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*
Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, and
Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114;
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC;
§
Department of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712; and
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Division of Gastroenterology, Albany Medical College, Albany, NY 12208
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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was decreased, corresponding to a Th2 pattern of T cell
activation. In CCR2-deficient mice, the infiltration of Th2-type T
cells in the lamina propria was absent, but increased levels of IL-10
and decreased levels of IFN- may have down regulated mucosal
inflammation. Our data indicate that CCR5 may be critical for the
promotion of intestinal Th1-type immune responses in
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of chemokines have been assumed to contribute to the regulation of intestinal immune responses and mucosal inflammation, largely based on their increased expression during mucosal inflammation and their known properties as leukocyte chemoattractants and activators in vitro (4). However, determination of the role of individual chemokines and/or receptors in the regulation of mucosal immune cells in vitro has been hampered by the apparent redundancy in the chemokine/chemokine receptor network (5). Thus, the function of specific chemokine ligand/receptor systems in the attraction of leukocyte subpopulations in the healthy mucosa and during intestinal inflammation remains largely unclear.
Chemokines exert their effects through specific receptors that are
differentially expressed on cell populations of hemopoietic origin
(1). The CC chemokine receptors CCR2 and CCR5 are involved
in both the regulation of monocyte- and macrophage-mediated immune
responses and in the modulation of T cell migration and activation. In
humans, two related receptors, CCR2A and CCR2B (6),
mediate responses to monocyte chemoattractant protein-1
(MCP-1),3 MCP-2, and
MCP-3. Interestingly, they also serve as necessary coreceptors for
several HIV isolates (7, 8). Increased expression of MCP-1
and MCP-3 has been found in colonic tissue from patients with
inflammatory bowel disease (IBD), and these chemokines possess
functional activity that could contribute to the inflammatory process
in the affected mucosa (9, 10). MCP-1 is a potent
chemoattractant and activator of monocytes (11, 12), NK
cells (13), and memory T cells (14) in vitro.
MCP-3 is able to induce activation of dendritic cells, eosinophils,
basophils, and neutrophils, whereas MCP-2 stimulates both eosinophils
and basophils (15). Expression of CCR2 on leukocytes is
highly regulated. CCR2 expression is decreased on monocytes after
stimulation with IFN-, TNF-
plus IL-1, or LPS
(16, 17, 18), whereas IL-2 can induce CCR2 expression on T
cells (19).
CCR5 binds the CC chemokines macrophage-inflammatory protein
(MIP)-1, MIP-1ß, and RANTES (20), which are released
from intestinal epithelial cells upon exposure to inflammatory
mediators (21, 22). Expression of both MIP-1 and RANTES
is increased in the intestinal mucosa in IBD (23, 24, 25).
Experiments performed in vitro suggest that all three CCR5 ligands are
involved in the generation of Th1 immune responses (26), a
property which may be relevant to both murine colitis models and human
Crohn’s disease in which increasing evidence suggests a predominant
Th1 response. In addition, CCR5-binding chemokines preferentially
attract memory and activated CD4+ and
CD8+ T cells (27, 28), which is
consistent with the presence of the CCR5 receptor on these lymphocytes
(29). In this study, mutant mice lacking either CCR2 or
CCR5 expression were used to determine the role of these chemokine
receptors in intestinal inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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129/Ola mice with a targeted deletion of the CCR2 gene were created as previously described (30) and subsequently backcrossed onto a C57BL/6J background. CCR5-deficient mice were generated by the insertion of a neomycin resistance gene in the first intron of the CCR5 gene, causing a deletion of the entire second exon and a portion of the 3' untranslated region. Correctly targeted embryonic stem cells were selected, and male chimeras were generated and mated to C57BL/6J females. F1 heterozygotes were mated to obtain homozygous CCR5-deficient mice (W. A. Kuziel, T. Dawson, and N. Maeda, manuscript in preparation). Control animals were generated by crossing F1 wild-type progeny of 129/Ola and C57BL/6J matings. The presence of a BglII restriction-length polymorphism was used to confirm that the CCR2 and CCR5 gene cluster in the control animals were derived from the 129/Ola strain (30).
