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【摘要】
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nerve system that is considered a T helper type 1 (Th1)-mediated autoimmune disease. EAE currently serves as an experimental animal model for multiple sclerosis in human. Cytokines, such as interferon- and interleukin-10, play a key role in the development and remission of EAE. Recent studies have also shown a role for B cells in the pathogenesis of EAE. Therefore, we examined the role of CD19, a B cell-specific surface molecule that defines signaling thresholds critical for B-cell responses and autoimmunity, on the development of EAE. Following immunization with myelin oligodendrocyte glycoprotein (MOG) peptide, CD19-deficient (CD19C/C) mice exhibited higher clinical and pathological severity scores of EAE than wild-type mice. The increased severity of EAE in CD19C/C mice was associated with polarized Th1 cytokines in the inflamed central nerve system but not with anti-MOG antibodies in the serum. MOG-primed CD19C/C B cells produced high levels of interferon-, and transfer of MOG-primed CD19C/C B cells to wild-type mice worsened the disease. Thus, CD19 modulates the Th1/Th2 cytokine balance in B cells and plays a critical role as a suppressive molecule in the development of EAE.
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Lymphocytes accomplish a complex balance between proper response to foreign antigens and minimized reaction to self-antigens. Disruption of this balance can result in the induction of autoimmune diseases. Recent assessments of the role of B cells in the immune system have indicated that B cells have more essential functions in regulating immune responses than had previously been appreciated.1-6 B-cell functions include immunoglobulin (Ig) secretion, antigen-presentation, production of various cytokines, and regulation of lymphoid organogenesis, T effector cell differentiation, and antigen-presenting dendritic cell function.7 Abnormalities of these B-cell functions could contribute to the induction or development of autoimmunity. Thus, B cells are now considered a potential therapeutic target in a wider range of autoimmune disorders.8 For example, B-cell depletion by anti-CD20 antibody (Ab) has shown unexpected impacts by dramatic effectiveness in treating patients with not only autoantibody-mediated diseases but also other various autoimmune disorders, including rheumatoid arthritis.9,10 On the other hand, recent studies have shown that B cells can play a protective role against autoimmune diseases in certain circumstances.1,2 Collectively, B cells have multiple critical roles in autoimmune disease expression through various functions.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nerve system (CNS) that is primarily mediated by CD4+ T cells specific for CNS autoantigens and is considered a prototypic T helper type 1 (Th1)-mediated autoimmune disease.11 Based on similarities in disease susceptibility, course, and histology, EAE is currently regarded as an experimental animal model for human multiple sclerosis, a common inflammatory and demyelinating disease of the CNS. Cytokines play a key role in the development and remission of EAE. The inflammatory lesion in the CNS requires a Th1 response, producing proinflammatory cytokines interferon- (IFN-) and tumor necrosis factor- (TNF-).12 Recovery is associated with production of Th2 cytokines interleukin 4 (IL-4) and IL-10.13,14 Although EAE has long been considered a T cell-mediated disease, because adoptive transfer of neural antigen-specific T cells alone is sufficient to induce the disease,15 recent studies have clarified roles of B cells and humoral immune response in the pathogenesis of EAE.1,16-20
B-cell fate and function are largely determined by signal transduction through a B-cell antigen receptor (BCR), which is further regulated by signal transduction molecules that amplify or inhibit BCR signaling during responses to self and foreign antigens. These regulatory molecules include a subset of functionally interrelated cell-surface receptors, such as CD19, CD21, CD22, CD40, CD72, and FcRIIb.21 CD19, in particular, regulates basal signaling thresholds and accelerates BCR signal, thus serving as a general "rheostat" that defines signaling thresholds critical for B-cell responses and autoimmunity.22-24 Modulating CD19 expression and/or function has been shown to have great effects on normal immune responses and autoimmunity.3,25-28
In the current study, we have assessed the roles of CD19 in EAE. Remarkably, CD19 expression influenced T-cell differentiation and cytokine profile of the tissue. CD19 loss resulted in increased severity of the disease as well as delayed recovery, suggesting an inhibitory role of CD19 in the etiology of EAE.
