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The Journal of Immunology, 2004, 172: 6388-6397.
Copyright © 2004 by The American Association of Immunologists

Ectopic CD40 Ligand Expression on B Cells Triggers Intestinal Inflammation 1

Takahiro Kawamura2,*, Takanori Kanai2,*, Taeko Dohi{ddagger}, Koji Uraushihara*, Teruji Totsuka*, Ryoichi Iiyama*, Chikara Taneda*, Motomi Yamazaki*, Tetsuya Nakamura*, Tetsuya Higuchi{dagger}, Yuichi Aiba{dagger}, Takeshi Tsubata{dagger} and Mamoru Watanabe3,*

* Department of Gastroenterology and Hepatology, Graduate School, and {dagger} Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and {ddagger} Department of Gastroenterology, Research Institute, International Medical Center of Japan, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several studies indicate that CD4+ T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8–15 wk of age. In contrast, CD40L/B TgxCD40–/– double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-{gamma} production. Furthermore, although mice transferred with CD4+ T cells alone or with both CD4+ T and B220+ B cells, but not B220+ cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220+ B cells from diseased CD40L/B Tg mice and CD4+ T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Crohn’s disease (CD) 4 and ulcerative colitis (UC) are the two major forms of chronic inflammatory bowel disease (IBD). Although their etiopathology remains unknown, increasing evidence has shown that an immune mechanism plays an important role in their pathogenesis (1, 2, 3). These include increased T cell infiltrates into gut, the production of cytokines by lamina propria (LP) T cells, and remission after treatment with immunosuppressants or by targeting proinflammatory cytokines. Although many animal experimental models clinically mimicking human IBD have been established, all these models induce diseases characterized by the common features of T cell- or macrophage/dendritic cell-dependent immune responses (4, 5).

One particular molecule that may be a potent target for the treatment of IBD is CD40 ligand (CD40L; CD154). This is found predominantly on activated CD4+ T cells and interacts with CD40, which is expressed on B cells, APCs, and endothelial cells (6, 7, 8). In patients with IBD, increased numbers of T cells expressing CD40L have been detected in the LP, and these contribute to induce proinflammatory cytokines, including IL-12, by macrophages/dendritic cells (9, 10). Furthermore, it is known that mice with overexpressed CD40L on T cells develop chronic colitis with the infiltration of CD40L+ T cells and CD40+ cells into disease tissues (11). In both 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced (12) and SCID-transferred colitis models (13, 14), neutralizing anti-CD40L Abs prevent colitis. These data suggest that CD40/CD40L interactions are probably relevant in the development of colitis in humans and mice.

In contrast, CD40-expressing B cells, which interact with CD40L-expressing T cells, are able to promote the proliferation and survival of B cells, Ig isotype switching, and germinal center reaction (7, 15, 16). B cells possess a variety of immune functions, including the production of Igs and various cytokines, the presentation of Ags, and the regulation of dendritic cell function. Interestingly, functionally distinct roles of B cells have been reported in autoimmune diseases such as systemic lupus erythematosus (SLE) (17). B cells drive the development of several autoimmune disorders through the production of pathogenic autoantibodies. Similarly, circulating autoantibodies to colon epithelial cells have been consistently identified in patients with UC or CD by many investigators in reports that span at least 2 decades (18, 19, 20). Interestingly, accumulating evidence suggests that CD40L is ectopically expressed on B cells in the same range of their T cells in patients with SLE, although the pathogenic role of CD40L on B cells in SLE is unknown (21). At present, however, to our knowledge, there have been no reports describing the expression of CD40L on B cells in patients with IBD.

Based on the fact that CD40L can be inducible on B cells in some autoimmune disorders and also several reports that described the association between IBD and SLE (22, 23), we investigated the possibility that intestinal inflammation may be induced by the primarily dysregulated B cells. To this end, we used transgenic (Tg) mouse lines expressing CD40L ectopically on B cells (CD40L/B Tg) (24). At 8–12 wk of age, CD40L/B Tg mice show increased number of B cells in spleen and increased serum IgG and IgM concentrations by ~4- and ~5-fold, respectively. In addition, B cells in CD40L/B Tg mice are resistant to apoptosis induced in vitro, probably due to constitutive CD40 signaling in B cells via CD40 (our unpublished observations). Interestingly, these mice developed SLE-like autoimmune disease, probably due to the production of many kinds of autoantibodies, such as anti-DNA Abs (24).

