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CCR 7 Ligands Are Required for Development of Experimental Autoimmune Encephalomyelitis through Generating IL-23-Dependent Th17 Cells

Taku Kuwabara, Fumio Ishikawa, Takuwa Yasuda, Kentaro Aritomi, Hideki Nakano, Yuriko Tanaka, Yayoi Okada, Martin Lipp and Terutaka Kakiuchi
J Immunol August 15, 2009, 183 (4) 2513-2521; DOI: https://doi.org/10.4049/jimmunol.0800729
Taku Kuwabara
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Fumio Ishikawa
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Takuwa Yasuda
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Kentaro Aritomi
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Hideki Nakano
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Yuriko Tanaka
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Yayoi Okada
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Martin Lipp
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Terutaka Kakiuchi
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Abstract

CCL19 and CCL21 are thought to be critical for experimental autoimmune encephalomyelitis (EAE) induction, but their precise role is unknown. We examined the role of these chemokines in inducing EAE. C57BL/6 mice lacking expression of these chemokines (plt/plt mice) or their receptor CCR7 were resistant to EAE induced with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) and pertussis toxin. However, passive transfer of pathogenic T cells from wild-type mice induced EAE in plt/plt mice, suggesting a defect independent of the role of CCR7 ligands in the migration of immune cells. Examination of draining lymph node (DLN) cells from MOG35–55-immunized plt/plt mice found decreased IL-23 and IL-12 production by plt/plt dendritic cells (DCs) and a concomitant defect in Th17 cell and Th1 cell generation. In contrast, production of the Th17 lineage commitment factors IL-6 and TGF-β were unaffected by loss of CCR7 ligands. The adoptive transfer of in vitro-generated Th17 cells from DLN cells of MOG35–55-immunized plt/plt mice developed EAE in wild-type recipient mice, whereas that of Th1 cells did not. Pathogenic Th17 cell generation was restored in plt/plt DLNs with the addition of exogenous IL-23 or CCL19/CCL21 and could be reversed by inclusion of anti-IL-23 mAb in cultures. Exogenous CCL19/CCL21 induced IL-23p19 expression and IL-23 production by plt/plt or wild-type DCs. Therefore, CCR7 ligands have a novel function in stimulating DCs to produce IL-23 and are important in the IL-23-dependent generation of pathogenic Th17 cells in EAE induction.

Experimental autoimmune encephalomyelitis (EAE)5 is an autoimmune disease of the CNS in mouse and rat that serves as a disease model for human multiple sclerosis (1). EAE is induced through sensitization with neuroantigens such as myelin oligodendrocyte glycoprotein (MOG) that activates neuroantigen-reactive T cells in the peripheral lymphoid organs. Subsequent migration of these T cells into the CNS leads to their reactivation upon encountering endogenous Ag leading to nerve demyelination. Th17 cells, a helper CD4+ T cell lineage generated by IL-6, TGF-β, and IL-23, are the pathogenic cells in EAE and are capable of inducing EAE in recipient mice by passive transfer (2). The activation and activity of pathogenic T cells appear dependent on the coordinated migration of several cell types, a phenomena regulated by chemokines (3). Indeed, many chemokines have been shown to be critical for the development of EAE, mainly in the context of recruitment of immune cells into the CNS (4). Chemokines may regulate induction of pathogenic T cells independent of their role in migration of immune cells but their role in inducing pathogenic T cells in EAE is unclear.

Using mutant mice lacking the expression of CCL19 and CCL21 (plt/plt mice), we previously showed that these CC chemokine ligands, which share the CCR7, not only play a critical role in the migration of naive T cells and dendritic cells (DCs) into secondary lymphoid organs, but also regulate in vivo T cell responses to an Ag (5, 6, 7, 8). Based on these findings, we hypothesized that these chemokines regulate not only the migration of pathogenic T cells into the CNS, which was suggested previously (9), but also the induction of pathogenic T cells. In the present study, we examined the role of the CCL19 and CCL21 chemokines in the induction of EAE using plt/plt mice. Deficiency in the expression of these chemokines was protective against development of EAE due to a defect in IL-23-dependent induction of Th17 cells; however, passively transferred pathogenic T cells could initiate EAE in plt/plt mice. Thus, chemokines CCL19 and CCL21 are crucial for the induction of pathogenic T cells, independent of their role in immune cell migration. In this study, we provide the first description of a novel function of these CCR7 ligands, namely, the stimulation of DCs to produce IL-23, resulting in the generation of Th17 cells.

Materials and Methods

Mice

C57BL/6 mice were obtained from Charles River Laboratories Japan and C57BL/6-plt/plt and C57BL/6-CCR7−/− mice were bred and kept at the Toho University School of Medicine animal facility (7) under specific pathogen-free conditions in accordance with the institutional guidelines. All animal experiments were approved by the institutional review board. Mice were used at 8–12 wk of age. Where indicated, CD45.1+ C57BL/6-Ly5.1 mice were also used. These mice were maintained in our animal facility. The peripheral lymph node (LN) was much smaller in plt/plt mice than in wild-type (WT) mice (5). The mean cell number in an experiment was 4.2 × 105 cells/LN in the inguinal and axillary LNs from six naive plt/plt mice, whereas 1.5 × 106 cells/LN in naive WT mice. Nine days after EAE induction as described below, the mean cell numbers were 6.7 × 106 and 2.1 × 107 cells/LN in plt/plt and WT mice, respectively. The LN size was always macroscopically monitored before preparing LN cells.