【关键词】 inhibitory progression experimental autoimmune encephalomyelitis regulating cytokine response
Materials and Methods
Mice
CD19C/C (C57BL/6 x 129) mice were generated as described26 and backcrossed 12 generations onto the C57BL/6 background before use in this study. Wild-type C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Lack of cell-surface CD19 expression was verified by two-color immunofluorescence staining with flow cytometric analysis. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science.
Peptide
Myelin oligodendrocyte glycoprotein (MOG) peptide (P35C55: MEVGWYRSPFSRVVHLYRNGK) was synthesized by Multiple Peptide Systems (San Diego, CA).
Induction and Evaluation of EAE
Active EAE was induced in female mice (6 to 8 weeks old) by subcutaneous immunization with 100 µg of MOG peptide emulsified in complete Freund??s adjuvant (CFA) containing 1 mg/ml heat-killed Mycobacterium tuberculosis H37RA (Sigma-Aldrich, St. Louis, MO) on days 0 and 7. Additionally, mice received 200 ng of pertussis toxin (Sigma-Aldrich) intraperitoneally in 0.5 ml of phosphate-buffered saline (PBS) on days 0 and 2. Clinical signs of EAE were assessed daily with a 0 to 6 scoring system (0, no signs; 1, flaccid tail; 2, impaired righting reflex and/or gait; 3, partial hind limb paralysis; 4, total hind limb paralysis; 5, hind limb paralysis with partial fore limb pa-ralysis; and 6, moribund or dead). Differences in total disease burdens between groups were analyzed with Mann-Whitney U-test.
Serological Evaluation of MOG Peptide-Specific Ig Production
Serum was obtained 14 and 28 days after immunization. Enzyme-linked immunosorbent assay (ELISA) was used for detection of MOG peptide-specific Ab. Briefly, 96-well microtiter plates (Costar, Cambridge, MA) were coated with 10 µg/ml MOG peptide. Plates were incubated with serum samples diluted 1:100. The bound Abs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-mouse IgM Abs (Southern Biotechnology Associates, Inc., Birmingham, AL).
Histology
MOG-induced EAE mice were euthanized on day 28 and perfused by intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde in PBS. Transverse sections of the cervical, upper thoracic, lower thoracic, and lumbar region of the spinal cord were stained with hematoxylin and eosin (H&E) or Luxol Fast Blue-Periodic acid. Each spinal cord section was further subdivided into an anterior, posterior, and two lateral columns. Each subdivided area displaying either lymphocyte infiltration or demyelination was assigned a score of 1; thus, each animal had a potential maximum score of 16.
For immunohistochemistry, frozen tissue sections of the spinal cord were acetone-fixed and then incubated with 10% normal rabbit serum in PBS (10 minutes, 37??C) to block nonspecific staining. Sections were then incubated with rat monoclonal antibodies (mAbs) specific for mouse macrophages (F4/80), CD4 (Clone RM4C5; BD Pharmingen, San Diego, CA), and CD8 (clone 53-6.7; BD Pharmingen). Rat IgG (Southern Biotechnology Associates, Inc.) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 minutes, 37??C) with a biotinylated rabbit anti-rat IgG and then horseradish peroxidase-conjugated avidin-biotin complexes (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide and then counterstained with methyl green.
Isolation of Total RNA and Real-Time Reverse Transcription-PCR
After perfusion with PBS, spinal cord from each MOG-induced EAE mouse was collected. Total RNA from spinal cord was extracted using Qiagen RNeasy spin columns (Qiagen Ltd., Crawley, UK) and digested with DNase I (Qiagen Ltd.) to remove chromosomal DNA in accordance with the manufacture??s protocol. Total RNA was reverse transcribed to cDNA using Reverse Transcription System with random hexamers (Promega, Madison, WI). Real-time quantitative reverse transcription-polymerase chain reaction (PCR) was performed using the TaqMan system (Applied Biosystems, Foster City, CA) on an ABI Prism 7000 Sequence Detector (Applied Biosystems) according to the manufacturer??s instructions. TaqMan probes and primers for IFN-, IL-12p40, TNF-, IL-6, IL-10, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems. Relative expression of real-time PCR products was determined using the CT technique.29 Briefly, each set of samples was normalized using the difference in threshold cycle (CT) between the target gene and housekeeping gene (GAPDH): CT = (CT target gene C CT GAPDH). Relative mRNA levels were calculated by the expression 2CCT, where CT = CT sample C CT calibrator. Each reaction was performed in, at least, triplicate.
