Prevention of Experimental Colitis in SCID Mice Reconstituted with CD45RB^high CD4⁺ T Cells by Blocking the CD40-CD154 Interactions¹

Crohn’s disease and ulcerative colitis are two major forms of human inflammatory bowel disease (IBD).³ Their etiopathology is unknown, but increasing evidence indicates that immune mechanisms play an important role (1). These include increased T cell infiltrates in inflamed intestinal mucosa, the production of cytokines by lamina propria T cells, and remission after treatment with immunosuppressants or by targeting proinflammatory cytokines. Moreover, experimental colitis clinically mimicking human IBD can be induced in murine models by a disrupted T cell-regulatory system (1, 2). However, the initiating event for signal-mediated lymphocyte activation in the intestinal mucosa is still poorly understood.

There is increasing evidence that interactions between CD40 ligand (CD40L) expressed on activated T cells and CD40 expressed on APC play a critical role in humoral and cellular immune responses (3, 4). CD40 signaling provides a signal for B cell activation, proliferation, and Ig isotype switching. It also stimulates monocyte/macrophage activation and cytokine production and up-regulates expression of costimulatory molecules such as B7 family molecules and of MHC class II on the surface of APC. Moreover, CD40 ligation induces expression of adhesion molecules such as CD54, CD62E, and CD106 on endothelial cells, suggesting that CD40 signaling plays an important role in leukocyte extravasation to sites of inflammation (5, 6). In recent years, greater attention has been paid to the role of CD40-CD40L interactions in the immunopathogenesis of some autoimmune diseases, where T or B cells have a prominent role, such as systemic lupus erythematosus (7), rheumatoid arthritis (8), and multiple sclerosis (9) as well as in allograft rejection (10). Administration of mAb directed against CD40L has been shown to effectively inhibit inflammatory responses in a variety of animal models such as experimental allergic encephalomyelitis (9, 11), lupus nephritis (12), collagen-induced arthritis (13), atherosclerosis (14), and spontaneous autoimmune diabetes (15). This Ab also dramatically prevents murine pancreatic islet allograft rejection (16) and CD4⁺ T cell-mediated graft-vs-host disease and marrow allograft rejection (17). These results have led to the conclusion that CD40-CD40L interactions play a central role in the pathogenesis of several autoimmune diseases and that target treatment against CD40 signaling may lead to improvement of ongoing disease.

The role of the CD40 stimulatory pathway in intestinal immunopathology has been suggested by the effect of anti-CD40L treatment in murine colitis induced by 2,4,6-trinitrobenzenesulfonic acid (18) and by data on CD40L-transgenic mice (19). More recently, we have reported that expression of CD40 and CD40L is considerably increased in inflamed mucosa from IBD patients and that CD40L expressed on lamina propria T cells is able to induce proinflammatory cytokine production by monocytes (20). These data indicate that CD40-CD40L interactions are probably relevant to the pathogenesis of human IBD. To further analyze the role of CD40-CD40L interactions, we used an experimental murine model for colitis, which has been shown to be useful for the investigation of the pathogenesis of human IBD (21, 22, 23, 24). SCID mice are reconstituted with syngeneic CD45RB^highCD4⁺ T cells. These SCID recipients develop a wasting disease 6–10 wk after T cell reconstitution. The disease is characterized by diarrhea, weight loss, transmural inflammation in the proximal colon, and a Th1 immune response by lamina propria CD4⁺ T cells in the diseased colon. The clinical, histopathological, and immunological features resemble those observed in human Crohn’s disease (1, 2). In the present study, treatment with anti-CD40L neutralizing mAb was performed in this model to determine the role of CD40 signaling in the pathogenesis of this CD4⁺ T cell-mediated colitis in vivo.

Specific pathogen-free female BALB/c mice were obtained from the Harlan Company (Zeist, The Netherlands). Female BALB/c SCID mice were bred under standard pathogen-free conditions and maintained in the Animal Care Facility of the Faculty of Medicine, Gasthuisberg, Catholic University of Leuven (Leuven, Belgium). BALB/c SCID mice were raised under specific pathogen-free conditions in microisolator cages with filtered air and were fed autoclaved food and water. Mice were used at 6–8 wk of age.

