International Immunology

International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(2):399-407; doi:10.1093/intimm/dxh379

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Abnormal T cell activation caused by the imbalance of the IL-1/IL-1R antagonist system is responsible for the development of experimental autoimmune encephalomyelitis

Taizo Matsuki¹, Susumu Nakae², Katsuko Sudo³, Reiko Horai⁴ and Yoichiro Iwakura

Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
¹ Present address: ERATO Yanagisawa Orphan Receptor Project, Japan Science and Technology Agency, Koto-ku, Tokyo 135-0064, Japan
² Present address: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5176, USA
³ Present address: Animal Research Center, Tokyo Medical University, Sinjyuku-ku, Tokyo 160-8402, Japan
⁴ Present address: National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA

Correspondence to: Y. Iwakura; E-mail: iwakura{at}ims.u-tokyo.ac.jp

	Abstract

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IL-1 is a pro-inflammatory cytokine that plays an important role in inflammation and host responses to infection. We have previously shown that imbalances in the IL-1 and IL-1R antagonist (IL-1Ra) system cause the development of inflammatory diseases. To explore the role of the IL-1/IL-1Ra system in autoimmune disease, we analyzed myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in mice bearing targeted disruptions of the IL-1, IL-1ß, IL-1 and IL-1ß (IL-1) or IL-1Ra genes. IL-1/ß double-deficient (IL-1^–/–) mice exhibited significant resistance to EAE induction with a significant reduction in disease severity, while IL-1^–/– or IL-1ß^–/– mice developed EAE in a manner similar to wild-type mice. IL-1Ra^–/– mice also developed MOG-induced EAE normally with pertussis toxin (PTx) administration. In contrast to wild-type mice, however, these mice were highly susceptible to EAE induction in the absence of PTx administration. We found that both IFN- and IL-17 production and proliferation were reduced in IL-1^–/– T cells upon stimulation with MOG, while IFN-, IL-17 and tumor necrosis factor- production and proliferation were enhanced in IL-1Ra^–/– T cells. These observations suggest that the IL-1/IL-1Ra system is crucial for auto-antigen-specific T cell induction and contributes to the development of EAE.

Keywords: autoimmunity, cytokines, dendritic cells, knockout mouse, T cells

	Introduction

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Multiple sclerosis (MS) is a chronic inflammatory disease associated with the demyelination of the central nervous system (CNS). Approximately, one million individuals in the world are afflicted by MS (1). Although significant progress has been made elucidating the causes of MS and improving patient outcomes over the past decade (2), definitive therapies either reducing the number of attacks or slowing the progression of disease are not yet available.

Experimental autoimmune encephalomyelitis (EAE) is regarded as an animal model mimicking several aspects of the pathogenesis of human MS, which is clinically characterized by paralysis and lethargy (3). Immunization with self-neuronal antigens, such as MBP, myelin-associated glycoprotein, proteolipid protein or myelin oligodendrocyte glycoprotein (MOG) (3, 4), results in inflammation within the CNS primarily mediated by CD4⁺ T_h1 cells (1, 2).

Systemic or local induction of cytokines is critical in the initiation, enhancement or perpetuation of CNS disease (5). T_h1 cell-derived IFN-, which contributes to the etiology of a wide range of diseases, is markedly elevated within the CNS during EAE. IFN--deficient and IFN-R^–/– mice, however, remain highly susceptible to EAE (6–9). In fact, the over-expression of IFN- in the CNS ameliorated the severity of EAE (10). Tumor necrosis factor (TNF), a potent pro-inflammatory cytokine, is produced by a variety of cell types, including T_h1 cells. Mice over-expressing TNF within the CNS exhibit neuronal demyelination (11, 12), while the development of EAE in TNF^–/– mice is partially suppressed (13, 14). Other group reported, however, that in TNF^–/– mice, the course of EAE was exacerbated by the abnormal regression and expansion of myelin-specific T cells (15). Clinically, anti-TNF therapy resulted in more severe MS (16). Thus, the contribution of pro-inflammatory cytokines, such as IFN- and TNF, cannot fully explain the precise molecular mechanisms underlying EAE development.

IL-1 is produced by a variety of cells, including monocytes/macrophages, epithelial and endothelial cells and glial cells (17). Through the up-regulation of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, this cytokine plays a crucial role in leukocyte extravasation into inflammatory sites (18). Dysregulation of IL-1 function leads to autoimmune and abnormal immune responses, such as arthritis and aortitis in mouse models (19, 20). Furthermore, exogenous IL-1 administration exacerbated the course of EAE, while administration of soluble IL-1R type-I (IL-1RI) or IL-1R antagonist (IL-1Ra) significantly suppressed EAE in Lewis rats (21, 22). Consistent with these observations, mice deficient in IL-1RI or IL-1R-associated kinase 1, which is involved in IL-1-mediated signal transduction, fail to develop inflammatory lesions or any evidence of EAE (23). These observations suggest that IL-1 may initiate or promote local and/or systemic inflammation during EAE pathogenesis. In vitro, IL-1 can augment the activation of encephalitogenic T lymphocytes, contributing to the development of EAE induced by adoptive transfer (24). Thus, IL-1 likely contributes to the activation of auto-antigen-specific immune cells, including T cells. Indeed, IL-1 can influence antigen-specific T cell activation directly (25) or indirectly via modulation of dendritic cell (DC) function (26). The importance of IL-1 in DC function, including migration, activation and acquisition of T_h1-inducing ability, has been demonstrated previously (27, 28). The precise effect of IL-1 on DCs and/or T cells during the development of EAE, however, has yet to be elucidated.