Isolation of Mononuclear Cells from the CNS and Analysis by Flow Cytometry
After perfusion with PBS, cells from the CNS were isolated as previously described.30 Briefly, tissues were dissociated by passing through a 100-µm cell strainer and then centrifuged at 400 x g for 10 minutes at 4??C. The pellet was resuspended in 70% isotonic Percoll (Amersham Biosciences, Piscataway, NJ), overlaid with equal volumes of 37% and 30% isotonic Percoll, and centrifuged at 500 x g for 20 minutes at room temperature. Cells were collected from the 37%:70% interface and washed.
For intracellular IFN- staining, collected cells were stimulated for 4 hours at 37??C with 25 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) in 1 ml of complete medium containing the intracellular transport inhibitor brefeldin A (10 µg/ml; Sigma-Aldrich). The cells were stained with anti-CD4 (RM4C5; BD Pharmingen) and anti-CD8 (53-6.7; BD Pharmingen) Abs and then washed and treated with fluorescence-activated cell sorting (FACS)-permeabilizing solution (BD Biosciences, San Jose, CA) for 10 minutes at room temperature. These cells were incubated for 30 minutes in the dark with anti-IFN- Ab (XMG1.2; BD Biosciences). The cells were analyzed on a FACScan flow cytometer (BD Biosciences).
Production of IL-10 and IFN- by Splenic B Cells
Splenic B cells from MOG-induced EAE on day 28 were purified (>95% B220+) by removing T cells with anti-Thy1.2 Ab-coated magnetic beads (Dynal Inc., Lake Success, NY). Purified splenic B cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 x 10C5 mol/L ß-mercaptoethanol, and 2 mmol/L L-glutamine (Invitrogen, Paisley, UK). B cells were cultured in 96-well flat-bottom plates (BD Biosciences, Franklin Lakes, NJ) at 4 x 105 cells per well with 40 µg/ml MOG peptide and 10 µg/ml of anti-CD40 mAb (1C10; R&D Systems, Minneapolis, MN) for 5 days. Control cultures were stimulated with MOG alone or anti-CD40 mAb alone. Each sample was performed in triplicate. IL-10 and IFN- accumulation in the culture medium was measured by ELISA according to the manufacturer??s protocol (R&D Systems).
For intracellular IL-10 and IFN- staining, purified splenic B cells were stimulated at 1 x 106 cells/ml with 40 µg/ml MOG peptide and 10 µg/ml anti-CD40 mAb for 72 hours and then restimulated with 25 ng/ml phorbol 12-myristate 13-acetate and 1 µg/ml ionomycin for 12 hours. Brefeldin A (10 µg/ml) was added 8 hours after the cultures were initiated. The cells were stained with anti-B220 (RA3C6B2; BD Pharmingen) Abs and then washed and treated with FACS-permeabilizing solution for 10 minutes at room temperature. These cells were incubated for 30 minutes in the dark with anti-IL-10 (JES5C16E3; BD Biosciences) and anti-IFN- Abs. The cells were analyzed on a FACScan flow cytometer (BD Biosciences).
Adoptive Transfer of MOG-Primed B Cells
CD19C/C and wild-type mice were immunized with MOG peptide to induce EAE. MOG-primed splenic B cells were collected from MOG-induced EAE on day 28 and purified (>95% B220+) by removing T cells with anti-Thy1.2 Ab-coated magnetic beads (Dynal Inc.). Then 107 B cells from CD19C/C and wild-type mice were transferred intravenously into wild-type and CD19C/C mice, respectively. One day later, the recipient mice were immunized with MOG peptide to induce EAE.
Statistical Analysis
All data are expressed as the mean value ?? SEM. Mann-Whitney U-test was used for determining the level of significance of differences between sample means.