Reagents

MOG35–55 peptide was prepared and provided by Dr. S. Imajoh-Ohmi (Institute of Medical Science, University of Tokyo, Tokyo, Japan). Allophycocyanin- or FITC-labeled anti-CD4 (RM4-5, IgG2a, κ), PE-labeled anti-IL-17 (TC11–18H10, IgG1, κ), PE-labeled rat IgG1 (R3-34, IgG1, κ) as an isotype control, and anti-IFN-γ mAb (XMG1.2, IgG1, κ) were purchased from BD Biosciences. Anti-IL-23 mAb (G23-8, IgG1, κ), anti-IL-4 mAb (11B11, rat IgG1, κ), anti-CD45.1 mAb (A20, mouse IgG2a, κ), and anti-CD45.2 mAb (104, mouse IgG2a, κ) were from eBioscience. Recombinant mouse (rm) GM-CSF was purchased from PeproTech/Tebu. rIL-12, rIL-23, rmCCL19, and rmCCL21 were from R&D Systems. rmIL-6 and TGF-β were obtained from eBioscience.

EAE induction

Mice were immunized s.c. on the flanks on day 0 with 150 μg of MOG35–55 peptide in CFA containing 5 mg/ml H37RA (Difco Laboratories). Two hundred nanograms of pertussis toxin (PT; List Laboratories) was injected i.v. on days 0 and 2. Mice were monitored for EAE development and graded on a clinical 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, severe morbidity. For passive transfer of EAE, donor mice were immunized as described above. Nine days later, draining LN (DLN) cells were cultured at 4 × 106cells/ml with 10 μM MOG35–55 peptide for 3 days in RPMI 1640 culture medium as previously described (7). Then, 1 or 3 × 107 CD4+ T cells were purified with a negative selection kit on a MACS system (Miltenyi Biotec) and were transferred i.v. into naive and 500-rad x-irradiated mice. X-irradiation was conducted using a MBR-1505R2 irradiator (Hitachi Koki). Where indicated, DLN cells from plt/plt mice were stimulated with 10 μM MOG35–55 peptide in the presence of IL-23 (10 ng/ml) and anti-IL-4 and anti-IFN-γ mAbs (5 μg/ml each) for generating Th17 cells or in the presence of IL-12 (10 ng/ml) and anti-IL-4 and anti-IL-23 mAbs (5 μg/ml each) for generating Th1 cells.

Histological analysis

On day 21 after immunization, animals were sequentially intracardially perfused with saline containing heparin (Aventis) and 4% paraformaldehyde. Spinal cords were embedded in paraffin. Transverse sections (5 μm) were prepared and stained with H&E.

In vitro recall response to MOG35–55

DLN cells from immunized mice (5 × 105cells/0.2 ml/well) were cultured for 72 h with MOG35–55. They were pulsed for 6 h with [3H]thymidine (1.25 μCi/ml; Amersham Biosciences) and assayed for incorporation of [3H]thymidine using a Matrix96 direct beta counter (Packard Instrument). Supernatants were collected at 24 h and assayed for IL-12 and IL-23 or at 72h for IL-4, IL-6, IL-10, IL-17, TGF-β, and IFN-γ with OptEIA ELISA kits (BD Biosciences). For intracellular cytokine staining, DLN cells were incubated for 48 h with 10 μM MOG35–55, followed by the addition of GolgiStop (BD Biosciences) for 5 h. Then the treated cells were stained with anti-CD4 and fixed and permeabilized using BD Biosciences Cytofix/Cytoperm followed by intracellular staining for anti-IL-17, anti-IFN-γ, or an appropriate isotype control. These cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences).

Real-time RT-PCR

Total cellular RNA was isolated from cells using EASYPrep RNA (Takara Bio). RNA (500 ng/reaction) was reverse transcribed using a High-Capacity cDNA Archive Kit (Applied Biosystems). For quantitative analysis, real-time PCR was conducted using TaqMan Gene Expression Assay Kits (Applied Biosystems), Mm00434165_m1 for IL-12 and Mm00518984_m1 for IL-23 on an Applied Biosystems Prism 7000 Sequence Detector System. GAPDH was used as an endogenous reference for normalization. Quantitative real-time PCR experiments were repeated twice in triplicate.

To analyze the expression of IL-23p19 mRNA and IL-12p35 mRNA in DLN DCs, CD11c+ cells were enriched from DLN cells using a magnetic positive selection kit (BD Biosciences).

Bone marrow-derived DCs (BMDCs)

BMDCs were prepared from bone marrow of naive C57BL/6 mice as previously described (10). Where indicated, 4 × 106 cells in 10 ml of RPMI 1640 containing rmGM-CSF (PeproTech/Tebu) were pulsed with rmCCL19 or rmCCL21 (R&D Systems).

