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Departments of
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Surgery and
Microbiology and Immunology, University of Illinois, Chicago, IL 60612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 production. In contrast, mice treated with GM-CSF, either before or after immunization with mTg, showed suppressed T cell response to mTg and failed to develop thyroiditis. Lymphocytes from these mice, upon activation with mTg in vitro, produced higher levels of IL-4 and IL-10. Additionally, GM-CSF-treated mice showed an increase in the frequency of CD4+/CD25+ T cells, which suppressed the mTg-specific T cell response. Neutralization of IL-10, but not IL-4, or depletion of CD4+/CD25+ cells resulted in increased mTg-specific in vitro T cell proliferation suggesting that IL-10 produced by the Ag-specific CD4+/CD25+ regulatory T cells might be critical for disease suppression. These results indicate that skewing immune response toward Th2, through selective activation of dendritic cells using GM-CSF, may have therapeutic potential in Th1 dominant autoimmune diseases including Hashimoto’s thyroiditis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and elicit Th1 types of responses, while CD8a- DCs produce little or no IL-12 and IFN-, but preferentially induce Th2 responses (e.g., IL-4 production) (10, 11, 12). In mice, polarization of immune responses toward either Th1 or Th2 type can be induced by injecting cytokines, different subsets of DCs or ligands, such as fms-like tyrosine kinase receptor 3 ligand (Flt3-L) and GM-CSF that can induce activation of different subtypes of DCs (13, 14, 15, 16, 17). Flt3-L mobilizes both CD8a+ and CD8a- subsets of DCs, but promotes a Th1 type of immune response against protein Ags (14, 15). GM-CSF is a potent growth factor for CD8a- DCs, which can induce a Th2 type of response (17, 18). Because of predominance of either Th1 or Th2 types of T cells in autoimmune diseases (19, 20, 21), we reasoned that it might be feasible to use Flt3-L or GM-CSF to modulate DC subsets and prevent autoimmune diseases including experimental autoimmune thyroiditis (EAT).
EAT is a chronic inflammatory organ-specific autoimmune disease of the thyroid and serves as a model for Hashimoto’s thyroiditis. The disease is characterized by lymphocytic infiltration of the thyroid and production of mouse thyroglobulin (mTg)-specific autoantibodies eventually leading to follicular destruction (22, 23). Although the mechanism by which CD4+ T cells cause destruction of the thyroid in EAT is not fully understood, the cytokines produced by Th1-type cells (e.g., IFN-) likely play an important role (24, 25). A combination of IFN-
and IFN- produced in the thyroid and increased levels of IFN- produced by the thyroid-infiltrating lymphocytes have been shown to facilitate apoptosis of thyroid follicular cells through caspase activation (26, 27, 28).
EAT can be exacerbated through administration of IL-12 or prevented by the continuous administration of IL-4 (29, 30). The predominance of Th1 responses in EAT has been shown through noninvolvement of IL-4 and IL-10 (31). Studies also demonstrated that increased levels of IL-12 at the early stages of disease can aggravate, while sustained exposure to IL-12 can reduce the severity of EAT (29). Skewing the response in favor of a Th2 type in EAT and experimental autoimmune encephalomyelitis has been found to be protective (32, 33, 34, 35, 36, 37, 38, 39) and the degree of protection appears to depend on the mode as well as the time of initiation of the treatment. For example, while continued presence of IL-10 or IL-4 had a protective effect on both EAT and experimental autoimmune encephalomyelitis (20, 40, 41), blocking or elimination of Th1 cytokines to promote Th2 responses has resulted in varying outcomes (35, 42, 43, 44). Collectively, these studies indicate autoimmune responses are regulated by a delicate balance between Th1 and Th2 cytokines and that a careful manipulation of this balance could be exploited to either induce or prevent the development of EAT.
Cytokines produced in the early phase of an autoimmune response can profoundly affect subsequent responses leading to disease development. Because DCs are involved in the earliest phase of immune responses, we explored the effects of mobilization of different subsets of DCs using either Flt3-L or GM-CSF on the development of EAT. Our results showed that treatment with Flt3-L, which enhanced Th1 responses, resulted in a more severe disease. In contrast, treatment with GM-CSF, even at later stages of the disease, enhanced Th2 responses and either prevented or significantly suppressed disease development.