In this report, we investigate the contribution of IL-1 to the development of EAE using IL-1^–/– and IL-1Ra^–/– mice. We determined that IL-1 is responsible for the induction of autoreactive T cells. Our data provide evidence that the IL-1/IL-1Ra system is critical for the development of CNS autoimmune disease by modulating T cell-mediated immunity.

	Methods

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Mice
IL-1^–/–, IL-1ß^–/–, IL-1/ß^–/– (IL-1^–/–) and IL-1Ra^–/– mice were generated as described (29). Mice were backcrossed to the C57BL/6 strain mice for eight generations. C57BL/6 mice (wild-type mice) were purchased from Clea (Tokyo, Japan). Age- and gender-matched wild-type mice were used as controls in each experiment. Mice were kept under specific pathogen-free conditions in an environmentally controlled clean room at the Center for Experimental Medicine, Institute of Medical Science, University of Tokyo. Animals were housed in an ambient temperature of 24°C on a daily cycle of 12 h of light and darkness (8:00 a.m. to 8:00 p.m.). All the experiments were performed according to the institutional ethical guidelines for animal experimentation.

MOG peptide
MOG 35–55 (MEVGWYRSPFSRVVHLYRNGK), corresponding to the murine sequence, was synthesized on a peptide synthesizer using fluorenylmethoxycarbonyl chemistry and purified by HPLC by Ohmi (Institute of Medical Science, University of Tokyo, Japan).

Induction and evaluation of EAE
Eight- to twelve-week-old mice were subcutaneously immunized with 100 µg MOG 35–55 emulsified in CFA (1 : 1) supplemented with 400 µg Mycobacterium tuberculosis H37RA (DIFCO Lab., Detroit, MI, USA) in both flanks. Pertussis toxin (PTx) (500 ng) (Alexis Corp., San Diego, CA, USA) was injected intravenously into animals on the day of immunization as well as 2 days later.

Mice were inspected daily for the clinical signs of EAE for up to 30 days after immunization. Scores were determined 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 and 5, moribund state. The mean clinical score was calculated by averaging the score of all of the mice in each group, including animals that did not develop EAE.

Titer for anti-MOG antibodies in serum
Detection of anti-MOG 35–55 antibodies was performed as described (25) with the following modifications. Briefly, MOG 35–55 peptide (0.5 µg per 96 well) was coated onto 96-well plates and incubated at 4°C overnight. After substantial washing and blocking, diluted sera (30 µl per well) were added to the wells for 2 h at room temperature. A series of serum dilutions were examined in preliminary experiments. After washing, alkaline phosphatase-conjugated goat anti-mouse Igs (Zymed, San Francisco, CA, USA) were added for 1 h at room temperature, followed by incubation in p-nitrophenyl phosphate substrate (Sigma–Aldrich, St Louis, MO, USA) as the substrate. The anti-MOG antibody titer is given as an OD₄₁₅ value. Samples were measured in duplicate.

T cell and DC purification and proliferation assay
Mice were immunized subcutaneously with 100 µg MOG 35–55 emulsified CFA (1 : 1) with or without PTx. Ten days later, T cells were prepared from multiple lymph nodes (LNs) (axillary, inguinal, branchial, cervical and poplitial). Cells were washed, treated with anti-mouse Thy1.2 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and passed through a MACS^® column to collect Thy1.2⁺ T cells.

DCs were prepared from the spleen. Spleens were collected, minced and digested with 1 mg ml^–1 collagenase (Sigma–Aldrich) and 1 mg ml^–1 DNase I (Sigma–Aldrich) in HBSS for 30 min at 37°C. Following the addition of EDTA (20 mM final concentration), cells were incubated for 5 min at room temperature, passed through a 70-µm nylon mesh, layered over RPMI 1640–10% FCS–14.5% metrizamide (Cedarlane Labs., Ontario, Canada) and centrifuged at room temperature for 30 min at 500 x g. The low buoyant density cells at the interface were collected and washed twice. Cells were then treated with anti-mouse CD11c magnetic beads (Miltenyi Biotec) and passed through a MACS^® column. The positively selected fraction was collected, washed and re-suspended for use.

Purified DCs (1 x 10⁴ cells) in the presence or absence of T cells (1 x 10⁵ cells) were plated on 96-well plates coated with MOG 35–55 in a final volume of 200 µl RPMI 1640–10% FCS. After 72 h of culture, cells were pulsed with [³H]thymidine ([³H]TdR) (0.25 µCi ml^–1; Amersham Biosciences, Tokyo, Japan) for 6 h. Cells were then harvested with a Micro 96 cell harvester (Skatron, Lier, Norway). The incorporated [³H]TdR radioactivity was measured using a Micro Beta System (Amersham Biosciences, Piscataway, NJ, USA). Culture supernatants were collected prior to [³H]TdR incorporation to measure cytokines levels.

ELISA of cytokine levels
The levels of IL-4, IL-17 and TNF were measured as described (30, 31). IFN- levels were measured with OptEIA® Set mouse IFN- kit (BD PharMingen). All assays were done in duplicate.