Results
Increased Severity of EAE in CD19C/C Mice
To assess whether CD19 expression plays a role in the pathogenesis of EAE, we immunized CD19C/C mice and wild-type C57BL/6 mice with MOG peptide in CFA and then observed the course of the clinical disease. In both lines of mice, EAE was induced at days 12 to 14, with a peak at days 18 to 23, followed by plateau phases (Figure 1) . However, compared with wild-type mice, CD19C/C mice showed significantly higher score at the peak and the plateau phase. Thus, CD19C/C mice were more susceptible to EAE. This was consistent with histological findings of the inflamed CNS tissue, which revealed that CD19C/C mice developed more severe inflammation (Figure 2A) . To further evaluate the pathological severity, transverse sections of the cervical, upper thoracic, lower thoracic, and lumbar regions of the spinal cord were stained with H&E and Luxol Fast Blue-Periodic acid and scored for the degree of inflammation and demyelination. Areas of inflammation and demyelination were more widespread in CD19C/C mice when compared with those in wild-type mice at day 28 (Figure 2E) . The pathological scores in the CD19C/C mice were significantly higher with 11.4 ?? 1.1 for inflammation and 7.4 ?? 1.8 for demyelination, whereas in the wild-type mice the scores for inflammation and demyelination were 5.2 ?? 0.7 and 2.8 ?? 0.5, respectively (P < 0.005 and P < 0.05, respectively; Figure 2E ). Collectively, CD19C/C mice developed more severe EAE, both clinically and pathologically, than wild-type mice.
Figure 1. Increased severity of EAE in CD19C/C mice. CD19C/C and wild-type mice (10 per group) were immunized with MOG peptide in CFA and intravenously injected with pertussis toxin. Mice were scored for the severity of EAE using the scale described in Materials and Methods. The EAE score is shown as mean ?? SEM. *P < 0.05.
Figure 2. CD19C/C mice had higher pathological severity scores of EAE. Spinal cords were harvested from CD19C/C and wild-type mice (five per group) 28 days after immunization. A representative section from each group shows lymphocyte infiltration stained by H&E (A), demyelination stained by Luxol Fast Blue (B), immunohistochemistry for CD4+ T cells (C) and CD8+ T cells (D), and the mean pathological score of demyelination and infiltration of each group (E). Original magnifications: x200. Arrows point to the foci of demyelination. Data are the mean ?? SEM. *P < 0.05; P < 0.005.
CD8+ T Cells Were Selectively Increased in the Inflamed CNS from CD19C/C Mice with EAE
To further evaluate the pathology of EAE in CD19C/C mice, the infiltrating cells in the inflamed CNS were examined by immunostaining. As shown in Table 1 , the frequency of infiltrating CD4+ T cells was not significantly different between CD19C/C and wild-type mice, whereas the frequency of CD8+ T cells was significantly 1.9-fold higher in CD19C/C mice than in wild-type mice. Representative photomicrographs of CNS-infiltrating CD4+ and CD8+ T cells are shown in Figure 2 (C and D). The ratio of CD4/CD8 T cells in CD19C/C mice was 1.4 ?? 0.5, significantly lower than that found in wild-type mice (3.3 ?? 1.5). The CD4/CD8 T-cell ratio was not altered in the peripheral blood in CD19C/C mice with or without EAE when compared with wild-type mice (data not shown). Additionally, the number and percentage of macrophages were not different between the two groups (Table 1) , and B-cell infiltration was not found in either line of mice (data not shown). CD19 expression was not detected in infiltrating T lymphocytes or macrophages (data not shown). Thus, CD8+ T cells were selectively increased in the inflamed CNS from CD19C/C mice with EAE.
Table 1. Infiltrating Cell Profile in the Inflamed CNS with CD19C/C and Wild-Type Mice
CD19 Loss Attenuated Anti-MOG Ig Production in MOG-Induced EAE
To dissect the mechanism by which CD19-deficiency enhanced disease severity, serum IgM and IgG anti-MOG Ab levels were measured by ELISA. Sera were collected from CD19C/C and wild-type mice with EAE on days 14 and 28. Serum IgM anti-MOG Abs levels in wild-type mice with EAE were significantly increased compared to control mice immunized with CFA alone 28 days after immunization (P < 0.005; Figure 3A ). Additionally, serum IgG anti-MOG Abs levels in wild-type mice with EAE were significantly elevated compared to normal controls 14 and 28 days after immunization (P < 0.005 and P < 0.005, respectively; Figure 3B ). By contrast, the serum levels of IgM and IgG anti-MOG Abs remained significantly lower in CD19C/C mice with EAE, which did not exhibit significantly higher levels than wild-type mice without MOG immunization (Figure 3) . Thus, the loss of CD19 expression dramatically inhibited anti-MOG Ab production in EAE.