The clinical course of the dextran sodium sulfate (DSS) colitis in the
animal groups (Figs. 1
-4) was determined
in wild type (n = 10), CCR2 (n = 9),
and CCR5 (n = 9) mice. Flow cytometry for the
characterization of lymphocyte phenotypes was conducted in wild type
(n = 16), CCR2 (n = 17), and CCR5
(n = 14). The determination of the expression of
lymphocyte differentiation markers, chemokines, and cytokines in
colonic tissues was conducted in at least five animals per group. All
mice were female and 10–12 wk old at the beginning of the trial. All
animal experiments were performed in accordance with National
Institutes of Health guidelines and protocols approved by the
Subcommittee on Research Animal Care at Massachusetts General Hospital
and Harvard Medical School.
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Colitis was induced in all groups by the addition of DSS (2.5% weight to volume ratio dissolved in distilled water; m.w. 40,000; ICN Biomedicals, Aurora, OH; lot no. 3073B) to their drinking water, as described previously (31). The mean DSS/water consumption and daily weights were recorded. Stool consistency was assessed daily using the following four-point scale: 0, normal; 1, soft; 2, very soft but formed; and 3, liquid. Fecal blood was assessed daily as follows: a single fecal pellet was suspended in 400 µl of H2O and centrifuged at maximum in a bench-top microcentrifuge ( Eppendorf, model 5415C, Brinkman Instruments, Westbury, NY). A 40-µl aliquot was then added to a 0.5 x 0.5-cm piece of Hemocult SENSA paper, allowed to air dry, and developed with one drop of the provided SENSA developer solution (SmithKline Diagnostics, San Jose, CA). The presence of blood results in a white to blue color change of the SENSA paper that is proportional to the amount of blood present in the stool sample. The intensity of the SENSA color change was scored by observers blinded to treatment and was incorporated into the following five-point fecal blood scale: 0, nil; 1, faintly blue; 2, moderately blue; 3, dark blue; and 4, blood visible in the stool to the naked eye. Serial dilutions of whole blood showed that the above assay could reliably detect 0.02–0.03 µl of blood per ml, and the ranges for assigned scores were: score of 1–1.5, 0.005–0.16 µl blood/ml; score of 2–3, 0.16–0.31 µl blood/ml; score of 4, minimum of 0.63 µl of blood/ml (based on the assessment of 52 samples by two independent investigators). The reliability and reproducibility of both the fecal blood and diarrhea scales were assessed in 32 consecutive samples. The Pearson correlation coefficient was greater than 0.9 when the scores obtained by two independent investigators (P.L.B. and P.G.A.), blinded to the identity of the samples, were compared.
General assessment of colitis
All animals were sacrificed by carbon dioxide narcosis. The entire colon was removed, and the length was recorded as previously described (32). The number of adhesions between the colon and other peritoneal structures was also recorded. The extent of adhesions was scored as follows: 0, nil; 1, adhesions to one organ; 2, adhesions to two organs and/or two distinct areas of adhesions; 3, adhesions to three organs and/or three distinct areas of adhesions. Colons were subsequently opened longitudinally, and the extent and severity of the colitis was assessed macroscopically as follows: 0, normal; 1, erythema of the mucosa; 2, presence of ulcers; and 3, diffuse ulceration. The extent of disease was determined by measuring the percentage of the colon and cecum that had macroscopic evidence of colitis.
The colons were divided into three sections: distal, mid-colon, and proximal colon. The distal colon was used for myeloperoxidase (MPO) assay, the mid-colon for histology, and the proximal colon for isolation of RNA. Tissue sections were examined in a blind fashion. Each section was scored for severity and extent of ulceration, and the tissue thickness from the muscularis propria to the luminal border was determined. Lesion severity was graded using a modification of a previously defined scoring system (33) with a scale of 0–3: 0, normal; 1, mild; 2, moderate; and 3, severe. Mild lesions contained small, focal, or widely dispersed areas of inflammation and/or fibrosis above the muscularis mucosa. Moderate lesions were multifocal or locally extensive and contained inflammation or fibrosis extending into the submucosa. Severe was defined as inflammatory cells extending into the muscularis propria. The reliability and reproducibility of this semiquantitative evaluation was assessed by comparing the scores of two independent investigators (P.L.B. and P.G.A.); the Pearson correlation coefficient was 0.787 (95% confidence interval (CI), 0.580–0.898).
