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Am J Physiol Gastrointest Liver Physiol 292: G1263-G1271, 2007. First published February 1, 2007; doi:10.1152/ajpgi.00409.2006
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INFLAMMATION/IMMUNITY/MEDIATORS

MIP-3{alpha} neutralizing monoclonal antibody protects against TNBS-induced colonic injury and inflammation in mice

Kianoosh Katchar,1 Ciarán P. Kelly,1 Sarah Keates,1 Michael J. O'Brien,2 and Andrew C. Keates1

1Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, and 2Mallory Institute of Pathology, Boston University School of Medicine, Boston, Massachusetts

Submitted 1 September 2006 ; accepted in final form 29 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A characteristic feature of human inflammatory bowel disease, particularly Crohn's disease, is the presence of activated CD4+ T cells. Recently, we have shown that colonic epithelial cell production of macrophage inflammatory protein (MIP)-3{alpha}, a CD4 T cell-directed chemokine, is elevated in inflammatory bowel disease. However, the functional relevance of MIP-3{alpha} production during intestinal inflammation is poorly understood. The aim of this study was to determine whether MIP-3{alpha} production is increased during murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and to examine the effect of anti-MIP-3{alpha} neutralizing monoclonal antibody administration in this model. We found that the administration of TNBS significantly increased colonic MIP-3{alpha} protein levels in Balb/c mice. Consistent with this, a marked increase in the number of CCR6-bearing lamina propria CD4+ and CD8+ T cells was also observed in TNBS-treated animals. Treatment of mice with an anti-MIP-3{alpha} neutralizing monoclonal antibody significantly reduced TNBS-mediated increases in colonic weight-to-length ratio, mucosal ulceration, histological damage, and myeloperoxidase activity. TNBS-mediated increases in the number of CCR6-bearing lamina propria T cells were also substantially reduced by anti-MIP-3{alpha} neutralizing monoclonal antibody treatment. Taken together, our findings indicate that blockade of MIP-3{alpha} bioactivity can significantly reduce TNBS-mediated colonic injury and T cell recruitment, suggesting a role for this chemokine in the pathophysiology of intestinal inflammation.

chemokine; inflammatory bowel disease; colitis; CD4; T lymphocyte; 2,4,6-trinitrobenzene sulfonic acid

LEUKOCYTE RECRUITMENT to areas of inflammation is a multistep process that includes endothelial cell activation, expression of leukocyte adhesion molecules, and production of chemoattractant cytokines (chemokines) that direct leukocyte migration to the inflammatory focus (28). Under normal conditions, infiltrating leukocytes are an important defense against pathogenic microorganisms and help to initiate repair of tissue damage following infection. In contrast, inappropriate leukocyte activation and recruitment causes tissue damage and is likely to underlie many inflammatory disorders including inflammatory bowel disease (IBD).

To date, over 40 chemokines have been identified and can be classified into 1 of 4 subfamilies according to the number and arrangement of conserved cysteine residues (C, C-C, C-X-C, or C-X3-C) (2, 3, 18, 23). Macrophage inflammatory protein (MIP)-3{alpha} (also known as CCL20) is a C-C chemokine that is predominantly expressed at extralymphoid sites, including the small intestine and colon, and is upregulated by proinflammatory stimuli (31). Binding of MIP-3{alpha} to its receptor, CCR6, induces migratory responses in memory CD4+ T lymphocytes and immature dendritic cells (5, 8, 22, 25). Moreover, MIP-3{alpha} has also be reported to induce T lymphocyte adhesion to the gastrointestinal-specific vascular addressin MAd-CAM-1 (5). In recent studies, we and others have shown that colonic mucosal MIP-3{alpha} production is upregulated in IBD (20). We have also shown that epithelial cell MIP-3{alpha} protein levels are elevated in both ulcerative colitis and Crohn's disease. In keeping with these findings, Scheerens et al. (30) have shown that colonic MIP-3{alpha} mRNA levels are significantly elevated in murine IL-10 knockout colitis as well as CD45RBhigh CD4+ T cell transfer colitis. Taken together, these data suggest that increased colonic MIP-3{alpha} production may play an important role in the recruitment of CD4+ T cells and/or immature dendritic cells to the epithelial layer during intestinal inflammation.