Surprisingly, we found that CD40L/B Tg mice developed severe transmural intestinal inflammations. Remarkably, the inflammatory infiltrates consisted predominantly of B cells. Although T cells also infiltrated inflamed lesions, they did so to a much lesser degree than B cells, and the CD4/CD8 ratio of LP T cells was decreased compared with that in control mice. Furthermore, we demonstrated that mice transferred with CD4+ T cells alone or with both CD4+ T and B220+ B cells, but not B220+ cells alone from diseased CD40L/B Tg mice, develop colitis similar to the original disease, indicating that the final cause of colitis is dysregulated CD4+ T cells. However, we demonstrated that mice transferred with B220+ B cells from diseased CD40L/B Tg mice and CD4+ T cells from wild-type (WT) mice also developed colitis, albeit to a lesser extent than those transferred with CD4+ T and B220+ B cells from diseased CD40L/B Tg mice, whereas mice reconstituted with both B220+ B cells and CD4+ T cells from WT mice did not. This indicates that Tg B cells should be involved as a trigger for this colitis model. These observations demonstrate that the CD40-CD40L interaction among B cells may introduce abnormal Ig isotype switching and consecutive B cell expansion and may contribute to the infiltration in gut. Thus, this experimental murine model seems to be sharply distinct from any established murine IBD models in which dysregulative function of T cells and macrophages/dendritic cells should be an essential pathogenic mechanism.


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

CD40L/B Tg mice expressing CD40L on B cells were previously established by injecting the DNA fragment containing the CD40L cDNA, a VH promoter, the IgH intron enhancer, and the Ig{kappa}3' enhancer into C57BL/6 fertilized eggs (24). CD40–/– mice were described previously (25). In the present study male, 4- to 20-wk-old CD40L/B Tg mice and WT littermate controls were used. CD40L/B TgxCD40–/– double-mutant mice were also generated. The animals were housed under specific pathogen-free conditions. All experiments were approved by the regional animal study committees. Cross-breeding of these mice was performed, and the presence of each transgene was identified by tail DNA.

Histopathology and immunohistochemical analysis

Tissue specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were stained with H&E. For the immunohistochemistry, sections were embedded in OCT compound (Tissue-Tek; Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at –80°C. Six-micron sections were incubated with primary Abs. These included anti-murine CD4 (RM4-5, rat IgG1; BD PharMingen, San Diego, CA), anti-murine CD8 (53-6.7, rat IgG2a; BD PharMingen), anti-murine B220 (RA3-6B2, rat IgG2a; BD PharMingen), anti-murine CD11c (HL-3, hamster IgG; BD PharMingen), and anti-F4/80 (A3-1, rat IgG2b; Serotec, Oxford, U.K.). Isotype-matched control Abs (BD PharMingen) were also used. Biotinylated goat anti-hamster IgG or rat anti-mouse IgG (BD PharMingen) was chosen as second Abs. After three washes with PBS, positively stained cells were detected by streptavidin-biotinylated HRP complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA), and visualized by diaminobenzidine. Then the sections were counterstained with hematoxylin.

Immunofluorescence analysis

In some experiments frozen sections of colon were prepared and fixed with cold acetone and then incubated with Block Ace (Dainippon-Pharmaceuticals, Tokyo, Japan). Sections were incubated with FITC-labeled goat anti-mouse IgM (magnification, x500; Southern Biotechnology Associates, Birmingham, AL), tetramethylrhodamine (TRITC)-labeled goat anti-mouse IgG (magnification, x500; Southern Biotechnology Associates), and biotin-labeled goat anti-mouse IgA (magnification, x400; BD PharMingen), followed by incubation with aminocumarin-labeled streptavidin (magnification, x500; Southern Biotechnology Associates) (26). Anti-colon autoantibodies in sera from 12- to 15-wk-old colitic CD40L/B Tg mice were detected by indirect immunofluorescence using colonic tissues from 6-wk-old nondiseased CD40L/B Tg mice. Sections were blocked with 10% goat serum in PBS for 30 min at room temperature and incubated with mouse serum diluted in 10% goat serum in PBS for 1 h, and then bound Ab was detected with TRITC-labeled anti-mouse IgG or FITC-labeled anti-mouse IgM. Sections were examined with a fluorescence microscope (BX50/BXFLA; Olympus, Tokyo, Japan) equipped with a charge-coupled device camera (Olympus) and an image capture system (Olympus). Combination images for multicolor staining were performed using Photoshop 4.0 (Adobe Systems, San Jose, CA).

Serum Ig

Total amounts of serum IgM, IgG, and IgA were measured by standard sandwich ELISA analysis using goat anti-IgM or IgG or IgA Abs (Southern Biotechnology Associates). Abs bound to the plates were detected using alkaline phosphatase-labeled anti-IgG, anti-IgM (BD PharMingen), and anti-IgA Abs (Southern Biotechnology Associates).

Cell preparation

LP mononuclear cells (LPMC) were isolated from the colon as described previously (27, 28). Briefly, the entire colon was opened longitudinally, washed with PBS, and cut into small pieces. The pieces were then incubated with Ca2+- and Mg2+-free HBSS containing 2.5% FBS and 1 mM DTT (Sigma-Aldrich, St. Louis, MO) for 30 min to remove mucus and then serially incubated twice in Ca2+- and Mg2+-free HBSS containing 2.5% FBS and 0.75 mM EDTA (Sigma-Aldrich) for 1 h each. The supernatants from these incubations were collected, pooled, and treated with 1 mg/ml collagenase (Worthington Biomedical, Freehold, NJ) and 0.01% DNase (Worthington Biomedical) in medium for 2 h. The cells were pelleted twice through a 40% isotonic Percoll solution and then further purified by Ficoll-Hypaque density gradient centrifugation (40/75%) at the interface.