Anti-mouse CD3ε mAb (clone 500A2, hamster IgG), biotinylated anti-mouse CD4 mAb (clone H129.19, rat IgG2a), anti-mouse CD40 mAb (clone 3/23, rat IgG2a), FITC-conjugated anti-mouse CD45RB mAb (clone 16A, rat IgG2a), and PE-conjugated anti-mouse CD4 mAb (clone GK1.5, rat IgG2b) were purchased from PharMingen (San Diego, CA). Anti-mouse CD54 (clone KAT-1, rat IgG2a) was obtained from BioSource International (Nivelles, Belgium). Biotinylated anti-mouse F4/80 mAb (clone CI:A3–1, rat IgG2b), directed specifically against a murine macrophage-restricted cell surface glycoprotein, was purchased from Serotec (Oxford, U.K.). Hamster anti-mouse CD40L mAb (clone MR1) was purified from culture supernatants of the MR1 hybridoma (25), which was obtained from the American Type Culture Collection (Manassas, VA). Hamster IgG (HIg) used as a control Ab was purchased from BioTrend (Cologne, Germany).

Spleen cells from BALB/c mice were used as a source of CD4⁺ cells for reconstitution of SCID recipient mice. CD4⁺ T cells subsets from the spleen of BALB/c mice were isolated as described previously (22) with some modifications. Briefly, a single cell suspension was prepared in cold PBS from BALB/c spleen. CD4⁺ T cell subsets were purified by positive selection using mouse CD4 Dynabeads (L3T4) and mouse CD4 DETACHaBEAD (Dynal, Oslo, Norway). These procedures resulted in ≥98% CD4⁺ T cells as assessed on a FACSort (Becton Dickinson, San Jose, CA). For preparation of CD45RB^high and CD45RB^lowCD4⁺ T cell subsets, CD4⁺ T cells were labeled with FITC-conjugated anti-CD45RB and PE-conjugated anti-CD4 mAbs and fractionated into CD4⁺ CD45RB^high and CD4⁺CD45RB^low fractions under sterile conditions by two-color sorting on a FACS Vantage (Becton Dickinson). The CD45RB^high and CD45RB^low populations were defined as the brightest staining 40–50% and the dullest staining 15–20% CD4⁺ T cells, respectively. Intermediate staining populations were discarded. All populations were >99% pure on reanalysis.

BALB/c SCID mice were injected i.p. with sorted syngeneic CD45RB^high or CD45RB^low CD4⁺ T cells (4 × 10⁵/mouse of each cell). Disease activities were monitored weekly on the basis of body weight, soft stool or diarrhea, and anorectal prolapse. SCID mice, reconstituted with CD45RB^high T cells, were treated by i.p. injection with 250 μg anti-CD40L mAb (MR1) in 200 μl PBS twice weekly, from the beginning of T cell reconstitution over a period of 8 wk. An equivalent amount of HIg was administrated in control mice under identical conditions. Because previous work has reported loss of body weight in colitic SCID recipients starting 3–5 wk after T cell reconstitution (21, 22, 23, 24), we treated another group of SCID mice by i.p. injection with 250 μg anti-CD40L mAb in 200 μl PBS twice weekly, from 5 wk after T cell reconstitution up to wk 8, to determine the effect of delayed treatment with anti-CD40L. Mice were sacrificed 8 wk after T cell reconstitution and analyzed for bowel inflammation.

Tissue samples were fixed in PBS containing 6% neutral-buffered formalin. Paraffin-embedded sections (5 μm) were stained with hematoxylin and eosin. The sections were analyzed without prior knowledge of the type of T cell reconstitution or treatment. Microscopic sections were graded by the number and severity of lesions. The mean degree of inflammation in the colon was calculated using a modification of a previously described scoring system (Table I) (26).

Colonic samples for immunohistochemistry were embedded in OCT compound, snap-frozen in liquid nitrogen, and stored at −80°C. Cryostat sections (5 μm) of colonic tissue were fixed and stained for the presence of CD40, CD40L, CD54, CD4, and F4/80 using an avidin-biotin-peroxidase complex technique (20). Serial sections were incubated for 30 min with 5–10 μg/ml mAb directed against CD40L, CD40, or CD54. The biotinylated goat anti-hamster IgG (Jackson Immunoresearch Laboratories, West Grove, PA; 1:400) or rabbit anti-rat IgG (Dako, Glostrup, Denmark; 1:400) was chosen as second Ab. After three washes with PBS, the avidin-biotin-peroxidase complex (Dako) was added, and sections were incubated for 30 min. Staining for CD4 or F4/80 Ag was performed using biotinylated anti-CD4 or anti-F4/80 as primary Ab at a concentration of 5–10 μg/ml, followed by incubation with the avidin-biotin-peroxidase complex. The color reaction was developed with 0.05% 3-amino-9-ethylcarbazole (Janssen, Beerse, Belgium). The slides were counterstained with hematoxylin. Negative control experiments were performed by incubating sections with irrelevant isotype-matched HIg or rat IgG, or by omitting the primary Ab.