Results

plt/plt mice lacking expression of CCL19 and CCL21 are resistant to EAE induction

To examine the role of the CCR7 ligands CCL19 and CCL21 in EAE induction, C57BL/6 WT and C57BL/6-plt/plt mice were immunized s.c. with MOG35–55 peptide in CFA on day 0 and injected i.v. with PT on days 0 and 2. As shown in Fig. 1⇓A (left), WT mice developed EAE with 100% disease incidence with onset at day 14, whereas plt/plt mice failed to develop EAE during the 42 days following immunization. Confirming the requirement for CCR7 ligand in EAE development, similarly treated CCR7−/− mice did not develop EAE (Fig. 1⇓A, right). Histological analysis of the lumbar spinal cord from WT mice 21 days after immunization clearly demonstrated cellular infiltration in the spinal parenchyma, as shown in Fig. 1⇓B. No such infiltration was observed in the spinal cord from plt/plt mice, consistent with the lack of EAE development (Fig. 1⇓B).

FIGURE 1.
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FIGURE 1.

Failure of plt/plt mice and CCR7−/− mice to develop EAE. A, Mice were s.c. immunized with MOG35–55 in CFA in the flanks and i.v. injected with PT on days 0 and 2 (10 mice/group). Clinical symptoms were monitored for 42 days after immunization. Mean clinical score ± SD is shown. Results from WT and plt/plt mice are shown in the left panel and those from WT and CCR7−/− mice in the right panel. Representative results of three independent experiments are shown. B, The lumbar spinal cords were prepared from WT and plt/plt mice 21 days after immunization as described above and stained with H&E. Similar findings were obtained from at least five different sections from each mouse. C, DLN cells were prepared from WT or plt/plt mice 9 days after immunization and incubated with the MOG35–55 peptide for 3 days. WT CD4+ T cells or plt/plt CD4+ T cells (1 × 107) prepared from the treated cells were i.v. transferred into naive and 500-rad x-irradiated WT or plt/plt mice (10 mice/group). Representative results of three independent experiments are shown as the mean EAE clinical score ± SD.

That EAE did not develop in plt/plt mice might be due to the failure of pathogenic T cells to migrate into the CNS because of the lack of CCR7 ligand expression, as suggested previously (9). To examine this possibility, 9 days after s.c. immunization, DLN cells from WT mice were incubated for 3 days with MOG35–55 peptide and then CD4+ T cells were adoptively transferred i.v. into WT and plt/plt mice. As shown in Fig. 1⇑C, both WT and plt/plt recipients developed EAE with 100% disease incidence with similar clinical scores and time courses. As expected, DLN cells from immunized plt/plt mice did not develop EAE in naive WT mice (Fig. 1⇑C). These results strongly suggest that pathogenic T cells can infiltrate the CNS to induce EAE despite the absence of CCR7 ligands but that pathogenic cells fail to be generated in plt/plt mice immunized with MOG35–55 peptide.

Deficient IL-17 and IFN-γ production by DLN cells from mice lacking CCR7 ligand expression

To examine whether pathogenic cells were generated in plt/plt mice, we first compared the in vitro recall responses of DLN cells from primed WT and plt/plt mice. DLN cells were prepared 9 days after immunization when EAE symptoms were not observed in WT mice and 14 days after immunization when the symptoms became evident. The proliferative recall responses to various doses of MOG35–55 peptide were similar between DLN cells from WT and plt/plt mice prepared 9 days and 14 days after immunization (Fig. 2⇓, upper panels), suggesting T cell responses were similarly elicited in WT and plt/plt mice. Next, we analyzed recall cytokine production to the MOG35–55 peptide. IL-4 and IL-10 were similarly produced by DLN cells from WT and plt/plt mice (Fig. 2⇓, second and third panels). Dose-dependent production of IFN-γ or IL-17 was detected in cultures of DLN cells from WT and plt/plt mice, but production of each of these cytokines was severely diminished in plt/plt DLNs (Fig. 2⇓, fourth and bottom panels). These results suggest that plt/plt T cells could be primed by immunization with the MOG35–55 peptide, but that the pattern of cytokine responses differed from that of WT mice.

FIGURE 2.
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FIGURE 2.

In vitro response to the MOG35–55 peptide of DLN cells from WT and plt/plt mice. WT and plt/plt mice were immunized as described in the legend for Fig. 1. DLN cells prepared 9 or 14 days after immunization were incubated with the MOG35–55 peptide at the indicated doses and assessed for proliferation by [3H]thymidine incorporation on day 3. The amounts of IL-4, IL-10, IFN-γ, and IL-17 in the culture supernatants were determined by ELISA using OptEIA kits (BD Biosciences). Each result is expressed as the mean ± SD.