Statistical analysis
All values were calculated as the average ± SD. Comparisons were made using the Student's t-test, one-way analysis of variance (ANOVA), Fisher's protected least significant difference test and Mann–Whitney's U-test. Differences among the three groups were tested by Kruskal–Wallis one-way ANOVA.

	Results

Top Abstract Introduction Methods Results Discussion References

 
IL-1^–/– mice are resistant to EAE
To examine the role of the IL-1/IL-1Ra system in the development of EAE, we immunized C57BL/6 wild-type, IL-1^–/– and IL-1RI^–/– mice with MOG 35–55 emulsified in CFA. Following the injection of PTx on days 0 and 2, the clinical signs of EAE were monitored daily and scored as described in Methods. IL-1RI^–/– mice are known to demonstrate resistance to the development of EAE (23), suggesting that IL-1 is involved in EAE pathogenesis. We confirmed that IL-1^–/– mice exhibit significant resistance to EAE and that IL-1RI^–/– mice demonstrate a reduction in disease severity (Fig. 1A and data not shown) (23). The onset of EAE in IL-1^–/– mice was also delayed from that of wild-type mice (Table 1). In contrast, mice deficient in either IL-1 or IL-1ß developed EAE with a comparable severity and time course to wild-type mice (Fig. 1B). The incidence of disease, day of onset and maximal clinical score were not significantly different between wild-type, IL-1^–/– and IL-1ß^–/– mice (Table 1). All genotypes mice exhibited >90% disease incidence. These observations suggest that, while IL-1 plays a principal role in the development of EAE, the presence of either IL-1 or IL-1ß alone is sufficient to initiate development of the disease.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Attenuated EAE induction in IL-1–/– mice. The clinical scores after EAE induction were determined as described in Methods. The averages of clinical scores are shown from the day of MOG immunization (day 0) to day 28 post-immunization in (A) wild-type (open squares, n = 9) and IL-1–/– (filled triangles, n = 7) mice and (B) wild-type (open squares, n = 8), IL-1–/– (filled squares, n = 9) and IL-1ß–/– (filled triangles, n = 7) mice. The data indicate the averages of each group. Statistical significances were determined by Mann–Whitney's U-test (A) and Kruskal–Wallis one-way ANOVA (B). *P < 0.05 versus wild-type mice.

 

View this table: [in this window] [in a new window]   Table 1. Clinical features of MOG 35–55-induced EAE in IL-1–/– and IL-1Ra–/– mice

 
Development of EAE is exacerbated in IL-1Ra^–/– mice without PTx administration
We immunized IL-1Ra^–/– mice with MOG 35–55 emulsified in CFA. After an injection of PTx on days 0 and 2, IL-1Ra^–/– mice developed EAE that was comparable in time of onset and severity to the EAE course observed in wild-type mice (Fig. 2A and Table 1). PTx is routinely used to facilitate the induction of experimental autoimmune diseases in animals. Previous reports using IL-10^–/– and TNF^–/– mice suggested that co-administration of PTx veiled the effects of cytokines as an inflammatory factor in EAE (15, 32, 33). Therefore, to address the contribution of IL-1Ra to EAE without the complications of PTx co-administration, we examined the susceptibility of wild-type and IL-1Ra^–/– mice to EAE in the absence of PTx. The severity of EAE was reduced in wild-type mice that were not treated with PTx (Fig. 2B). IL-1Ra^–/– mice, however, developed severe EAE in both the absence and presence of PTx (Fig. 2B). Without PTx, IL-1Ra^–/– mice developed more severe EAE at earlier time points than wild-type mice (Table 1). These results indicate that dysfunction of IL-1 signaling mediated by IL-1Ra deficiency contributes to EAE induction in the absence of PTx. In wild-type mice, PTx may be necessary to overcome the function of IL-1 in EAE induction.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Exacerbated EAE induction without PTx injection in IL-1Ra–/– mice. Clinical scores after MOG immunization in the (A) presence or (B) absence of PTx (500 ng) injection in wild-type [open squares, (A) n = 13 and (B) n = 8], and IL-1Ra–/– mice [filled diamonds, (A) n = 12 and (B) n = 9]. Data show the average from each group. Statistical significances were determined by Mann–Whitney's U-test. *P < 0.05 versus wild-type mice.

 
T_h1-type antibody production against MOG 35–55 is increased in sera of IL-1Ra^–/– mice
Auto-antigen-specific Igs were detected in the sera of mice with EAE. At 37 days after immunization with MOG 35–55, blood samples were collected from mice for the measurement of MOG-specific auto-antibody levels in the sera. The levels of MOG-specific IgG and IgM classes and the IgG1 subclass in sera, as well as those of IgG2b and IgG3 (data not shown), were comparable among IL-1^–/– and wild-type mice given PTx and among IL-1Ra^–/– and wild-type mice in the absence of PTx co-administration (Fig. 3A and B). In contrast, the levels of MOG-specific IgG2a, whose production depends on T_h1 cytokines, were significantly increased in sera from IL-1Ra^–/– mice in comparison with those from wild-type and IL-1^–/– mice (Fig. 3A and B). These results suggest that IL-1 signaling promotes the polarization of T_h1 immune responses toward the production of high levels of auto-antigen-specific IgG2a, as seen in IL-1Ra^–/– mice during the development of EAE.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Titer of anti-MOG antibodies in serum of IL-1–/– and IL-1Ra–/– mice. Thirty-seven days after EAE induction (A) with PTx injection (wild-type, open bars and IL-1–/– mice, filled bars) or (B) without PTx injection (wild-type, open bars and IL-1Ra–/– mice, filled bars), we collected serum samples. The levels of IgG, IgG1, IgG2a and IgM specific for the MOG 35–55 peptide are shown as OD values. Data show the average ± SD from each group. Statistical significances were determined by Student's t-test. *P < 0.05 versus wild-type mice.