Figure 3. CD19 loss attenuates anti-MOG Ig production. CD19C/C and wild-type mice were immunized with MOG peptide in CFA. Sera (five per group) were collected on days 14 and 28, IgM (A) and IgG (B) anti-MOG Ab levels were measured by ELISA. The data are presented as the mean values of three separate measurements of each sample. Controls (n = 12) were wild-type mice immunized with CFA alone.
To test whether decreased anti-MOG Ab enhanced the disease severity in CD19C/C mice, sera from wild-type mice with EAE were transferred to CD19C/C mice with EAE. However, no alteration of disease course was observed in CD19C/C mice receiving sera from wild-type mice with EAE (data not shown), suggesting that decreased anti-MOG Ab is not solely responsible for the increased severity of EAE in CD19C/C mice.
The Inflamed CNS Tissue in CD19C/C Mice Displays a Th1 Bias
To assess the cytokine expression profile of CNS-infiltrating cells, spinal cords were harvested from wild-type mice and CD19C/C mice 28 days after MOG immunization, and cytokine expression was analyzed by real-time PCR (Figure 4A) . The mRNA expressions of Th1 cytokines, IFN- and IL-12p40, in CD19C/C mice were increased compared with wild-type mice (2.5-fold; P < 0.05 and 2.0-fold, respectively). The mRNA expression of TNF-, a proinflammatory cytokine, in CD19C/C mice was also slightly higher by 1.5-fold than in wild-type mice. IL-6 mRNA expression in CD19C/C mice was comparable to wild-type mice. By contrast, spinal cords from CD19C/C mice showed dramatically decreased mRNA expression of IL-10, a Th2 cytokine, compared with wild-type mice (77% decrease, P < 0.05; Figure 4A ). To address what cell types may be accounting for excess IFN- in the CNS of CD19C/C mice when compared to wild-type mice, flow cytometric intracellular cytokine staining was performed. IFN- production from CNS-infiltrating CD4+ T cells in CD19C/C mice was augmented compared to wild-type mice (Figure 4B) . In addition, IFN- production from CD8+ T cells in CD19C/C mice was also augmented (Figure 4C) . Thus, it was likely that excess IFN- in the CNS of CD19C/C mice was generated by CD4+ and CD8+ T-cell collaboration. These findings indicate that the inflamed CNS tissue of CD19C/C mice with EAE displays a Th1 bias and that this skewed cytokine balance may contribute to the augmented disease manifestation.
Figure 4. The inflamed CNS tissue in CD19C/C mice displays Th1-biased cytokine production. A:RNA was isolated from PBS-perfused spinal cords of CD19C/C and wild-type mice (five per group) 28 days after immunization. The mRNA levels of IFN-, IL-12p40, TNF-, IL-6, and IL-10 were analyzed by real-time reverse transcription-PCR and normalized with internal control glyceraldehyde-3-phosphate dehydrogenase. Data are shown as mean ?? SEM. *P < 0.05. B, C:Mononuclear cells were isolated from pooled CNS tissue in CD19C/C and wild-type mice (three per group) 28 days after immunization by Percoll gradient centrifugation and incubation with phorbol 12-myristate 13-acetate and ionomycin. The cells were stained with anti-CD4 and anti-CD8 monoclonal antibodies. After permeabilization, cells were stained with anti-IFN- antibody. Percentages of IFN-+ cells are shown in the upper right quadrants. These data are representative of three independent experiments.