For the optimal induction of IL-17-producing cells, IL-6, TGF-β, and IL-23 are required (11, 12, 13). IL-12 is critical for inducing IFN-γ-producing cells (14). Deficient production of IL-17 and IFN-γ suggested that these cytokines were insufficiently produced in DLN cells from plt/plt mice. As shown in Fig. 3⇓A, in DLN cells prepared from WT and plt/plt mice 4 or 9 days after immunization similar levels of IL-6 and TGF-β production were observed following incubation with the MOG35–55 peptide. In contrast, as shown in Fig. 3⇓, B and C, the expression of IL-23p19 mRNA and IL-12p35 mRNA and production of IL-23 and IL-12 were much lower in the cells from plt/plt mice than in WT mice, suggesting that the defect in production of IL-17 and IFN-γ in DLN cells from plt/plt mice was due to insufficient provision of IL-23 and IL-12.

FIGURE 3.
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FIGURE 3.

Decreased production of IL-12 and IL-23 in DLN cells from plt/plt mice. DLN cells were prepared 4 or 9 days after immunization as described in the legend for Fig. 1. A, DLN cells were incubated for 3 days with MOG35–55 at the indicated doses. Culture supernatants were assessed for TGF-β and IL-6. Results are plotted as the mean ± SD. B, Expression of IL-23p19 mRNA and IL12p35 mRNA in CD11c+ cells was estimated by quantitative RT-PCR in DLN cells from WT and plt/plt mice. The expression is shown as the mean ± SD of the ratio to GAPDH, an internal control. These experiments were repeated five times with similar results. C, DLN cells from naive mice or 4 days after immunization were incubated with 10 μM MOG35–55 for 24 h. Culture supernatants were assessed for IL-23 and IL-12. Results of triplicate assay are presented as the mean ± SD.

Requirement for CCR-7 ligand in generation of IL-17 or IFN-γ-secreting T cells

Reduced in vitro IL-17 and IFN-γ production by DLN cells from plt/plt mice suggested a defect in Th17 and Th1 cell generation. To examine this possibility, DLN cells were prepared 9 days after immunization, incubated with MOG35–55 peptide, and assessed for intracellular IL-17 or IFN-γ staining. As shown in Fig. 4⇓A, CD4+IL-17+ Th17 cells were found at a much lower frequency in DLN cells from plt/plt mice than in those from WT mice (0.4% vs 4.2%). Addition of CCL19 or CCL21 to DLN cells from plt/plt mice during incubation with the MOG35–55 peptide restored Th17 cell generation from 0.4% to 3.0 or 4.1%, respectively (Fig. 4⇓A). Also, the frequency of CD4+IFN-γ+ Th1 cells was much lower in plt/plt mice than in WT mice (0.4% vs 4.4%). The addition of CCL19 or CCL21 restored Th1 cell generation in plt/plt mouse DLN cells from 0.4% to 3.1 or 3.2%, respectively (Fig. 4⇓A). These results support the hypothesis that the defect in generating Th17 or Th1 cells in plt/plt mice was due to the lack of CCR7 ligand expression.

FIGURE 4.
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FIGURE 4.

Analysis of the T cell response in DLNs from WT and plt/plt mice immunized for EAE induction and generation of Th17 or Th1 cells by the CCR7 ligand or IL-23. DLN cells were prepared from WT and plt/plt mice 9 days after immunization as described in the legend for Fig. 1. A, DLN cells were incubated with MOG35–55 in the presence or absence of CCL21 or CCL19 (100 ng/ml), then assessed for intracellular IL-17 or IFN-γ expression. Numbers in the right quadrants are the percentage of the total cells. B, CD4+ DLN cells enriched from immunized plt/plt mouse spleen were stimulated with immobilized anti-CD3 and anti-CD28 mAbs for 2 days in the presence or absence of rIL-23 (10 ng/ml) and analyzed for intracellular IL-17. C, DLN cells from plt/plt mice were incubated with the MOG35–55 peptide for 2 days in the presence or absence of CCL21 or CCL19 alone or with anti-IL-23 mAb. The cells were analyzed for CD4 expression and intracellular IL-17.

Previous reports demonstrated that the neuroantigen-specific Th17 or Th1 cell is responsible for EAE induction (2, 15, 16). To determine which defect in generating Th17 or Th1 cells was more critical in the resistance to EAE development, DLN cells from plt/plt mice were stimulated in vitro with the MOG35–55 peptide under the conditions for generating Th17 cells or Th1 cells, enriched for CD4+ T cells, and transferred into WT mice. As shown in Fig. 5⇓A, CD4+ T cells containing Th17 cells (CD4+IL-17+cells, 9.2%; CD4+IFN-γ+ cells, 0.1%; Fig. 5⇓C) induced EAE in the recipient mice with 100% disease incidence, whereas those containing Th1 cells (CD4+IL-17+cells, 0.1%; CD4+IFN-γ+ cells, 11.0%; Fig. 5⇓C) did not, suggesting that Th1 cells are less efficient at inducing EAE. The cell preparation containing Th17 or Th1 cells was confirmed to predominantly produce IL-17 or IFN-γ, respectively (Fig. 5⇓, D and E). The cells similarly prepared from WT mice and enriched for Th1 cells (CD4+IL-17+cells, 0.6%; CD4+ IFN-γ+ cells, 20.3%) also failed to elicit EAE in the recipient mice, whereas those containing Th17 cells (CD4+IL-17+cells, 19.2%; CD4+IFN-γ+ cells, 0.4%) elicited EAE (Fig. 5⇓B). These findings strongly support our interpretation that the defect in generating Th17 cells is crucial in the resistance to EAE development in plt/plt mice under the conditions used.