 
IL-1 is involved in auto-antigen-specific T cell activation during EAE
EAE is considered to be a T cell-mediated autoimmune disease model (3). Abnormal EAE induction in IL-1^–/– and IL-1Ra^–/– mice may be due to abnormal control of MOG-specific effector T cells. DCs also play a significant role in (auto)immune responses through the induction of T_h1 cell activation (26). In the EAE animal model, we examined if dysfunction of the IL-1/IL-1Ra system affected T cell or DC function using IL-1^–/– and IL-1Ra^–/– mice. We examined in vitro the activation of T cells derived from wild-type, IL-1^–/– and IL-1Ra^–/– mice immunized with MOG 35–55/CFA in the absence of PTx co-administration. Ten days after MOG 35–55 immunization, Thy1.2⁺ T cells and CD11c⁺ DCs were isolated from the draining LNs and spleen, respectively. LN T cells were then co-cultured with DCs in the presence of MOG 35–55. No proliferative responses were observed in DCs of gene-deficient and/or wild-type mice treated with MOG 35–55 in the absence of T cells (data not shown). Low proliferative responses of T cells were observed even without DCs in the absence or presence of MOG 35–55 (data not shown). When cultured with DCs in the presence of MOG 35–55, MOG-specific T cell proliferative responses were induced in a manner dependent on MOG 35–55 concentration (0, 10, 50 and 100 µg ml^–1) (data not shown). In these co-cultures, the MOG-specific proliferative responses of wild-type T cells were comparable among wild-type, IL-1Ra^–/– and IL-1^–/– DCs (Fig. 4A and B), suggesting that IL-1 or IL-1Ra deficiency of DCs did not result in any defects in antigen presentation or cytokine production that would influence the induction of MOG-specific T cell recall responses in vitro. Interestingly, the proliferative responses of MOG-specific IL-1Ra^–/– T cells were significantly hyperactive following co-cultured with either wild-type (Fig. 4D) or IL-1Ra^–/– (data not shown) DCs in comparison with wild-type T cells. In contrast, the responses of IL-1^–/– T cells after co-culture with either wild-type (Fig. 4C) or IL-1^–/– (data not shown) DCs were profoundly impaired, despite comparable non-specific proliferative responses of T cells against mitogenic stimuli Con A 1 µg ml^–1) (Fig. 4). These results indicate that intrinsic IL-1 is responsible for the activation of auto-antigen-specific T cells during the priming process in vivo.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Abnormal T cell activation in IL-1–/– and IL-1Ra–/– mice immunized with MOG. Wild-type, IL-1–/– and IL-1Ra–/– mice were immunized with a MOG 35–55/CFA emulsion without PTx co-administration. Ten days after MOG immunization, LN T cells were cultured with splenic DCs in the absence (medium) or presence MOG of MOG 35–55 (100 µg ml–1) or Con A (1 µg ml–1) for 72 h. MOG-sensitized T cells from wild-type mice were co-cultured with DCs from (A) wild-type or IL-1–/– mice and (B) wild-type or IL-1Ra–/– mice. MOG-sensitized T cells from (C) wild-type or IL-1–/– mice and (D) wild-type or IL-1Ra–/– mice were co-cultured with DCs from wild-type mice. The genotypes of the T cells (T) and DCs (DC) are indicated as WT: wild-type mice, IL-1–/–: IL-1–/– mice and Ra–/–: IL-1Ra–/– mice. Data indicate the averages. These data were reproducible in three independent experiments. Statistical significances were determined by one-way ANOVA and Fisher's protected least significant difference test. *P < 0.01 versus wild-type mice.