CD19 Deficiency Suppressed IL-10 Production from B Cells
Because a recent study has demonstrated that IL-10 from B cells is important for EAE recovery,1 we next investigated the difference between IL-10 production in B cells from CD19C/C mice and wild-type mice. Splenic B cells were purified from CD19C/C mice and wild-type mice at day 28, followed by in vitro stimulation. Incubation with MOG alone resulted in modest and comparable IL-10 production both in CD19C/C and wild-type B cells (Figure 5A) . By contrast, an agonistic Ab to CD40 induced higher levels of IL-10 production in wild-type B cells than in CD19C/C B cells (Figure 5A) . Concomitant stimulation with MOG and anti-CD40 Ab further increased IL-10 production in wild-type B cells, whereas it did not increase IL-10 production in CD19C/C B cells. In addition, IL-10 production from B cells was confirmed using intracellular cytokine staining. Wild-type B cells produced IL-10 with MOG and anti-CD40 Ab stimulation, whereas CD19C/C B cells did not (Figure 5B) . Thus, anti-CD40-induced IL-10 production was impaired in CD19C/C B cells with or without antigen stimulation.
Figure 5. Production of high IFN- and low IL-10 by splenic B cells in CD19C/C mice with EAE. Splenic B cells were purified from CD19C/C and wild-type mice (five per group) 28 days after immunization. Purified splenic B cells were incubated in vitro in media alone (Med) or in MOG peptide, anti-CD40, or both. IL-10 (A) and IFN- (B) accumulation in the culture medium was measured by ELISA. Data are the mean ?? SEM. *P < 0.05. For intracellular IL-10 (C) and IFN- (D) staining, purified splenic B cells were stimulated with MOG peptide and anti-CD40 monoclonal antibody for 72 hours and then restimulated with phorbol 12-myristate 13-acetate and ionomycin for 12 hours. Intracellular cytokine staining was performed using anti-B220 antibody to identify B lineage cells. Percentages of IL-10+ and IFN-+ cells are shown in the upper right quadrants. These data are representative of three independent experiments.
CD19 Deficiency Increased IFN- Production from B Cells
We also examined in vitro B-cell production of IFN- using purified splenic B cells from CD19C/C and wild-type mice 28 days after immunization. B cells from wild-type or CD19C/C mice incubated with MOG alone did not produce IFN- (Figure 5B) . Anti-CD40 Ab induced only slight increase of IFN- production, if any, in wild-type B cells, whereas B cells from CD19C/C mice demonstrated increased IFN- production following anti-CD40 Ab stimulation (Figure 5B) . Furthermore, addition of MOG to anti-CD40 Ab greatly increased IFN- production in CD19C/C B cells, whereas wild-type B cells showed only modest IFN- production by MOG and anti-CD40 Ab stimulation (Figure 5B) . In addition, IFN- production from B cells was confirmed using intracellular cytokine staining. IFN- production from CD19C/C B cells with MOG and anti-CD40 Ab stimulation was augmented compared to wild-type B cells (Figure 5C) . Unlike IFN-, B cells from CD19C/C and wild-type mice with MOG and anti-CD40 Ab stimulation produced scant but comparable amounts of IL-12, another Th1 cytokine (data not shown). Therefore, anti-CD40-induced IFN- production was augmented in CD19C/C B cells and was further exaggerated by the presence of antigen. Collectively, CD19C/C B cells showed skewed cytokine response with increased IFN- production and decreased IL-10 production.
Adoptive Transfer of CD19C/C B Cells Increased the Severity of EAE in Wild-Type Mice
We next assessed whether the altered B-cell functions, especially cytokine production, were responsible in vivo for increased severity of EAE in CD19C/C mice. First, MOG-primed B cells (107 cells, >95% B220+), with T cells removed anti-Thy1.2 Ab, from wild-type mice with EAE were adoptively transferred to CD19C/C mice before EAE induction. However, CD19C/C mice that received wild-type B cells developed EAE with almost the same severity as CD19C/C mice receiving no B cells (Figure 6A) . Therefore, the failure of IL-10 production from B cells alone did not explain the increased severity of EAE in CD19C/C mice. Next, MOG-primed B cells (107 cells, >95% B220+) from CD19C/C mice with EAE were adoptively transferred to wild-type mice before EAE induction. Wild-type mice that received CD19C/C B cells developed EAE with significantly higher severity than wild-type mice receiving no B cells and exhibited as severe EAE as CD19C/C mice (Figure 6B) . To exclude dendritic cell or natural killer cell contamination in adoptive transfer of B cells, we obtained high purity B cells (>99% B220+) by removing T cells, natural killer cells, monocytes, macrophages, dendritic cells, granulocytes, and erythrocytes with antibodies for mouse CD43, CD4, and Ter-119. The results of adoptive transfer of B cells using high purity B cells (>99% B220+) were comparable to using conventional purity B cells (>95% B220+) by removing T cells with anti-Thy1.2 Ab (data not shown). Thus, exaggerated B-cell functions, especially excessive IFN- production from B cells, may increase severity of EAE in CD19C/C mice.