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Materials and Methods |
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Six-week-old female CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in the Biological Resources laboratory facility at the University of Illinois (Chicago, IL) and provided food and water ad libitum. All mice were used at 8 wk of age and cared for in accordance with the guidelines set forth by the University of Illinois Animal Care and Use committee.
Flt3-L and GM-CSF, Abs and thyroglobulin
Recombinant human Flt3-L and polyethylene-modified GM-CSF were provided by Immunex (Seattle, WA). FITC-conjugated anti-CD11c and PE-conjugated anti-I-Ak (MHC class II) anti-CD8a, anti-CD80, and anti-CD86 (BD PharMingen, San Diego, CA); PE-conjugated anti-CD4, anti-CD8a, and anti-CD25 (Caltag Laboratories, San Francisco, CA) were used in flow cytometry. Paired Abs and required cytokine standards for detecting mouse IL-2, IL-4, IFN-, and IL-12 (Caltag Laboratories) and Abs for detecting IL-10 and TGF-
1 (BD PharMingen) were used in ELISA. Neutralizing Abs to mouse IL-4 (rat IgG1, clone 11B11), IL-10 (rat IgG1, clone JES5-2A5), and normal rat IgG1 isotype controls were purchased from E-Bioscience (San Diego, CA). PE-labeled anti-mouse CD25 Ab (BD PharMingen), magnetic bead-conjugated anti-PE and anti-CD4, and anti-CD3 Abs (Miltenyi Biotec, Auburn, CA) were used to isolate CD4+/CD25+, CD4+, and CD3- cells. HRP-labeled anti-mouse IgG, IgG1, and IgG2a (Caltag Laboratories) were used to detect mTg-specific Abs. Normal mouse thyroids were obtained from BiochemMed (CA) and thyroglobulin (mTg) was prepared as described earlier (Winchester, VA).
Effects of Flt3-L and GM-CSF on the induction of EAT
Mice were divided into four groups of 10 mice each, (viz, 1, control; 2, mTg; 3, Flt3-L/mTg; and 4, GM-CSF/mTg). Mice in the Flt3-L group were injected i.p. with 10 µg of Flt3-L/mouse/day for 9 consecutive days. Mice from the GM-CSF group were injected with PBS from days 1 to 4 and then with 2 µg of GM-CSF/mouse/day for 5 consecutive days from days 5 to 9. Control animals (control and mTg groups) were injected with PBS for 9 days. All groups of mice, except the control group, were immunized s.c. with mTg (100 µg/mouse) emulsified in CFA on day 10. Mice in groups 3 and 4 were further treated with the same doses of Flt3-L and GM-CSF, respectively, for 5 consecutive days from day 19. On day 24, mice in all groups, except the control group, were given a second dose of mTg (100 µg/mouse) emulsified in CFA. Control mice received PBS emulsified in CFA on days 10 and 24. Mice were bled on days 23, 31, and 38. They were then sacrificed on day 38 and lymph nodes, spleens, and thyroids were collected. Parallel to this experiment, another set of mice were treated and immunized as described above but two mice from each group were sacrificed on days 10, 13, and 27, and spleen and lymph nodes were collected for analyzing the DCs.
Treatment after immunization with mTg
In a second set of experiments, five groups of seven mice per group (viz, 1, control; 2, mTg; 3, GM-CSF-group1; 4, GM-CSF-group 2; and 5, GM-CSF-group 3) were immunized with mTg and CFA as mentioned above but on days 1 and 14. Animals in GM-CSF group 1 were given 2 µg of GM-CSF/day/mouse from days 3 to 7 and again from days 15 to 19. Mice in GM-CSF group 2 were given 2 µg of GM-CSF from days 15 to 19 and again from days 28 to 32. Mice in GM-CSF group 3 were injected with GM-CSF from days 28 to 32. Control animals received PBS. Mice were bled on days 15, 28, and 45 to obtain sera. Mice were sacrificed on day 45, lymph nodes and spleens were collected to test for ex vivo T cell proliferation and cytokine responses. Thyroids were collected for histochemical staining.