MPO assay
This assay was performed as previously described (34). Absorbance at 460 nm was determined for three separate 30-s intervals. One unit of MPO activity was defined as 1 µmol H2O2 broken down to H2O and O- by MPO (a change in absorbance of 1.13 x 102).
Immunohistochemistry
Tissue samples were frozen in OTC compound (Ames, Elkhart, IN). Sections cut from these tissues were then fixed in acetone and incubated with primary and subsequently secondary Abs as previously described (35). Purified primary Abs against CD4 (RM4–5) were purchased from PharMingen (San Diego, CA). Anti-F4/80 Ab was purchased from Serotec (Raleigh, NC). The secondary Ab was a biotinylated rabbit anti-rat Ig (Vector, Burlingame, CA). Each immunohistologic section was subsequently examined in a blind fashion by two investigators (P.G.A. and H.-C.R.), who counted the number of positive cells per five separate high-powered fields. Statistical analysis was performed as described below.
Lamina propria leukocyte isolation and flow cytometry
Leukocytes were extracted from the lamina propria of the large
intestine as previously described (36). For flow
cytometry, 2 x 105 colonic lamina propria
cells were washed in PBS containing 0.2% BSA and 0.1% sodium azide.
They were subsequently incubated first with blocking buffer (10%
normal hamster, rat, and mouse serum; and 1 µg anti-CD16/32 mAb)
at 4°C for 20 min, and then with FITC- and PE-tagged mAbs at 4°C
for 30 min. After washing, cells were analyzed using Lysis II software
on FACScan (Becton Dickinson, Mountain View, CA). The mAbs (PharMingen)
used in this study were FITC-CD3 (145-2C11) and -B220 (RA3-6B2), and
PE-CD4 (RM4-4), -CD8 (53-6.7), and -NK-1.1 (PK136).
RNA analysis
Whole colonic tissue RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the instructions of the manufacturer. RNase protection assays (RPAs) were performed using the Riboquant multiprobe RPA system (PharMingen) according to the instructions of the manufacturer. Mouse cluster of differentiation-1 or mouse cluster of chemokine-5 probes were synthesized and incubated with 10–15 mg of RNA for 12–16 h. The samples were analyzed by autoradiography. Bands were quantitated using NIH Image 1.61 scanning software. Data are presented after correction for the average intensity of the L32 and GAPDH bands (relative band intensity).
RT-PCR
RT-PCR was performed as previously described (9).
The PCR reaction was performed using 1 µl of the cDNA product
produced by the reverse transcription reaction. This product was
amplified in a final concentration of 1x PCR buffer
(Perkin-Elmer-Cetus, Norwalk, CT), 0.8 µmol of each primer, 0.2 mM
dNTPs, and 1 U of Taq polymerase (Perkin-Elmer-Cetus) in a
total volume of 50 µl. The primers, Mg2+
concentration, and cycle parameters used for each individual PCR
amplification are shown in Table I.
Amplification was conducted in a PTC-200 Peltier Thermal Cycler (MJ
Research, Watertown, MA).
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Data are presented as the mean ± SEM or as indicated. Parametric data were analyzed using a one-way ANOVA and then a Dunnett multiple comparisons post test. Nonparametric data (scoring) were analyzed using a Kruskal-Wallis test (nonparametric ANOVA) and then a Dunn’s multiple comparisons posttest. An associated probability (p value) of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mean consumption of 2.5% DSS in water was equal in all animals
throughout the duration of the study: wild-type mice, 5.53 ± 1.6
ml/day; CCR2-deficient, 5.25 ± 1.3 ml/day
(p > 0.1); and CCR5-deficient, 4.62 ±
1.7 ml/day (p > 0.1). Over the 7-day course of
DSS, wild-type mice developed diarrhea and hemocult-positive stools,
which were also often bloody. They became progressively more lethargic
and lost almost 15% of their body weight (Fig. 1
, A–C). In contrast, the CCR5-deficient animals
remained active throughout the course of the study and experienced
minimal weight loss (Fig. 1A). This difference became
statistically significant after day 5 of the trial. Diarrhea and
hemocult scores of the CCR5-deficient mice were also significantly
lower than those of the wild-type animals (Fig. 1, B and
C). In aggregate, these findings suggest that CCR5-deficient
mice experience clinically less severe DSS-mediated colitis than
wild-type mice. By day 7, the CCR2-deficient mice lost an amount of
weight comparable to that of wild-type animals and developed
quantitatively similar stool diarrhea and hemocult scores. However, the
development of diarrhea and bloody stools was delayed (Fig. 1, B and C), with a significantly reduced hemocult
score on day 4 of the trial and significantly diminished diarrhea on
days 1, 3, and 4.
