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The Journal of Immunology, 2006, 177: 566-573.
Copyright © 2006 by The American Association of Immunologists

IL-17 Plays an Important Role in the Development of Experimental Autoimmune Encephalomyelitis1

Yutaka Komiyama, Susumu Nakae2, Taizo Matsuki3, Aya Nambu, Harumichi Ishigame, Shigeru Kakuta, Katsuko Sudo4 and Yoichiro Iwakura5

Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-17 is a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines, and cell adhesion molecules. This cytokine is augmented in the sera and/or tissues of patients with contact dermatitis, asthma, and rheumatoid arthritis. We previously demonstrated that IL-17 is involved in the development of autoimmune arthritis and contact, delayed, and airway hypersensitivity in mice. As the expression of IL-17 is also augmented in multiple sclerosis, we examined the involvement of this cytokine in these diseases using IL-17–/– murine disease models. We found that the development of experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis, was significantly suppressed in IL-17–/– mice; these animals exhibited delayed onset, reduced maximum severity scores, ameliorated histological changes, and early recovery. T cell sensitization against myelin oligodendrocyte glycoprotein was reduced in IL-17–/– mice upon sensitization. The major producer of IL-17 upon treatment with myelin digodendrocyte glycopritein was CD4+ T cells rather than CD8+ T cells, and adoptive transfer of IL-17–/– CD4+ T cells inefficiently induced EAE in recipient mice. Notably, IL-17-producing T cells were increased in IFN-{gamma}–/– cells, while IFN-{gamma}-producing cells were increased in IL-17–/– cells, suggesting that IL-17 and IFN-{gamma} mutually regulate IFN-{gamma} and IL-17 production. These observations indicate that IL-17 rather than IFN-{gamma} plays a crucial role in the development of EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The cytokine IL-17 can activate the expression of a variety of proinflammatory cytokines, chemokines, and cell adhesion molecules throughout a wide range of cell types, including macrophages, dendritic cells, T cells, synovial cells, and endothelial cells (1). Augmented expression of this cytokine is observed in patients with various diseases, such as rheumatoid arthritis (RA)6 (2), systemic lupus erythematosus (3), Behcet’s disease (4), allograft rejection (5), nephritic syndrome (6), asthma (7), and multiple sclerosis (MS) (8), suggesting the involvement of IL-17 in the development of these diseases. We have also demonstrated the contribution of IL-17 to the development of allergic and autoimmune diseases in mice, including contact dermatitis, airway inflammation, and arthritis (9, 10, 11). IL-17 also plays an important role in the host defense mechanisms protecting against Klebsiella pneumoniae infection (12). However, the role of IL-17 has only been elucidated in a few diseases and the role of this cytokine in the pathogenesis of most diseases remains largely unknown.

IL-17 is produced by a variety of cell types. A subset of Th0 and Th1 cell clones, but not Th2 cell clones, that were established from the synovial tissues of RA patients produced IL-17 (13). A number of Th0, Th1, and Th2 clones established from patients with allergic contact dermatitis also produce this cytokine (14). IL-17 is also produced by TNF-{alpha}- and/or GM-CSF-producing CD4+ T cells isolated from the synovial fluid of patients with Lyme arthritis, which exhibit neither a Th1 nor a Th2 phenotype (15). Eosinophils from patients with asthma are also reported to produce this cytokine (16). Both lung neutrophils from mice treated with LPS and CD8+ T cells derived from mice infected with Klebsiella pneumoniae are producers of IL-17 (17, 18). Thus, the producer cells of IL-17 differ in a manner dependent on the disease.

Experimental autoimmune encephalomyelitis (EAE), a rodent model of human MS, is induced by immunization of mice with encephalitogenic myelin Ags in the presence of adjuvants. EAE pathogenesis is characterized by inflammation of the CNS associated with demyelination and the infiltration of inflammatory cells including neutrophils and encephalitogenic myelin Ag-specific CD4+ T cells. In MS patients, IL-17 mRNA and protein are increased in both brain lesions and mononuclear cells isolated from blood and cerebrospinal fluids (8, 19). IL-17 is also increased in lymphocytes derived from mice with EAE (20). Although these observations suggest that IL-17 may contribute to the development of MS and EAE, the precise role of this cytokine in the pathogenesis of these diseases is still poorly understood.

In this report, we have investigated the role of IL-17 in the development of the EAE using IL-17–/– mice. We demonstrated that the development of EAE was markedly suppressed in IL-17–/– mice. We also determined that IL-17 was important for the optimal activation of myelin oligodendrocyte glycoprotein (MOG)-specific T cells. In this model, CD4+ T cells were the major producers of IL-17 in this system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

IL-17–/– mice (9), generated as described previously, were backcrossed to the C57BL/6J strain (six or nine generations). C57BL/6J and IFN-{gamma}–/– mice, both on the C57BL/6J background were purchased from Japan SLC and from The Jackson Laboratory, respectively. Mice were kept under pathogen-free conditions in an environmentally controlled clean room at the Center for Experimental Medicine, Institute of Medical Science, University of Tokyo. All experiments were conducted according to the institutional ethical guidelines for animal experimentation and the safety guidelines for genetic manipulation experiments.