FIGURE 5.
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FIGURE 5.

A Th17-enriched, rather than Th1-enriched, cell population was responsible for EAE development in recipient mice. A and B, DLN cells from primed plt/plt (A) or WT (B) mice were incubated with the MOG35–55 peptide for 3 days in the presence of CCL19, IL-12, and anti-IL-4 and anti-IL-23 mAbs for developing Th1 cells, in the presence of CCL19, IL-23, and anti-IL-4 and anti-IFN-γ mAbs for developing Th17 cells, or in the presence of IL-23 alone. CD4+ T cells (1 × 107) prepared from the treated cells were i.v. transferred into naive and 500-rad x-irradiated WT mice (10 mice/group). EAE development is shown as a mean EAE clinical score ± SD. C, Before transfer, Th1-skewed or Th17-skewed cells prepared from plt/plt mice as described above were assessed for intracellular IL-17 or IFN-γ expression on a flow cytometer. Numbers in the right lower and left upper quadrants are the percentage of the total cells. D and E, Aliquots of these cells were incubated in the presence of MOG35–55 and assessed for production of IL-17 (D) and IFN-γ (E) as described in the legend for Fig. 2.

IL-23-dependent induction of pathogenic Th17 cells

Deficient IL-23 production in DLN cells from plt/plt mice led us to evaluate the role of IL-23 in inducing Th17 cells. The addition of exogenous IL-23 to CD4+ DLN cells from immunized plt/plt mice stimulated with immobilized anti-CD3 and anti-CD28 mAbs increased the frequency of Th17 cells from 0.18 to 1.34% (Fig. 4⇑B), supporting the idea that the defect in developing Th17 cells in plt/plt mice was due to reduced production of IL-23. To confirm that stimulation with IL-23 was able to induce pathogenic T cells in EAE induction, DLN cells from immunized plt/plt mice were incubated with the MOG35–55 peptide in the presence of IL-23, enriched for CD4+ T cells, and adoptively transferred into naive WT mice, which resulted in the development of EAE in the recipient mice (Fig. 5⇑A). These results suggested that exogenous IL-23 was able to stimulate plt/plt mouse DLN cells along with the MOG35–55 peptide to induce pathogenic Th17 cells, consistently, with the critical role of IL-23 in the induction phase of EAE (17). Taken all together, these findings suggest that the defect in plt/plt mice is likely a defect in Th17 cell generation due to deficient IL-23 production.

CCL19 and CCL21 stimulate DCs to produce IL-23

DCs are known to produce IL-23 (18). The reduced production of IL-23 in the incubation of plt/plt DLN cells with MOG35–55 suggests the dependency of the IL-23 production on CCR7 ligands. To confirm this possibility, we prepared BMDCs and stimulated the cells with CCR7 ligands or other chemokines. LPS (100 ng/ml) was used as a positive control for induction of IL-23p19 mRNA (18). CCL19 or CCL21 at 100 ng/ml increased IL-23p19 mRNA expression, although not to the same extent as LPS (Fig. 6⇓A, left and middle). The chemokines CCL5 and CXCL12 did not stimulate BMDCs to produce IL-23 (Fig. 6⇓A, left). Confirming that CCL19 and CCL21 stimulate DCs through CCR7 to express IL-23p19mRNA, BMDCs from CCR7−/− mice did not respond to the chemokines (Fig. 6⇓A, right).

FIGURE 6.
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FIGURE 6.

CCR7 ligands stimulate DCs to express IL-23p19 mRNA and to produce IL-23. A, BMDCs were prepared from WT, plt/plt, and CCR7−/− mice and stimulated with LPS or the indicated chemokines at 100 ng/ml for 6 h. Cellular RNA was prepared from each cell population and IL-23p19 mRNA expression was evaluated by quantitative RT-PCR. The expression is shown as the mean ± SD of the ratio to GAPDH, an internal control. B, DLN cells were prepared 4 days after immunization from WT, plt/plt, and CCR7−/− mice and incubated with 10 μM MOG35–55 peptide in the presence or absence of CCL19 or CCL21 for 6 h. CD11c+ cells were enriched with a positive selection kit (BD Biosciences) by MACS. CD11c+ cells were 89.2, 92.2, and 90.4% for WT, plt/plt, and CCR7−/− mice, respectively. Cellular RNA was prepared from each cell population and assessed for IL-23p19 mRNA expression by quantitative RT-PCR. Controls were LN cells from naive mice. Expression is shown as the mean ± SD of the ratio to GAPDH as an internal control. C and D, BMDCs (C) or DLN cells (D) from WT and plt/plt mice were stimulated as described above for 24 h. The supernatants were assessed for IL-23 using an ELISA kit. Results are shown as the mean ± SD of triplicate assays.

