Acquired thymic tolerance to autoimmune encephalomyelitis is associated with activation of peripheral IL-10-producing macrophages/dendritic cells
Yoshio Okura1,
Youngheun Jee1 and
Yoh Matsumoto1
1 Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6 Fuchu, Tokyo 183-8526, Japan
Correspondence to: Y. Matsumoto; E-mail: matyoh{at}tmin.ac.jp.
Transmitting editor: H. R. MacDonald
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Abstract
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Antigen injection into the thymus of adult animals induces systemic tolerance and protects animals from subsequent challenge for the autoimmune disease. However, its mechanisms are not well understood. In this study, we analyzed tolerance to experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by intrathymic (i.t.) injection of myelin basic protein (MBP). Intrathymic injection of MBP 7 days before immunization with MBP/complete Freunds adjuvant resulted in complete suppression of clinical signs of EAE in most animals and markedly reduced the histological severity in the central nervous system lesion. However, immunohistochemical examination and the TCR repertoire analysis revealed that there was no significant difference in the T cell composition in the lesion and the TCR spectratype pattern between MBP and saline i.t. rats, suggesting that encephalitogenic T cell activation occurs equally in both protected and symptomatic rats. In contrast, quantitative analysis of cytokine mRNA and flow cytometry revealed a marked increase of IL-10 production in the splenic macrophages/dendritic cell (Mø/DC) population of MBP i.t. rats. Adoptive transfer of this population significantly suppressed the clinical course of EAE in recipients. Taken together, IL-10-secreting Mø/DC in peripheral lymphoid organs activated by MBP i.t. injection may play a critical role in the induction and maintenance of tolerance.
Keywords: acquired thymic tolerance, dendritic cell, experimental autoimmune encephalomyelitis, IL-10
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Introduction
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The majority of autoreactive T cells are deleted during maturation in the thymus by negative selection. Although some T cells that react with autoantigens escape from this deletion process, undergo differentiation and move to the periphery, they are in an unresponsive state by several mechanisms (1,2). First, regulatory cells including CD25+CD4+ T and NK cells are involved to maintain this steady sate because deletion of these types of regulatory cells induces spontaneous development of organ-specific autoimmune diseases (3,4) and exacerbates experimental autoimmune encephalomyelitis (EAE) (5,6). A second group of regulatory cells plays an important role in antigen-specific tolerance induced by oral, nasal or intrathymic (i.t.) administration of antigen. Transforming growth factor (TGF)-ß-secreting Th3 cells induced by oral tolerance (7) and IL-10-producing Tr1 cells (8) are reported to be responsible for the maintenance of antigen-induced tolerance. In addition, dendritic cells (DC) also play an important role for the induction of tolerance by direct tolerization (9) or by differentiating other regulatory cells (10). In contrast to CD25+CD4+ T and NK cells, the characterization of antigen-specific tolerance-associated T cells and DC has been performed mainly using cultured cells or cell lines. Therefore, their roles in situ remain poorly understood.
Acquired thymic tolerance is induced and maintained by several immunological processes. Injection of antigen into the thymus induces profound apoptosis of antigen-reactive T cells in the thymus (11). During this process, not all the responding T cells undergo apoptosis and some surviving T cells migrate to the periphery. However, these antigen-specific T cells are in an anergic state and do not respond to the injected antigen (12). These findings strongly suggest that the suppressive machinery is induced by i.t. injection of antigen and exists in the periphery.
To analyze the mechanisms of acquired thymic tolerance in more detail, we induced tolerance in Lewis rats by i.t. injection of guinea pig myelin basic protein [MBP (GPBP)]. These rats become resistant to EAE and do not develop clinical signs after challenge with GPBP/complete Freunds adjuvant (CFA). In the present study, we tried to analyze in vivo immunological events as much as possible and found that IL-10, but not TGF-ß, mRNA is generally up-regulated in peripheral lymphoid organs of protected animals and that the major IL-10-producing cell population in the spleen is macrophages (Mø)/DC. Moreover, adoptive transfer of the non-T cell, but not of the T cell, population isolated from the spleens of GPBP i.t. rats to naive rats reduced the severity of active EAE in recipients. These findings suggest that GPBP i.t. injection induced the production of IL-10 by Mø/DC in the peripheral organ, and that these resident Mø/DC play a central role in the induction and maintenance of acquired thymic tolerance.
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Methods
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Animals and antigen
Female Lewis rats were purchased from Seiwa (Fukuoka, Japan) and used at 68 weeks of age. GPBP was purified as described previously (13).
GPBP and saline i.t.
The i.t. injections were performed under ether anesthesia. The thymus was exposed through a small anterior sternal incision. Then, 150 µg GPBP dissolved in 50 µl saline was injected into each thymus lobe (total 300 µg in 100 µl) via a 30-G needle. In control groups, the same volume of saline was injected.