Induction of EAE

Active EAE. The MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized and purified by HPLC at our institute (Dr. S. Imajoh-Ohmi, Division of Molecular Biology, Institute of Medical Science, University of Tokyo). Mice (8–13 wk of age) were immunized s.c. in one flank on day 0 and in the other on day 7 with 300 µg of MOG35–55 peptide emulsified in CFA (1:1), which consisted of IFA with 5 mg/ml Mycobacterium tuberculosis H37RA (Difco Laboratories). Pertussis toxin (PTx; Alexis) (200 ng) was injected i.v. on days 0 and 2. Following the first immunization, the severity of EAE was monitored and graded on a scale of 0–5: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and fore limb paralysis; 5, moribundity and death.

Passive EAE. Mice were immunized s.c. with MOG/CFA. Ten days after the immunization, the spleen and inguinal and axillary lymph nodes (LNs) were collected and a single-cell suspension was prepared. Pooled lymphocytes (4 x 106 cells/ml) were cultured in the presence of 50 µg/ml MOG35–55 peptide in RPMI 1640 medium containing 50 µM 2-ME, 50 µg/ml streptomycin, 50 µg/ml penicillin, and 10% heat-inactivated FBS (Sigma-Aldrich) for 4 days. After harvesting, CD4+ T cells were purified by positive selection using an AutoMACS system (Miltenyi Biotec). Isolated CD4+ T cells (4 x 106) were then transferred i.v. into naive C57BL/6J mice.

Histology

On day 42 after the first immunization with MOG/CFA and PTx, spines were harvested and fixed with neutral 10% formalin. Spinal cords were then extracted and embedded in paraffin. Sections (5 µm) were stained with H&E.

MOG-specific LN cell proliferation assay

Mice were immunized s.c. with MOG/CFA. Ten days after immunization, the inguinal and axillary LNs were collected and a single-cell suspension was prepared. LN cells (1–4 x 105 cells/well) were cultured for 72 h in the absence or presence of various concentrations of MOG35–55 peptide as described above. Isolated cells were then pulsed for 6 h with [3H]thymidine (0.25 µCi/ml; Amersham Biosciences), and harvested using a Micro 96 cell harvester (Skatron). Levels of radioactivity were measured using a Micro beta system (Pharmacia Biotech).

Measurement of cytokine levels by ELISA

To detect IFN-{gamma} and IL-4 in culture supernatants, we used mouse IFN-{gamma} OptEIA kit (BD Pharmingen) and IL-4 ELISA kit (Endogen). Detection of IL-17 by ELISA was performed as described previously (9).

Flow cytometry

To examine LN cell population, inguinal and axillary LN cells were harvested 10 days after immunization with MOG/CFA. After incubation of cells on ice with anti-mouse CD16/CD32 mAb (2.4G2) in a staining buffer (Hank’s buffer containing 2% FCS and 0.1% sodium azide) on ice for 15 min, cells were incubated on ice for 45 min with either FITC-anti-mouse CD45RB (C363.16A) or FITC anti-mouse CD62L (MEL-14) in the presence of PE anti-mouse CD44 (Pgp-1) and allophycocyanin anti-mouse CD4 (RM4-5). 7-Aminoactinomycin D (Sigma-Aldrich)-negative, CD4+ cells were examined by a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). To detect IL-17 production in lymphocytes (T cells and B cells), LN cells were harvested 10 days after immunization with MOG/CFA. Isolated cells were cultured in the presence or absence of 50 µg/ml MOG35–55 peptide for 72 h as described above. To examine IL-17 production by Gr-1+ neutrophils, we prepared a single-cell suspension from the spleens of EAE-affected wild-type mice (day 42). LN cells (72 h after cultivation) and spleen cells were stimulated for 6 h with 20 ng/ml PMA (Sigma-Aldrich), 1 µM ionomycin (Sigma-Aldrich), and 2 µM monensin (Sigma-Aldrich) for 6 h. After harvesting, cells were incubated on ice with anti-mouse CD16/CD32 mAb (2.4G2) in a staining buffer on ice for 15 min. Cell samples were then incubated on ice for 45 min with either FITC-anti-mouse B220 (RA3-6B2), FITC anti-mouse Gr-1 (RB6-8C5), or FITC anti-mouse CD3{epsilon} (145-2C11), allophycocyanin anti-mouse CD4 (RM4-5 or GK1.5), or allophycocyanin anti-mouse CD8{alpha} (53-6.7) Abs. After washing, the cells were fixed in a fixation buffer (2% paraformaldehyde in PBS) at room temperature for 10 min. Samples were then permeabilized with permeabilization buffer (staining buffer containing 0.1% saponin) and incubated for 30 min with PE-conjugated anti-mouse IL-17 mAb (TC11-18H10) or isotype-matched control rat IgG1 (R3-34) at 4°C. The cells were analyzed on a FACSCalibur flow cytometer as described above. All mAbs were purchased from BD Pharmingen.