We also assessed IL-23p19 mRNA expression by DCs in DLNs in response to CCR7 ligands. DLN cells from immunized WT, plt/plt, or CCR7−/− mice were incubated with the MOG35–55 peptide for 6 h in the presence or absence of CCL19 or CCL21. Then CD11c+ cells were enriched and assayed for IL-23p19 mRNA expression. As shown in Fig. 6⇑B (left), CD11c+ cells from WT mice expressed much higher IL-23p19 mRNA than those from naive mice, and the addition of CCL19 did not further enhance IL-23p19 mRNA expression in these cells from immunized WT mice, probably because they had been exposed to CCL19 produced in DLNs. In CD11c+ cells from plt/plt mice, however, the addition of exogenous CCL19 or CCL21 increased IL-23p19 mRNA expression (Fig. 6⇑B, middle). As expected, cells from CCR7−/− mice did not respond to the addition of CCR7 ligands (Fig. 6⇑B, right).

CCR7 ligands also stimulated IL-23 production by BMDCs from WT and plt/plt mice and by DLN cells from plt/plt mice (Fig. 6⇑, C and D). DLN cells alone from immunized WT mice produced much more IL-23 than those from naive WT mice, probably because endogenous CCR7 ligands induced a sufficient level of IL-23 production (Fig. 6⇑D). Taken together, the results shown in Fig. 6⇑ demonstrate that CCL19 or CCL21 is necessary and sufficient to induce IL-23 production from DCs. Confirming that IL-23 production in response to a CCR7 ligand plays a critical role in Th17 induction, in a dose-dependent fashion anti-IL-23 mAb inhibited Th17 cell generation following incubation of DLN cells from plt/plt mice with the MOG35–55 peptide in the presence of CCL19 or CCL21 (Fig. 4⇑C).

IL-12p35 mRNA expression and IL-12 production in BMDCs from plt/plt mice were also induced by the addition of exogenous CCL19 or CCL21 (data not shown).

It was also possible CCR7 ligands directly stimulated CD4+ T cells to produce IL-17. However, this seemed unlikely since CD4+ T cells isolated from naive plt/plt mice or plt/plt mice primed with the MOG35–55 peptide were not induced to produce IL-17 in response to immobilized anti-CD3 and anti-CD28 mAbs in the presence of exogenously added CCL19 or CCL21 (data not shown). We concluded that CCR7 ligands stimulated DCs to produce IL-23, which in turn resulted in Th17 differentiation.

Pathogenic T cell induction by incubation of DLN cells from primed plt/plt mice with CCR7 ligands

To determine the pathogenicity of DLN T cells from plt/plt mice that had been incubated with CCR7 ligands under EAE-inducing conditions, 9 days after immunization, DLN cells were incubated for 3 days with the MOG35–55 peptide in the presence of CCL19 or CCL21. CD4+ T cells were enriched from the treated cells and i.v. injected into naive WT mice. As shown in Fig. 7⇓A, the recipient mice developed EAE with >70% disease incidence. Thus, stimulation of DLN cells from primed plt/plt mice with the peptide in the presence of a CCR7 ligand increased pathogenicity.

FIGURE 7.
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FIGURE 7.

Restoration of pathogenic T cells by incubation with CCR7 ligands. DLN cells from immunized plt/plt mice (CD45.2+) were incubated at 4 × 106 cells/ml with 10 μM MOG35–55 peptide in the presence of CCR7 ligands (100 ng/ml) for 3 days. A, CD4+ T cells (3 × 107) prepared from the treated cells were i.v. transferred into naive and 500-rad x-irradiated WT mice and mice were monitored for EAE (10 mice/group). A mean ± SD of EAE clinical score is plotted. The EAE incidence was 0% for recipients of cells incubated in the absence of CCR7 ligands, 70% for those in the presence of CCL19, and 80% for those in the presence of CCL21. Data representative of three independent experiments are shown. B, CD4+ T cells prepared from the treated cells were analyzed for expression of intracellular IL-17 and IFN-γ on a flow cytometer (pretransfer). They were transferred into CD45.1+ C57BL/6-Ly5.1 mice. Fourteen days after (posttransfer), spleen cells from recipient mice were prepared and donor cells (CD45.2+) were gated and analyzed as above. Numbers in the left upper and right lower quadrants are the percentage of the total CD4+ or CD45.2+ cells for pretransfer or posttransfer, respectively.

Finally, CD4+ T cells recovered from WT recipient mice were analyzed for intracellular IFN-γ and IL-17. DLN cells from primed plt/plt mice (CD45.2+) were incubated with MOG35–55 alone or in the presence of CCL21 or IL-23 and enriched for CD4+ T cells. In these cells, IL-17+ cells were 0.1, 3.5, or 4.5% and IFN-γ+ cells were 0.2, 4.7, or 0.4%, respectively (Fig. 7⇑B, left column). Then, treated cells were transferred into CD45.1+ WT mice. Fourteen days after, in CD45.2+ spleen cells in the recipients of plt/plt DLN cells incubated with MOG35–55 alone or in the presence of CCL21 or IL-23, CD4+IL-17+ were 0.1%, 1.3%, or 1.2%, and CD4+IFN-γ+ T cells were 0.1%, 1.3%, or 0.1%, respectively (Fig. 7⇑B, right column). These results support that Th17 cells transferred survived in recipient mice at least until EAE was detectable in the recipient mice and could be pathogenic for EAE development.