Although colonic MIP-3{alpha} levels are elevated in human IBD, particularly Crohn's disease, the functional relevance of increased MIP-3{alpha} production during gut inflammation has not been extensively studied. Varona et al. (35) reported recently that colonic inflammation is reduced in CCR6-deficient C57BL/6 mice treated with dextran sulfate sodium but increased in animals treated with low doses of 2,4,6-trinitrobenzene sulfonic acid (TNBS). In this study, we investigated whether MIP-3{alpha} participates in TNBS-induced colitis in mice. We found that, following TNBS administration, colonic MIP-3{alpha} production was significantly upregulated in Balb/c mice and that this was associated with elevated numbers of CCR6+CD4+ and CCR6+CD8+ T cells in the lamina propria. We also showed that treatment of mice with a MIP-3{alpha} neutralizing monoclonal antibody (mAb) can markedly reduce TNBS-mediated colonic injury and inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TNBS-induced colitis. Male BALB/c mice weighing ~20 g were obtained from Charles River Laboratories and housed in the animal research facility at Beth Israel Deaconess Medical Center under standard environmental conditions. All animals received standard pelleted chow and tap water ad libitum.

TNBS colitis was induced in Balb/c mice as described previously with slight modifications (17). Briefly, mice were sedated by an intraperitoneal injection of ketamine (45 mg/kg) and xylazine (5 mg/kg). A 50-µl enema of 100 mg/kg TNBS (Sigma-Aldrich) in 50% ethanol was then infused into the colonic lumen (~3 cm from the anal verge) via a 1-ml syringe (Becton Dickinson) fitted with a polyethylene canula (Intramedic PE-20 tubing, Becton Dickinson). Preliminary experiments showed that this dose of TNBS produced a colitis in this strain of mice. Lower doses of TNBS (50–75 mg/kg) gave nonreproducible inflammatory responses, whereas higher doses (125–150 mg/kg) were associated with an unacceptable level of mortality (~50%). In some experiments, this initial dose was followed by a second treatment with 100 mg/kg TNBS after 7 days. Control mice received either ethanol alone (vehicle group) or were untreated. To determine whether MIP-3{alpha} participates in TNBS-induced colitis, some mice were treated via an intraperitoneal injection with 1 mg/kg neutralizing antibody to murine MIP-3{alpha} (MAB7601, R&D Systems) or with a nonspecific rat IgG1 isotype control mAb (MAB005, R&D Systems) at 1 day before and 1, 3, 6, and 9 days after TNBS administration. All animal studies were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.

Assessment of inflammation. Three days following the second treatment with either TNBS or control enemas (day 10), mice were killed by carbon dioxide asphyxiation. Body weights were then recorded, and the colons were removed, opened longitudinally, and washed with saline to remove fecal material. Each colon was then assigned a macroscopic damage score (0–10) as previously described (17), and the mean ulcer index was recorded. In brief, scores were determined as follows: 0, no damage; 1, hyperemia without ulcers; 2, hyperemia and thickening of the bowel wall without ulcers; 3, one ulcer without thickening of the bowel wall; 4, two sites of ulceration or inflammation; 5, more than two sites of inflammation or one site extending over 0.5 cm; and 6–10, damage extending at least 1 cm with the score increasing by 1 for each additional 0.5 cm of involvement.

To assess histological damage, full-thickness colonic tissues were fixed in 10% neutral buffered formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Histological damage in each section was then graded (1–7) by a "blinded" gastrointestinal pathologist (M. J. O'Brien) as described by Gurtner et al. (13). In brief, scores were determined as follows: 1, no evidence of inflammation; 2, low level of inflammatory cell infiltration with infiltration seen in <10% high-power fields (HPFs) and no structural changes observed; 3, moderate inflammatory cell infiltration with infiltration seen in 10–25% HPFs, crypt elongation, bowel wall thickening that did not extend beyond mucosal layer, and no evidence of ulceration; 4, high level of inflammatory cell infiltration with infiltration seen in 25–50% HPFs, high vascular density, and thickening of bowel wall that extended beyond the mucosal layer; 5, marked degree of inflammatory cell infiltration with infiltration seen in >50% HPFs, high vascular density, crypt elongation with distortion, and transmural bowel wall thickening with ulceration; 6, complete loss of mucosal architecture (crypts) with ulceration covering >1 low-power field and loss of mucosal vasculature; and 7, coagulation necrosis of at least the mucosal layer.