Colonic samples were washed thoroughly with PBS to remove debris and blood and were cut into 0.5-cm pieces. The epithelium was removed from the lamina propria by incubation with 2 mM DTT and 1 mM EDTA in PBS at 37°C for 2 × 20 min under gentle shaking. Tissues were subsequently minced into 2- × 2-mm pieces and digested with 0.5 mg/ml collagenase A (Boehringer Mannheim, Mannheim, Germany) in 5% CO₂ humidified air at 37°C for 90–120 min. Lamina propria mononuclear cells were harvested by discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradients. CD4⁺ T cells were then purified from lamina propria mononuclear cells by positive selection using mouse CD4 Dynabeads (L3T4) and mouse CD4 DETACHaBEAD as described above. The purity of cell population was >98% CD4⁺ T cells as assessed on a FACSort.

Ninety-six-well culture plates (Nunc, Roskilde, Denmark) were precoated with anti-CD3ε mAb (final concentration, 5 μg/ml) in 100 μl PBS at 37°C for 4 h and washed with PBS three times to remove unbound Ab. Lamina propria CD4⁺ T cells (5 × 10⁵/ml) were incubated in the presence of coated anti-CD3ε and mitomycin C-treated (50 μg/ml for 30 min at 37°C) mouse mastocytoma P815 cells transfected with mouse CD80 (5 × 10⁶/ml) at 37°C in 5% CO₂ humidified air. Samples were performed in quadruplicate in a total volume of 200 μl/well. After 48 h of culture, supernatants were harvested and assayed for IL-2, IFN-γ, and IL-4. IL-2 and IL-4 were measured by sandwich ELISA using paired matched Abs according to the manufacturer’s instructions (BioSource International). IFN-γ was determined by sandwich ELISA, as previously described (27).

Colonic tissue was obtained from SCID mice either anti-CD40L-treated or control HIg-treated or unmanipulated 8 wk after of CD45RB^highCD4⁺ T cell reconstitution. Colonic samples from naive SCID mice and those reconstituted with CD45RB^lowCD4⁺ T cells were also obtained as control. All colonic samples were immediately frozen in liquid nitrogen after dissection and stored at −80°C until used.

Total RNA was extracted using TRIzol reagents (Life Technologies, Gaithersburg, MD). A constant amount of 1 μg target RNA was used for cDNA synthesis. The cDNA reaction was conducted at 42°C for 80 min, using 100 U of Superscript II reverse transcriptase and 5 mM oligo(dT)₁₆ (Life Technologies). The sequences of the primers and probes for IFN-γ, IL-4, IL-5, IL-12, TNF, and β-actin have been previously reported (28). Sense/antisense PCR primers for murine CD40 and CD40L were 5′-GTCATCTGTGGTTTAAAGTCCCG-3′/5′-AGAGAAACACCCCGAAAATGG-3′ and 5′-CTCAAACTCTGAACAGTGCGCT-3′/5′-GGCAGGTCCTAACTGACTTGCT-3′, respectively. The probes for murine CD40 and CD40L were modified to incorporate a reporter dye at the 5′-end (6-carboxyfluorescein (FAM)) and a quencher at the 3′-end (6-carboxytetramethylrhodamine (TAMRA)): 5′-FAM-AGCCCTGCTGGTCATTCCTGTCGTG-TAMRA-3′ and 5′-FAM-AGGGAAGACTGCCAGCATCAGCCCT-TAMRA-3′, respectively. All primers and probes were designed with the assistance of the computer program Primer Express and purchased from PE Applied Biosystems (Foster City, CA). Real-time quantitative PCR was performed in the ABI prism 7700 sequence detector (PE Applied Biosystems), as previously described (28). The 5′-nuclease activity of the Taq polymerase was used to cleave a nonextendable dual-labeled fluorogenic probe. Fluorescent emission was measured continuously during the PCR reaction. PCR amplifications were performed in a total volume of 25 μl containing 0.5 μl cDNA sample, 50 mM KCl, 10 mM Tris-HCl, 10 mM EDTA, 60 nM Passive Reference 1, 200 μM dNTP, 3–9 mM MgCl₂, 100–200 nM concentrations of each primer, 0.625 U AmpliTaq Gold (PE Applied Biosystems), and 100 nM concentrations of the corresponding detection probe. Each PCR amplification was performed in triplicate wells using the following conditions: 50°C for 2 min and 94°C for 10 min, followed by 40 or 45 cycles at 94°C for 15 s and 60°C for 1 min. All results were normalized to β-actin to compensate for differences in the amount of cDNA in all samples.

Data were statistically analyzed by Student’s t test. p < 0.05 was considered significant.

