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

Animals

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.

Reagents and mAbs

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).

Isolation of CD4⁺ T cell subpopulations

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.

Reconstitution of SCID mice with T cell subpopulations and Ab treatment

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.

Histological examination and immunohistochemistry

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.

Isolation of lamina propria CD4⁺ T cells

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.

Lamina propria CD4⁺ T cell cytokine production

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).

RNA extraction and quantitative PCR

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.

Statistical analysis

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

BALB/c SCID mice reconstituted with CD45RB^highCD4⁺ T cells develop a wasting disease with severe colitis

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).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

BALB/c SCID mice reconstituted with CD45RB^highCD4⁺ T cells develop a wasting disease. BALB/c SCID mice were reconstituted with either CD45RB^high or CD45RB^lowCD4⁺ T cells (4 × 10⁵/mouse). The change of weight over an 8-wk period of observation is expressed as a percent of the original weight at the start of the experiment. Data represent the mean ± SEM of both groups from three experiments.

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%).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Expression of CD40 and CD40L in inflamed colon from colitic SCID mice. BALB/c SCID mice were reconstituted with T cell subpopulations as described in Fig. 1⇑. Colonic section from a CD45RB^highCD4⁺ T cell-reconstituted mouse showing severe inflammation, leukocyte infiltration, and ulceration (A). Colonic section from a CD45RB^lowCD4⁺ T cell-reconstituted mouse showing little leukocyte infiltration in the lamina propria (B). Cryostat sections (5 μm) of colonic samples from colitic SCID mice were stained immunohistochemically with mAbs directed against CD40L (C) and CD40 (D), showing increased expression of CD40 and CD40L in the inflamed mucosa from colitic SCID mice. Original magnifications: A and B, ×20; C and D, ×50.

Increased expression of CD40 and CD40L in inflamed colon from SCID mice reconstituted with CD45RB^highCD4⁺ T cells

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.

Early administration of anti-CD40L mAb prevents the onset and severity of experimental colitis

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.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Effects of early and delayed anti-CD40L mAb treatment on colitis. A, Early anti-CD40L mAb treatment prevents the development of colitis in SCID mice. Colitic SCID mice were treated for 8 wk with either anti-CD40L mAb or HIg starting at the time of CD45RB^highCD4⁺ T cell reconstitution. B, Delayed administration of anti-CD40L mAb improves experimental colitis in colitic SCID mice. Colitic SCID mice were treated with either anti-CD40L mAb or HIg starting 5 wk after CD45RB^highCD4⁺ T cell reconstitution and continuing to wk 8. The change of weight over time is expressed as percent of the original weight. The arrow indicates the start of treatment. Data represent the mean ± SEM of each group from three experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Early anti-CD40L administration prevents intestinal mucosal inflammation and diminishes leukocyte infiltration and CD54 expression in the inflamed colon. Colonic sections from colitic SCID mice treated with either HIg (left panels) or anti-CD40L (right panels) starting at the time of CD45RB^high T cell reconstitution up to 8 wk were stained with hematoxylin and eosin (A and B) or were stained immunohistochemically with anti-CD4 (C and D), mAb F4/80 (E and F), and anti-CD54 (G and H), respectively. Colonic sections from HIg-treated mice show severe colitis and increased numbers of CD4⁺, F4/80⁺, and CD54⁺ cells in lamina propria, whereas colonic sections from anti-CD40L-treated mice show normal intestinal mucosa and a few CD4⁺, F4/80⁺, and CD54⁺ cells in lamina propria. Original magnifications: A and B, ×50; C–H, ×150.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Anti-CD40L treatment down-regulates but does not abolish lymphocyte repopulation in the spleen of colitic SCID mice. Splenic sections from naive SCID mice (A), or colitic SCID mice given early treatment with either HIg (B) or anti-CD40L (C) were stained with hematoxylin and eosin. Splenic sections from HIg-treated mice show that lymphocytes are present in the follicles, whereas sections from anti-CD40L-treated mice demonstrate small numbers of lymphocytes in the white pulp area. Only few lymphocytes are present in the follicles from naive SCID mice. Extramedullary hemopoiesis is prominent in all animals as shown by the presence of megakaryocytes (M). Arrows indicate the white pulp area. The micrographs are representative of two different experiments with two mice per group. Original magnification, ×325.

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.

Anti-CD40L improves clinical manifestations when started 4 wk after T cell reconstitution

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⇑).

Cytokine secretion by lamina propria CD4⁺ T cells

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.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Delayed treatment with anti-CD40L decreases IL-2 and IFN-γ production by lamina propria CD4⁺ T cells. Lamina propria CD4⁺ T cells were isolated from either untreated colitic SCID mice (n = 6) or colitic SCID mice treated with anti-CD40L (n = 6) or HIg (n = 5) starting 5 wk after CD45RB^highCD4⁺ T cell reconstitution and continued till wk 8. These T cells (5 × 10⁵/ml) were incubated with coated anti-CD3ε (5 μg/ml) plus CD80-transfected P815 cells (5 × 10⁵/ml). Supernatants were harvested after 48 h of culture, and IL-2 and IFN-γ were assayed by ELISA. ∗, p < 0.005 vs controls.

Transcriptional levels of cytokines, CD40, and CD40L in colonic tissue

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.

Colitis relapses after withdrawal of anti-CD40L treatment

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.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Colitis relapses after withdrawal of anti-CD40L treatment. Colitic SCID mice (n = 6) were treated with anti-CD40L mAb starting after CD45RB^highCD4⁺ T cell reconstitution and continuing over an 8-wk period. Three mice were then sacrificed for histological examination. The three remaining mice were observed for the following 7 wk, and clinical features were monitored. The arrow indicates the end of anti-CD40L treatment. Each line represents an individual mouse.