Flow cytometric analysis
Single cell suspensions of spleen and lymph nodes were prepared from mice sacrificed on days 10, 13, and 27. Cells were washed with PBS supplemented with 2% FBS, blocked with anti-CD16/CD32 Fc-Block (BD PharMingen) on ice for 30 min. Cells were stained with PE-labeled anti-mouse I-Ak, CD8a, CD80, CD86, CD25 Abs and FITC-labeled anti-mouse CD11c and CD4 Abs on ice for 15 min, washed, and analyzed using a FACS analyzer (BD Biosciences, San Jose, CA) and the CellQuest software. Control cells were stained with isotype-matched control Abs and analyzed. At least 10,000 cells were analyzed in all experiments and all experiments were repeated three times with similar results.
Measurement of cytokine production
Spleen and lymph node cells, obtained on days 10, 13, and 27 were tested for spontaneous cytokine production without in vitro stimulation. However, cells collected on days 38 and 45 were tested both with and without mTg stimulation in vitro. For in vitro stimulation, 5 x 106 spleen cells/well (12-well plate) were incubated in the presence of mTg (20 µg/ml) in 1.5 ml of RPMI 1640 medium supplemented with 2% normal mouse serum in a CO2 incubator. Cell-free culture supernatants were collected after 36 h by centrifugation. The cytokine levels in culture supernatants were assayed by ELISA using paired Abs for the detection of IL-2, IL-10, IL-12, TGF-1, IL-4, and IFN-, following manufacturer’s instructions. Cytokines were detected by adding HRP-labeled streptavidin followed by washing and addition of tetramethyl benzidine-H2O2 substrate (BD PharMingen) for 5–10 min. The OD450 was read using a Microplate reader (Bio-Rad, Hercules, CA). The amount of cytokine was determined using appropriate cytokine-specific standard curves.
ELISPOT assay
ELISPOT plates were coated with 5 µg/ml anti-IL4 or anti-IL-10 Ab in sterile PBS, pH 7.2, at 4°C overnight. The plates were washed once and blocked with PBS-10% FBS for 2 h at room temperature (RT). After two washes, varying numbers of lymph node cells (1 x 104–1 x 105/well) were incubated in 100 µl of RPMI 1640 containing 2% normal mouse serum in the presence or absence of mTg (2 µg/well) for 18 h at 37°C. The plates were washed once with distilled water and then with PBS-0.05% Tween (PBS/T) twice and incubated with 0.5 µg/ml biotin-labeled anti-IL-4 or anti-IL-10 detection Abs. After a 2-h incubation at 37°C, plates were washed and HRP-conjugated streptavidin was added and allowed to incubate for another 1 h. The plates were washed and the enzyme reaction was detected using 4-ACE-H2O2 substrate for 30 min. Plates were washed with water and spots were counted under a dissection microscope.
Quantitation of anti-mTg Abs
Serum levels of mTg-specific IgG, IgG1, and IgG2a Abs were determined using an ELISA. Ninety six-well plates (Nunc, Roskilde, Denmark) were coated with 0.5 µg/well (100 µl) mTg in 0.01 M carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. Wells were blocked with 300 µl of PBS containing 1% BSA for 1 h at RT. Two-fold serial dilutions of serum samples were added to the wells in triplicate and incubated for 1 h at RT. After washing, the plates were further incubated with 100 µl/well PBS/T containing an optimal concentration of HRP-labeled anti-mouse IgG, IgG1, or IgG2a for 1 h at RT. The enzyme reaction was developed as described above.