Induction of EAE
EAE was induced by immunization with an emulsion containing 100 µg GPBP and CFA (Difco, Detroit, MI) in bilateral footpads. At 1, 3, and 7 days after GPBP or saline i.t., rats were challenged for EAE and clinical signs were observed daily. The clinical stage of EAE was divided into four grades (grade 1, floppy tail; grade 2, mild paraparesis; grade 3, severe paraparesis; grade 4, tetraparesis or moribund condition) (14).
Histological studies
Rats were killed under ether anesthesia on days 11 and 14 post-immunization. Three segments of the lumbar spinal cord were snap-frozen in OCT compound. Sections (10 µm) stained with hematoxylin & eosin were used for histological examination. Histological findings were graded into four categories (grade 1, leptomeningeal and adjacent subpial cell infiltration; grade 2, mild perivascular cuffing; grade 3, extensive perivascular cuffing; grade 4, extensive perivascular cuffing and severe parenchymal cell infiltration) (14).
Immunohistochemical staining procedures
A single immunoperoxidase staining was performed as described previously (14,15). Briefly, frozen sections were air-dried and fixed in ether for 10 min. After incubation with normal horse serum, sections were allowed to react with one of the following mAb; R73 (anti-TCR
ß), R78 (anti-Vß8.2), G101 (anti-Vß10), ED1 (anti-Mø) and OX62 (anti-DC) (16). After washing, sections were incubated with biotinylated horse anti-mouse IgG (Vector, Burlingame, CA) followed by a horseradish peroxidase (HRP)-labeled Vectstain Elite ABC kit (Vector). HRP binding sites were detected in 0.005% diaminobenzidine and 0.01% hydrogen peroxide.
cDNA synthesis and PCR amplification
Total RNA was extracted from the thymus, lymph node, spleen, peripheral blood lymphocytes (PBL) and spinal cord using RNAzol B (Biotecx, Houston, TX). cDNA was then synthesized by reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan) and amplified in a thermal cycler (Perkin-Elmer, Norwalk, CT) using primer pairs for TCR and cytokines. The primers for TCR and cytokines were the same as those used in previous studies (17,18). Cycling conditions for PCR were as follows: 93°C for 1 min for denaturation, 55°C for 1 min for annealing and 72°C for 1 min for extension, followed by 40 cycles of 93°C for 1 min, 55°C for 1 min and 72°C for 1 min. PCR products were then electrophoresed on 1.5% agarose gels containing ethidium bromide and analyzed with a fluorescence image analyzer on the FMBIO software (Hitachi Software Engineering, Yokohama, Japan).
Complementarity-determining region (CDR) 3 spectratyping analysis
CDR3 spectratyping was performed as described previously (1921). cDNA was amplified with Vß-specific and rhodamine-labeled Cß primers, and undiluted PCR products were added an equal volume of formamide/dye loading buffer and heated at 94°C for 2 min. The samples were applied to 6% acrylamide sequencing gels. Gels were run at 30 W for 2 h 30 min at 50°C. Then, the fluorescence-labeled DNA profile on the gels was directly recorded using a FMBIO fluorescence image analyzer (Hitachi Software Engineering).
Establishment of GPBP-specific T cell lines (TCL)
GPBP-specific TCL were established from lymph node cells taken from GPBP-immunized rats by cycle stimulation with GPBP and mitomycin C-treated thymocytes as antigen-presenting cells (APC). Between antigen stimulations, T cells were propagated in culture medium containing 5% concanavalin A supernatant.
Proliferative responses of lymph node cells against MBP
Proliferative responses of lymph node cells were assayed in microliter wells by uptake of [3H]thymidine. The lymph nodes were taken on 14 days post-immunization from the i.t, injected rats and single-cell suspensions were prepared. After being washed with PBS, lymph node cells (2 x 105 cells/well) were cultured with the indicated concentrations of GPBP for 3 days, with the last 18 h in the presence of 0.5 µCi [3H]thymidine (Amersham Pharmacia Biotech, Tokyo, Japan). In some assays, rat IL-2 (Collaborative Research, Bedford, MA) was added at a concentration of 10 U/ml.
Using GPBP-specific TCL, the antigen-presenting ability of spleen cells taken from rats that had received GPBP or saline i.t, was evaluated in the presence of various concentrations of GPBP. TCL (3 x 104 cells/well) were cultured with the antigens and spleen cells (5 x 105 cells/well). The cells were harvested on glass fiber filters and the label uptake was determined using standard liquid scintillation techniques.