Colonic myeloperoxidase (MPO) levels, an index of neutrophil infiltration, were assayed as previously described (17). Whole colons were homogenized in a solution containing 50 mM KH2PO4 (pH 6.0) and 0.5% hexadecyltrimethylammonium bromide, frozen (–70°C) and thawed, and then centrifuged at 40,000 g for 15 min at 4°C. Duplicate aliquots of the supernatant were then assayed for MPO activity using a microtiter plate colorimetric assay. The MPO activity of each sample was determined by comparison with a standard curve generated using purified human MPO (Calbiochem). Results are expressed as MPO units per milligram of total protein.

Analysis of murine MIP-3{alpha} mRNA levels by real-time RT-PCR. Total RNA was isolated from frozen colonic tissue using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA (1 µg) was then reversed transcribed using random hexamer primers and Moloney murine leukemia virus reverse transcriptase as previously described (20), and the resulting cDNA was stored at –20°C. Murine MIP-3{alpha} mRNA levels and GAPDH mRNA levels were quantified by real-time RT-PCR assay using a GeneAmp 5700 sequence detection system (ABI/Perkin-Elmer). For each sample, duplicate reactions containing 5 µl cDNA were incubated for 2 min at 50°C, denatured for 10 min at 95°C, and subjected to 40 cycles of annealing at 60°C for 1 min, followed by denaturation at 95°C for 15 s. To detect murine MIP-3{alpha} mRNA, the following gene-specific primers and a dual-labeled fluorogenic probe containing FAM (at the 5'-end) and TAMRA (at the 3'-end) were synthesized (Genosys): sense primer, 5'-CCTTGCTTTGGCATGGGTACT-3'; antisense primer, 5'-CAGTCGTAGTTGCTTGCTGCTT-3'; and probe, 5'-TGGCTCACCTCTGCAGCCAGGC-3'. Analysis of murine GAPDH mRNA levels in each sample was performed using a commercially available dual-labeled fluorogenic probe and gene-specific primers (Applied Biosystems). MIP-3{alpha} levels in each sample were then normalized to GAPDH expression, and the relative change in mRNA level was expressed as fold induction compared with control using the –{Delta}{Delta}CT method, where CT is threshold cycle.

Analysis of murine MIP-3{alpha} protein levels by ELISA. For the cytokine ELISA, frozen full-thickness colonic tissue specimens were homogenized in PBS containing a cocktail of protease inhibitors (Mini-Complete, Roche) supplemented with 1 mM PMSF. After a centrifugation at 20,000 g for 10 min at 4°C, the total protein content of the supernatants was determined by the BCA protein assay (Pierce). Murine IL-1beta, TNF-{alpha}, and MIP-3{alpha} protein levels in colonic mucosal homogenates were then determined using commercially available ELISA kits (R&D Systems). Results are expressed as picograms per milligram of total protein.

Isolation of lamina propria lymphocytes. Lamina propria lymphocytes were isolated from whole colons according to the method of LeFrancois and Lycke (21). Briefly, colons were removed, opened longitudinally, and washed six times in ice-cold Ca2+- and Mg2+-free HBSS (CMF)-HEPES solution (CMF with 15 mM HEPES, pH 7.2) to remove fecal matter. Colons were then cut into 5-mm pieces, and the pieces were stirred (~100 rpm) for 15 min at room temperature in CMF-FBS-EDTA solution (CMF-HEPES solution with 10% heat-inactivated FBS, 5 mM EDTA, and 100 mg/ml gentamycin, pH 7.2) five times. After the final incubation in CMF-FBS-EDTA solution, tissue pieces were stirred for 15 min at room temperature in complete RPMI-10 medium (RPMI-1640 medium with 10% heat-inactivated FBS and 100 mg/ml gentamycin) to remove residual EDTA. Colonic tissue pieces were then stirred (~200 rpm) for 1 h at 37°C in complete RPMI-10 medium containing 300 U/ml type VIII collagenase (Sigma-Aldrich). After passage through a 70-µm nylon cell strainer (Fisher Scientific), released cells were collected by centrifugation (850 g for 10 min) and washed two times with CMF-HEPES solution, and lamina propria mononuclear cells were then purified by fractionation through a discontinuous Percoll density gradient as previously described (24).