T cell proliferation assay
Mouse splenocytes (5 x 105 cells/well) or lymph node cells (2 x 105 cells/well) were plated in 96-well flat-bottom tissue culture plates in triplicate in RPMI 1640 containing 1% normal mouse serum at a final volume of 0.25 ml/well. mTg was added at a concentration of 20 µg/ml. Con A (2 µg/ml) was used as a positive control. Plates were incubated for 48 h at 37°C in a CO2 incubator and the cells were then pulsed with 1 µCi [3H]thymidine/well and incubated for an additional 18 h. Cells were harvested onto glass-fiber filter papers using a 96-well cell harvester (Tomtec, Hamden, CT) and counted using a microbeta counter (PerkinElmer Wallac, Gaithersburg, MD).
Cytokine neutralization assay
Cells were cultured in the presence of mTg as described above for T cell proliferation. To these cultures, varying concentrations of neutralizing rat anti-mouse IL-4 (10 ng to 2000 ng/ml) and/or anti-mouse IL-10 (10–5000 ng/ml) mAbs (IgG1) or isotype (rat IgG1) matched control Abs were added and incubated for 48 h, pulsed with [3H]thymidine, harvested, and counted as described above.
Isolation of CD4+/CD25+ T cells
CD4+/CD25+ cells were isolated using Abs conjugated to magnetic beads and a magnetic separation column by following the manufacturer’s directions. Pooled mouse spleen and lymph node cells were incubated with anti-CD16/32 for 15 min on ice to block FcRs; subsequently, they were incubated with PE-labeled anti-mouse CD25 Ab for 30 min on ice. Cells were washed and incubated with magnetic bead-conjugated anti-PE Ab for 15 min, washed, and separated using an autoMACS (Miltenyi Biotec). An unbound CD25- fraction was incubated with anti-mouse CD4 Abs, coupled to magnetic beads, for 30 min and fractionated as described above. Isolated cells were subjected to repurification, washed, and stained with FITC-labeled anti-CD4 and PE-labeled anti-CD25 Abs, and tested for purity using a flow cytometer. Spleen cells were depleted of CD3+ cells by negative selection using magnetic beads coated with anti-mouse CD3 Abs.
Cocultivation of lymphocytes with CD4+/CD25+ cells
CD4+/CD25- cells from untreated mice were mixed with CD4+/CD25+ cells from GM-CSF-treated mice and vice versa at a ratio of 30:1. These mixtures were used in a T cell proliferation assay that was conducted, either in the presence or absence of mTg, as described above. Mitomycin C-treated CD3- spleen cells from naive mice (2 x 105 cells/well) were used as APCs in all assays.
Histological examination of thyroids
Thyroids collected from mice, at the time of sacrifice, were fixed in formalin, embedded in paraffin, and sectioned. Tissue sections were stained with H&E and subjected to microscopic examination. The severity of thyroiditis was graded based upon the degree of lymphocytic infiltration and thyroid destruction.
Statistical analysis
Mean, geometric mean titer (GMT), SD, and statistical significance were calculated using an SPSS application (Chicago, IL). Statistical significance was determined using the nonparametric Wilcoxon signed test. In most cases, values of an individual-treated and immunized group was compared with that of an untreated but immunized group. Differences in the percentage of fluorescence-positive cells between untreated and individual-treated groups were tested using the nonparametric sign test. A p value of 
0.05 was considered significant.
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Results |
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As shown in Fig. 1a, spleens and lymph nodes from mice treated with Flt3-L or GM-CSF ligand showed increased numbers of CD11c+ cells, compared with control mice. Flt3-L-treated mice showed increases in both CD8a+ and CD8a- DCs, while GM-CSF-treated mice showed an increase in CD8a- DCs. There was a 2-fold increase in the total number of spleen cells in Flt3-L-treated mice (1.4 x 108–1.6 x 108) and a 5-fold increase in GM-CSF-treated mice (2.9 x 108–3.4 x 108) compared with the controls (6.6 x 107–8.3 x 107) indicating a significant increase in the total number of DCs. The range of absolute numbers of DCs present in the spleens was 2.2 x 106–2.3 x 106 in control mice, 2.4 x 107–2.7 x 107 in Flt3-L-treated mice, and 2.1 x 107–2.38 x 107 in GM-CSF-treated mice. A larger proportion of DCs from both Flt3-L- and GM-CSF-treated mice showed surface expression of CD80 and CD86. Further, when cultured in vitro in the absence of Ag, cells from Flt3-L-treated mice produced higher levels of IL-12 and IFN- compared with cells from mice either untreated or treated with GM-CSF (Fig. 1b). However, there was no detectable difference in the levels of IL-4 produced by different groups.