Competitive PCR analysis of cytokines
We determined the amount of mRNA of pro-inflammatory cytokines [tumor necrosis factor (TNF)-
and IFN-
] and anti-inflammatory cytokines (IL-4, IL-10 and TGF-ß1) by competitive PCR as described previously (18). In this analysis, the competitor DNA and target templates were amplified using the same primer pair in the same reaction. After amplification, PCR products were electrophoresed and analyzed with the fluorescence image analyzer. The amount of target mRNA was estimated from the amount of the competitor DNA added to the reaction. The densities of the target and competitor bands for each reaction were measured, and the ratios of target DNA to competitor DNA were calculated. The concentration of target DNA was determined as the point at which the ratio of the target to the competitor was 1:1. In some experiments, T cells, B cells and Mø/DC were isolated by positive selection using BioMag (Polysciences, Warrington, PA) conjugated with R73, OX33 and OX42 respectively, and subjected for the cytokine analysis.
Cytokine ELISA
For cytokine assay at the protein level, rats were injected i.t, with GPBP or saline (n = 3 in each group). Seven days later, spleens were removed and spleen cells at a dose of 2 x 106 cells/well were cultured in a 48-well plate in the presence or absence of lipopolysaccharide (10 ng/ml) for 48 h. Culture supernatants were collected and the levels of IL-10 were determined using an IL-10 ELISA kit (Biosource, Camarillo, CA) according to manufacturers instructions.
Flow cytometry
Flow cytometric analysis was performed to identify the cell type of IL-10-secreting cells. Intracellular staining of IL-10 was performed using CytoStain kits (PharMingen, San Diego, CA) with a few modifications. Spleen cells isolated from rats that had been injected with GPBP i.t. 7 days earlier were stimulated with 500 ng/ml ionomycin (Sigma), 25 ng/ml phorbol myristate acetate (Sigma) and 1 µl/ml GogiPlug for 2 h at 37°C. After stimulation, cells were washed and stained with FITC- conjugated R73 (anti-TCR
ß), OX42 (anti-Mø/DC) or OX62 (anti-DC) for 15 min. Then, cells were fixed with 4% paraformaldehyde and incubated with biotinylated mouse anti-rat IL-10 mAb (PharMingen) in Perm/Wash solution for 15 min followed by incubation with streptavidinPerCp (Becton Dickinson, Mountain View, CA). The percentages of IL-10-secreting cells which were stained positively with mAb against T cells, Mø/DC and DC were determined using a FACScan (Becton Dickinson).
Adoptive transfer of spleen cells from GPBP i.t. rats
Adoptive transfer experiments were performed to determine whether spleen cells from thymic tolerant donors are able to suppress the development of EAE in naive recipients. Donor rats were inoculated i.t. with GPBP. Seven days later, spleen cell suspensions were prepared, and sorted into T cell and non-T cell populations by negative selection using BioMag conjugated with R73 (anti-TCR
ß) and with OX33 (anti-B cell) plus OX42 (anti-Mø/DC) respectively. These cell populations (25 x 108 cells) were injected i.v. to naive rats. Then, recipients were immunized with GPBP/CFA on the following day to observe the onset of the disease and the clinical score of EAE.
Statistical analysis
Statistical analysis was performed by Students t-tests. Significant differences were judged to be at P < 0.05.
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Results
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GPBP i.t. 7 days before immunization effectively protects animals from EAE
In initial studies, the dose of GPBP to be injected in the thymus and timing of administration were determined according to the findings reported previously (22,23). However, sufficient suppressive effects were not obtained. Then, we tried to determine the optimal protocol for the induction of effective acquired thymic tolerance. GPBP was injected into the thymus 7, 3 and 1 days before immunization, and the animals were challenged with GPBP/CFA and observed daily for clinical signs until day 23. As seen in Fig. 1, GPBP i.t. on day 1 did not protect animals from EAE at all (Fig. 1A). The clinical course of this group was essentially the same as that of saline i.t. control rats. GPBP i.t. on day 3 showed weak suppression of EAE (Fig. 1B). Although all rats exhibited the clinical signs, the maximum severity and duration of the disease was significantly reduced compared with those of control rats. In contrast, GPBP i.t. 7 days before the challenge suppressed the development of EAE almost completely. The majority of immunized rats did not exhibit the clinical signs (Fig. 1C). These results were different from those reported previously (23) in terms of duration that was needed for tolerance induction. This may be attributable to the difference in antigen used. The whole MBP molecule used in the present study would need more time for processing and presentation than MBP peptide used in the previous study.