After 7 days of DSS ingestion, macroscopic examination showed
significantly fewer intraabdominal adhesions in CCR5- and
CCR2-deficient mice compared with those in wild-type mice (Fig. 2A). Their colons were also
less erythematous and less ulcerated (Fig. 2A). This was
especially notable in the CCR5-deficient mice, whose colons frequently
looked normal. These observations paralleled the extent of measured
affected colon. CCR5- and CCR2-deficient mice exhibited significantly
less macroscopic disease in both the cecum and distal colon than
wild-type mice (Fig. 2B).
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). As
demonstrated in Fig. 4A,
panels 1–3, wild-type animals often had extensive deep
ulcerations characterized by near obliteration of the mucosa and rarely
had areas of normal-appearing mucosa. In contrast, the colonic mucosa
of CCR5-deficient mice was characterized by a diminished inflammatory
infiltrate, and many areas appeared normal. The average percentage of
ulcerated tissue in both the CCR5- and CCR2-deficient animals was less
than that in the wild-type controls at 3 and 7 days of DSS ingestion
(Fig. 3A). The difference between the CCR5 and wild-type
mice was statistically significant at both day 3 and day 7, whereas the
difference between CCR2 knockout mice and wild-type mice was
significant only at day 3. Assessment of mucosal inflammation revealed
a significantly diminished average depth of inflammatory cell
penetration in CCR5- and CCR2-deficient animals at both 3- and 7-day
time points (Fig. 3B).
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Induction of DSS colitis in mice has been associated with the influx of macrophages into the intestinal mucosa. Therefore, we determined whether the diminished severity of DSS-mediated colitis in the CCR5-deficient and CCR2-deficient mice was associated with alterations in the composition of immune cell populations in the intestinal mucosa. Macrophage infiltration into the lamina propria was assessed by immunohistochemistry. Mucosal frozen tissue sections were stained with anti-F4/80 Abs to detect mucosal macrophages and with Abs to detect CD4+ T cells.
As demonstrated in Fig. 4, DSS-induced mucosal inflammation in all
animal groups was characterized by a significant increase in
F4/80+ macrophages in the lamina propria. The
number of macrophages in the CCR2- and CCR5-deficient animals was not
statistically different when compared with the wild-type controls at
either day 3 or day 7 (Fig. 4, A panels 4–6 and
B). In contrast, the number of mucosal
CD4+ T cells during DSS-induced colitis in CCR2-
and CCR5-deficient mice differed significantly from that in wild-type
mice. The intestinal mucosa of CCR5-deficient mice was characterized by
an enhanced infiltration of CD4+ lymphocytes
after 7 days of DSS administration compared with either wild-type or
CCR2-deficient mice (Fig. 4, A panels 7–9 and
B). The intestinal mucosa of CCR2-deficient mice did not
contain a significantly increased number of CD4+
T cells (Fig. 4B).
To quantitate mucosal neutrophil infiltration, colonic MPO activity was
determined after 7 days of DSS (Fig. 4C). No difference in
colonic MPO activity between CCR2- and wild-type mice was observed:
wild-type, 32.4 ± 9.2 U; CCR2-deficient, 32.8 ± 6.4 U. MPO
activity in the colon of CCR5-deficient mice (28.7 ± 6.3 U)
appeared to be lower when compared with wild-type and CCR2-deficient
mice, but this difference did not reach statistical significance (Fig. 4C).