CD4+ T cell cultures

CD4+ T cells (< 95%) from wild-type mouse spleen were purified by MACS system as described elsewhere (9), then stimulated with 1.0 µg/ml plate-coated anti-CD3 mAb (145-2C11; BD Pharmingen) in the presence or absence of various concentration of recombinant mouse IFN-{gamma} (rmIFN-{gamma}; PeproTech), rmIL-12 (R&D Systems), rmIL-17 (R&D Systems), and rmIL-23 (R&D Systems) for 48 h. Then, culture supernatants were collected and IFN-{gamma} or IL-17 levels in the supernatants were determined by ELISA as described above.

Measurement of MOG-specific serum Ab levels by ELISA

A 96-well flat-bottom plate (Falcon 3912 Micro test III Flexible Assay Plates; BD Biosciences) was coated with 10 µg/ml MOG35–55 peptides at 4°C overnight. After washing the wells with 0.05% Tween 20 in PBS, the wells were blocked with PBS containing 1% skim milk, 5 mM EDTA, 0.02% NaN3 for 1 h at room temperature. After washing, diluted serum samples were added and incubated for 2 h at room temperature. Then, after washing the wells, alkaline phosphatase-conjugated goat anti-mouse Igs (IgG, IgG1, IgG2a, IgG2b, IgG3, IgM; Zymed Laboratories) were added and incubated for 1 h at room temperature. Alkaline phosphatase activity was measured using Substrate Phosphatase SIGMA104 (Sigma-Aldrich). The anti-MOG Ab titer was shown as OD415 values.

Statistics

The Student t test, the Mann-Whitney’s U test, or the {chi}2 test was used for the statistical evaluation of the results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Development EAE was suppressed in IL-17–/– mice

To investigate the role of IL-17 in the pathogenesis of EAE, we examined the effect of IL-17-deficiency on the development of EAE using IL-17–/– mice. To induce EAE, mice were treated with MOG35–55 peptide emulsified in CFA and injected with PTx. The initial signs of EAE were observed 10 days after the first immunization of wild-type mice (Fig. 1). In contrast, the onset of EAE was significantly delayed in IL-17–/– mice until day 15 (Fig. 1). Twenty days after the first immunization, however, IL-17–/– mice exhibited a similar incidence of EAE as that seen in wild-type mice (Fig. 1A), although the severity of disease in IL-17–/– mice was milder than that in wild-type mice (Fig. 1B). After day 20 from the immunization, the severe signs of disease continued in wild-type mice, while early amelioration was observed in IL-17–/– mice (Fig. 1A). Consistent with these observations, a massive infiltration of mononuclear cells was observed within the spinal cords of wild-type mice 42 days after the first immunization (Fig. 2, B and D). In contrast, the cellular infiltration was significantly reduced in IL-17–/– mice (Fig. 2, A and C).


Figure 1
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  		FIGURE 1. Development of EAE was reduced in IL-17–/– mice. EAE was induced in mice by immunization with MOG/CFA coinjected with PTx. A, Incidence of EAE. B, Clinical scores of diseased mice. {circ}, Wild-type mice (n = 10); •, IL-17–/– mice (n = 10). Data are the averages ± SD for each group. {dagger}, p < 0.05 and *, p < 0.001 vs IL-17–/– mice by Mann-Whitney’s U test (A) and by {chi}2 test (B), respectively.

 

Figure 2
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  		FIGURE 2. Local inflammation of the CNS was suppressed in IL-17–/– mice during EAE. On day 42 after MOG/CFA and PTx immunization, spinal cords were removed. The tissue sections were stained with H&E. The sections at the lumbar level are shown. A and C, IL-17–/– mice. B and D, Wild-type mice. A and B, x40. C and D, x200.

 
PTx is widely used to enhance the development of T cell-mediated organ-specific autoimmune diseases, including EAE. TNF-{alpha}–/– mice develop EAE normally when high doses of PTx are injected, while in the presence of low doses of PTx, mutant mice exhibit significantly reduced development of EAE symptoms (21). Thus, PTx may sometimes mask or compensate for the pathological functions of some proinflammatory mediators, such as TNF-{alpha}, in the pathogenesis of EAE. Therefore, to more clearly observe the effect of IL-17 deficiency, we next induced EAE in IL-17–/– mice in the absence of PTx treatment. Under these conditions, disease onset in IL-17–/– mice delayed compared with wild-type mice similarly to that seen in the presence of PTx (Fig. 3). Interestingly, in the absence of PTx, the disease also gradually ameliorated in wild-type mice after 22 days of induction, as seen for IL-17–/– mice, although the maximal severity score of the wild-type mice remained significantly higher (Fig. 3). These results demonstrate that IL-17 contributes to the development of EAE.