 
T cells from IL-1Ra^–/– mice produce high levels of pro-inflammatory cytokines
We measured cytokine production by MOG-specific T cells by assaying the supernatants of proliferative response cultures. The levels of IFN- and TNF in supernatants from wild-type T cells co-cultured with either IL-1^–/– or IL-1Ra^–/– DCs were similar to those cultured with wild-type DCs. In contrast, the levels of IFN- and IL-17, but not TNF, in the supernatants of IL-1^–/– T cells co-cultures with wild-type DCs was reduced from the levels seen in wild-type T cells co-cultures with wild-type DCs (Fig. 5A and data not shown). In correlation with MOG-specific T cell proliferative responses, IFN-, IL-17 and TNF levels measured in the supernatants of IL-1Ra^–/– T cells co-cultures with wild-type or IL-1Ra^–/– DCs were significantly increased in comparison with those from wild-type or IL-1^–/– T cells co-cultured with wild-type DCs (Fig. 5B and data not shown). The levels of IL-4, a T_h2-skewing cytokine, were below the limits of detection in the supernatants from any of the culture conditions (data not shown). These results suggest that excess IL-1 signaling breaks tolerance for auto-antigens in peripheral lymphoid tissues, resulting in hyperresponsive effector T cell activation and auto-antigen-specific T cell proliferation and inflammatory cytokine production as seen in IL-1Ra^–/– mice during EAE pathogenesis.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Abnormal cytokine production from IL-1–/– T cells and IL-1Ra–/– T cells. MOG-sensitized T cells from (A) wild-type or IL-1–/– mice and (B) wild-type or IL-1Ra–/– mice were co-cultured with wild-type DCs in the absence (medium) or presence MOG of MOG 35–55, as shown in Fig. 4. IFN-, IL-17 and TNF levels in culture supernatants were determined by ELISA. The genotypes of the T cells are indicated as WT: wild-type mice, IL-1–/–: IL-1–/– mice and Ra–/–: IL-1Ra–/– mice. Data indicate the averages. These data were reproducible in two independent experiments. Statistical significances were determined by one-way ANOVA and Fisher's protected least significant difference test. *P < 0.01 versus wild-type mice.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Using IL-1^–/–, IL-1ß^–/–, IL-1^–/– and IL-1Ra^–/– mice, we demonstrate that IL-1 is responsible for the development of EAE. Either IL-1 or IL-1ß alone was sufficient to induce EAE; excess IL-1 signaling resulting from the lack of IL-1Ra augmented EAE severity in the absence of PTx injection. These findings suggested that the adjuvant effect of PTx exerts a related function as IL-1 in the induction of EAE. We clearly demonstrated that, while IL-1 controls optimal antigen-specific T cell activation, dysfunction of the IL-1/IL-1Ra system leads to excess T cell activation by breaking peripheral tolerance for auto-antigens during the pathogenesis of EAE.

In a series of inflammatory response models, we have previously shown that antigen-presenting cell (APC)-derived IL-1 was required for (auto)antigen-specific T cell activation. We previously showed that IL-1 plays an important role in the interaction between T cells and APCs in priming process through inducing CD40L (CD154) and OX40 (CD134) on T cells (25). CD40L and OX40 expressions were enhanced in T cells stimulated with antigen-bearing IL-1Ra^–/– APCs compared with wild-type APCs (25). Thus, upon interaction with antigens, APCs produce IL-1, and IL-1 activates T cells, resulting in the induction of CD40L (34, 35). Then, CD40L–CD40 interaction activates APCs to produce TNF (34). This TNF induces OX40 on T cells (36), that leads to enhancement of cytokine production, especially IL-17 (37). With these mechanisms, APCs-derived IL-1 contributes to the development of allergic and/or autoimmune diseases in mice (28, 36, 38). IL-1RI^–/– DCs demonstrate impaired cytokine production, leading to insufficient CD4⁺ T cell activation (26). Thus, IL-1 can modulate T cell function both directly and indirectly by influencing DC activation. These findings suggest that IL-1 may play a role in the induction and/or activation of autoreactive T cells in EAE. Despite comparable non-specific T cell proliferation upon stimulation with mitogen Con A among wild-type, IL-1^–/– and IL-1Ra^–/– mice (Fig. 4C and D), the proliferation of MOG-specific IL-1^–/– T cells co-cultured with wild-type DCs, which can produce IL-1, was markedly impaired. The proliferation of IL-1Ra^–/– T cells co-cultured with wild-type DCs, which could produce IL-1Ra, was greatly enhanced (Fig. 4C and D). The proliferation of MOG-specific wild-type T cells co-cultured with IL-1^–/– DCs was similar to that observed with wild-type DCs (Fig. 4A), indicating that DC-derived IL-1 is not essential for the activation of MOG-specific memory T cells. Instead, IL-1 is likely involved in the induction of MOG-specific memory T cells in vivo. Thus, insufficient induction of MOG-specific T cells resulting from IL-1 deficiency may lead to the attenuated development of EAE as observed in IL-1^–/– mice. In contrast, excess MOG-specific T cell activation observed in IL-1Ra-deficient mice may explain the exacerbation of EAE in IL-1Ra^–/– mice.

Despite the normal development of EAE following PTx injection, IL-1Ra^–/– mice exhibited more severe MOG-induced EAE in the absence of PTx injection than wild-type mice. Similarly, the development of EAE in TNF^–/– mice was completely suppressed in the presence of low doses of PTx, although susceptibility to the disease in TNF^–/– mice was normal at high doses of PTx (33). PTx is widely used to enhance T_h1-mediated organ-specific autoimmune disease through inhibition of the Gi/o protein signaling pathways that negatively regulate IL-12 production (39) and induction of pro-inflammatory cytokines, MHC class II, CD80, CD86 and CD40 on APCs (40, 41). These observations imply that PTx exerts a similar function as the pro-inflammatory cytokines IL-1 and TNF. We, as well as others, previously observed that the function of IL-1 in ovalbumin-induced airway hypersensitivity responses could be substituted for by a potent adjuvant, aluminum potassium sulfate (42, 43). Therefore, the physiological function of IL-1 (and TNF) may be masked by the excessive adjuvant-dependent artificial activation of the immune system observed in MOG–EAE with PTx injection and ovalbumin-induced airway hypersensitivity responses with aluminum potassium sulfate.