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and IL-12, and IL-4 and IL-10, respectively (Fig. 2b). In vitro stimulation of these cells with mTg did not significantly alter the cytokine expression pattern by different groups, but the levels were significantly higher (results not shown). This indicated that major differences observed between treated and untreated mice are primarily due to the effects of Flt3-L and GM-CSF and that they can differentially activate Th1 and Th2 types of immune responses, respectively.
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Next, we analyzed mTg-induced cytokine responses by spleen and lymph node cells obtained on day 38 from different groups of mice (Fig. 3). Spleen cells from Flt3-L-treated mice produced significantly higher levels of IL-2 (p = 0.027) and IFN- (p = 0.019), and reduced levels of IL-10 (p = 0.015) compared with the mTg group. In contrast, GM-CSF-treated mice produced significantly higher amounts of IL-4 (p = 0.011) and IL-10 (p = 0.017), and lower levels of IL-2 (p = 0.019) and IFN- (p = 0.027), relative to the mTg group. There was a reduction in TGF-1 production in Flt3-L-treated mice when compared with the other groups but did not attain statistical significance.
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The effects of treatments on the anti-mTg Ab response were monitored in sera obtained at different time points (Fig. 4a). Treatment with Flt3-L before immunization with mTg increased the anti-mTg IgG Ab response throughout the observation period compared with mTg alone. Although the mTg group showed a geometric mean anti-mTg IgG titer (GMT) of 96,149 on day 38, the GMT in the Flt3-L group was 291,185. In contrast, treatment with GM-CSF resulted in a significant decrease in IgG Ab levels (GMT = 27,923) compared with mTg alone. Although the differences in Ab production were evident throughout the observation period, they were more profound on day 38. Treatment with GM-CSF and Flt3-L had no significant effect on the IgG1 Ab response (Fig. 4b). Although treatment with Flt3-L enhanced, GM-CSF diminished IgG2a autoantibody production by day 38. (Fig. 4c).
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Both spleen and lymph node cells from mice treated with Flt3-L showed significantly higher (p = 0.011 and 0.015), and cells from GM-CSF treated mice showed significantly reduced, mTg-specific proliferation (p = 0.011 and 0.019) (Fig. 5a). These results are consistent with higher and lower IL-2 responses seen in Flt3-L- and GM-CSF-treated mice, respectively.
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Although there was a significant difference in the pattern of cytokine responses in mice treated with Flt3-L and GM-CSF on day 18, no significant infiltration and/or apparent difference in the thyroid pathology was observed on day 18 (results not shown). However, as shown in Fig. 5b and Table I, relative to untreated mice, the thyroids obtained on day 38 from Flt3-L-treated mice showed massive infiltration of lymphocytes and follicular destruction with severity ranging from 3+ to 4+. Untreated mice showed infiltration ranging from 1+ to 3+, while thyroids from mice treated with GM-CSF showed minimal to no lymphocytic infiltration (0 and 1+) and follicular destruction, and in most cases the appearance of thyroids was comparable to that noted in nonimmunized control mice (Table I). When compared with untreated mice, the increase and decrease in disease severity in Flt3-L- and GM-CSF-treated mice, respectively, were statistically significant.
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Encouraged by the profound suppression of thyroiditis induced by GM-CSF when given before Ag inoculation, next we tested to see whether a similar effect can be elicited if the mTg-immunized mice were treated with GM-CSF at different stages of disease development. Irrespective of the time of treatment, relative to mTg mice, GM-CSF-treated mice showed reduced IgG responses, which was most likely due to a reduction in IgG2a response, as the IgG1 responses were similar in all groups. This effect was maximal when the mice were treated early after immunization with mTg (Fig. 6).