Discussion

In the present study, mice lacking the expression of the CCR7 ligands CCL19 and CCL21 (plt/plt mouse) or lacking expression of CCR7 failed to develop EAE following a standard immunization protocol with which WT mice developed EAE. The dependency of EAE development on CCR7 ligands is not due to a defect in the migration of pathogenic T cells in plt/plt mice, since adoptive transfer of pathogenic CD4+ T cells prepared from DLN cells of WT mice result in EAE development in plt/plt and WT recipient mice with similar time course and disease severity. Instead, a defect was observed in the ability of mice lacking CCL19 and CCL21 or lacking CCR7 to develop pathogenic Th17 cells. Consistently, expression of IL-23p19mRNA and IL-23 production were decreased in DLN cells from immunized plt/plt mice (Fig. 3⇑). IL-23 has been shown to be a critical Th17 growth and survival factor (11, 12, 13). The expression of IL-23p19mRNA and production of IL-23 by DCs from plt/plt mice were recovered by the addition of exogenous CCL19 or CCL21 (Fig. 6⇑). T cell responses to the MOG35–55 peptide in DLNs from plt/plt mice were not too low to generate pathogenic T cells. Proliferation and IL-4 production in recall responses were quite similar in plt/plt and WT mice. We conclude that the failure to generate pathogenic T cells in plt/plt mice was due to a defect in induction of pathogenic effecter T cells rather than an insufficient response to the MOG35–55 peptide. Thus, chemokines CCL19 and CCL21 play a critical role for the IL-23-dependent generation of pathogenic Th17 cells, rather than in the migration of pathogenic T cells into the CNS.

IFN-γ and IL-12 production was also decreased in DLN cells from plt/plt mice (Figs. 2⇑ and 3⇑, B and C). The decrease in the frequency of intracellular IFN-γ+CD4+ T cells and in IL-12 production by DCs was recovered by the addition of exogenous CCL19 or CCL21 (Fig. 4⇑A and data not shown), as shown previously (19), suggesting that the defect to generate MOG35–55-specific Th1 cells could be included in the resistance to EAE development in plt/plt mice as reported very recently (15, 16). However, this possibility is unlikely since DLN cells prepared from plt/plt mice and incubated under the conditions for generating Th1 cells could not develop EAE in naive WT recipient mice, whereas those incubated under the Th17-generating conditions did develop EAE (Fig. 3⇑B). Based on these findings, we conclude that the failure to develop EAE in plt/plt mice was due to a defect in generating IL-23-dependent Th17 cells. The reason for the discrepancy between our results and those in which IL-12-dependent Th1 cells are able to transfer EAE (16) is presently unknown. It might be due to different conditions for immunization. Our priming mice once, not twice, with the MOG35–55 peptide might be suboptimal for EAE induction, which resulted in the inability for Th1-skewed T cells to transfer EAE to recipient mice, as described previously (15). Th17 cells have been shown to induce greater encephalitogenic responses on a cell-cell basis, following passive transfer, when compared with Th1 cells (2). Several recent reports support the interpretation that pathogenic Th17 cells induce EAE more efficiently than IL-12-dependent Th1 cells (15, 17, 20, 21, 22).

In a previous report where mice were immunized twice with the MOG35–55 peptide in CFA and injected i.v. with PT on days 7 and 9, CCR7−/− mice developed EAE with very similar disease severity to WT mice (23). In plt/plt mice, we observed EAE development with two immunizations of the MOG35–55 peptide, but the mean clinical score for disease severity in plt/plt mice was less than half that of WT mice (data not shown). The reason for the discrepancy between our results and those by Pahuja et al. (23) is presently unknown; however, low-level expression of CCL21-leucine in plt/plt mice (24) might account for the discrepancy. The difference in immunizing protocols also might be an explanation. Repeated immunization of plt/plt mice may increase production of IL-6 and TGF-β, thereby contributing to further generation of Th17 cells and development of EAE (9, 11, 12). Alternatively, stimulation with CXCL9, CXCL10, and/or CXCL11 via CXCR3, an additional receptor for mouse CCL21 (25, 26, 27), might partially substitute for CCR7 stimulation in heavily immunized plt/plt or CCR7−/− mice. These possibilities might also explain why DLN cells from plt/plt mice produced a small but detectable amount of IL-17 (Fig. 2⇑).