CCR5-deficient mice are characterized by an increase of CD4+ and NK1.1+ lymphocytes in the intestinal mucosa
To determine the DSS-induced regulation of lamina propria
lymphocytes in wild-type, CCR2-deficient, and CCR5-deficient mice,
lymphocytes were isolated from the colons of all three groups of mice
before and at days 3 and 7 of DSS. The phenotype and the composition of
mucosal lymphocyte before and during the development of DSS-induced
colitis were assessed by FACS analysis (Fig. 5 and Table II). The expected number of isolated
cells was proportional to the degree of inflammation in the colon. The
CD4+ and CD8+ T cell
populations were differentially regulated in the wild-type,
CCR2-deficient, and CCR5-deficient animals. As demonstrated in Table II, CCR5-deficient animals were characterized by an increased number of
CD4+ lymphocytes and a reduced number of
CD8+ lymphocytes before and during DSS-induced
colitis. The resulting CD4/CD8 ratio increased from 1.8 ± 0.3
before DSS treatment to 5.8 ± 2.3 at day 7 in CCR5-deficient animals.
In contrast, the lamina propria of CCR2 deficient mice was
characterized by an increase in the CD4/CD8 ratio from 0.9 ± 0.1 to
1.5 ± 0.4, whereas in wild-type mice the CD4/CD8 ratio changed from
1.0 ± 0.3 to 1.4 ± 0.1 over the 7-day DSS treatment. These data
indicate that CCR5-deficient mice have an increased percentage of
CD4+ cells in the lamina propria lymphocyte
population, and this population may be further expanded during the
course of the DSS-induced colitis. In all animal groups, we observed a
decrease in CD4+ T cells in the intestinal mucosa
at day 3 of the DSS treatment. This decrease was accompanied by an
increased number of CD8+ cells in the wild-type
and the CCR2-deficient animals. This increase in
CD8+ T cells was absent in the CCR5-deficient
animals (Table II). The total number of isolated
CD3+ T cells was not different in the animal
groups.
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, the percentage of NK
1.1+ lymphocyte increased from a mean ± SD of
4.7 ± 5.8% to 14.5 ± 6.1% at day 3 to reach 13.8 ± 1.3% percent
after 7 days of DSS treatment. In contrast, the percentage of NK
1.1+ lymphocyte increased in the wild-type mice from 2.7 ±
1.0% to 7.7 ± 3.2% and 6.1 ± 0.8% and in the CCR2-deficient group
from 4.7 ± 4.3% to 8.2 ± 4.5% and 5.4 ± 3.0% before DSS
treatment, at day 3, and at day 7, respectively. In all animal groups,
the strongest influx or expansion of NK1.1+ lymphocyte in the
intestinal mucosa occurred on day 3 of the DSS treatment.
The percentage of B cells in the lamina propria was also differentially
regulated during DSS colitis in wild-type and chemokine
receptor-deficient animals. Although the number of B cells in the
wild-type mice decreased from 45.2 ± 5.7% down to 34.5 ± 5.4% at
day 3 and rebounded back to 38.9 ± 4.7% at day 7, the percentage of
lamina propria B cells increased from 27.4 ± 4.0% to 43.5 ± 3.8% in
the CCR2-deficient mice (Table II). In the CCR5-deficient mice, the B
cell population initially decreased from 34.0 ± 14.2% down to 18.8 ±
9.3% at day 3 and then increased back to 34.8 ± 2.6% after 7 days of
DSS treatment (Table II).
To further characterize lymphocytes within the intestinal mucosa,
additional analysis was performed by RPA using specific RNA probes for
TCR
, TCR, CD3
, CD4, CD8, CD8ß, CD19, F4/80, and CD45 as
well as for the two housekeeping genes L32 and GAPDH (Fig. 6). Quantification of gel band density
revealed that in untreated animals, CCR5-deficient mice had a 2-fold
increase in the expression of CD4 mRNA compared to controls. Baseline
expression of the other markers of T cell and macrophage
differentiation was equivalent between the mouse groups (Fig. 6). After
7 days of DSS, CCR5-deficient mice had a 2-fold elevation in CD4
mRNA expression compared with wild-type controls, which is
consistent with data obtained by flow cytometry. CCR5-deficient mice
had a 2-fold increase in mucosal CD8 and CD8ß mRNA
expression (Fig. 6). The amount of TCR, CD3, CD19,
CD45, and F4/80 mRNA expression after DSS exposure was comparable to
that of wild-type mice. The equivalent F4/80 mRNA expression suggests
the presence of similar numbers of macrophages in the lamina propria of
CCR5-deficient and wild-type mice.