BALB/c SCID mice were reconstituted with either CD45RB^high or CD45RB^lowCD4⁺ T cells, and clinical manifestations were monitored weekly up to 8 wk. As shown in Fig. 1, SCID mice that received CD45RB^highCD4⁺ T cells developed progressive weight loss after 3–5 wk of reconstitution. These mice had diarrhea with increased mucus in the stool, anorectal prolapse, and hunched posture by 6–8 wk. In contrast, SCID mice reconstituted with CD45RB^lowCD4⁺ T cells appeared healthy with gradual increase of body weight and absence of diarrhea during the period of observation. Additionally, SCID mice were reconstituted with CD45RB^high plus CD45RB^lowCD4⁺ T cells or total CD4⁺ T cells (n = 8 for each group). These mice, similar to those reconstituted with CD45RB^lowCD4⁺ T cells, showed gradual increase of body weight, and no detectable pathological changes in the intestine were seen (data not shown).

Macroscopically, the colon from SCID mice reconstituted with CD45RB^highCD4⁺ T cells was enlarged and had a greatly thickened wall. These changes were due to inflammation as shown by microscopy. Transmural inflammation was common in the ascending and transverse colon. The cellular inflammatory infiltrate was composed of large numbers of lymphocytes and macrophages mixed with a small population of neutrophils and eosinophils. Neutrophils were common in ulcerative areas (10–15% of the leukocytes) but rare in other areas (<5%), whereas monocytes/macrophages and lymphocytes were present in almost equal proportions. In the mucosa, the infiltrates showed a transmucosal distribution with diffuse basal lymphocytes. Epithelial lesions included ulceration, occasionally with a mountain-peak appearance, and less severe lesions such as mucin depletion, loss of goblet cells, and crypt abscesses (Fig. 2,A). In addition, architectural changes including crypt hyperplasia, crypt elongation, villous transformation of the surface, and crypt branching were regularly observed. The mucosal abnormalities were also seen in the cecum and terminal ileum. In these areas the lesions were characterized by focal aggregates of lymphocytes and macrophages in the lamina propria and by crypt hyperplasia. Splenomegaly with large amounts of lymphocytes was present in these colitic SCID mice. Moreover, infiltration of lymphocytes was commonly observed in the lamina propria and submucosa of the stomach, the periportal areas of liver, and mesenteric lymphoid nodes. In contrast, in colonic sections from CD45RB^lowCD4⁺ T cell-reconstituted mice, only few lymphocytes and macrophages were observed in the lamina propria (Fig. 2 B). The mean inflammatory score was significantly higher for CD45RB^high T cell-reconstituted mice (6.75 ± 1.75) compared with CD45RB^low T cell-reconstituted recipients (0.11 ± 0.08) (p < 0.005). In the lamina propria of the inflamed colon, the majority of CD4⁺ T cells displayed an activated memory phenotype with CD45RB^low and high expression of CD69 (65–76.8%) and CD25 (27.5–38.4%).

In situ expression of CD40 and CD40L in colonic sections was analyzed by immunohistochemistry. The number of cells expressing CD40 and CD40L was significantly increased in samples from the inflamed colon from all SCID mice reconstituted with CD45RB^highCD4⁺ T cells (Fig. 2, C and D). The majority of positive cells were located in the lamina propria and submucosa, but some were also observed in the tunica muscularis and subserosa. The positive cells were present as a diffuse population but also in aggregates, suggesting a granulomatous type of inflammation. A membranous CD40 expression was occasionally observed in endothelial cells. No expression of CD40 and CD40L was detected in epithelial cells. In contrast, CD40 and CD40L were not or only weakly expressed in colonic sections from SCID mice reconstituted with CD45RB^low, CD45RB^high plus CD45RB^low, or unseparated CD4⁺ T cells (data not shown). These findings indicate that many of the infiltrating CD4⁺ T cells express CD40L, whereas the majority of the infiltrating macrophages express CD40.