Figure 3
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  		FIGURE 3. Development of EAE was reduced in IL-17–/– mice. Mice were induced EAE by immunization with MOG/CFA, but without PTx. A, Incidence of EAE. B, Clinical score of diseased mice. {circ}, Wild-type mice (n = 15), and •, IL-17–/– mice (n = 15). Data are shown as an average + SD in each group. {dagger}, p < 0.05 and *, p < 0.001 vs IL-17–/– mice by Mann-Whitney’s U test (A) and by {chi}2 test (B).

 
MOG- specific T cell sensitization was impaired in IL-17–/– mice

We previously showed that IL-17 plays an important role in Ag-specific T cell activation during the development of multiple allergic and autoimmune diseases (9, 10, 11). To compare our previous results to the models in the current study, we next assessed the role of IL-17 in the activation of MOG-specific T cells during the development of EAE. Ten days after immunization with MOG/CFA alone (without PTx), hypertrophy of inguinal and axillary lymph nodes was observed. Although the total number of pooled inguinal and axillary LN cells was significantly decreased in IL-17–/– mice in comparison with wild-type mice (Fig. 4A), the content of memory CD4+ T cells (CD62L–CD44high or CD45RBlowCD44high) was comparable (Fig. 4, B and C). Then, the draining LN cells were cultured in the presence or absence of MOG peptide. When a large excess of LN cells (4 x 105 cells) was present in a well, the observed MOG-specific LN cell proliferative responses in IL-17–/– mice were similar to those seen in wild-type LN cells (Fig. 4D). In the presence of an optimal number of LN cells (2 x 105 and 1 x 105 cells), the proliferative responses of cells derived from IL-17–/– mice were markedly decreased compared with those in wild-type mice despite similar number of memory T cells was contained in wild-type and IL-17–/– mouse culture (Fig. 4D). IL-17 was detected in the supernatants of wild-type LN cell cultures (2 x 105 cells), and its levels increased in a manner dependent on the dose of MOG peptide. IL-17 was undetectable in IL-17–/– LN cell cultures (2 x 105 cells) (Fig. 4D). The MOG-specific proliferative responses of LN T cells were reduced in IL-17–/– mice (Fig. 4D). Nevertheless, IFN-{gamma} production in the LN cell culture supernatants was similar in wild-type and IL-17–/– mice (Fig. 4E). IL-4 levels in the LN cell culture supernatants from both wild-type and IL-17–/– mice were below the limit of detection (Fig. 4E). These results suggest that the delayed onset of the EAE response in IL-17–/– mice is caused by insufficient T cell sensitization against the MOG peptide.


Figure 4
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  		FIGURE 4. MOG-specific lymphocyte activation was impaired in IL-17–/– mice. Mice were immunized once with MOG/CFA in the absence of PTx, and 10 days after immunization, the inguinal and axillary LNs were collected and pooled. A, Total LN cell number. B and C, Percentage of CD62L–CD44high or CD45RBlowCD44high CD4+ T cells in LNs. In A and C, each circle represents a value from an individual mouse, and the column represents the average for each group. Representative FACS results are shown in B. *, p < 0.05 and vs the corresponding values for wild-type mice. D, Isolated LN cells were cultured in the absence or presence of MOG peptide for 72 h, and MOG-specific LN cell proliferation as measured by [3H]thymidine incorporation (D) and IFN-{gamma}, IL-17, and IL-4 levels in the culture supernatants (2 x 105 cells) (E) are shown. {circ}, Wild-type mice, and •, IL-17–/– mice. Averages ± SD of triplicate wells are shown. All results are representative at least in three experiments. D, Data are averages ± SD from three independent experiments. N.D., Not detected.

 
CD4+ T cells produced IL-17 in LN cells during EAE

Different subsets of CD4+ Th cells and eosinophils are known to produce IL-17 in patients with dermatitis, RA, Lyme arthritis, and asthma (13, 14, 15, 16). Neutrophils and CD8+ T cells can also produce IL-17 during certain infectious diseases in mice (17, 18). These observations suggest that the IL-17 producer cells may differ from those known to produce this cytokine in other diseases. Thus, we next analyzed the IL-17 producer cells in the LNs of wild-type mice following MOG immunization. Ten days after immunization, inguinal and axillary LNs were collected and LN cells were cultured in the presence of MOG peptide for 72 h. After MOG stimulation, IL-17 production was detected in CD3+ T cells, but not in granulocytes or B cells (Fig. 5A). Within the T cell population, IL-17 was predominantly produced in CD4+ T cells, but at low levels in CD8+ T cells (Fig. 5B). Thus, CD4+ T cells, rather than CD8+ T cells, were the major producer of IL-17 within LNs during the development of EAE.