Flow cytometry

The isolated LPMC were preincubated with an Fc{gamma}R-blocking mAb (CD16/32; 2.4G2; BD PharMingen) for 20 min, followed by incubation with FITC-, PE-, or biotin-labeled mAb for 30 min on ice. Biotinylated Abs were detected with PE-streptavidin (BD Biosciences, Mountain View, CA). Two-color flow cytometric analysis was performed on a FACSCalibur (BD Biosciences) using CellQuest software. Background fluorescence was assessed by staining with isotype-matched control mAbs.

Cytokine analysis

Ninety-six-well culture plates were precoated with anti-CD3 mAb (5 µg/ml; 145-2C11; BD PharMingen) and anti-CD28 mAb (2 µg/ml; 37.51; BD PharMingen) in 100 µl of PBS at 37°C for 4 h and washed with PBS three times to remove unbound Abs. LP CD4+ T cells (5 x 105/well) were then incubated at 37°C in 5% CO2 humidified air for 48 h. Culture supernatants of littermate mice and Tg mice after onset of disease were harvested and assayed for IFN-{gamma} and IL-4. Cytokine concentrations were determined by a specific ELISA kit for mouse IFN-{gamma} and IL-4 (Endogen, Cambridge, MA). ODs were measured on an Intermet ELISA reader at a wavelength of 490 nm.

Adoptive transfer experiment

Adult female C57BL/6J SCID mice (6–8 wk old) were used as recipients in the following experiments. Diseased CD40L/B Tg and control WT littermates were euthanized at 18 wk of age. To obtain CD4+ T or B220+ B cells from the pooled splenocytes and mesenteric lymph node cells, CD4+ T cells were purified using the anti-CD4 (L3T4) MACS beads (Miltenyi Biotec, Auburn, CA), and thereafter B220+ cells were purified from the negatively selected CD4– cells using anti-B220 (RA3-6B2) MACS beads (Miltenyi Biotec). After the enriched CD4+ T cells (96–97% pure, as estimated by FACSCalibur) and B220+ B cells (95–96% pure) were labeled with PE-conjugated anti-mouse CD4 (RM4-5) and FITC-conjugated anti-B220 (RA3-6B2) mAbs, and the purified CD4+ T cells and B220+ B cells were then sorted by two-color sorting on a FACS Vantage (BD Biosciences). All populations were >99.0% pure on reanalysis. SCID mice were then injected i.p. with one or two subpopulations of the sorted CD4+ T cell and/or B220+ B cells in PBS: 1) CD40L/B Tg B220+ (5 x 105/mouse), 2) CD40L/B Tg CD4+ (5 x 105/mouse), 3) CD40L/B Tg B220+ (5 x 105/mouse) and CD40L/B Tg CD4+ (5 x 105/mouse), 4) CD40L/B Tg B220+ (5 x 105/mouse) and WT CD4+ (5 x 105/mouse), 5) WT B220+ (5 x 105/mouse) and WT CD4+ (5 x 105/mouse), 6) WT B220+ (5 x 105/mouse), 7) WT CD4+ (5 x 105/mouse), or 8) no transfer control. Mice were killed and analyzed 4 wk after transfer, and the colons were removed and evaluated histologically. For the histological score, the area of the proximal colon was scored on a scale of 0–3 in each of three criteria: cell infiltration, crypt elongation, and crypt abscesses. Histological grades were assigned in a blind fashion by one pathologist (R.I.).

Statistical analysis

Data were expressed as the mean and SD and are plotted in corresponding figures. The differences between experimental groups were statistically analyzed with the Mann-Whitney U test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40L/B Tg mice developed a wasting disease with severe intestinal inflammation

Clinical manifestation was monitored for up to 30 wk. IBD symptoms were first observed between 8 and 15 wk of age. Approximately 90% of CD40L/B Tg mice had diarrhea and developed significant weight loss until 20 wk of age (p < 0.05; Fig. 1A). In contrast, their WT littermates appeared healthy, with a gradual increase in body weight and absence of diarrhea during the period of observation (Fig. 1A). Macroscopic appearance showed enlarged colon with a greatly thickened wall in CD40L/B Tg mice (Fig. 1C), and the colonic weight of 15-wk-old CD40L/B Tg mice was significantly increased compared with that of littermates (p < 0.005; Fig. 1B). In addition, the spleen and mesenteric lymph node enlargement was also present in diseased CD40L/B Tg mice (data not shown). Consistent with these data, the numbers of mononuclear cells recovered from the colon and spleen of 15-wk-old CD40L Tg mice were significantly increased compared with those of their littermates (Fig. 1, D and E), indicating extensive mononuclear cell proliferation in the inflamed colon and spleen of CD40L/B Tg mice.