IFN-, TNF and IL-17, T cell-derived inflammatory cytokines, play critical roles in multiple pathological inflammatory responses. TNF has a similar biological activity to IL-1 as a potent pro-inflammatory cytokine. As seen in studies using TNF^–/– mice, TNF is also involved in the development of EAE (13, 14). Interestingly, TNF production is normal in IL-1^–/– mice after MOG/CFA immunization (Fig. 5A), despite the profound suppression of EAE development in IL-1^–/– mice (Fig. 1 and Table 1). In contrast, IL-1Ra^–/– mice exhibited elevated TNF production (Fig. 5B) and exacerbated development of EAE (Fig. 2 and Table 1). Thus, TNF is not essential for, but contributes to, the development of EAE (15, 23, 44). Excess TNF production resulting from excessive IL-1 activity may explain the synergistic exacerbation of EAE development in IL-1Ra^–/– mice. These results suggest, however, that TNF alone is not sufficient to induce adequate responses in the absence of IL-1, as observed in IL-1^–/– mice.

While IFN--producing T_h1 cells are crucial for the induction of autoimmune diseases, IFN-^–/– and/or IFN-R^–/– mice develop autoimmune diseases, such as EAE and collagen-induced arthritis (6–9, 45). Currently, T cell-derived IL-17, rather than IFN-, is suspected to be critical in the pathogenesis of EAE. In support of this hypothesis, increased levels of IL-17 were observed in the lesions of MS patients (46). Otherwise, IL-12 has been well characterized as a potent activator of IFN--producing T_h1 cells, while IL-23, a member of the IL-12 family consisting of IL-23 p19 and IL-12 p40, can induce IL-17 production by T cells (47). IL-23, but not IL-12, is crucial for the development of EAE (48). As seen with IFN-^–/– and IL-12^–/– mice, IL-12Rß2^–/– mice exhibited exacerbated EAE development and increased IL-17 production (49). IL-12 administration, however, led to the inhibition of IL-17 mRNA expression during EAE pathogenesis (50). Currently, the contribution of IL-17 to the pathogenesis of EAE was suggested in mice treated with anti-IL-17-neutralizing antibody (51). We determined that, in MOG-stimulated T cells, IL-17 production was reduced in IL-1^–/– mice and increased in IL-1Ra^–/– mice (Fig. 5A and B). Thus, our data suggest that IL-1 plays an important role in the activation of both IFN--producing T_h1 and IL-17-producing CD4⁺ T cells, contributing to the development of EAE.

In conclusion, our findings suggest that dysregulation of the IL-1/IL-1Ra balance leads to the failure of peripheral lymphoid tolerance for self-antigens, resulting in the severe inflammation seen in EAE. These observations may provide a clue to develop new therapeutics against MS.

	Acknowledgements

 
We would like to thank Ohmi for providing the MOG peptide. We would also like to thank K. Habu and Y. Komiyama for their technical support and critical comments. We thank all the members of our laboratory for their kind discussion and help in animal care. This work was supported by grants from the Ministry of Education, Science, Sport and Culture of Japan, the Ministry of Health and Welfare of Japan, the Japan Society for the Promotion of Science and Pioneering Research Project in Biotechnology.

	Abbreviations

 

ANOVA	analysis of variance
APC	antigen-presenting cell
CNS	central nervous system
DC	dendritic cell
EAE	experimental autoimmune encephalomyelitis
IL-1Ra	IL-1R antagonist
IL-1RI	IL-1R type-I
LN	lymph node
MOG	myelin oligodendrocyte glycoprotein
MS	multiple sclerosis
PTx	pertussis toxin
[3H]TdR	[3H]thymidine
TNF	tumor necrosis factor