Figure 5
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  		FIGURE 5. IL-17 was primarily produced by CD4+ LN T cells during the development of EAE. To detect IL-17-producing cells in the lymphocyte or granulocyte populations, LN cells from mice immunized with MOG/CFA were cultured in the presence of 50 µg/ml MOG peptide for 72 h, followed by stimulation with PMA + ionomycin in the presence of monensin; then IL-17-producing cells were detected by FACS. Gr-1+ cells were stained using spleen cells from mice with EAE. A, The IL-17+ populations in CD3+ or B220+ cell populations within the LN cells, or the Gr-1+ cells of the spleen. B, IL-17 production by CD3+CD4+ or CD3+CD8+ T cells.

 
The efficiency of EAE induction of IL-17–/– CD4 T cells was low in comparison with wild-type T cells

To examine the effect of IL-17 deficiency on T cell sensitization against the MOG peptide, we adoptively transferred CD4+ T cells into recipient mice of the same genetic background. Lymphocytes from MOG/CFA-immunized wild-type or IL-17–/– mice were stimulated with MOG peptide for 4 days in vitro, and CD4+ T cells were then purified and transferred into naive wild-type mice. The development of EAE in mice that received IL-17–/– CD4+ T cells was markedly reduced in comparison to those animals receiving wild-type CD4+ T cells (Fig. 6). These observations indicate that MOG-specific T cells from IL-17–/– mice cannot efficiently induce EAE in recipient mice.


Figure 6
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  		FIGURE 6. CD4+ T cells were responsible for the induction of EAE. Mice were immunized once with MOG/CFA alone. At 10 days postimmunization, the spleen and the inguinal and axillary LNs were collected and pooled. The pooled cells were cultured in the presence of MOG peptide for 72 h, and then CD4+ T cells were purified and transferred into naive, wild-type mice. A, The incidence of EAE. B, The clinical scores of the diseased mice. {circ}, Mice that received wild-type CD4+ T cells (n = 11), and, •, mice that received IL-17–/– CD4+ T cells (n = 10). In B, data are shown as the averages ± SD for each group. {dagger}, p < 0.05 and *, p < 0.001 vs IL-17–/– mice by the Mann-Whitney’s U test (A) and by the {chi}2 test (B), respectively.

 
IL-17 production was enhanced in IFN-{gamma}–/– mice

MS and EAE are typically classified as Th1 cell-mediated autoimmune diseases. It has been shown, however, that the development of EAE is exacerbated in IFN-{gamma}–/– and/or IFN-{gamma}R–/– mice (22, 23, 24, 25), indicating that IFN-{gamma} serves a protective role in the disease pathogenesis. Therefore, we next examined whether IFN-{gamma}-deficiency influences IL-17 production by CD4+ T cells during EAE development. Consistent with previous reports, the LN (inguinal and axillary) cell number of IFN-{gamma}–/– mice was significantly increased compared with that of wild-type mice 10 days after MOG/CFA immunization (Fig. 7A). When LN cells from MOG-immunized mice were cultured in the presence of MOG peptides, proliferating cells were predominantly observed in a region indicated as "R2", while nonproliferating cells were observed in a region indicated as "R1" (Fig. 7B), as determined by CFSE labeling (data not shown). Thus, to detect MOG-reactive, IL-17-producing T cells in LN cells, cells were selectively gated to the R2 region. The percentage of IL-17-producing CD4+ T cells in the draining LN cells of IFN-{gamma}–/– mice was greatly increased in comparison to that in wild-type mouse T cells, irrespective of MOG restimulation (Fig. 7, C and D). A large proportion of CD8+ T cells, as well as CD4+ T cells from IFN-{gamma}–/– mice immunized with MOG/CFA, produced IL-17, although only a small proportion of CD8+ cells from wild-type mice produced IL-17 (Fig. 7, C and D).


Figure 7
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  		FIGURE 7. The proportion of IL-17-producing CD4+ and CD8+ T cells was increased in IFN-{gamma}–/– mice after MOG immunization. LN cells from mice immunized with MOG/CFA were cultured for 72 h in the presence or absence of 50 µg/ml MOG peptide. IL-17-producing cells were then analyzed by FACS, and the percentage of IL-17+CD3+CD4+ T cells is shown. A, Total LN cell number from inguinal and axillary LNs from wild-type (n = 6) and IFN-{gamma}–/– (n = 6) mice. **, p < 0.01 vs wild-type mice. B, Gating in FACS analysis, R2 rather than R1 contained MOG-specific proliferating cells using CFSE labeling. C, Staining of intracellular IL-17 in CD3+CD4+ or CD3+CD8+ T cells stimulated with or without MOG peptides. Shaded areas, staining with isotype-matched control Ab; bold lines, anti-mouse IL-17 staining. The percentage of IL-17-positive cells (upper bold figures) and percentage of cells which were stained with an isotype-matched control IgG (lower figures) are shown. D, Each circle represents a value from an individual mouse, and the column represents the average for each group in C. {square}, Wild-type mice (n = 6). {blacksquare}, IFN-{gamma}–/– mice (n = 6). IgG, isotype-matched control IgG staining. IL-17, anti-mouse IL-17 staining. {dagger}, p < 0.05 vs the corresponding values for control IgG staining. *, p < 0.05 and **, p < 0.01 vs the corresponding values for the cultures in the absence of MOG peptide (medium alone). ¶, p < 0.05 vs the corresponding values of wild-type mice. All p values were determined by the Student’s t test.