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  		FIGURE 1. CD40L/B Tg mice developed the wasting disease with local and peripheral expansive cell growth. A, Approximately 90% of CD40L/B Tg mice (n = 29) had diarrhea and showed significant weight loss compared with WT littermates (n = 24) at 15 wk of age. B, The colonic weight of CD40L/B Tg mice was markedly larger than that of WT littermates at 15 wk of age. C, The macroscopic appearance of colon of CD40L/B Tg mice was enlarged, shortened, and had a greatly thickened wall compared with that of WT littermates. D, LPMCs were isolated from the colon of 15-wk-old CD40L/B Tg mice or their littermates, and the number of LPMCs was determined. Data are the mean ± SD of seven mice in each group. E, Splenocytes (Sp) were isolated from the colon of 15-wk-old CD40L/B Tg mice or their littermates, and the number of Sp was determined. *, p < 0.05.

 
Histopathologic examination of colonic tissue revealed the development of severe colitis in 15-wk-old CD40L/B Tg mice (Fig. 2, B–D). Large numbers of mononuclear cells infiltrated transmurally in colonic mucosa (Fig. 2B). Prominent epithelial hyperplasia with glandular elongation (Fig. 2C) and goblet cell depletion (data not shown) were common in whole colon. Extensive leukocytic infiltrates were seen in the LP and submucosa and, to a lesser degree, in the muscularis, serosa, and mesentery (Fig. 2, B and D). The mucosal abnormalities, such as thickening of the intestinal wall, virous atrophy, crypt hyperplasia, and infiltrations of mononuclear cells were also seen in the ileum of CD40L/B Tg mice (Fig. 2G). Splenomegaly with large amounts of lymphocytes and markedly enlarged mesenteric lymph nodes were present in these colitic mice (data not shown). In contrast, colonic and ileac sections from WT littermates showed few lymphocytes and macrophage infiltration in LP (Fig. 2, A and F). To determine whether these abnormalities in CD40L/B Tg mice was really due to the direct CD40-CD40L interaction in vivo, we next generated CD40L/B TgxCD40–/– double-mutant mice. These mice were monitored for up to 30 wk, but they appeared healthy with a gradual increase in body weight and the absence of diarrhea during the period of observation (data not shown). In addition, colonic and ileac sections from age-matched CD40L/B TgxCD40–/– mice showed few lymphocytes and macrophage infiltration in the LP (Fig. 2, E and H).



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  		FIGURE 2. Histopathologic examination revealed the development of severe enterocolitis in CD40L/B Tg mice. Colons from WT littermates (A), CD40L/B Tg mice (B–D), and CD40L/B TgxCD40–/– (G) mice and small intestines from WT littermates (E), CD40L/B Tg mice (F), and CD40L/B TgxCD40–/– mice (H) were stained with H&E. Original magnification: A, B, E–G, and H, x 40; C and D, x100.

 
To examine the phenotypical cell surface markers of infiltrated mononuclear cells, the sections of colon were immunohistochemically stained with Abs against B220, CD4, CD8, F4/80, and CD11c. The major population of cell infiltrates was B220+ cell aggregates, which were structurally different from colonic patches. B cells were markedly infiltrated in the LP from diseased CD40L/B Tg mice as well as serosa and mesentery (Fig. 3). In addition, CD4+, CD8+, F4/80+, and CD11c+ cells in the inflamed mucosa from diseased CD40L/B Tg mice were increased in the inflamed colonic mucosa compared with those from WT littermates (Fig. 3). Unlike most reported animal models of IBD, CD8+ T cells seemed to infiltrate to a greater extent in inflamed mucosa compared with CD4+ T cells. In contrast, the littermates and age-matched CD40L/B TgxCD40–/– mice showed no obvious infiltration of B220+, CD4+, CD8+, F4/80+, or CD11c+ cells in the colon (Fig. 3).



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  		FIGURE 3. Immunohistochemical analysis showed the massive B220-positive cell infiltration to colonic tissue in CD40L/B Tg mice. A large number of aggregated B220+ cells, which were structurally different from B cells in colonic patches, were infiltrated in the LP of diseased CD40L/B Tg mice. In addition, CD4+, CD8+, F4/80+, and CD11c+ cells were increased in the inflamed colonic mucosa of CD40L/B Tg mice compared with those of WT littermates or CD40L/B TgxCD40–/– mice. Original magnification, x100.

 
Autoimmune diseases are often associated with the involvement of other organs, in particular the kidneys and sometimes the lungs, during disease progression. Therefore, cryostat sections of various tissues were analyzed for the presence of inflammation. Approximately half the CD40L/B Tg mice showed apparent glomerulonephritis and hyperplastic bronchus-associated lymphoid tissue in lungs (data not shown). However, no inflammation in other organs, such as skin, submaxillary gland, heart, liver, pancreas, and stomach, were observed (data not shown).