To study whether CD40-CD40L interactions participate in the inflammatory response in this model, we administered an anti-CD40L neutralizing mAb to SCID mice. SCID mice, reconstituted with CD45RB^highCD4⁺ T cells, were randomly assigned to receive either anti-CD40L neutralizing mAb or control HIg, respectively, at the dose of 250 μg/mouse by i.p. injection twice weekly starting at the time of T cell reconstitution and continuing over an 8-wk period. As shown in Fig. 3,A, control HIg-treated mice developed a severe colitis 6–8 wk postreconstitution, characterized by significant weight loss and diarrhea, with thickening of the colonic wall because of inflammation. Microscopic analysis of colonic sections showed transmural inflammation with high numbers of leukocytes in the lamina propria and submucosa, and prominent epithelial hyperplasia with loss of goblet cells, crypt abscesses, and extensive ulceration (Fig. 4,A). In contrast, anti-CD40L-treated mice appeared healthy and did not exhibit any signs of colitis, with gradual increase of body weight and absence of diarrhea. No detectable pathological changes were observed in the bowel wall (Fig. 4,B). The effect of anti-CD40L treatment was also illustrated by comparison of histological activity scores from colonic sections being 1.21 ± 0.42 in anti-CD40L-treated mice compared with 6.70 ± 1.24 in HIg-treated recipients (p < 0.005). A further quantitative evaluation of CD4⁺ T cell infiltration was provided by isolating the CD4⁺ T cells from the resected bowels. Only a few CD4⁺ T cells were recovered from the colonic tissue of anti-CD40L-treated mice as compared with those in HIg-treated mice (Table II). Because the number of cells recovered from the colon of colitic SCID mice was higher than the number of cells injected i.p., these results also suggest an extensive T cell proliferation in the inflamed colon. Additionally, anti-CD40L treatment resulted in a significant reduction of splenomegaly and of the lymphocyte infiltration in the spleen (Fig. 5), the liver and lymphoid nodes of SCID recipients.

Colonic sections from reconstituted SCID mice treated with either anti-CD40L or HIg were stained immunohistochemically for CD4, F4/80, and CD54. As shown in Fig. 4, the number of CD4⁺ T cells, F4/80⁺ macrophages, and CD54⁺ cells was considerably increased in the inflamed mucosa from HIg-treated mice. CD54⁺ cells were predominantly lymphocytes and macrophages and some were endothelial cells. In the colonic sections from anti-CD40L-treated mice, a low frequency of CD4⁺ T cells, F4/80⁺ macrophages, and CD54⁺ cells was found in lamina propria and submucosa, and no expression was observed in the muscularis and subserosa. These data indicate a critical role for the CD40-CD40L costimulatory pathway in the initial stage of intestinal inflammation.

We next evaluated whether delayed anti-CD40L treatment would improve ongoing disease. Given that our results had demonstrated that wasting disease started 3–5 wk after T cell reconstitution, and because infiltration of leukocytes and colitis were detectable from 5 wk on (data not shown), we started anti-CD40L treatment 4 wk after T cell reconstitution. Colitic SCID mice were randomly assigned to be injected i.p. with either anti-CD40L or control HIg, respectively, at the dose of 250 μg/mouse twice weekly from wk 5 after CD45RB^highCD4⁺ T cell reconstitution up to 8 wk. Fig. 3,B shows that anti-CD40L-treated mice had a significantly clinical and histological improvement with intermediate weight loss and absence of diarrhea. Histopathological analysis of colonic sections from anti-CD40L-treated mice revealed significantly diminished granulomatous inflammation, leukocyte infiltration, and epithelial hyperplasia. The inflammatory changes appeared to be focal with only mild leukocyte infiltration in the lamina propria, occasionally in the submucosa, and absence in muscular layers (data not shown). Histological scores in the colonic sections were significantly decreased in mice with delayed anti-CD40L treatment (3.75 ± 1.28) compared with those in HIg-treated mice (6.84 ± 1.44) (p < 0.05). The average CD4⁺ T cell recovery in the inflamed colon from delayed HIg-treated colitic SCID mouse was (1.53 ± 0.38) × 10⁶/colon; whereas in those that received delayed anti-CD40L treatment, CD4⁺ T cell recovery was (0.81 ± 0.20) × 10⁶/colon (Table II).

Previous work has shown that intestinal inflammation in colitic SCID mice is driven by a Th1 type of immune response with elevated levels of IFN-γ (22, 23). Because CD40-CD40L interaction plays a critical role in induction of Th1 immune responses (3, 4), we next analyzed whether production of proinflammatory cytokines was inhibited after anti-CD40L treatment. Colonic lamina propria CD4⁺ T cells were isolated from colitic SCID mice that had received delayed treatment with either anti-CD40L or HIg and were stimulated with coated anti-CD3ε mAb and CD80 transfectants. The levels of IL-2, IFN-γ, and IL-4 secreted in the supernatants were examined by ELISA. As shown in Fig. 6, lamina propria CD4⁺ T cells from HIg-treated mice produced high levels of IL-2 and IFN-γ. Of particular interest, delayed administration of anti-CD40L significantly down-regulated CD4⁺ T cell IL-2 and IFN-γ production as compared with controls (p < 0.001). IL-4 levels were very low in all groups (data not shown). These data suggest that colonic T cells in anti-CD40L-treated mice are less preactivated in vivo or that anti-CD40L mAb binding in vivo provides a long lasting negative signal for cytokine secretion on further stimulation.