 
Next, we assessed the effect of IL-17 on IFN-{gamma} production during MOG immunization. As shown in Fig. 4, D and E, although MOG-specific T cell proliferation was impaired in IL-17–/– mice, IFN-{gamma} levels in culture supernatants were normally observed. These observations suggested that the IFN-{gamma}-producing cell population is increased in IL-17–/– mice. In support of this, the percentage of IFN-{gamma}-producing CD4+ T cells in the draining LN cells of IL-17–/– mice was significantly increased compared with that in wild-type mice after MOG stimulation (Fig. 8). Similar results were also obtained in CD8+ T cells (Fig. 8). Thus, these data suggest that IL-17 production is regulated by IFN-{gamma} while IFN-{gamma} is regulated by IL-17.


Figure 8
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  		FIGURE 8. The proportion of IFN-{gamma}-producing CD4+ and CD8+ T cells was increased in IL-17–/– mice after MOG immunization. LN cells from mice immunized with MOG/CFA were cultured for 72 h in the presence or absence of 50 µg/ml MOG peptide. IFN-{gamma}-producing cells were then analyzed by FACS, and the percentage of IFN-{gamma}+CD3+CD4+ or CD3+CD8+ T cells is shown. A, Staining of intracellular IFN-{gamma} in CD3+CD4+ or CD3+CD8+ T cells stimulated with or without MOG peptides. Shaded areas, staining with isotype-matched control Ab; bold lines, anti-mouse IFN-{gamma} staining. The percentage of IFN-{gamma}-positive cells (upper bold figures) and percentage of cells which were stained with an isotype-matched control IgG (lower figures) are shown. B, Each circle represents a value from an individual mouse, and the column represents the average for each group in A. {square}, Wild-type mice (n = 6). {blacksquare}, IL-17–/– mice (n = 6). IgG, isotype-matched control IgG staining. IFN-{gamma}, anti-mouse IFN-{gamma} staining. {dagger}, p < 0.05 vs the corresponding values for control IgG staining. *, p < 0.05 vs the corresponding values for the cultures in the absence of MOG peptide (medium alone). ¶, p < 0.05 vs the corresponding values of wild-type mice. All p values were determined by the Student t test.

 
Then, we examined whether IL-17 and IFN-{gamma} can directly regulate IFN-{gamma} and IL-17 production, respectively. When purified splenic CD4+ T cells were stimulated with plate-coated anti-CD3 mAb in the presence of various concentration of IL-12, IFN-{gamma} production was enhanced in a dose-dependent manner (Fig. 9A). However, IL-17 did not influence IFN-{gamma} production by CD4+ T cells in the absence or presence of IL-12 (Fig. 9, B and C). IL-23 could promote IL-17 production dose dependently (Fig. 9D), while IFN-{gamma} did not show any effects on IL-17 production irrespective of the presence of IL-23 (Fig. 9, E and F). These observations indicate that IFN-{gamma} or IL-17 cannot directly modulate IL-17 or IFN-{gamma} production.


Figure 9
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  		FIGURE 9. Exogenous IFN-{gamma} and IL-17 did not directly affect IL-17 and IFN-{gamma} production by CD4+ T cells. CD4+ T cells from spleen of wild-type mice were stimulated with plate-coated anti-CD3 mAb in the presence or absence of rmIL-17 or rmIFN-{gamma} with or without rmIL-12 or rmIL-23 for 48 h. Then, IFN-{gamma} or IL-17 levels in culture supernatants were determined by ELISA. IFN-{gamma} levels in culture supernatants from CD4+ T cells: stimulated with anti-CD3 mAb plus the indicated amount of rmIL-12 (A), rmIL-17 (B), and 1 ng/ml rmIL-12 + indicated amount of rmIL-17 (C). IL-17 levels in culture supernatants from CD4+ T cells: stimulated with anti-CD3 mAb plus rmIL-23 (D), rmIFN-{gamma} (E), or 1 ng/ml rmIL-23 + indicated amount of rmIFN-{gamma} (F). Data showed the average ± SD from three mice, and a representative result from two independent experiments.