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and CD8 compared with that in wild-type
controls. After DSS exposure, CD8 mRNA expression was further
increased and was comparable to that of wild-type mice (Fig. 6).
Moreover, CCR2-deficient mice had a 10-fold increase in CD19 mRNA
expression after 3 days of DSS that returned to baseline by day 7. The
expression of mRNA of all other cell markers studied, including F4/80
and CD45, was not altered after DSS administration in either
CCR2-deficient or wild-type animals, suggesting that the absence of
CCR2 does not affect macrophage and overall leukocyte recruitment
in this model (Fig. 6). All animal groups developed a DSS-dependent
increase in TCR mRNA expression, although this pattern was less
pronounced in the CCR2-deficient mice (Fig. 6). CCR5-deficient mice develop a Th2 pattern of cytokine expression in DSS-induced colitis
To determine the pattern of cytokine expression in the knockout
and wild-type animals, we performed RT-PCR with RNA derived from whole
colonic tissue (Fig. 7). The pattern of
Th1- (IFN-) and Th2- (IL-4, IL-5, and IL-10) type cytokine mRNA
expression differed significantly among the groups of mice. After
correction for GAPDH, CCR5-deficient animals expressed one-fifth as
much IFN- mRNA in the intestinal mucosa as wild-type animals after 3
and 7 days of DSS (Fig. 7). The reduced expression of this Th1 cytokine
was accompanied by a 75-fold increase in IL-4 mRNA, and a two-fold
increase in IL-5 and IL-10 mRNA (Fig. 7). CCR2-deficient mice also had
one-fifth as much IFN- mRNA expression as wild-type animals, a level
comparable to that observed in CCR5-deficient mice. The level of IL-4
mRNA was much less than that of CCR5-deficient mice, but it was still
3-fold greater than that of wild-type animals. IL-10 mRNA expression in
CCR2-deficient mice was 9-fold greater than in wild-type mice and over
4-fold more than in CCR5-deficient mice (Fig. 7). IL-5 mRNA expression
in CCR2-deficient mice was equivalent to that of wild-type mice. All
mouse types developed increased IL-1ß, TNF-, and IL-6 mRNA
expression upon DSS exposure, although the level of IL-6 was 3-fold
less in the CCR2-deficient mice. By contrast, the level of IL-15 mRNA
expression remained relatively stable throughout the course of DSS
treatment (Fig. 7). Together these data indicate that CCR2 and CCR5
regulate the composition of lamina propria mononuclear cells and
determine the development of Th1- vs Th2-type immune responses in the
intestinal mucosa.
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Because the migration of leukocytes into the intestinal mucosa may
be in part regulated by chemokines, RPAs were used to assess intestinal
chemokine mRNA expression in CCR2- and CCR5-deficient mice during
DSS-mediated colitis (Fig. 8). RPAs were
conducted to detect expression of lymphotactin (LTN), RANTES, eotaxin,
MIP-1ß, MIP-1, MIP-2, IP-10, MCP-1, TCA-3, L32, and GAPDH.
Densitometry analysis revealed that baseline IP-10 mRNA expression was
2-fold less in both CCR2- and CCR5-deficient animals compared with that
in controls. After DSS treatment, CCR5-deficient mice had a 3- to
4-fold increase in RANTES mRNA expression compared with that of
wild-type animals. By densitometry, the average expression levels of
the other chemokines before and during DSS treatment were comparable to
those of the controls (Fig. 8). The strong induction of RANTES may have
contributed to the enhanced migration of T cells into the lamina
propria of CCR5-deficient mice after challenge with DSS
(37).