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(p < 0.01), and increased amounts of Th2 cytokines, IL-4 and IL-10 (p < 0.01) (Fig. 7). Similarly, there was a significant reduction (p < 0.01) in mTg specific proliferation of spleen and LN cells from treated mice (Fig. 8a). As expected, these data clearly showed that earlier the initiation of treatment more profound the suppression.
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GM-CSF-treated mice produced high levels of IL-4 and IL-10 compared with untreated mice (Fig. 7, c and d) indicating that these cytokines might be important for suppression of mTg-specific immune responses and the disease. We determined the frequency of IL-4- and IL-10-producing cells in GM-CSF-treated mice in the presence and absence of mTg by ELISPOT. Table III shows that cells capable of producing IL-4 and IL-10 are present in both treated and untreated mice, but the numbers are significantly higher in GM-CSF-treated mice. Upon stimulation with mTg, the number of cells producing IL-10 and IL-4 in GM-CSF-treated mice increased by 
4- and 3-fold, respectively. This indicated that a large number of IL-10- and IL-4-secreting T cells are mTg-specific and thus might be present in the thyroids of these mice where they are likely to be continuously stimulated by the endogenous thyroglobulin.
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To see whether treatment with GM-CSF increased the regulatory T cells, we tested for the expression of different cell surface markers by FACS. Most interestingly, we observed an increase in the number of CD4+/CD25+ T cells in all three GM-CSF-treated groups which is statistically significant (p < 0.018), compared with the untreated mice (Fig. 10), and suggested that these cells could have an important regulatory function in GM-CSF-mediated suppression of the disease.
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CD4+ cells from GM-CSF-treated mice showed much reduced proliferative response to mTg relative to cells from the untreated group (Fig. 11a). Because we had seen an increase in the number of CD4+/CD25+ T cells in GM-CSF-treated mice, we tested to see whether these cells have a role in down-regulating mTg-specific T cell responses. CD4+/CD25+-depleted CD4+ cells from GM-CSF-treated mice, but not untreated mice, showed enhanced proliferative response to mTg (Fig. 11a). When we cocultured CD4+/CD25+ cells from mice treated with GM-CSF with CD4+/CD25- spleen cells from untreated mice, we noted that the response to mTg was significantly suppressed (Fig. 11b). However, addition of CD4+/CD25+ T cells from untreated mice to CD4+/CD25- cells from GM-CSF-treated mice failed to suppress the T cell response to mTg (Fig. 11b). As seen in Fig. 11, c and d, both IL-4 and IL-10 production was significantly reduced (p values, 0.011 and 0.015, respectively) in CD4+/CD25- T cell cultures from GM-CSF-treated mice relative to the levels in CD4+ T cell cultures, and indicated that CD4+/CD25+ cells were the source of these cytokines.
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Discussion |
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DCs are not only highly efficient APCs but are also a major source of several cytokines through which they can profoundly influence other cells in the vicinity, including T cells (10). Because DC subsets can help determine whether a Th1 or a Th2 response is induced against a given Ag (8), we treated CBA/J mice with Flt3-L and GM-CSF before Ag inoculation to induce different subsets of DCs. Similar to earlier findings (6, 7), mice treated with Flt3-L had increased numbers of CD11c+ cells consisting of both CD8a+ and CD8a- DCs, and mice treated with GM-CSF had increased numbers of CD11c+/CD8a- DCs. Because the total number of cells in both treated groups was much higher than in the controls, these mice had higher absolute numbers of DCs as well.