	Notes

 
Transmitting editor: Okumura

Received 5 September 2005, accepted 30 November 2005.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Steinman, L., Martin, R., Bernard, C., Conlon, P. and Oksenberg, J. R. 2002. Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy. Annu. Rev. Neurosci. 25:491.[CrossRef][Web of Science][Medline]
2. Keegan, B. M. and Noseworthy, J. H. 2002. Multiple sclerosis. Annu. Rev. Med. 53:285.[CrossRef][Web of Science][Medline]
3. Kuchroo, V. K., Anderson, A. C., Waldner, H., Munder, M., Bettelli, E. and Nicholson, L. B. 2002. T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Annu. Rev. Immunol. 20:101.[CrossRef][Web of Science][Medline]
4. Hemmer, B., Archelos, J. J. and Hartung, H. P. 2002. New concepts in the immunopathogenesis of multiple sclerosis. Nat. Rev. Neurosci. 3:291.[CrossRef][Web of Science][Medline]
5. Owens, T., Wekerle, H. and Antel, J. 2001. Genetic models for CNS inflammation. Nat. Med. 7:161.[CrossRef][Web of Science][Medline]
6. Ferber, I. A., Brocke, S., Taylor-Edwards, C. et al. 1996. Mice with a disrupted IFN- gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
7. Krakowski, M. and Owens, T. 1996. Interferon- confers resistance to experimental allergic encephalomyelitis. Eur. J. Immunol. 26:1641.[Web of Science][Medline]
8. Willenborg, D. O., Fordham, S., Bernard, C. C., Cowden, W. B. and Ramshaw, I. A. 1996. IFN- plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
9. Chu, C. Q., Wittmer, S. and Dalton, D. K. 2000. Failure to suppress the expansion of the activated CD4 T cell population in interferon -deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:123.[Abstract/Free Full Text]
10. Furlan, R., Brambilla, E., Ruffini, F. et al. 2001. Intrathecal delivery of IFN- protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes. J. Immunol. 167:1821.[Abstract/Free Full Text]
11. Probert, L., Akassoglou, K., Pasparakis, M., Kontogeorgos, G. and Kollias, G. 1995. Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor . Proc. Natl Acad. Sci. USA 92:11294.[Abstract/Free Full Text]
12. Powell, M. B., Mitchell, D., Lederman, J. et al. 1990. Lymphotoxin and tumor necrosis factor- production by myelin basic protein-specific T cell clones correlates with encephalitogenicity. Int. Immunol. 2:539.[Abstract/Free Full Text]
13. Sedgwick, J. D., Riminton, D. S., Cyster, J. G. and Korner, H. 2000. Tumor necrosis factor: a master-regulator of leukocyte movement. Immunol. Today 21:110.[CrossRef][Web of Science][Medline]
14. Sean Riminton, D., Korner, H., Strickland, D. H., Lemckert, F. A., Pollard, J. D. and Sedgwick, J. D. 1998. Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice. J. Exp. Med. 187:1517.[Abstract/Free Full Text]
15. Kassiotis, G. and Kollias, G. 2001. Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: implications for pathogenesis and therapy of autoimmune demyelination. J. Exp. Med. 193:427.[Abstract/Free Full Text]
16. Kollias, G. and Kontoyiannis, D. 2002. Role of TNF/TNFR in autoimmunity: specific TNF receptor blockade may be advantageous to anti-TNF treatments. Cytokine Growth Factor Rev. 13:315.[CrossRef][Web of Science][Medline]
17. Tocci, M. J. and Schmidt, J. A. 1997. Interleukin-1: structure and function. In Remick, D. G. and Friedland, J. S., eds, Cytokines in Health and Disease, 2nd edn, p. 1. Dekker Encyclopedias, Taylor and Francis Group, KY, USA.
18. Tamaru, M., Tomura, K., Sakamoto, S., Tezuka, K., Tamatani, T. and Narumi, S. 1998. Interleukin-1ß induces tissue- and cell type-specific expression of adhesion molecules in vivo. Arterioscler. Thromb. Vasc. Biol. 18:1292.[Abstract/Free Full Text]
19. Horai, R., Saijo, S., Tanioka, H. et al. 2000. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J. Exp. Med. 191:313.[Abstract/Free Full Text]
20. Nicklin, M. J., Hughes, D. E., Barton, J. L., Ure, J. M. and Duff, G. W. 2000. Arterial inflammation in mice lacking the interleukin 1 receptor antagonist gene. J. Exp. Med. 191:303.[Abstract/Free Full Text]
21. Jacobs, C. A., Baker, P. E., Roux, E. R. et al. 1991. Experimental autoimmune encephalomyelitis is exacerbated by IL-1 and suppressed by soluble IL-1 receptor. J. Immunol. 146:2983.[Abstract]
22. Martin, D. and Near, S. L. 1995. Protective effect of the interleukin-1 receptor antagonist (IL-1ra) on experimental allergic encephalomyelitis in rats. J. Neuroimmunol. 61:241.[CrossRef][Web of Science][Medline]
23. Schiffenbauer, J., Streit, W. J., Butfiloski, E., LaBow, M., Edwards, C., III and Moldawer, L. L. 2000. The induction of EAE is only partially dependent on TNF receptor signaling but requires the IL-1 type I receptor. Clin. Immunol. 95:117.[CrossRef][Web of Science][Medline]
24. Mannie, M. D., Dinarello, C. A. and Paterson, P. Y. 1987. Interleukin 1 and myelin basic protein synergistically augment adoptive transfer activity of lymphocytes mediating experimental autoimmune encephalomyelitis in Lewis rats. J. Immunol. 138:4229.[Abstract]
25. Nakae, S., Asano, M., Horai, R., Sakaguchi, N. and Iwakura, Y. 2001. IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells. J. Immunol. 167:90.[Abstract/Free Full Text]
26. Eriksson, U., Kurrer, M. O., Sonderegger, I. et al. 2003. Activation of dendritic cells through the interleukin 1 receptor 1 is critical for the induction of autoimmune myocarditis. J. Exp. Med. 197:323.[Abstract/Free Full Text]