 
Increased MOG-specific Ab production in IL-17–/– mice during EAE

To elucidate the role of IL-17 in MOG-specific Ab production, we measured the level of anti-MOG-specific serum Abs in wild-type and IL-17–/– mice during EAE. Before immunization with MOG peptides, the level of MOG-specific IgG was very low in both wild-type and IL-17–/– mice (Fig. 10A). On day 20 after MOG/CFA immunization with PTx injection as shown in Fig. 1, the level of MOG-specific IgG in IL-17–/– mice was slightly higher than that in wild-type mice (Fig. 10A). In chronic inflammatory phases during EAE induced by MOG/CFA with PTx, the levels of MOG-specific IgG and IgG1 in IL-17–/– mice were profoundly increased compared with these in wild-type mice, although these IL-17–/– mice did not show any sign of EAE (Fig. 10). Similarly, the levels of MOG-specific IgG2a and IgG2b were also slightly, but not significantly, increased in IL-17–/– mice, while those of MOG-specific IgG3 and IgM were not different between wild-type and IL-17–/– mice (Fig. 10B). These results indicated that IL-17 has an influence upon MOG-specific Ab production by B cells. However, our findings suggest that no correlation exists between the susceptibility and severity of EAE and the levels of anti-MOG Abs in IL-17–/– mice.


Figure 10
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  		FIGURE 10. Enhanced MOG-specific Ig production in IL-17–/– mice during EAE. MOG-specific Ig levels in sera from wild-type and IL-17–/– mice that developed EAE were determined by ELISA. A, As performed in Fig. 1A, sera were collected from mice before (–1 day) and after MOG immunization (20 and <40 days). Then, MOG-specific IgG levels in sera were measured. B, On day 42 after MOG immunization, MOG-specific IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3 levels in sera were determined. Each circle represents the value from an individual mouse, and a column shows the average of each group. {square}, Wild-type mice (n = 7). {blacksquare}, IL-17–/– mice (n = 10). *, p < 0.05 and **, p < 0.01 for the comparisons shown in brackets by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have demonstrated using IL-17–/– mice that IL-17 plays an important role in the development of EAE induced by MOG/CFA. We found that, upon immunization with MOG/CFA, T cell sensitization was defective in IL-17–/– mice, and CD4+ T cells from IL-17–/– mice did not induce EAE efficiently compared with wild-type T cells. These observations suggest that IL-17 plays an important role in the activation of encephalitogenic T cells during the sensitization phase of EAE. In contrast, it was recently reported that IL-17-producing CD4+ T cells enhance the disease severity of EAE and that treatment with anti-IL-17 neutralizing Abs during the elicitation phase suppressed disease development (26). These observations strongly suggest that IL-17 is involved in the pathogenesis of EAE during both the sensitization and elicitation phases.

As Th1 cells, which are the major producers of IFN-{gamma}, infiltrate the inflamed lesions of EAE or collagen-induced arthritis (CIA) (27, 28), it was suspected that IFN-{gamma} may have a pathological role in the development of these autoimmune diseases. However, administration of neutralizing Abs for IFN-{gamma} leads to exacerbation of these diseases (29). The development of CIA is enhanced in IFN-{gamma}–/– mice and that of EAE is also exacerbated in both IFN-{gamma}–/– and IFN-{gamma}R–/– mice compared with wild-type mice (22, 23, 24, 25). Thus, IFN-{gamma} may have a protective role in these diseases, rather than a pathogenic role. Consistent with this notion, the development of EAE is also exacerbated in mice deficient for IL-12 p35, a subunit of IL-12 that is required for the differentiation of IFN-{gamma}-producing Th1 cells (30, 31). In a similar manner, the severity of EAE was exaggerated in mice deficient in IL-12Rbeta2. Interestingly, we found that the IL-17-producing T cell population was increased in IFN-{gamma}–/– mice in comparison to that seen in wild-type mice upon immunized with MOG/CFA (Fig. 7). Similar observations were also currently reported by other groups (32, 33). IL-17 production was also augmented in the splenocytes of IL-12Rbeta2–/– mice (20). These observations suggest that IFN-{gamma} plays a beneficial role during the development of EAE by regulating IL-17 production. However, we demonstrated that IFN-{gamma} did not directly influence IL-17 production by CD4+ T cells (Fig. 9, E and F), suggesting that the suppressive effect of IFN-{gamma} on IL-17 production may be due to the suppression of the development of IL-17-producing cells. In this context, it was recently reported that IL-17-producing cells are induced by IL-23, while IL-12/IFN-{gamma} suppresses the production of IL-17 (34).

We also found that IFN-{gamma}-producing CD4+ and CD8+ T cells were markedly increased in IL-17–/– mice stimulated with MOG peptides, although IL-17 did not show any direct effect on IFN-{gamma} production by CD4+ T cells (Fig. 9, B and C) (Fig. 8). These observations suggest that IL-17 negatively regulates the development of IFN-{gamma}-producing Th1 cells. Thus, IL-17 and IFN-{gamma} may mutually regulate the development of these cytokine producer cells during immune responses.