Increased expression of CD40L on B cells in diseased CD40L/B Tg mice

Flow cytometric analysis of LPMC from colitic mice showed that the percentages of B220+ LPMC were significantly increased compared with those in WT littermates (Fig. 4). Consistent with immunohistochemical studies, unlike other T cell-mediated colitis models, the percentages of CD8+ cells and the CD8:CD4 ratio were significantly increased compared with those in WT littermates (Fig. 4). In diseased CD40L/B Tg mice, the expression of CD40L on B220-expressing LP B cells was significantly up-regulated. The mean fluorescein intensity of B220+CD40L+ LP B cells in CD40L/B Tg mice was much higher than that in young nondiseased CD40L/B Tg mice (data not shown). In contrast, there were no differences in CD40 expression on B cells between CD40L/B Tg mice and littermates (Fig. 4). Although we used the B cell-specific promoter and enhancer to establish CD40L/B Tg mice, CD40L on LP T cells in diseased, but not in nondiseased, CD40L/B Tg mice was also up-regulated compared with that in littermates (Fig. 4). These data indicated that CD40L expression on T cells could be secondary to the response to inflammatory actions in colon.



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  		FIGURE 4. Flow cytometric analysis of LPMC from diseased CD40L/B Tg mice and WT littermates. The percentages of CD8+ cells and the CD8/CD4 ratio were significantly increased in LPMC from diseased CD40L/B Tg mice compared with those from WT littermates. In CD40L Tg mice, the percentages of B220+ LPMC were significantly increased and the expression of CD40L on B220-expressing LP B cells was also significantly up-regulated compared with those in WT littermates. In contrast, there were no differences in CD40 expression on LP B cells between CD40L/B Tg mice and littermates. Although we used the B cell-specific promoter and enhancer to establish CD40L/B Tg mice, CD40L/B on LP T cells in diseased mice was also slightly up-regulated compared with that in littermates. These data were representative of three experiments.

 
To further investigate the role of CD40L-expressing B cells in the development of colitis in CD40L/B Tg mice, we assessed Ig-producing cells using confocal fluorescent immunohistochemistry. As shown in Fig. 5, diseased and nondiseased CD40L/B Tg mice had almost similar numbers of IgA plasma cells in colon compared with WT littermates. In contrast, it was of note that there was markedly increased number of surface IgM-positive cells were observed in inflamed mucosa in diseased CD40L/B Tg mice (Fig. 5). In addition, surface IgG-producing cells, which were rarely present in LP, were often detected in diseased CD40L/B Tg compared with nondiseased CD40L/B Tg mice (Fig. 5).



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  		FIGURE 5. Colonic Ig-producing cells in CD40L/B Tg mice. Fifteen-week-old WT littermates (A), 6-wk-old nondiseased CD40L Tg mice (B), and 15-wk-old diseased CD40L/B Tg mice (C and D) were stained for IgG (red), IgM (green), and IgA (blue) by fluorescent immunohistochemistry. Diseased and nondiseased CD40L/B mice had almost similar numbers of IgA plasma cells in colon as WT littermates. In contrast, a markedly increased number of IgM+ cells was observed in inflamed mucosa in diseased CD40L/B Tg mice. In addition, the number of IgG-producing cells in diseased CD40L/B Tg mice was slightly, but significantly, increased compared with those in nondiseased CD40L/B Tg mice and WT littermates. Original magnification: A–C, x200; and D, x1000. These data were representative of three experiments.

 
To next assess the systemic Ig production in CD40L/B Tg mice, we tested concentrations of serum IgG, IgM, and IgA using the specific sandwich ELISA. As shown in Fig. 6, concentrations of serum IgG and IgM were significantly increased by 4- and 5-fold, respectively, in diseased CD40L/B Tg mice compared with WT littermates (IgG; p < 0.005, IgM; p < 0.05). In some diseased CD40L/B Tg mice, serum IgA was increased, but not significantly different (p = 0.29).



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  		FIGURE 6. Serum concentrations of Igs from CD40L/B Tg mice (n = 4) and WT littermates (n = 4) at 18 wk of age. Serum IgG, IgM, and IgA were measured using the specific sandwich ELISA. The average concentrations of serum IgG and IgM were significantly increased by 4- and 5-fold, respectively, in diseased CD40L/B Tg mice compared with WT littermates. In some diseased CD40L/B Tg mice, serum IgA was markedly increased, but the difference was not significant (p = 0.29). *1, p < 0.05, *2, p < 0.005.

 
Anti-colon autoantibodies

To assess the involvement of anti-colon autoantibodies in the pathogenesis of colitis, we tested whether sera obtained from colitic mice react with epithelial cells in the uninflamed colon of young nondiseased CD40L/B Tg mice. As shown in Fig. 7, both anti-colon IgG and IgM autoantibodies were detected in sera from colitic mice, IgM autoantibody reacted with crypt epithelium, and IgG autoantibody showed strong reaction with surface epithelium of the colon. These reactivities were seen with 1/125 diluted sera from diseased CD40L/B Tg mice. In contrast, staining of epithelial cells was not seen with sera from nondiseased mice and WT littermates at 1/25 or 1/125 dilution (Fig. 7).