Flow cytometric analysis of colonic lamina propria lymphocytes. For flow cytometric analysis, colonic lamina propria lymphocytes were washed with FACS buffer (Ca2+- and Mg2+-free PBS with 4% heat-inactivated FBS, pH 7.4) and then incubated with anti-mouse CD16/32 antibody (clone 93, E-Bioscience) for 15 min at 4°C to block nonspecific binding of test antibodies to Fc receptors. To assess the number of CCR6-bearing T lymphocytes in each preparation, cells were triple stained with a FITC-labeled CD3{epsilon} antibody (clone 145-2C11, E-Bioscience), an Alexa Fluor 647-labeled CCR6 antibody (clone 140706, Pharmingen), and phycoerythrin-labeled CD4 or CD8 antibodies (clones GK1.5 and 53-6.7, E-Bioscience) for 20 min at 4°C. To assess the number of CCR6-bearing B lymphocytes in each preparation, cells were doubled stained with the Alexa Fluor 647-labeled CCR6 antibody and a phycoerythrin-labeled CD19 antibody (clone MB19-1, E-Bioscience) for 20 min at 4°C. Antibody-labeled lamina propria lymphocytes were then analyzed using a LSR II flow cytometer and FACS Diva software (Becton Dickinson). CD4+CCR6+ and CD8+CCR6+ cell numbers are expressed as percentages of the total numbers of T lymphocytes (i.e., CD3{epsilon}+ cells) in each sample. CD19+CCR6+ cell numbers are expressed as percentages of the total numbers of CD19+ B lymphocytes in each sample.

Statistical analyses. Statistical analyses were performed using SigmaStat version 3.1 (Jandel Scientific Software). ANoVA followed by post hoc t-tests were used for intergroup comparisons except where otherwise stated.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Colonic mucosal MIP-3{alpha} protein levels are increased in murine TNBS-induced colitis. A previous study from our group (20) has shown that MIP-3{alpha} mRNA and protein levels are elevated in colonic tissue from patients with IBD. To determine whether MIP-3{alpha} may play a functional role in murine colitis, we first examined whether colonic MIP-3{alpha} production was elevated in mice treated with the haptenizing agent TNBS.

As shown in Fig. 1, colonic mucosal MIP-3{alpha} protein levels were significantly increased in mice treated with TNBS for 3, 7, or 10 days (by ~25%, ~40%, and ~100%, respectively) compared with untreated control animals or animals treated with vehicle. In keeping with these findings, a 2.6-fold increase in colonic mucosal MIP-3{alpha} mRNA levels (measured by real-time RT-PCR) was also detected 10 days after TNBS treatment (data not shown).


Figure 1
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  		Fig. 1. Colonic macrophage inflammatory protein (MIP)-3{alpha} protein levels are elevated during 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: control (Ctl), vehicle [i.e., 50% ethanol (EtOH)], 3-day TNBS, 7-day TNBS, and 10-day TNBS. Colonic MIP-3{alpha} protein levels in tissue homogenates were measured by ELISA. Results are expressed as means ± SE; n = 3–6 mice/group.

 
CCR6-bearing lamina propria T cells are elevated in TNBS-induced colitis. A number of previous studies (5, 12, 22) have shown that binding of MIP-3{alpha} to its receptor, CCR6, induces the migration of memory lymphocytes and adhesion of these cells to MAd-CAM-1, a gastrointestinal-specific vascular addressin. These findings suggest that binding of MIP-3{alpha} to CCR6 can induce the expression of {alpha}4beta7-integrin, the counterreceptor for MAd-CAM-1, on T cells and thereby direct their recruitment to gastrointestinal tissues. Since TNBS-induced colitis has been shown to be mediated by activated T helper type 1 (Th1) T lymphocytes (32), we next decided to investigate whether the increase in MIP-3{alpha} protein levels observed during TNBS treatment was associated with the recruitment of CCR6-bearing lymphocytes to the colonic lamina propria.

To analyze whether CCR6-bearing lymphocyte numbers were elevated during TNBS-induced colitis, lamina propria lymphocytes were isolated and subjected to three-color flow cytometric analysis (Fig. 2). In control and vehicle-treated mice, 18% and 23% of the colonic lamina propria CD4+ T cells were found to express CCR6 (Fig. 2, B and C). Following treatment with TNBS for 3 days, the proportion of lamina propria CD4+ T cells bearing CCR6 significantly increased to 45% compared with control mice. A further elevation in CCR6-bearing CD4+ T cell numbers (to 52%) was observed after 7 days of TNBS treatment, in keeping with the increased levels of colonic MIP-3{alpha} in these animals. A similar pattern to that seen with CCR6+CD4+ lamina propria T cells was also seen with CCR6+CD8+ T cells (Fig. 2, B and D). As before, no significant differences in the numbers of CCR6-bearing CD8+ T cells were seen in vehicle-treated mice compared with control animals. Similarly, compared with untreated control mice, the numbers of CCR6-bearing CD8+ T cells were increased ~2-fold in animals treated with TNBS for 3 days and ~2.5-fold in mice treated with TNBS for 7 days, consistent with increased mucosal MIP-3{alpha} production. Interestingly, the expression of CCR6 by lamina propria B lymphocytes (i.e., CD19+ cells) was not significantly altered by TNBS treatment (Fig. 2E).