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Fig. 1. GPBP i.t. 7 days, but not 3 and 1 day, before immunization with GPBP/CFA protects animals from the development of EAE. To determine the optimal protocol for induction of acquired thymic tolerance, Lewis rats were injected i.t. with 300 µg of GPBP dissolved in 100 µl saline on days 1, 3 and 7 of immunization, and challenged with GPBP/CFA (closed circles). For control studies, the same volume of saline was injected i.t. (open circles). Each point represents the mean clinical score for the group (n = 46 in each group). Injection of GPBP on day 1 showed no suppressive effect (A). When rats were pretreated with GPBP on day 3, all the rats developed clinical signs of EAE after the challenge (B). However, the clinical score of GPBP i.t. rats was significantly lower than that of saline i.t. rats (P < 0.05). In contrast, the majority of rats that received GPBP on day 7 did not develop the clinical sign of EAE (C).
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For i.t. injection, special attention was paid to minimize the leakage of the antigen from the thymus into blood circulation. However, there is the possibility that a small amount of leaked antigen plays a role in disease suppression. To test this, we injected GPBP i.v. at the same concentration and volume (total 300 µg in 100 µl) as used for i.t. injection, and challenged animals 7 days later. We found that rats that had received i.v. injection of the antigen developed full-blown EAE (data not shown), indicating that GPBP leaked into blood circulation, if any, plays no role in the disease suppression. Furthermore, this suppression was shown to be antigen-specific since i.t. injection of other antigens such as ovalbumin could not suppress the development of EAE (data not shown). Based on these findings, subsequent experiments were performed using this protocol, i.e. i.t. injection on day 7 of immunization.
Histological examination of the central nervous system (CNS)
Using hematoxylin & eosin-stained sections of the lumbar spinal cord, pathology of the CNS lesion was evaluated on days 11 and 14 post-immunization (Table 1). On day 14 post-immunization, saline i.t. rats (n = 6) showed severe inflammation characterized by the presence of extensive perivascular cuffing and severe parenchymal cell infiltration (data not shown). All the lumber spinal cord segments examined showed inflammation with a mean histological score of 2.53 ± 0.25. Interestingly, 94.1% of the segments from GPBP i.t. rats (n = 6) on day 14 exhibited mild inflammation with a mean histological score of 1.44 ± 0.16, although the majority of rats did not develop clinical signs (Table 1).
To characterize the nature of the inflammatory lesions in GPBP and saline i.t. rats in more detail, immunohistochemical studies were performed using mAb against TCR
ß (R73), Vß8.2 (R78), Vß10 (G101), Mø (ED1) and DC (OX62). TCR
ß+ T cells were the major components (Fig. 2A and B) and Vß8.2+ T cells (Fig. 2C and 2D) were more frequently found than Vß10+ T cells (Fig. 2E and F). As reported previously, encephalitogenic T cells mainly use Vß8.2 (24) and Vß8.2+ T cells are the predominant population in the CNS with the EAE lesion (17). Although inflammation in saline i.t. rats were far more severe than in the GPBP i.t. rats, there was no significant difference in the proportion of Vß8.2+ and Vß10+ T cells in the T cell population between the two groups. We also examined ED1+ and OX62+ cells in the spinal cord, and observed that a considerable number of Mø/DC infiltrated the CNS parenchyma of saline i.t. rats, while a few Mø/DC were found in the CNS of GPBP i.t. rats (data not shown). The number of Mø/DC in each group correlated well with the histological severity and there was no morphological evidence suggesting that Mø/DC play an active role in the suppression of inflammation in the CNS of protected animals.

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Fig. 2. Immunohistochemical staining of the lumbar spinal cord from saline i.t. (A, C and E) and GPBP i.t. rats (B, D and F). Although the histological severity of GPBP i.t. rats was significantly milder than that of saline i.t. control rats (see Table 1), the composition of infiltrating TCR ß (R73)+ T cells was essentially the same between the two groups (A and B). Vß8.2 (R78)+ T cells were the predominant population (C and D) and Vß10 (G101)+ T cells were few in number (E and F). Original magnification x110.
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The TCR repertoire analysis of spinal cord and lymph node T cells by CDR3 spectratyping
Then, we examined the TCR repertoire of the spinal cord and lymph node T cells taken from GPBP and saline i.t. rats on days 11 and 14 post-immunization (n = 3 in each group). Previous studies demonstrated that activation of MBP-reactive and Vß8.2+ T cells is responsible for the development of EAE, and that Vß8.2 spectratype expansion persists throughout the course of EAE in Lewis rats (21,24). The results are summarized in Fig. 3. Spleen cells taken from normal rats showed a Gaussian distribution without any spectratype expansion (Fig. 3A). In contrast, spinal cord T cells from saline i.t. rats showed marked Vß8.2 expansion (arrow in Fig. 3B). As reported previously, this activation pattern represents that of encephalitogenic T cells and treatment of immunized animals with anti-Vß8.2 mAb normalizes the spectratype pattern (19,21,25). Interestingly, the Vß8.2 spectratype pattern of spinal cord T cells from GPBP i.t. rats (Fig. 3A) was indistinguishable from that of saline i.t. rats (Fig. 3C). Essentially the same findings were obtained using lymph node T cells (data not shown). These findings strongly suggest that encephalitogenic T cells are activated equally in both GPBP and saline i.t. rats, although GPBP i.t. protected animals from both clinical and histological EAE.