To further confirm that anti-CD40L treatment down-regulates proinflammatory cytokine production in the colonic tissue, we also investigated the mRNA levels of cytokines and CD40 and CD40L in colonic tissues using real-time quantitative PCR technique. As shown in Table III, the mRNA levels of IFN-γ, TNF, and IL-12 p40 were greatly increased in SCID mice reconstituted with CD45RB^high CD4⁺ T cells under unmanipulated or HIg-treated conditions. The mRNA levels of CD40 and CD40L were also markedly increased in inflamed colon, in agreement with immunohistochemical results. Interestingly, mRNA levels of cytokines as well as CD40 and CD40L were significantly decreased in the early anti-CD40L-treated group (p < 0.05) and they were also down-regulated in the delayed treatment group. Relatively low mRNA levels of IL-4, IL-5, and IL-10 were seen in all groups (data not shown), concurrent with earlier reports (22, 23). All cytokine mRNA levels were found to be low in colonic samples from naive SCID mice and CD45RB^low T cell-reconstituted recipients. It should be stressed that the values given for cytokine mRNA levels in Table III do not allow a quantitative comparison between the cytokines but only comparison for the same cytokine among groups of mice. These findings indicate that the CD40-CD40L costimulatory pathway plays a critical role in the induction of a Th1 response in colitic SCID mice.

We next explored the development of experimental colitis after withdrawal of anti-CD40L treatment. Six SCID mice were treated for 8 wk with 250 μg anti-CD40L mAb twice weekly starting immediately after CD45RB^high CD4⁺ T cell reconstitution. After 8 wk, none of them had developed wasting disease (Fig. 7). Three mice were sacrificed for histological examination, showing that leukocyte infiltration, granulomatous inflammation, and epithelial hyperplasia were effectively prevented. Splenomegaly was significantly reduced and some lymphocytes were observed on the splenic sections, similar to findings shown in Fig. 5,C. Strikingly, the remaining three mice had weight loss by 4 wk after withdrawal of anti-CD40L administration and developed severe colitis by 6–7 wk after MR1 cessation with marked loss of body weight and diarrhea (Fig. 7). Anorectal prolapse was found in one of three. Macroscopically, the colon was significantly enlarged and had a thickened wall. Concurrent with this, there was an extensive leukocyte infiltrate in inflamed colon, mainly in the lamina propria and submucosa. Epithelial hyperplasia with glandular elongation was common. Ulceration was found in one colonic sample. Histological scores reached to 6.2 ± 1.1, as seen in HIg-treated recipients. These data suggest that after withdrawal of anti-CD40L and its degradation in vivo, CD4⁺ T cells that persist in the spleen and other organs (e.g., lymphoid nodes, liver, intestine) will be primed again by luminal Ags and expand into cytokine secreting effector cells in the intestine.

To study the role of CD40-CD40L interactions in intestinal inflammation, we have used BALB/c SCID mice that develop a wasting disease with severe colitis after reconstitution with syngeneic CD45RB^highCD4⁺ T cells. The disease was characterized by weight loss, soft stools, and diarrhea; anorectal prolapse; chronic inflammatory and epithelial lesions in the colon; and a local Th1 cytokine pattern, which is in accordance with earlier studies (21, 22, 23, 24). The number of CD40⁺ and CD40L⁺ cells and their mRNA levels were significantly increased in the inflamed colon. Early administration of anti-CD40L neutralizing mAb (starting at the time of disease induction) effectively prevented the onset and progression of colitis and significantly abrogated infiltration of leukocytes (CD4⁺ T cells and macrophages), expression of CD54, and local proinflammatory cytokine production (e.g., IL-2, IFN-γ, IL-12, TNF) in the inflamed colon. Moreover, delayed treatment with anti-CD40L still improved clinical and histological features of ongoing CD4⁺ T cell-mediated colitis and down-regulated local proinflammatory cytokine secretion. Colitis relapses were observed 6–7 wk after withdrawal of anti-CD40L treatment. Our present studies on anti-CD40L treatment in an experimental colitis in SCID mice thus stress the important role of the CD40-CD40L costimulatory pathway in CD4⁺ T cell-dependent inflammatory responses and tissue damage in intestinal mucosa in colitic SCID mice.