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  		FIGURE 7. Anti-colon Abs were detected in sera from diseased CD40L/B Tg mice. Sera obtained from 12- to 15-wk-old colitic mice stained some surface epithelial cells in the uninflamed colon of young nondiseased CD40L/B Tg mice (C and F), but this was not observed when sera from WT littermate mice (A and D) and nondiseased young CD40L/B Tg mice (B and E) were used. For the detection of IgM (A–D) and IgG type (E–H) autoantibodies, FITC-labeled anti-mouse IgM and TRITC-labeled anti-mouse IgG, respectively, were used for the second antibodies. Original magnification: A–C and E–G, x200; and D and H, x1000. These data were representative of three experiments.

 
B1/B2 differentiation in CD40L/B Tg mice

Based on the involvement of autoantibodies against intestinal epithelial cells in sera derived from diseased CD40L/B Tg mice, we further assessed B1 B cells in inflamed mucosa, because B1 cells appears to produce Abs in a T cell-independent manner (29). Flow cytometric analysis of LPMC from colitic mice showed no significant absolute increase in CD5+IgM+ B1 B cells on LPMC from diseased CD40L/B Tg mice (percentage of CD5+IgM+/lymphocyte gate: WT, 0.4 ± 0.2%; CD40L/B Tg, 0.9 ± 0.5%; p = 0.18), indicating that autoantibody production could require T cell-dependent help or the involvement of nonmucosal B1 B cells, which may be located in peritoneal and pleural cavities.

Th1-mediated immune responses in CD40L/B mice

As mentioned above (Fig. 4), CD4+ and CD8+ T cells were also increased in inflamed mucosa from diseased CD40L/B Tg mice. We then assessed the involvement of Th cells in the development of this colitis. Isolated LP CD4+ T cells were cultured, and the supernatants were analyzed for concentrations of IFN-{gamma} and IL-4 by specific ELISA. IFN-{gamma} (p < 0.05), but not IL-4, production by anti-CD3/anti-CD28 mAbs-stimulated LP CD4+ T cells was significantly increased in diseased CD40L/B Tg compared with WT littermates (IFN-{gamma}: WT, 12.1 ± 3.0 ng/ml; CD40L/B Tg, 84.8 ± 12.1 ng/ml (p = 0.001); IL-4: WT, 575.2 ± 75.0 pg/ml; CD40L/B Tg, 469.0 ± 75.9 pg/ml (p = 0.68)), indicating that colitis induced in CD40L/B Tg mice could be mediated in secondary Th1-immune responses.

Adoptive transfer experiments

To establish which cell type mediates the inflammation in CD40L/B Tg mice, we next performed the adoptive transfer experiments using sorted B and T cells from diseased CD40L/B Tg and WT littermate mice in the indicated protocol (Fig. 8A). As shown in Fig. 8B, mice transferred with CD40L/B Tg CD4+ T cells alone or with both CD40L/B Tg CD4+ T and CD40L/B Tg B220+ B cells, but not CD40L/B Tg B220+ cells alone, develop colitis 4 wk after transfer, indicating that the final cause of colitis is the dysregulated CD4+ T cells. However, mice reconstituted with CD40L/B Tg B220+ B cells and WT CD4+ T cells also developed colitis, whereas mice reconstituted with both WT B220+ B cells and WT CD4+ T cells did not. These differences were confirmed by histological scoring of multiple colon sections obtained from five mice in each group (Fig. 8C). This indicates that Tg B cells should be a trigger for this colitis model, and thereafter, dysregulated CD4+ T cells are also needed as final effectors.



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  		FIGURE 8. Adoptive transfer of sorted T and B cells obtained from spleens and mesenteric lymph nodes. A, Transfer protocol. C57BL/6 SCID mice were injected i.p. with one or two of the indicated subpopulations of sorted CD4+ T cells and B220+ B cells from diseased CD40L/B Tg mice (18 wk old) and WT littermates: a, CD40L/B Tg B220+ (5 x 105/body); b, CD40L/B Tg CD4+ (5 x 105/body); c, CD40L/B Tg B220+ (5 x 105/body) and CD40L/B Tg CD4+ (5 x 105/body); d, CD40L/B Tg B220+ (5 x 105/body) and WT CD4+ (5 x 105/body); e, WT B220+ (5 x 105/body) and WT CD4+ (5 x 105/body); f, WT B220+ (5 x 105/body); g, WT CD4+ (5 x 105/body); or h, no transfer control. Mice were euthanized 4 wk after transfer. B, Histopathology of distal colon from each mice. Original magnification, x100. C, Histological scores were determined 4 wk after transfer as described in Materials and Methods. Data are presented as the mean ± SD (n = 5). *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report describes a novel model of intestinal pathology induced by primarily B cell-triggered mechanism. To date, studies in animal models of IBD have mainly focused on CD4+ T cells of unknown specificity and in part dendritic cells/macrophages (30) and CD8+ T cells (31). In this study we show that abnormal B cells expressing CD40L can also trigger chronic colitis and may provide a link with autoimmune intestinal inflammation.