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). When corrected for GAPDH and L32 mRNA
expression, the mRNA expression of LTN, eotaxin, MIP-1ß, MIP-1,
MIP-2, and TCA-3 was not different in CCR2-deficient mice and wild-type
mice before or during DSS treatment (Fig. 8). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CCR5-deficient mice, and to a lesser extent CCR2-deficient mice, developed less clinical and morphologic evidence of colonic injury after DSS administration, although the accumulation of neutrophils and macrophages within the intestinal mucosa was comparable to that in the wild-type mice. The continuous oral administration of DSS produces a colitis characterized by mucosal ulceration and accumulation of macrophages, neutrophils, and lymphocytes in the lamina propria and the serosa (43). Our data indicate that CCR2 and CCR5 play an important role in the induction of mucosal damage in response to DSS administration. Although CCR2 and CCR5 are not critical for macrophage migration into the colonic lamina propria, both chemokine receptors are able to regulate the migration and differentiation of mucosal T cells. Our experiments suggest that these intestinal T cells may determine the course and outcome of the DSS-induced colitis.
In the present study, DSS-induced colitis in wild-type mice was
characterized by a Th1-type response with a strong induction of IFN-
mRNA expression. In contrast, the reduced colonic damage observed in
CCR5-deficient mice was associated with an increase in IL-4 and IL-5
mRNA expression, a reduction in IFN- mRNA expression, and an
increase in the proportion of CD4+ T lymphocytes
in the lamina propria. Although it is unclear whether the expansion of
CD4+ T cells results from a proliferation of
these cells within the lamina propria or from an enhanced recruitment
from the peripheral blood, the data suggest that they represent cells
of a predominantly Th2 phenotype. The increased percentage of
CD4+ T cells in CCR5-deficient animals may be in
part the result of an enhanced influx or propagation of
NK1.1+ cells in the lamina propria of these mice.
NK1.1+CD4+ T cells have
been shown to produce IL-4 in vivo promptly upon injection of anti-CD3
and therefore have been speculated to play a role in initiating Th2
cell-mediated immunity (44). Furthermore,
NK1.1+ T cells have been shown to mediate immune
suppression through expression of TGF-ß (45). Our data
indicate that NK1.1+ T cells may be able to exert
a protective effect in the regulation of DSS-induced colitis. This is
consistent with a recent report in which NK cells were identified as
regulators of CD4+ T cells in a transfer model of
mouse colitis (46). In these experiments, NK cells
inhibited effector
CD4+CD45RBhigh T cells in
an effect that was dependent on perforin (46).
Prior studies have shown that CCR5 is involved in the regulation of Th1
lymphocyte function. CCR5 is expressed almost exclusively on Th1 cells
(47). The CCR5 binding chemokine MIP-1, and to a lesser
extent MIP-1ß and RANTES, induces selective Th1 lymphocyte migration
in transwell chemotaxis assays (48). In addition, CCR5 is
highly expressed in the T cell areas of the synovium of patients with
rheumatoid arthritis, a predominantly Th1-mediated disease
(49). Therefore, absence of CCR5 may result in diminished
Th1 responses in the intestinal mucosa, resulting in a Th2-type
cytokine profile. Consistent with this hypothesis, CCR5-deficient mice
generate high amounts of RANTES, which may signal through CCR3 in the
absence of CCR5 (37, 50), preferentially attracting Th2
cells (51, 52, 53). Decreased IFN- and increased IL-4 mRNA
expression in turn may have acted to suppress macrophage activation
(54).
In the DSS colitis model, a Th2-type mucosal immune response may result
in less severe colonic damage. Th2-inducing agents decrease the
severity of colitis in other murine models including
trinitrophenyl-kehole limpet hemocyanin-induced colitis in
IL-2-deficient mice and trinitrobenzene sulfonic acid colitis
(55). Moreover, in mice given DSS, anti-IFN- and/or
anti-TNF- Abs significantly attenuate the severity of the
colitis (56). In addition, BALB/c mice are characterized
by a preferential Th2 immune response and a susceptibility to
Leishmania infections (57) and develop less
severe colonic damage in response to DSS administration than other
mouse strains do (32).