Although colitis in this SCID model is clearly initiated by injection of pathogenic T cells and depends on the Th1-type cytokine secretion, this experimental colitis does not cover all pathological and symptomatic aspects of human Crohn’s disease. Genetic predisposition, persistent infection, a defective mucosal barrier and an abnormal host immune response to food Ags may all play a role in the etiology of human IBD. Crohn’s disease manifests primarily as a transmural inflammation involving the full thickness of the bowel wall that frequently leads to fissuring ulcers, fistulas, and abscess formation (29). Crohn’s disease is further characterized by the presence of typical aphthous ulcers, by transmural lymphoid hyperplasia, and by the presence of granulomas. In the present model, transmural inflammation and typical mucosal ulcers (mountain-peak ulcer) are present, whereas granulomas are not observed. Granulomas are, however, found in only 15–78% of the Crohn’s disease cases. Additionally, Crohn’s disease can be found anyplace in the gastrointestinal tract and has a predilection for the terminal ileum and ascending colon, whereas in the present model the ascending colon is predominantly involved. Crohn’s disease is also discontinuous, with areas of inflammation alternating with normal areas, and these features are also present in colitic SCID mice.

To date, there is little information on the mechanisms whereby SCID mice develop colitis after CD45RB^highCD4⁺ T cell reconstitution. CD45RB^lowCD4⁺ T cells do not induce colitis, and they prevent colitis development if coadministered with CD45RB^highCD4⁺ T cells. CD45RB^high (naive) CD4⁺ T cells, compared with CD45RB^low (memory) CD4⁺ T cells, produce high levels of IFN-γ and IL-2 but little IL-4 in response to primary mitogenic stimuli (30). The CD45RB^low cells on the other hand produce IL-10, and the latter is responsible for control of the disease if both subsets are coadministered (31). Increased infiltration of CD62L, CD69, α_E integrin, and mucosal addressin cell adhesion molecule-1 has also been found in inflamed colon (24, 26). Wasting disease in this model can be abrogated after reducing the intestinal bacterial flora (24, 26). Bacterial Ag-activated CD4⁺ T cells from spontaneous colitic C3H/HeJBir mice, showing a Th1 phenotype of immune response, induce colitis after transfer into SCID mice (32). Thus, luminal bacterial Ags are the potential triggers of the inflammatory response in the SCID model, although it is not currently defined as to how these Ags trigger the initial CD4⁺ T cell activation and expansion. A role for intestinal Ags is also suggested by the absence or amelioration of intestinal inflammation in several knockout and transgenic models of IBD raised under completely germfree conditions (33, 34, 35).

To obtain more insight into disease pathogenesis, we analyzed the phenotypic features of lamina propria CD4⁺ T cells and macrophages in the inflamed colon. The majority of CD4⁺ T cells were CD45RB^low cells, and they expressed high levels of CD25 and CD69, indicating that these CD4⁺ T cells were activated memory cells. We have also found increased expression of B7.1 and B7.2 molecules by immunohistochemistry in the inflamed colon (our unpublished data), although their role in the intestinal immunopathology is currently unknown. CD54 and B7 family molecules may play a role in ensuring sustained activation of Ag-primed CD4⁺ T cells (36, 37, 38). As evidenced by quantitative mRNA analysis, CD4⁺ T cells and macrophages secrete large amounts of proinflammatory cytokines such as IFN-γ, TNF, and IL-12. IL-12 plays a critical role in the generation of Th1 immune response (39, 40). TNF has an important role in recruitment of inflammatory cells, partly by up-regulation of adhesion molecules on endothelial cells. The effects of IFN-γ, TNF, and IL-12 have been considered to be central to the pathogenesis in most animal models of experimental colitis, as well as in patients with Crohn’s disease (1). Treatment targeted against these proinflammatory cytokines has been found to effectively prevent intestinal inflammation in experimental models of colitis and (at least for TNF) also in patients with Crohn’s disease (22, 23, 41, 42). Some reports have shown that treatment targeted against IFN-γ, TNF, or mucosal addressin cell adhesion molecule-1 can decrease homing of leukocytes into the intestine and ameliorate wasting disease in these SCID recipients (22, 23, 26).

Most importantly, we found increased expression of CD40 and CD40L (by immunohistochemistry and PCR) in the colitis lesions. Early anti-CD40L treatment significantly decreased mRNA levels for the proinflammatory cytokines IFN-γ and TNF. Moreover, IL-12 p40 mRNA, which is abundant in infiltrates in inflamed colonic tissue, was effectively diminished after anti-CD40L treatment. These results were consistent with previous reports demonstrating that anti-CD40L administration inhibited Th1-driven inflammatory responses by reducing IL-12 production (11, 17, 18). Increased IL-12 production by mucosal macrophages or dendritic cells triggered by CD40 signaling can favor maturation of Th1 cells and development of cell-mediated immunity in the inflamed colon. CD40-CD40L interactions are indeed known to play a critical role in the induction of Th1 immune responses. Both CD40L transfectants and CD40L⁺ Th1 clones induce TNF and IL-12 secretion by CD40-expressing monocytes/macrophages in vitro (43, 44), whereas activated T cells and a Th1 clone derived from CD40L-deficient mice fail to induce IL-12 and TNF production (44, 45). CD40L-deficient mice not only show a defect in Ag-specific CD4⁺ T cell priming characteristic of a dramatic reduction of IFN-γ and IL-4 production (46) but also demonstrate markedly impaired production of TNF, nitric oxide, and IL-12, correlating with impaired cell-mediated immune responses against an intracellular parasite (e.g., Leishmania major) and enhanced susceptibility to infection (47, 48).