It is generally established that the interaction of CD40L on activated T cells with CD40 on APC leads to the secretion of IL-12 by APC, including dendritic cells and macrophages (32, 33, 34, 35, 36, 37), and that such interactions are critical for IL-12 production in Ag-driven responses of animal models of colitis (12, 38, 39) and human IBD (9). In contrast, there were no reports that mentioned the involvement of CD40L-expressing B cells in IBD despite some evidence that CD40L is expressed on B cells as well as T cells in patients with SLE (21), and ectopic expression of CD40L on B cells has been observed in lupus-prone BXSB mice (40). The expression of CD40L is activation dependent; CD40L appears on the cell surface within 1–3 h after Ag recognition, and then immediately disappears by 24 h (7). In fact, it was difficult to detect CD40L on any type of cell, probably because CD40L expression in nondiseased young CD40L/B Tg mice was down-regulated in the presence of CD40 as described previously (25, 41). Indeed, we could detect surface expression of CD40L using CD40L/B Tg mice crossed with CD40-deficient mice in the spleen, lymph nodes, and bone marrow (Y. Aiba, details will be described elsewhere). Further study will be required to determine whether CD40L could be expressed on B cells in inflamed mucosa from patients with IBD as well as activated T cells, although it could be technically hard to determine this. Of importance, isolated LPMC from IBD mucosa have been reported to contain more B cells, particularly B cells producing IgG, compared with noninvolved IBD mucosa or control mucosa (42). Furthermore, circulating autoantibodies produced by abnormal B cells, such as colon epithelial cells and leukocyte nuclei, have been consistently identified in patients with UC or CD (43). The accumulating evidence led us to investigate the possible role of CD40L on B cells in the primary development of colitis using CD40L/B Tg mice expressing CD40L ectopically on B cells.

In the present study we demonstrate that CD40L/B Tg mice expressing CD40L on B cells develop chronic enterocolitis, which is first observed between 8 and 15 wk of age with massive infiltration of aggregated IgM-producing B cells in LP, and produce autoantibodies against colonic epithelial cells. First, we postulated that abnormal CD40-CD40L B-B interaction might be induced by ectopic expression of CD40L on B cells and may introduce 1) abnormal Ig isotype switching, 2) consecutive IgM-positive B cell proliferation, and 3) infiltration in the gastrointestinal tract. In contrast, T cells also infiltrate inflamed lesions, but the degree is much less than that of B cells. Interestingly, unlike most other animal models of colitis (1, 5), the CD4/CD8 ratio of LP T cells of inflamed colon was significantly decreased compared with that in WT littermates. These results indicated that CD40L/B Tg mice are sharply distinct from any established T cell/macrophages/dendritic cell-mediated murine models of intestinal inflammation.

Similarly, Clegg et al. (11) reported that CD40L Tg mice overexpressing CD40L on T cells (CD40L/T Tg) acquire a lethal chronic colitis marked by the infiltration of CD40L+ T cells and CD40+ cells into diseased tissues. Interestingly, they showed abnormal thymus development in CD40L/T Tg, such as a transgene copy-dependent decline in thymocyte number, loss of cortical epithelium, and expansion of CD40+ medullary cells (11). In their model they postulated that CD40L/T Tg failed to develop regulatory T cells in thymus that suppress the development of colitis by abnormal T cell development. In addition to a defect in thymic negative selection in CD40L/T Tg, the chronic inflammation in colon might be caused by the continuation of transgene expression in peripheral T cells (11). In contrast, our CD40L/B Tg expressing CD40L on B cells showed no abnormalities in thymus in either flow cytometric or histological analysis (data not shown), indicating that not only could chronic intestinal inflammation in our CD40L/B Tg mice expressing CD40L on B cells be mediated in a thymus-independent manner, but also the CD40L transgene system does not introduce CD40L expression into T cells because of the use of a B cell-specific promoter. Nevertheless, the fact that we detected CD40L protein on LP CD4+ T cells from diseased CD40L/B Tg mice suggested that the expression of CD40L on LP CD4+ T cells might be secondary in the reaction against the local intestinal inflammation. Indeed, like most CD40L-mediated animal models of IBD (9, 10, 12, 13, 14), LP CD4+ T cells in inflamed mucosa from diseased CD40L/B Tg mice showed IFN-{gamma}-dominant, rather than IL-4-dominant, cytokine production, indicating that our mice might be a good Th1-mediated chronic colitis model. It is also possible that CD40L-expressing LP B cells could directly stimulate CD40-expressing dendritic cells/macrophages to produce IL-12 and to introduce Th1-mediated immune responses. To determine the role of CD40L on B cells in the development of colitis in this model, we next performed an adoptive transfer of isolated T or B cells from diseased CD40L/B Tg and WT littermate mice into SCID mice. We demonstrated that mice transferred with CD4+ T cells alone or with both CD4+ T and B220+ B cells, but not B220+ cells alone, from diseased CD40L/B Tg mice develop colitis, indicating that the final cause of colitis is the dysregulated CD4+ T cells. However, we also demonstrated that mice transferred with B220+ B cells from diseased CD40L/B Tg mice and CD4+ T cells from WT mice also developed colitis, whereas mice transferred with both B220+ B cells and CD4+ T cells from WT mice did not. This indicates that the Tg B cells should be involved in this colitis as a trigger for this colitis model. It should be noted that this transfer experiment also clearly excluded the possibility that the Tg CD40L was expressed on T cells in our Tg system.