Earlier studies have shown that CD11c+/CD8a+ DCs produce IL-12, IFN-, and IFN- in response to various stimuli at amounts greater than those produced by CD11c+/CD8a- DCs and promote Th1 responses while inhibiting Th2 responses (8, 10, 11, 12). In contrast, CD8a- DCs produce less IL-12 and IFN-, but more IL-10, upon activation leading to Th2 responses characterized by the production of IL-4 (17, 18), and suppression of Th1 responses. Although Flt3-L induced expansion of both CD8a+ and CD8a- DCs, there was no significant increase in the production of IL-4, but there was enhanced production of IL-12 and IFN-, indicating a predominantly Th1 response against mTg. It has been reported that CD8a+ DCs from IL-12-/- or IFN--/- mice failed to induce Th1 T cells, but instead induced the production of Th2 responses against specific Ags (1, 2). Further, CD11c+/CD8a- DCs in the presence of IFN- caused a less effective Th2 differentiation (8). These studies showed the importance of cytokines produced by CD11c+ DCs, in regulating Th1/Th2 differentiation. The increases in the levels of IL-2, IFN-, and IL-12 were even more profound on day 18, when the mice had been immunized twice with mTg and had received a second course of Flt3-L. These results are consistent with earlier reports on the effectiveness of Flt3-L as an adjuvant for the induction of Th1 types of responses against tumor, microbial, and allo-antigens (46, 47, 48).
Cytokine milieu in GM-CSF-treated mice was significantly different compared with that in Flt3-L-treated mice. These mice showed little or no increase in IFN- but showed a significantly higher IL-4 response after immunization with mTg. Interestingly, immunization with mTg also induced enhanced IL-10 production in GM-CSF-treated mice that was not apparent in Flt3-L-treated mice. These results suggested that little or no increase in Th1 cytokines, coupled with a significant increase in IL-4, had shifted the immune response preferentially to Th2 type. This is consistent with earlier reports of GM-CSF promoting Th2 responses to specific Ags in vivo (17, 18).
Modulation of early cytokine responses induced by Flt3-L and GM-CSF had a profound effect on subsequent immune responses against mTg and development of the disease. Thyroid histology clearly indicated that on day 18, there was little or no lymphocytic infiltration or follicular damage. However, by day 38, massive infiltration of lymphocytes into thyroid and destruction of thyroid follicles were seen in mice treated with Flt3-L, and suggested that increased Th1 cytokine production (i.e., IFN- and IL-2) by T cells infiltrating the thyroid, where they most likely remain activated by exposure to endogenous thyroglobulin, might play an important role in follicular destruction. This speculation is based on earlier reports which showed that IFN- and TNF- produced by thyroid resident immune cells can up-regulate CD95 expression on thyroid follicles resulting in apoptotic cell death (26, 27, 28). Previous studies also demonstrated that Ag-specific T cells preferentially migrate to the site of inflammation (viz, thyroid in the case of EAT), where the resident APCs capture and present Ag to infiltrating T cells (49).
In contrast, GM-CSF-treated mice were essentially euthyroid with no significant lymphocytic infiltration. In these mice, an increased level of IL-4 and IL-10 production was most likely sustained throughout the observation period. Further, as evident from the ELISPOT assay, relative to the other two groups, a higher number of IL-4- and IL-10-producing cells were present in GM-CSF-treated mice. Upon Ag stimulation in vitro, both the frequency of cells capable of producing IL-4 and IL-10, and the amounts of these cytokines increased suggesting that these cytokines might be contributing to the suppression of the disease. An earlier study has shown that a significant increase in IL-4 levels in the thyroid induce the expression of higher levels of antiapoptotic molecules (28) on thyrocytes. Additional studies are required to establish whether a similar increase in antiapoptotic molecules results from GM-CSF treatment.
GM-CSF given after mTg administration could also confer protection. Initiation of treatment as late as day 28, by which time mice had already received two doses of mTg (on days 1 and 14) significantly reduced lymphocytic infiltration in the thyroid, and T cell and Ab responses against mTg. The pattern of cytokines produced by mice treated with GM-CSF, either pre- or postimmunization, was similar and suggested that a common mechanism of disease suppression might be involved.