Our data thus underline the critical role of CD40 signaling in the intestinal immune pathology in this model at several levels. Anti-CD40L may prevent T-APC interactions so that T cells receive inadequate signals from residual host APC. Moreover, it can abrogate Th1 differentiation and effector function by blocking IL-12 production. Finally, anti-CD40L diminishes emigration of leukocytes at sites of inflammation in the intestine at least partly as a result of down-regulated TNF production, resulting in turn in decreased expression of CD54 on endothelial cells.

Delayed treatment with anti-CD40L also improved experimental colitis and inhibited local Th1 cytokine production, although to a lesser extent than when started immediately after T cell reconstitution. This suggests that the maintenance of intestinal inflammation requires continuous or repeated CD40-CD40L interactions and continuous T cell activity. Similar results have also demonstrated the effectiveness of anti-CD40L at advanced stages of other experimental diseases such as murine experimental allergic encephalomyelitis (9, 11) and murine lupus (12). However, no response to administration of anti-CD40L mAb was seen in established experimental diseases such as spontaneous autoimmune diabetes in nonobese diabetic mice (15) and murine colitis induced by 2,4,6-trinitrobenzenesulfonic acid (18). There is no clear explanation yet for the difference in efficacy of anti-CD40L treatment in the development and maintenance of experimental diseases. For effective inhibition of Th1-mediated inflammatory responses at the advanced stage of disease in colitic SCID mice, it is reasonable to perform further studies using increased doses of anti-CD40L mAb or combined treatments with mAb to other important player in the immune and inflammatory response.

Colitis relapses were observed 6–7 wk after withdrawal of anti-CD40L, this period of colitis relapse being similar to the time needed for colitis induction in naive SCID mice reconstituted with CD45RB^highCD4⁺ T cells. Histological analysis revealed persistent presence of low numbers of CD4⁺ T cells in the spleen and the lymphoid nodes as well as in colonic mucosa despite anti-CD40L treatment. It is likely that after withdrawal of anti-CD40L, CD4⁺ T cells that persisted in the spleen or other organs (e.g., liver, lymphoid nodes) are again being transported to the colonic mucosa by the bloodstream. Therefore, one must accept that the initial stimulus that triggers lamina propria CD4⁺ T cell activation and proliferation and induces expression of CD40L is permanently present. CD4⁺ T cells are then activated by luminal Ags, expand into effector cells, and induce inflammation in the colon in a CD40-CD40L-dependent way. Whether anti-CD40L treatment in the mean time acts directly by interfering with T cell homing to the colon, by interfering with T cell-dependent macrophage activation and cytokine secretion, and/or by interfering with T cell expansion, is unclear at this moment. In view of disease recurrence after treatment has been stopped, it is, however, excluded that Ag-responsive T cells were pushed into apoptosis after interaction with Ags and anti-CD40L, as has recently been shown to occur in monkeys treated with anti-CD40L mAb 5C8 after organ transplantation (49).

A hypothetical mechanism that governs the intestinal pathology in colitic SCID mice can now be proposed. After CD45RB^highCD4⁺ T cells migrate into the intestine, they are activated due to continuous exposure to dietary and microbial products, or possibly by intraluminal Ags presented by APC such as macrophages, dendritic cells, and perhaps epithelial cells in the intestinal mucosa. This event results in expression of CD40L. Interaction of CD40L on activated CD4⁺ T cells with CD40 on macrophages induces IL-12 secretion and initiates a proinflammatory cascade that includes a Th1 immune response (i.e., IFN-γ) and macrophage cytokine secretion (i.e., TNF), which amplify inflammatory responses. Furthermore, CD40-CD40L interactions also induce expression of costimulatory molecules such as B7 family molecules on macrophages/dendritic cells to amplify immune responses, as well as expression of adhesion molecules such as CD54 and mucosal addressin cell adhesion molecule-1 (26) on endothelial cells to enhance leukocyte infiltration in the inflammatory sites.

This work may have important implications in understanding initiation of intestinal immunopathology and progression of established disease. Our studies also provide evidence for the potential use of anti-CD40L immunotherapy for human IBD, especially in newly diagnosed patients or in the early stages of disease flare-up.

We thank Dr. J. Plum (University of Gent, Gent, Belgium) for his help in cell sorting on a FACS Vantage.