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Fig. 3. CDR3 spectratyping of TCR ß chain of normal spleen cells (A), and spinal cord T cells from GPBP i.t. (B) and saline i.t. (C) rats. Spleen cells were isolated from normal rats. Spinal cord tissues were taken on day 14 post-immunization when control rats showed full-blown EAE. cDNA was amplified with TCR-specific primers and analyzed by CDR3 spectratyping. Regardless of the difference in clinical and histological severity, both GPBP and saline i.t. rats show oligoclonal expansion of the Vß8.2 spectratype (indicated by an arrow in B and C respectively). (A) Normal spleen cells, (B) GPBP i.t. rat, clinical grade 0, and (C) saline i.t. rat, clinical grade 3.
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Proliferation responses of lymph node cells taken from GPBP i.t. rats
To evaluate whether an anergy or deletion mechanism is involved in acquired thymic tolerance, proliferative responses of lymph node cells to GPBP were assayed in microliter wells by uptake of [3H]thymidine. The lymph node cells from saline i.t. control rats reacted strongly to GPBP (closed circles in Fig. 4). In contrast, lymph node cells from GPBP i.t. rats showed marginal proliferative responses to GPBP (open squares in Fig. 4). However, these responses recovered well with the addition of exogenous IL-2 (open circles in Fig. 4). These findings demonstrated that not all GPBP-specific T cells were eliminated by deletion and that acquired i.t. tolerance is, at least partly, due to anergy.

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Fig. 4. GPBP-specific proliferative responses of lymph node cells from GPBP or saline i.t. rats after challenge. Lymph node cell suspensions were prepared 14 days after immunization with GPBP. Lymphocytes from saline i.t. rats responded well to GPBP (closed circles). In contrast, those from GPBP i.t. rats showed marginal responses to GPBP (open squares), which recovered by the addition of exogenous IL-2 (10 U/ml) (open circles).
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Quantitative analysis of cytokine mRNA and protein in the lymphoid organ after GPBP or saline i.t.
Histological, the TCR repertoire and in vitro analyses revealed that a certain number of T cells were activated at the TCR level in the peripheral lymphoid organs of rats that had received GPBP i.t. followed by the challenge with GPBP/CFA, but that they were in the anergic state. These findings prompted us to examine the cytokine profile because cytokines play a pivotal role in the initiation, propagation and regulation of tissue-specific autoimmune inflammation and anergy induction. By competitive PCR analysis, we determined the cytokine mRNA levels in the spinal cord, lymph node, spleen, PBL and thymus. We first examined the tissues that were taken 7 days after GPBPor saline i.t. without immunization to clarify the effects of GPBP i.t. The results are shown in Fig. 5(A). The levels of IFN-
, TNF-
and TGF-ß1 in the lymph node (LN), spleen (Spl), PBL and thymus (Thy) of GPBP i.t. rats were not significantly different from those of saline control rats. In sharp contrast, the levels of IL-10 in the lymph node and spleen of GPBP i.t. rats were significantly up-regulated compared with those of saline control rats (asterisks in Fig. 5A, P < 0.05). It should be noted that there was no significant difference in the IL-10 level in the thymus between the two groups.
We then undertook a similar analysis on day 14 post-immunization using tissues taken from rats that had received GPBP or saline i.t. and the challenge (Fig. 5B). Saline i.t. rats showed full-blown EAE, whereas GPBP i.t. rats remained asymptomatic, as shown in Fig. 1(C). At this stage, the levels of pro-inflammatory cytokines, IFN-
and TNF-
, were generally higher in symptomatic control rats than in GPBP i.t. asymptomatic rats. Significant differences were noted in some tissues (indicated by asterisks in Fig. 5B). In sharp contrast, levels of IL-10 were significantly higher in all the tissues taken from GPBP i.t. rats than from saline control rats (Fig. 5B). Interestingly, there was no significant difference in the TGF-ß1 levels between the two groups. With regard to IL-4, only a small amount was detectable in all the organs examined and there was no significant difference between GPBP and saline i.t. rats (data not shown). In the thymus, there was no significant difference in the cytokine level (IFN-
, TNF-
, IL-10, TGF-ß1 and IL-4) between the two groups. We also examined the tissues taken on day 11 after immunization when saline i.t. rats showed mild clinical signs and obtained essentially the same results (data not shown).