Although the mechanism by which the DC-induced cytokine milieu, in GM-CSF-treated mice, suppressed EAT development is not clear, it was accompanied by an increase in CD4+/CD25+ regulatory T cells. Despite persistent exposure of T cells to endogenous thyroid Ags in both untreated and GM-CSF-treated mice, significant increase in CD4+/CD25+ T cell number was observed only in GM-CSF-treated mice. This suggested that treatment with GM-CSF leads to selective expansion of regulatory T cells that constitutively express IL-2R (CD25). Depletion of CD4+/CD25+ cells from GM-CSF-treated mice, but not control mTg-immunized mice, resulted in enhanced proliferation and reduced IL-4 and IL-10 production. Furthermore, CD4+/CD25+ cells from GM-CSF-treated mice could suppress the anti-mTg response of CD4+/CD25- T cells from untreated control mice. This is in stark contrast to a lack of inhibitory effect of CD4+/CD25+ T cells from untreated mice on CD4+/CD25- T cells from GM-CSF-treated mice. These results clearly indicate that CD4+/CD25+ cells from GM-CSF-treated mice could produce high levels of IL-4 and IL-10 and these cytokines are involved in inhibiting T cell proliferation in response to mTg. Although there is no conclusive evidence, a critical role for DCs in the induction of Ag-specific regulatory T cells has been proposed (50, 51). Our results suggest that GM-CSF-induced DCs, either directly or through cytokine production, may play an important role in selective expansion of Ag-specific CD4+/CD25+ regulatory T cells.
CD4+/CD25+ cells can modulate immune responses including induction of unresponsiveness to both nonself and self-Ags either through cytokines like TGF-1 and IL-10 or direct T cell-T cell interaction (50, 51, 52, 53). In this study, lymphocytes from GM-CSF-treated mice devoid of CD4+/CD25+ cells showed reduced IL-10 and IL-4 production. Moreover, neutralization of IL-10, and not IL-4, significantly enhanced mTg-specific T cell responses and suggested that IL-10 might play a more important role in suppressing mTg-specific T cell responses in GM-CSF-treated mice. Collectively, these studies suggest that the inhibitory properties of CD4+/CD25+ T cells on the anti-mTg response could be mediated through IL-10. In this context, it is interesting to note that a recent study demonstrated that IL-10 can increase Fas-L expression on thyrocytes and confer protection against autoimmune thyroiditis through the induction of Fas-mediated killing of infiltrating T cells (54, 55). Moreover, other studies have shown that IL-4 can induce antiapoptotic proteins in thyroid follicular cells and aid in their survival (27, 28). Thus these cytokines could prevent the disease by several mechanisms including direct suppression and/or elimination of mTg-specific T cells, and or activation of antiapoptotic proteins.
Several approaches have been used to skew immune responses in autoimmune thyroid conditions to suppress the disease (33, 34, 35, 36, 37, 38, 39, 56, 57, 58, 59). The disease outcome varied from complete recovery to severe exacerbation, most likely due to differences in the time and mode of treatment. Although continuous presence of IL-10 or IL-4 had curative effects (43, 54), either blocking Th1 cytokines or disrupting the expression of Th1 cytokines to promote a Th2 response resulted in conflicting results (30, 31). These reports strongly suggest that multiple factors are involved in autoimmune induction, and targeting one or a few cytokines may not be sufficient to suppress the disease. Our results suggest that targeting the afferent limb, which profoundly affects subsequent downstream responses, rather than the efferent limb of the immune response might be a more effective approach to modulate autoimmune responses. These results also indicate that differential activation of DCs by GM-CSF might be useful in treating T cell-mediated autoimmune conditions like Hashimoto’s thyroiditis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bellur S. Prabhakar, Department of Microbiology and Immunology, College of Medicine, University of Illinois, 835 South Wolcott, Chicago, IL 60612. E-mail address: bprabhak{at}uic.edu
3 Abbreviations used in this paper: DC, dendritic cell; Flt3-L, fms-like tyrosine kinase receptor 3 ligand; EAT, experimental autoimmune thyroiditis; mTg, mouse thyroglobulin; RT, room temperature; GMT, geometric mean titer.
Received for publication November 6, 2002. Accepted for publication March 25, 2003.
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) or mTg (
) and results are expressed as mean ± SD of triplicate values obtained from 10 individual mice. The p value (statistical significance) for the treated group was calculated by comparing each value with the corresponding value of the mTg group. b, Thyroid histology. Representative photomicrographs of H&E-stained thyroid sections are shown.
