lack of IFN- along with substantial amounts of IL-4 failed to either increase the severity of the disease or reduce the time required for its clinical onset. This later observation is consistent with our findings in mice treated with GM-CSF. Similarly, the results obtained from Flt3-L-treated mice show that mere skewing in favor of Th1 with reduced IL-4 levels is not sufficient to prevent the development of EAGD. Collectively, our results show that IL-4 is required for the development of EAGD. Although a reduction in the level of IL-4 does not appear to be sufficient to affect disease development, an absence of IL-4, as in IL-4^-/- mice, can prevent development of the disease, and this indicates that a more complete and prolonged suppression of IL-4 might be of therapeutic value in GD.

	Acknowledgments

 
We thank Drs. Charles R. Maliszewski and Prasad Kanteti for critical review of our manuscript.

	Footnotes

 
¹ This work was supported by the National Institutes of Health Grants DK47417, DK57938, and DK44972.

² Address correspondence and reprint requests to Dr. Bellur S. Prabhakar, Department of Microbiology and Immunology, 835 South Wolcott, University of Illinois College of Medicine, Chicago, IL 60612. E-mail address: bprabhak{at}uic.edu

³ Abbreviations used in this paper: GD, Graves’ disease; CTB, cholera toxin B subunit; EAGD, experimental autoimmune Graves’ disease; Flt3-L, fms-like tyrosine kinase receptor 3 ligand; hTSHR, human thyrotropin receptor; mETSHR, mouse extracellular domain of thyrotropin receptor; mTSHR, mouse thyrotropin receptor; T₃, 3,5,3-triiodothyroinine; T₄, thyroxine; TBII, TSH binding inhibition index; TSH, thyrotropin; TSHR, thyrotropin receptor; 293 cells, human embryonic kidney cells; WT, wild type.

Received for publication August 1, 2002. Accepted for publication December 6, 2002.

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