To determine the cell type of IL-10-producing cells, spleens were removed 7 days after GPBP or saline i.t., and T cells, B cells and Mø/DC were sorted by positive selection with magnetic beads. Then, the amount of IL-10 mRNA in each cell population was determined by competitive PCR. As clearly shown in Fig. 6(A), the IL-10 level of Mø/DC isolated from GPBP i.t. rats was significantly higher than that from saline control rats. There was no difference in the T and B cell populations between the two groups (Fig. 6A).

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Fig. 6. Identification of IL-10-producing cells in the spleen after GPBP i.t. (A) To identify the cell type of IL-10-producing cells in the spleen after thymic tolerance induction, T cells (R73+), B cells (OX33+) and Mø/DC (OX42+) were sorted by positive selection with magnetic beads, and the amount of IL-10 mRNA in each cell population was determined by competitive PCR. The expression of IL-10 in the Mø/DC population, but not in the T and B cell populations, of GPBP i.t. rats was significantly higher than that of saline control rats (*P < 0.05). (B) Up-regulation of IL-10 in the spleens from GPBP i.t. rats was also confirmed at the protein level. Seven days after GPBP or saline i.t., spleens were removed (n = 3 in each group) and single-cell suspensions were cultured in the presence of LPS (10 ng/ml) for 48 h. The supernatants were harvested and the levels of IL-10 were determined by a sandwich ELISA. The mean level of IL-10 in spleen cells from GPBP i.t. rats (hatched bars) was significantly higher than that from saline i.t. rats (shaded bars) (P < 0.05).
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We also measured IL-10 in spleen cells at the protein level. Spleen cells were isolated from rats that had received GPBP or saline i.t. and stimulated in vitro with LPS for 48 h. Then, the amount of IL-10 in the supernatant was measured by a sandwich ELISA. As shown in Fig. 6(B), the level of IL-10 in spleen cells from GPBP i.t. rats was significantly higher than that from saline i.t. rats (P < 0.05).
Flow cytometric analysis of IL-10-producing cells in the spleen
Since competitive PCR analysis suggested that IL-10-producing cells in the spleen are Mø/DC, we tried to confirm this finding by flow cytometry for IL-10 and cell-type markers. We examined three rats and obtained essentially the same results. A representative finding is shown in Fig. 7. Double staining with anti-IL-10 mAb and one of the cell-type markers, OX42 (anti-Mø/DC), OX62 (anti-DC) and R73 (anti-TCR
ß), revealed that 34.1% of OX42+ cells (Mø/DC) and 44.6% of OX62+ cells (DC) expressed IL-10 (Fig. 7A and B respectively), while only 1.7% of T cells were positive for IL-10 (Fig. 7C). Double-positive cells stained with OX42 and OX62 in the spleen accounted for
15% (data not shown), indicating that OX62 Mø as well as OX62+ DC produce IL-10. These findings are consistent with those obtained by IL-10 mRNA quantitation (Fig. 6) and indicate that the major IL-10-producing cell population in acquired thymic tolerance is Mø/DC and not T cells.

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Fig. 7. Identification of the cell type of IL-10-producing cells in the spleen of tolerant rats. Seven days after GPBP injection, spleen cells were isolated and double-stained with anti-IL-10 mAb and one of the cell-type markers, such as OX42 (anti-Mø/DC), OX62 (anti-DC) and R73 (anti-TCR ß). Fifty thousand cells were recorded, and the OX42 and OX62 profiles and the R73 profiles were obtained after monocyte and lymphocyte gating respectively. The numbers in parentheses represent percentages of IL-10+ cells to the total OX42+ (A), OX62+ (B) or R73+ (C) cells.
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Functional assays of spleen cells taken from GPBP i.t. rats
It was shown in the present study that OX42+ Mø/DC in the spleen of GPBP i.t. rats produced significant amounts of IL-10, which has a crucial role for the suppression of clinical signs of EAE. To clarify this finding in more detail, we performed two functional assays. First, we examined the effects of spleen cells taken from GPBP or saline i.t. rats on GPBP-specific T line cells. Seven days after GPBP or saline i.t., spleen cells were isolated, treated with mitomycin C and then added to the T cell culture. The results are shown in Fig. 8. Thymocytes, which show strong APC function in the rat system, taken from a naive animal induced vigorous responses of T cells (thy in Fig. 8). Spleen cells taken from a saline i.t. rat (S1) induced strong T cell responses as well as thymocytes did. In sharp contrast, spleen cells taken from a GPBP i.t. rat (M2) suppressed T cell proliferation. The rest of the spleen cells from one saline i.t. (S2) and one GPBP i.t. (M1) rats showed intermediate APC function. These results demonstrated that spleen cells taken from GPBP i.t. rats have a tendency to suppress T cell proliferation, but that the suppressive effects vary from case to case.