Figure 6. The increased severity of EAE in CD19C/C mice does not depend on the loss of IL-10 production from B cells. A: Wild-type mice were immunized with MOG peptide to induce EAE. Splenocytes were harvested 28 days after immunization. Then MOG-primed splenic B cells were purified (>95% B220+), and wild-type B cells (107 cells) were transferred intravenously into CD19C/C mice. One day later, the recipient mice were immunized with MOG peptide in CFA to induce EAE. The other groups, CD19C/C and wild-type mice, did not receive any cells. B: MOG-primed splenic CD19C/C B cells were transferred intravenously into wild-type mice, followed by immunization with MOG peptide in CFA. Mice were scored for the severity of EAE as in Figure 1 .
Discussion
In the current study, we assessed the role of CD19, a B-cell-specific cell-surface molecule, in the induction and recovery of EAE. CD19 loss resulted in both increased severity and prolonged recovery of the disease (Figure 1) . This result was striking because CD19C/C mice generally exhibit an immunodeficient phenotype.25,26,28 CD19C/C mice with EAE showed increased CD8+ T cell infiltration but not increased CD4+ T cell infiltration in the inflamed CNS (Table 1 and Figure 2 (C and D)). Furthermore, cytokine expression profiles were shifted to Th1 in the CNS from CD19C/C mice when compared with wild-type mice (Figure 4) . CD19C/C mice with EAE also showed decreased serum IgM and IgG anti-MOG Abs (Figure 3) , although serum transfer between CD19C/C and wild-type mice did not alter disease severity. Remarkably, adoptive transfer of B cells from CD19C/C mice with EAE to wild-type mice enhanced the disease severity in the recipient wild-type mice (Figure 6) . By contrast, wild-type B cells did not rescue EAE in CD19C/C mice. Therefore, the loss of a single B-cell-specific protein, CD19, was sufficient to modify the disease manifestation of EAE, suggesting an inhibitory role of CD19 in the etiology of the disease.
Although there are some discrepancies among studies, the importance of B cells in EAE has been reported, although the mechanisms are still controversial. Mice genetically deficient for B cells develop EAE16,31 but fail to resolve the disease.1,16 Therefore, B cells or Abs are not required for primary induction of EAE, whereas antigen-specific B cells are essential for the recovery from the disease. Fillatreau et al1 have demonstrated that IL-10 production from B cells plays a key role in resolving EAE, suggesting a regulatory role of B cells in the disease. Furthermore, another study using mice deficient for Lyn, an Src-family protein-tyrosine kinase expressed abundantly in B cells, has demonstrated that Lyn-deficient (LynC/C) mice show both increased severity and more sustained course of EAE and that serum transfer from LynC/C mice worsens EAE in wild-type mice.18 The current study has shown that the loss of CD19 expression increased severity of EAE at the peak and plateau phases. Taken together, B cells appear to have multiple roles during the process of EAE.