We demonstrated that CD4+ T cells are the predominant producers of IL-17 in LN cells after immunization with MOG/CFA. It has been reported that, in Lyme arthritis, IL-17 is primarily produced by a specific subpopulation of CD4+ T cells that are neither Th1 nor Th2 and that produce TNF-{alpha} and/or GM-CSF simultaneously. As IL-17 is produced by multiple cell types, including CD8+ T cells, {gamma}{delta} T cells, neutrophils, and eosinophils under different conditions (13, 14, 15, 16, 17, 18, 35), the production of IL-17 is not limited to a specific T cell population. Instead, the producer cells in a particular disease appear to be defined by a specific cell population. The mechanism by which these IL-17 producer cells are controlled in different diseases, however, remains to be elucidated.

In MS patients, elevation of anti-MOG Ab levels is detectable in cerebrospinal fluid (36, 37). In association with the elevation of anti-MOG Abs following immunization with MOG peptides (38), severe demyelination occurred in the Lewis rat, suggesting that the generation of myelin-specific Abs may be involved in the development of EAE. However, we observed that anti-MOG Ab levels were increased in IL-17–/– mice in comparison with the levels seen in wild-type mice after immunization with MOG, while the development of EAE was markedly suppressed in IL-17–/– mice (Fig. 10). Thus, in IL-17–/– mice, there is no apparent correlation between the severity of EAE and the elevation of anti-MOG Abs in IL-17–/– mice, suggesting that anti-MOG Abs are not directly involved in the development of this disease. In fact, the serum MOG-specific Ab levels in mice that developed EAE after the transfer of MOG-specific CD4+ T cells were below the limits of detection (data not shown). In support of this notion, B cell-deficient mice develop EAE normally (39, 40, 41). It was reported, however, that Abs are involved in the remyelination of the lesions in the CNS during disease resolution (42). We did not expect that Ab levels specific for MOG would be enhanced in IL-17–/– mice, because Ab production was suppressed in IL-17–/– mice during CIA and contact, delayed-type, and airway hypersensitivity (9, 10, 11). We do not currently understand the reasoning for this. The molecular nature of the Ags involved in the autoimmune disorders, however, appears to affect the sensitivity of the disease to IL-17. Additional experiments will be necessary to elucidate the mechanism.

We previously reported that IL-17 is not essential for the induction of graft-vs-host reaction (GVHR) (9), in which CD8+ T cell-derived FasL and perforin play important roles (43, 44). Likewise, IL-17-deficiency did not affect the incidence of hyperglycemia in NOD mice (T. Matsuki, S. Nakae, and Y. Iwakura, unpublished observations), although IL-17 mRNA expression is increased in NOD mice upon development of insulin-dependent diabetes mellitus (IDDM) (45). In this case, CD8+ cells, rather than CD4+ cells, are also suggested to be involved in the apoptosis of beta cells in the pancreatic Langerhans islands (46, 47). In both cases, IFN-{gamma} is involved in the pathogenesis of the diseases (48, 49) (50). Thus, these observations indicate that these two types of inflammatory responses are clearly different; EAE and CIA are IL-17 dependent, and IL-17-producing cells play a major role, while IDDM and GVHR are IFN-{gamma} dependent, and CD8+ cytotoxic T cells and/or CD4+ Th1 cells play important roles.

Taken together, our data demonstrate that IL-17 and IFN-{gamma}, produced by a distinct population of T cells, have different roles in the development of EAE, CIA, GVHR, and hyperglycemia. These results suggest that these cytokines may also be involved in the development of MS, RA, GVHD, and IDDM in humans. Elucidation of the roles of pathogenic cytokines and the mechanisms of cytokine dependency may provide potential targets for novel therapeutics to treat these diseases.


   Acknowledgments
 
We thank Dr. S. J. Galli (Stanford University School of Medicine, Stanford, CA) for his generous support for this study. We also thank Tomoko Hata and Hayato Kotaki for their excellent animal care.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Ministry of Education, Culture, Sports, and Science of Japan, and the Ministry of Health and Welfare of Japan. Back

2 Current address: Department of Pathology, Stanford University School of Medicine, 269 Campus Drive, Center for Clinical Sciences Research 3255, Stanford, CA 94305-5176. Back

3 Current address: ERATO Yanagisawa Orphan Receptor Project, Japan Science and Technology Agency, Koto-Ku, Japan. Back

4 Current address: Animal Research Center, Tokyo Medical University, Sinjyuku-ku, Tokyo 160-8402, Japan. Back

5 Address correspondence and reprint requests to Dr. Yoichiro Iwakura, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: iwakura{at}ims.u-tokyo.ac.jp Back

6 Abbreviations used in this paper: RA, rheumatoid arthritis; MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; PTx, pertussis toxin; LN, lymph node; rm, recombinant human; GVHR, graft-vs-host reaction; CIA, collagen-induced arthritis; IDDM, insulin-dependent diabetes mellitus. Back

Received for publication August 30, 2005. Accepted for publication April 7, 2006.


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