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Fig. 8. Effects of spleen cells taken from GPBP or saline i.t. rats on the proliferation of GPBP-specific T line cells. GPBP-specific T line cells were cultured with thymocytes, which show strong APC function in the rat system, taken from a naive animal, spleen cells taken from saline i.t. rats (S1 and S2) or spleen cells taken from GPBP i.t. rats (M1 and M2) in the presence of various concentrations of GPBP. The proliferative responses were measured by 3H uptake.
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Second, an adoptive transfer experiment was performed to determine whether splenic Mø/DC from GPBP i.t. rats really inhibit the development of EAE. Spleens were removed from GPBP i.t. rats and a single-cell suspension was prepared. The cell suspension was divided into two subsets, T cells and non-T cells, by negative selection with magnetic beads and each cell population was adoptively transferred into naive rats. Then, recipient rats were immunized with GPBP/CFA on the next day (Table 2). The clinical course of rats receiving the T cell population showed no difference from the untreated control EAE rats (Group C versus A). Disease onset occurred on day 12.0 ± 1.2 post-immunization, reached a maximal severity of 3.2 ± 0.3 and lasted 4.8 ± 0.5 days. This finding indicates that T cells from thymic tolerant donors are not able to inhibit pathogenic immune responses to GPBP in recipients. In contrast, adoptive transfer of the non-T cell population (Group B) showed a different result. All rats exhibited clinical signs of EAE, but the peak of EAE was evidently suppressed in the recipients of the non-T population. The cumulative index was significantly lower than that of untreated and T cell-injected control rats (Group A versus B and Group B versus C, P < 0.05). The significant difference between Groups A and B and between Groups B and C was noted more clearly in weight loss of rats after immunization (P < 0.01). In order to exclude the possibility that the suppressive effect found in the non-T population is non-specific and antigen independent, we performed a similar experiment using T and non-T populations taken from saline i.t. rats. As clearly shown in Groups D and E in Table 2, the non-T cell population from saline i.t. rats showed no suppressive effects on the development of EAE in recipients, indicating that the suppressive activity of splenic non-T cells was conferred by i.t. injection of antigen.
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Table 2. Splenic non-T cells isolated from rats that had previously received an GPBP i.t. possess the suppressive effects against EAEa
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Discussion
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In the present study, we induced systemic tolerance to MBP by i.t. injection of the antigen. The majority of pretreated animals were protected completely against the development of clinical EAE after challenge with MBP/CFA. Although, by CDR3 spectratyping, clonal expansion of encephalitogenic T cells was noticed in GPBP i.t. rats (Fig. 3), they were in an anergic state (Fig. 4). Cytokine analysis revealed several interesting findings. First, pro-inflammatory cytokines such as IFN-
and TNF-
were generally suppressed in lymphoid organs of GPBP i.t. rats. However, there was no significant difference in IFN-
and TNF-
expression in the spinal cord between GPBP and saline i.t. rats, reflecting the finding that there were a few, but significant number of inflammatory foci in the CNS of tolerant animals (Fig. 2). Second, and most importantly, IL-10 was significantly up-regulated in the lymphoid organs and, to lesser extent, in the target organ of tolerant animals, and IL-10-producing cells were judged to be Mø/DC that have the suppressive activity of developing EAE of recipient animals. Although neutralization studies using anti-IL-10 antibody is necessary to confirm that IL-10 is a key factor for acquired thymic tolerance, mAb that neutralize rat IL-10 is not available at present. In spite of the lack of such an experiment, several pieces of circumstantial evidence obtained in the present study suggest that IL-10-producing Mø/DC in the periphery are closely associated with the induction and maintenance of systemic tolerance induced by i.t. injection of antigen. First, among anti-inflammatory cytokines, only IL-10 was up-regulated in tolerant rats (Fig. 5). Second, spleen cells from GPBP i.t. rats showed suppressive effects on the proliferation of GPBP-specific T line cells (Fig. 8). Finally, adoptive transfer of non-T cells isolated from spleen cells of tolerant rats into naive animals ameliorated clinical EAE (Table 2).