In our CD40L/B Tg mice, serum IgM and IgG concentrations were 4- to 5-fold increased. Intestinal mucosal plasma cells producing IgG or IgM were apparently increased, but the number of IgA-producing cells was not significantly different compared with that in WT littermates. Comparison with the findings in human tissue from patients with IBD and in some experimental colitis models reveals some similarities. In human IBD, levels of IgG and IgM are increased along with disease progression. In contrast, it has been demonstrated the level of IgA is decreased in patients with chronic UC (44) and CD (45), but increased in the quiescent disease (46). Furthermore, we detected IgM- and IgG-type autoantibodies against colonic epithelial cells. T cell-dependent immune responses generally involve conventional B2 B cells. In contrast, the other subset of B cells, B1 B cells, appear to produce Abs in a T-independent manner (27, 47, 48). B1 B cells, originally defined by the surface expression of CD5 and high levels of IgM, arise early in ontogeny, home predominantly to the peritoneal and pleural cavities, have a capacity for self-renewal, and display different receptor specificities. B1 cells appear to recognize self-Ag as well as common bacterial Ags. Production of autoantibodies by B1 cells is also supported by the fact that the neoplastic expansion of B1 cells, such as in chronic lymphocytic leukemia, is often associated with autoimmune symptoms (26, 47, 48). Based on the evidence, we assessed whether autoantibodies are produced via T cell-dependent (B1; CD5+IgM+) or T cell-independent (B2; CD5–IgM+) fashion. We showed no increase in CD5+IgM+ B cells in inflamed mucosa from these mice compared with those in WT littermates, indicating that autoantibodies in diseased CD40L/B Tg mice might be produced by T cell-dependent B2 cells. Alternatively, the autoantibodies in this model could be generated in other tissues, such as peritoneal cavity. In addition, it should be noted that Mizoguchi and colleagues (49, 50) recently demonstrated that highly CD1d-expressing B cells were significantly increased in mesenteric lymph nodes from colitic TCR{alpha}–/– mice and had a unique ability to produce IL-10. They postulated that these B cells functioned as regulatory B cells to suppress the progression of intestinal inflammation by down-regulating inflammatory cascades associated with IL-1 up-regulation and STAT3 activation (49, 50). Although there might be a correlation between their regulatory B cells and our pathogenic B cells in the development of colitis at this point, it seems likely that our aggregated IgM-producing, activated B cells in inflamed mucosa from diseased CD40L/B Tg mice did not possess such a regulatory function, but differentiated to more pathogenic cells in the early stage of colitis development. Further study will be required to assess the features of CD40L-expressing B cells, such as cytokine production and APC function.

In conclusion, the present data suggested that under some conditions the CD40-CD40L interaction is constitutively generated on B cells by themselves, and IBD pathogenesis might be established by a primarily B cell-triggered mechanism without involving mainly T cells or macrophages/dendritic cells. Further investigation, such as establishing double-mutant mice crossed between CD40L/B Tg mice and nude mice, may be beneficial to elucidate the primary involvement of B cells in the pathogenesis of IBD.


   Acknowledgments
 
We express special thanks to Dr. Morio Koike for critical comments, and to Saeko Matsumoto for manuscript preparation.


   Footnotes
 
1 This work was supported in part by grants-in-aid for Scientific Research, Scientific Research on Priority Areas, Exploratory Research, and Creative Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology; the Japanese Ministry of Health, Labor, and Welfare; the Japan Medical Association; Foundation for Advancement of International Science; Terumo Life Science Foundation; Ohyama Health Foundation; Yakult Bio-Science Foundation; and Research Fond of Mitsukoshi Health and Welfare Foundation. Back

2 T. Kawamura and T. Kanai contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Mamoru Watanabe, Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address: mamoru.gast{at}tmd.ac.jp Back

4 Abbreviations used in this paper: CD, Crohn’s disease; IBD, inflammatory bowel disease; LP, lamina propria; LPMC, lamina propria mononuclear cell; UC, ulcerative colitis; SLE, systemic lupus erythematosus; Tg, transgenic; TNBS, 2,4,6-trinitrobenzene sulfonic acid; TRITC, tetramethylrhodamine; WT, wild type. Back

Received for publication June 25, 2003. Accepted for publication March 4, 2004.


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