27. Shornick, L. P., Bisarya, A. K. and Chaplin, D. D. 2001. IL-1ß is essential for langerhans cell activation and antigen delivery to the lymph nodes during contact sensitization: evidence for a dermal source of IL-1ß. Cell. Immunol. 211:105.[CrossRef][Web of Science][Medline]
28. Nakae, S., Naruse-Nakajima, C., Sudo, K., Horai, R., Asano, M. and Iwakura, Y. 2001. IL-1, but not IL-1ß, is required for contact-allergen-specific T cell activation during the sensitization phase in contact hypersensitivity. Int. Immunol. 13:1471.[Abstract/Free Full Text]
29. Horai, R., Asano, M., Sudo, K. et al. 1998. Production of mice deficient in genes for interleukin (IL)-1, IL-1ß, IL-1/ß, and IL-1 receptor antagonist shows that IL-1ß is crucial in turpentine-induced fever development and glucocorticoid secretion. J. Exp. Med. 187:1463.[Abstract/Free Full Text]
30. Nakae, S., Asano, M., Horai, R. and Iwakura, Y. 2001. Interleukin-1ß, but not interleukin-1, is required for T-cell-dependent antibody production. Immunology 104:402.[CrossRef][Web of Science][Medline]
31. Nakae, S., Komiyama, Y., Nambu, A. et al. 2002. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity 17:375.[CrossRef][Web of Science][Medline]
32. Bettelli, E., Das, M. P., Howard, E. D., Weiner, H. L., Sobel, R. A. and Kuchroo, V. K. 1998. IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J. Immunol. 161:3299.[Abstract/Free Full Text]
33. Kassiotis, G., Kranidioti, K. and Kollias, G. 2001. Defective CD4T cell priming and resistance to experimental autoimmune encephalomyelitis in TNF-deficient mice due to innate immune hypo-responsiveness. J. Neuroimmunol. 119:239.[CrossRef][Web of Science][Medline]
34. van Kooten, C. and Banchereau, J. 2000. CD40-CD40 ligand. J. Leukoc. Biol. 67:2.[Abstract]
35. Weinberg, A. D., Bourdette, D. N., Sullivan, T. J. et al. 1996. Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis. Nat. Med. 2:183.[CrossRef][Web of Science][Medline]
36. Horai, R., Nakajima, A., Habiro, K. et al. 2004. TNF- is crucial for the development of autoimmune arthritis in IL-1 receptor antagonist-deficient mice. J. Clin. Investig. 114:1603.[CrossRef][Web of Science][Medline]
37. Nakae, S., Saijo, S., Horai, R., Sudo, K., Mori, S. and Iwakura, Y. 2003. IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. Proc. Natl Acad. Sci. USA 100:5986.[Abstract/Free Full Text]
38. Saijo, S., Asano, M., Horai, R., Yamamoto, H. and Iwakura, Y. 2002. Suppression of autoimmune arthritis in interleukin-1-deficient mice in which T cell activation is impaired due to low levels of CD40 ligand and OX40 expression on T cells. Arthritis Rheum. 46:533.[CrossRef][Web of Science][Medline]
39. He, J., Gurunathan, S., Iwasaki, A., Ash-Shaheed, B. and Kelsall, B. L. 2000. Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses. J. Exp. Med. 191:1605.[Abstract/Free Full Text]
40. Shive, C. L., Hofstetter, H., Arredondo, L., Shaw, C. and Forsthuber, T. G. 2000. The enhanced antigen-specific production of cytokines induced by pertussis toxin is due to clonal expansion of T cells and not to altered effector functions of long-term memory cells. Eur. J. Immunol. 30:2422.[CrossRef][Web of Science][Medline]
41. Ryan, M., McCarthy, L., Rappuoli, R., Mahon, B. P. and Mills, K. H. 1998. Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7-1, B7-2 and CD28. Int. Immunol. 10:651.[Abstract/Free Full Text]
42. Nakae, S., Komiyama, Y., Yokoyama, H. et al. 2003. IL-1 is required for allergen-specific Th2 cell activation and the development of airway hypersensitivity response. Int. Immunol. 15:483.[Abstract/Free Full Text]
43. Schmitz, N., Kurrer, M. and Kopf, M. 2003. The IL-1 receptor 1 is critical for Th2 cell type airway immune responses in a mild but not in a more severe asthma model. Eur. J. Immunol. 33:991.[CrossRef][Web of Science][Medline]
44. Frei, K., Eugster, H. P., Bopst, M., Constantinescu, C. S., Lavi, E. and Fontana, A. 1997. Tumor necrosis factor and lymphotoxin are not required for induction of acute experimental autoimmune encephalomyelitis. J. Exp. Med. 185:2177.[Abstract/Free Full Text]
45. Manoury-Schwartz, B., Chiocchia, G., Bessis, N. et al. 1997. High susceptibility to collagen-induced arthritis in mice lacking IFN- receptors. J. Immunol. 158:5501.[Abstract]
46. Lock, C., Hermans, G., Pedotti, R. et al. 2002. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat. Med. 8:500.[CrossRef][Web of Science][Medline]
47. Aggarwal, S., Ghilardi, N., Xie, M. H., de Sauvage, F. J. and Gurney, A. L. 2003. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J. Biol. Chem. 278:1910.[Abstract/Free Full Text]
48. Cua, D. J., Sherlock, J., Chen, Y. et al. 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744.[CrossRef][Medline]
49. Zhang, G. X., Gran, B., Yu, S. et al. 2003. Induction of experimental autoimmune encephalomyelitis in IL-12 receptor-ß2-deficient mice: IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the central nervous system. J. Immunol. 170:2153.[Abstract/Free Full Text]
50. Gran, B., Chu, N., Zhang, G. X. et al. 2004. Early administration of IL-12 suppresses EAE through induction of interferon-. J. Neuroimmunol. 156:123.[CrossRef][Web of Science][Medline]
51. Langrish, C. L., Chen, Y., Blumenschein, W. M. et al. 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201:233.[Abstract/Free Full Text]

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