The mechanism by which i.t. injection of antigen results in systemic tolerance has been investigated by several groups (11,12,22,23,26). Injection of antigen induces massive apoptosis of thymic T cells reactive with the antigen (11). T cells escaped from this apoptosis emerge from the thymus to the periphery. However, these T cells are in an anergic state as shown previously (12) and confirmed here. Taken together, active suppression of antigen-reactive T cells by specialized cells is essential for anergy induction. So far, several types of cells including T cells (Th3, Tr1 and CD25+CD4+ T cells), DC and NK T cells were reported to be responsible for the maintenance of tolerance. In oral tolerance that has been most extensively investigated, Th3 cells secreting TGF-ß, but not IL-4, play an essential role in this type of tolerance [reviewed in (27)]. Clearly, it is not the case in acquired thymic tolerance because TGF-ß up-regulation was not observed in either target or lymphoid organ. Involvement of Tr1 cells was not supported strongly in this study, but is possible. Recently, Jonuleit et al. reported that human IL-10-producing Tr1-like cells generated by stimulation with immature DC suppress the proliferation of alloreactive T cells (28). Since only a few T cells produced a small amount of IL-10 in tolerant animals as shown in this study, Tr1 cells may not play an active role in acquired thymic tolerance. However, it is unknown whether a small number of Tr1 cells are sufficient for the tolerance induction and maintenance. In addition, the possibility that Tr1 cells activated by IL-10-producing DC maintain tolerance in an IL-10-independent manner (28) is not excluded. The involvement of CD25+CD4+ T cells, another type of regulatory cells, is less likely. Although there is concrete evidence indicating that depletion of this type of T cells from normal animals results in organ-specific autoimmune diseases such as thyroiditis and colitis (1,2,29), there is no known study directly demonstrating the involvement of the T cells in actively induced tolerance. In anterior chamber-associated immune deviation, IL-10 plays an essential role for its maintenance. While Sonoda et al. reported that NK T cells are the major producer of IL-10 (30), DOrazio and Niederkorn showed that APC are IL-10-producing cells (31). In the present study, there was no clear evidence supporting that NK T cells play an active role in IL-10 production. In addition, lack of IL-4 up-regulation in GPBP i.t. rats also supports this notion.
With regard to DC, circumstantial evidence suggesting that DC are actively involved in the induction and maintenance of peripheral tolerance has been accumulated (12,3234). In most cases, immature DC cultured only with granulocyte macrophage colony stimulating factor and IL-4 have been shown to possess a tolerogenic activity (28,32,34). In in vivo situations, immature DC phagocytose apoptotic cells (35,36) and induce tolerance to antigens expressed by apoptotic cells [reviewed in (9)]. Since i.t. injection of antigen induces apoptosis of thymic T cells reactive with the antigen (11), it is highly possible that DC phagocytosing apoptotic cells move to the periphery and play a role in the maintenance of tolerance. To obtain evidence, we injected GPBP with a labeling dye, PKH26, and examined the presence of labeled cells in the spleen. We constantly found that 34% of spleen cells were labeled with PKH26, demonstrating that some thymic Mø/DC migrate to the peripheral lymphoid organ (our unpublished observation). However, the percentage of thymus-derived IL-10-producing Mø/DC in the spleen was low and the majority of IL-10-producing Mø/DC were judged to be resident cells. This finding superficially contradicts that reported previously. Khoury et al. showed that transfer of thymic, but not of splenic, DC isolated from naive animals and pulsed in vitro with MBP protected recipient animals from the development of EAE after challenge with encephalitogenic antigen (12). Although the precise reason for this discrepancy is not clear, it would be attributable to the difference in the experimental conditions. In the previous study, freshly isolated DC were pulsed in vitro with antigen without any other accessory cells, while in the present study splenic DC were present with thymus-derived DC that had been primed with antigen in the presence of apoptotic antigen-reactive T cells in the thymus. Therefore, it is possible that a small number of thymus-derived DC stimulate resident splenic DC to produce IL-10 by presenting antigen as found in other experimental system (9,37,38).
In the present study, we have demonstrated that activation of IL-10-producing Mø/DC in the peripheral lymphoid organ is closely associated with the induction and maintenance of antigen-induced systemic tolerance. Understanding of the mechanism of DC-mediated tolerance facilitates the design of immunotherapy using genetically engineered DC.
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Acknowledgements
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We thank Dr K. Kawai (Department of Dermatology, Niigata University School of Medicine) for critical reading of the manuscript. We also thank Y. Kawazoe and K. Kohyama for technical assistance. This study was supported in part by Grants-in-Aid from the Ministry of Education, Japan.
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Abbreviations
|
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APCantigen-presenting cell
CDRcomplementary-determining region
CFAcomplete Freunds adjuvant
CNScentral nervous system
DCdendritic cell
EAEexperimental autoimmune encephalomyelitis
GPBPguinea pig myelin basic protein
HRPhorseradish peroxidase
i.t.intrathymic (injection)
MBPmyelin basic protein
Mømacrophage
PBLperipheral blood lymphocyte
TCLT cell line
TGFtransforming growth factor
TNFtumor necrosis factor
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