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*
Laboratory of Experimental Immunology,
Department of Pathology,
Laboratory of Experimental Transplantation, and
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Department of Gastroenterology, University Hospital Gasthuisberg, Leuven, Belgium;
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Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium; and
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Tanox Pharma, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF, and IL-12. When
colitic SCID mice were treated with anti-CD40L starting at 5 wk
after T cell transfer up to 8 wk, this delayed treatment still led to
significant clinical and histological improvement and down-regulated
proinflammatory cytokine secretion. These data suggest that the
CD40-CD40L interactions are essential for the Th1 inflammatory
responses in the bowel in this experimental model of colitis. Blockade
of CD40 signaling may be beneficial to human
IBD. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 CD45RBhighCD4+ 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 CD3
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 CD45RBhigh and
CD45RBlowCD4+ T cell
subsets, CD4+ T cells were labeled with
FITC-conjugated anti-CD45RB and PE-conjugated anti-CD4 mAbs and
fractionated into CD4+
CD45RBhigh and
CD4+CD45RBlow fractions
under sterile conditions by two-color sorting on a FACS Vantage (Becton
Dickinson). The CD45RBhigh and
CD45RBlow 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 CD45RBhigh or CD45RBlow CD4+ T cells (4 x 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 CD45RBhigh 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).
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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 x 20 min under gentle shaking. Tissues were subsequently minced into 2- x 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-CD3 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 x 105/ml) were incubated in the presence
of coated anti-CD3 and mitomycin C-treated (50 µg/ml for 30
min at 37°C) mouse mastocytoma P815 cells transfected with mouse CD80
(5 x 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-, 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- 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 CD45RBhighCD4+ T cell reconstitution. Colonic samples from naive SCID mice and those reconstituted with CD45RBlowCD4+ 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-, IL-4, IL-5, IL-12, TNF, and
ß-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 ß-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c SCID mice were reconstituted with either
CD45RBhigh or
CD45RBlowCD4+ T cells, and
clinical manifestations were monitored weekly up to 8 wk. As shown in
Fig. 1, SCID mice that received
CD45RBhighCD4+ 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
CD45RBlowCD4+ 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 CD45RBhigh plus
CD45RBlowCD4+ T cells or
total CD4+ T cells (n = 8 for
each group). These mice, similar to those reconstituted with
CD45RBlowCD4+ T cells,
showed gradual increase of body weight, and no detectable pathological
changes in the intestine were seen (data not shown).
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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 CD45RBlowCD4+ T
cell-reconstituted mice, only few lymphocytes and macrophages were
observed in the lamina propria (Fig. 2B). The mean
inflammatory score was significantly higher for
CD45RBhigh T cell-reconstituted mice (6.75
± 1.75) compared with CD45RBlow 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
CD45RBlow and high expression of CD69
(65–76.8%) and CD25 (27.5–38.4%).
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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
CD45RBhighCD4+ 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 CD45RBlow,
CD45RBhigh plus CD45RBlow,
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
CD45RBhighCD4+ 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. 3A, 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. 4A). 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. 4B). 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.
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, 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
CD45RBhighCD4+ T cell
reconstitution up to 8 wk. Fig. 3B 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) x
106/colon; whereas in those that received delayed
anti-CD40L treatment, CD4+ T cell recovery
was (0.81 ± 0.20) x 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- (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-CD3
mAb and CD80 transfectants. The levels of IL-2, IFN-, 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-. Of particular interest, delayed
administration of anti-CD40L significantly down-regulated
CD4+ T cell IL-2 and IFN- 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.
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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-, TNF, and
IL-12 p40 were greatly increased in SCID mice reconstituted with
CD45RBhigh 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 CD45RBlow 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.
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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 CD45RBhigh 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. 5C. 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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
CD45RBhighCD4+ T cell
reconstitution.
CD45RBlowCD4+ T cells do
not induce colitis, and they prevent colitis development if
coadministered with
CD45RBhighCD4+ T cells.
CD45RBhigh (naive) CD4+ T
cells, compared with CD45RBlow (memory)
CD4+ T cells, produce high levels of IFN- and
IL-2 but little IL-4 in response to primary mitogenic stimuli
(30). The CD45RBlow 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, 
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 CD45RBlow
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-, 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-, 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-, 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- 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- 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 CD45RBhighCD4+ 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
CD45RBhighCD4+ 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-) 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jan L. Ceuppens, Laboratory of Experimental Immunology, U.Z. Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium.
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; CD40L, CD40 ligand; HIg, hamster IgG; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.
Received for publication December 8, 1999. Accepted for publication March 13, 2000.
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