Failure to develop EAE in plt/plt mice was due to the inefficient generation of Th17 cells, which mainly resulted from deficient IL-23 production, indicating a critical role for CCR7 ligands in IL-23 production. Indeed, CCL19 and CCL21 increased IL-23 production by DCs (Fig. 5⇑). In WT mice, DCs constitutively secrete CCL19 (28, 29), which might stimulate these cells to produce IL-23 in an autocrine manner. Consistently, DCs in DLN cells from WT mice exhibited a higher background level of IL-23p19 mRNA expression and IL-23 production than those from plt/plt mice (Fig. 5⇑, B and C). In recent studies, several reagents, including whole pathogens, have been found to stimulate DCs to produce IL-23 (30). Serum amyloid A, an acute phase protein, PGE2, and phagocytosis of apoptotic neutrophils, each of which are found in inflammatory conditions, have been shown to generate IL-23 production by DCs (31, 32, 33, 34). These stimulators might enhance the constitutive production of CCL19 by DCs, which results in augmentation of IL-23 production in an autocrine manner. Thus, under these inflammatory conditions, CCR7 ligands might play an important role in generation or enhancement of IL-23 production by DCs.

Very recently, it has been reported that the enhancement of IL-13 production inhibits IL-23 production and increases the Th2-type immune response, which is accompanied by a reduction in EAE development (35). In the present study, IL-4 production by DLN cells from immunized plt/plt mice was comparable to that of WT mice, whereas IFN-γ production was much lower in plt/plt mice (Fig. 2⇑). The relative dominance of the Th2-type response might further suppress EAE development in plt/plt mice.

It has been also shown recently that IL-21 is required for full commitment of Th17 cells (36, 37, 38). In these reports, IL-21 is produced by Ag-stimulated and committed Th17 cells and increases IL-23 receptor expression on these cells in an autocrine manner. In the present study, the CCR7 ligand increased the induction of IL-23-dependent Th17 cells in plt/plt mouse DLN cells to levels seen in WT mice (Fig. 4⇑, A and D), indicating that the generation of IL-23 in response to CCR7 ligands is required for Th17 cell generation. Supporting this interpretation, recent reports have demonstrated that IL-23 is critically important in inducing Th17 cells (17, 39, 40). Taken together, it is possible that CCR7 ligands might also increase IL-21 production and IL-23 receptor expression for the optimal induction of Th17 cells. We cannot formally exclude this possibility although we could not detect a direct effect of the CCR7 ligand on CD4+ T cells stimulated with anti-CD3 and anti-CD28 Abs (data not shown).

In conclusion, plt/plt mice fail to develop EAE due to a defect in IL-23 production by DCs, which in turn results in inefficient generation of pathogenic Th17 cells. Thus, we have demonstrated a novel role for the chemokines CCL19 and CCL21 in the generation of Th17 cells and EAE induction independent of their role in the migration of immune cells. Our data suggest that manipulation of IL-23 production through regulation of the CCR7 ligands could be a new strategy to control human multiple sclerosis.

Acknowledgments

We thank Dr. T. Hasegawa (Ohno Chuo Hospital, Ichikawa, Japan) for support.

Disclosures

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 in part by Project Research of Toho University School of Medicine (to T.Ku., F.I., and Y.O.), the Research Promotion Grants from Toho University Graduate School of Medicine (Grant 05-02 to T.Ka., Grant 07-02 to T.Ku., and Grant 08-02 to Y.T.), and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to T.Ka. (Grants 17590900 and 19591013), to T.Ku. (Grant 18790605), and to Y.K. (Grant 19790695).

  • ↵2 Current address: Laboratory for Immunogenetics, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan.

  • ↵3 Current address: Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences. National Institutes of Health, 111 T.W. Alexander Drive, Building 101, E244, Research Triangle Park, NC 27709.

  • ↵4 Address correspondence and reprint requests to Dr. Terutaka Kakiuchi, Department of Immunology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan. E-mail address: tkaki{at}med.toho-u.ac.jp

  • ↵5 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; DC, dendritic cell; BMDC, bone marrow-derived DC; LN, lymph node; DLN, draining LN; MOG, myelin oligodendrocyte glycoprotein; rm, recombinant mouse; WT, wild type; PT, pertussis toxin.

  • Received March 6, 2008.
  • Accepted June 21, 2009.
  • Copyright © 2009 by The American Association of Immunologists, Inc.

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The Journal of Immunology: 183 (4)
The Journal of Immunology
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15 Aug 2009
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CCR 7 Ligands Are Required for Development of Experimental Autoimmune Encephalomyelitis through Generating IL-23-Dependent Th17 Cells
Taku Kuwabara, Fumio Ishikawa, Takuwa Yasuda, Kentaro Aritomi, Hideki Nakano, Yuriko Tanaka, Yayoi Okada, Martin Lipp, Terutaka Kakiuchi
The Journal of Immunology August 15, 2009, 183 (4) 2513-2521; DOI: 10.4049/jimmunol.0800729

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CCR 7 Ligands Are Required for Development of Experimental Autoimmune Encephalomyelitis through Generating IL-23-Dependent Th17 Cells
Taku Kuwabara, Fumio Ishikawa, Takuwa Yasuda, Kentaro Aritomi, Hideki Nakano, Yuriko Tanaka, Yayoi Okada, Martin Lipp, Terutaka Kakiuchi
The Journal of Immunology August 15, 2009, 183 (4) 2513-2521; DOI: 10.4049/jimmunol.0800729
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