CD19 positively regulates B cell response to a variety of transmembrane signals.23,24 B cells from CD19C/C mice are hyporesponsive to antigen, anti-CD40, lipopolysaccharide, and other stimuli.25,26,28 Consistent with this, CD19C/C mice immunized with MOG peptide produced lower levels of IgM and IgG anti-MOG Ab than wild-type mice. CD19 regulates B-cell signaling pathways by acting as a scaffold protein that recruits Lyn, phosphatidylinositol 3-kinase, Vav, and possibly other molecules.22 CD19C/C mice and LynC/C mice both show increased severity of EAE.18 CD19 regulates Lyn kinase activity in BCR signaling,22 although the mechanisms exaggerating EAE appear different between the two lines of mice, because augmented Ab response appears to be the main mechanism in LynC/C mice. In the case of CD19C/C mice, serum transfer did not alter disease severity. Additionally, mRNA levels of IFN- are decreased in the inflamed CNS from LynC/C mice. By contrast, B cells from CD19C/C mice with EAE produced significantly higher levels of IFN- in response to MOG and anti-CD40 Ab than wild-type mice, whereas MOG and anti-CD40 Ab induced less IL-10 production in B cells from CD19C/C mice with EAE than those from wild-type mice with EAE. Thus, CD19 positively regulates IL-10 production and negatively regulates IFN-, with this shifted balance likely affecting the disease severity of EAE. The mechanism by which CD19 regulates cytokine production is still unclear. Because CD19 has been shown to regulate BCR-induced signal transducers and activators of transcription 1 (STAT1) phosphorylation,32 which has a critical role in IFN-induced response, alterations in STAT1 function by CD19 loss may affect the signaling pathway. CD19 may also regulate other cytokine signaling pathways such as Janus tyrosine kinase/STAT. Therefore, CD19 may directly regulate IL-10 and/or IFN- production pathway(s). Alternatively, CD19 regulation of the signal strength in response to stimuli may indirectly affect the cytokine balance. In either manner, CD19 can control Th1/Th2 cytokine balance in B cells by the reciprocal regulation between IFN- and IL-10.
The adoptive transfer experiments have demonstrated that wild-type B cells did not rescue EAE in CD19C/C mice but that CD19C/C B cells worsened EAE in wild-type mice. Therefore, B cells deficient for CD19 have a positive role for driving the disease. The most likely explanation for this phenomenon is excessive production of IFN- by CD19C/C B cells. Recent studies have emphasized B cells as an important source of IFN-.33-40 IFN- production in B cells is induced by IL-12,35,37-40 whereas B cells down-regulate the Th1 response via the release of IL-10.1,2 Additionally, autoreactive B cells function as highly efficient antigen-presenting cells, enhancing the selective action of MOG-specific T cells.41 B-cell-T-cell interactions with B-cell antigen presentation favors Th2, rather than Th1, differentiation.42,43 Therefore, excessive production of IFN- from CD19C/C B cells can negatively regulate B-cell antigen presentation and then can commit T cells to a Th1 phenotype. Intriguingly, the inflamed CNS of CD19C/C mice exhibited an increase in TNF- and Th1 cytokines such as IFN- and IL-12p40 and a decrease in Th2 cytokine such as IL-10, although there was no B-cell infiltration. Thus, skewed cytokine production of B cells influenced the total cytokine profile in the tissue.
Another striking finding in the current study was a specific increase of CD8+ T cells in spinal cord of CD19C/C mice. EAE is considered to be CD4+ T cell-driven15 and associated with an inflammatory cytokine profile. However, recent studies have shown that CD8+ T cells also have a disease-promoting role.44 Furthermore, it has been demonstrated that T cells infiltrating into active human multiple sclerosis lesions are dominated by CD8+ T cells.45 Thus, the increase in CD8+ T cells in inflamed CNS of CD19C/C mice may be related to more severe EAE.
Previous studies have shown that MOG-specific antibodies enhance demyelination and inflammation, resulting in increased severity of EAE.19,20 Furthermore, plasma exchange can reduce clinical disease activity in a subset of multiple sclerosis patients46,47 ; therefore, humoral factors play an important role in multiple sclerosis and EAE. However, MOG-specific antibodies had no direct effect on EAE induction in the current study. Although the reasons for this discrepancy are not clear, B-cell functions for cytokine-producing and antigen-presenting cells may be more important than MOG-specific antibody production in the current study. It is possible that CD19C/C B cells act as antigen-presenting cells and stimulate autoantigen-specific CD8+ T cells, contributing to exacerbation of EAE.
In conclusion, the current study has shown that CD19 plays an important role in the development of EAE, and CD19 is a critical factor for T cell commitment to the Th2 phenotype. Thus, modulating CD19 function or its pathway may be a potential therapeutic target of multiple sclerosis and other Th1 diseases.
Acknowledgements
We thank M. Matsubara and Y. Yamada for technical assistance.
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作者单位：From the Department of Dermatology,* Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; the Department of Immunology, Duke University Medical Center, Durham, North Carolina; and the Department of Dermatology, Nagasaki University Graduate School of Biomedical Science, Nagasaki,