Although the pathogenesis of DSS-mediated colitis is incompletely understood, the recruitment and activation of macrophages seems to have a critical role (43). Prior studies have found that CCR5 plays an important role in macrophage activation. CCR5-deficient mice display a reduced ability to clear Listeria infection and a resistance to LPS-induced endotoxemia (58). CCR5 seems to have less effect on the regulation of macrophage chemotaxis, in that CCR5-deficient mice develop normal glucan-induced granulomas in the lung and liver (58). Consistent with these results, DSS-induced migration of macrophages into the colonic lamina propria of CCR5-deficient mice was similar to that observed in wild-type controls. Therefore, a defect in macrophage activation caused by the absence of CCR5 may have contributed to the reduced colonic damage observed in CCR5-deficient mice.
Clinical signs of colonic injury in response to DSS administration were delayed for the first 3 days of the study in CCR2-deficient mice. By day 7, the clinical severity of the disease was similar to that observed in wild-type mice. This transient period of reduced disease severity coincided with increased CD19 mRNA expression and lamina propria B cells. Because prior studies have suggested a role for B cells in the regulation of experimental colitis (59), it is possible that the increased number of B cells acts to reduce the severity of the colonic inflammation. Further studies of the role of B cells in the development of DSS-mediated colitis will be necessary to verify this hypothesis.
Several studies have shown CCR2 to be critical for the induction of
macrophage migration. CCR2-deficient mice show impaired macrophage
migration in response to Mycobacterium bovis Ag
(60), Schistosoma mansoni Ag (61)
in the lung, and in the thioglycollate model of peritoneal inflammation
(62). In contrast to these studies, CCR2-deficient mice
administered DSS were observed to develop colonic lamina propria
macrophage infiltration similar to that seen in wild-type controls.
Thus, in this model CCR2 does not play a critical role in macrophage
chemotaxis. It is possible that in DSS colitis, the absence of CCR2 is
compensated for by other receptor/ligand pairs, or that DSS itself may
provide such a strong chemical signal that it may directly overcome the
effects of CCR2 deficiency on macrophage chemotaxis. Although
infiltration of T cells and macrophages into the colonic lamina propria
of CCR2-deficient mice did not differ from that of wild-type mice,
CCR2-deficient mice exhibited a shift in cytokine mRNA expression
during DSS-induced colitis that was characterized by a reduction in
IFN- and an increase in IL-10. A defect in IFN- production by
CCR2-deficient mice has been demonstrated in the Schistosoma
Ag-induced model of pulmonary granuloma formation (61). In
addition, CCR2-deficient mice show a reduced Th1-type cytokine response
after Mycobacterium bovis Ag challenge in the
lung (60). This reduced Th1 response is associated with a
decrease in IFN- production by splenocytes activated by Con A,
suggesting that CCR2 may be directly involved in the induction of
IFN- expression by T cells (60).
The lack of a complete shift to a Th2-type mucosal immune response in
CCR2-deficient mice may explain the different clinical and
morphological responses to DSS administration in CCR2- and
CCR5-deficient mice. However, the diminished IFN- and increased
IL-10 expression observed in the CCR2-deficient mice may have been
sufficient to decrease attenuate the severity of the colonic damage by
reducing macrophage inflammatory function (54). The
proinflammatory role of IFN- in the activation of macrophages in
animal models of IBD and in Crohn’s disease has been well established
(55, 63). IL-10 is able to reduce macrophage secretion of
inflammatory mediators by directly inhibiting NF-
B activation
(64). Furthermore, IL-10-deficient mice demonstrate
enhanced production of colonic proinflammatory cytokines, including
IFN-, and develop a spontaneous chronic enterocolitis (65, 66).
In conclusion, our results demonstrate that the lack of CCR2 and CCR5 signaling protects mice from the severe inflammation and mucosal damage induced by DSS. Both CCR5 and CCR2 are regulators of intestinal Th1- and Th2-type immune responses, and their absence cannot be compensated for by the redundancy of the chemokine network.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hans-Christian Reinecker, Gastrointestinal Unit, Jackson 7, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114.
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; IBD, inflammatory bowel disease; MIP, macrophage-inflammatory protein; DSS, dextran sodium sulfate; MPO, myeloperoxidase; RPA, RNase protection assay; LTN, lymphotactin.
Received for publication September 9, 1999. Accepted for publication April 6, 2000.
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