Figure 2
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  		Fig. 2. Numbers of CCR6-bearing lamina propria CD4+ and CD8+ T cells are increased during TNBS-induced colitis. Colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 4 experimental groups: Ctl, vehicle (i.e., 50% EtOH), 3-day TNBS, and 7-day TNBS. At the end of the experiment, animals were killed, and lamina propria lymphocytes were isolated from each colon as described in MATERIALS AND METHODS. For flow cytometric analysis, the lymphocyte population was selected from a FSC vs. SSC plot (gate P1; not shown). For T lymphocyte analysis, cells in the P1 gate were subgated into either CD3{epsilon}+ (gate P2; blue) or CD3{epsilon}– (gate P3; red) populations. For B lymphocyte analysis, cells in the P1 gate were subgated into either CD19+ or CD19– populations. A: representative experiment showing lamina propria lymphocytes stained with CD3{epsilon}-FITC antibody (positive control for P2 gate; left and middle left) or a mixture of FITC, phycoerythrin (PE), and Alexa Fluor 647 isotype control antibodies (negative control; right and middle right). Percentage values represent the proportion of cells from the P2 and P3 gates in each quadrant. B: representative experiment showing lamina propria lymphocytes stained with CD3{epsilon}-FITC, CD4-PE, and CCR6-Alexa Fluor 647 antibodies (top) or with CD3{epsilon}-FITC, CD8{alpha}-PE, and CCR6-Alexa Fluor 647 antibodies (bottom). Percentage values represent the proportion of cells from the P2 gate (i.e., CD3{epsilon}+) in each quandrant. C: numbers of CD4+CCR6+ lamina propria lymphocytes were determined by flow cytometry and expressed as percentages of the total numbers of CD3{epsilon}+ cells. Results are expressed as means ± SE; n = 3 mice/group. D: numbers of CD8+CCR6+ lamina propria lymphocytes were determined by flow cytometry and expressed as percentages of the total numbers of CD3{epsilon}+ cells. Results are expressed as means ± SE; n = 3 mice/group. E: numbers of CD19+CCR6+ lamina propria lymphocytes were determined by flow cytometry and expressed as percentages of the total numbers of CD19+ cells in the lymphocyte (P1) gate. Results are expressed as means ± SE; n = 3 mice/group.

 
Neutralization of MIP-3{alpha} bioactivity protects against TNBS-mediated colonic inflammation and injury. Since the numbers of CCR6-bearing CD4+ and CD8+ T cells were markedly elevated in the lamina propria of mice treated with TNBS, we next decided to examine whether neutralization of MIP-3{alpha} bioactivity could protect mice against colonic injury in this model.

While body weight loss was not significantly affected by MIP-3{alpha} neutralizing mAb treatment, a marked reduction in the colonic weight-to-length ratio was observed. As shown in Fig. 3, mice given TNBS showed a marked increase in the colon weight-to-length ratio compared with untreated control or vehicle-treated mice. In contrast, in MIP-3{alpha} mAb-treated mice, the colonic weight-to-length ratio was reduced by 59% compared with animals treated with TNBS alone (P < 0.001) or animals given the isotype control mAb (P < 0.01).


Figure 3
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  		Fig. 3. Anti-MIP-3{alpha} monoclonal antibody (mAb) therapy significantly attenuates TNBS-mediated increases in colonic weight-to-length ratio. Colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: Ctl, vehicle (i.e., 50% EtOH), TNBS alone, TNBS + MIP-3{alpha} mAb, and TNBS + isotype control mAb. Animals were killed after 10 days, and the colonic weight-to-length ratio for each animal was recorded. Results are expressed as means ± SE; n = 6–11 mice/group.

 
To further examine whether neutralization of MIP-3{alpha} bioactivity could prevent the colonic injury associated with TNBS administration, we next examined mucosal ulceration using a macroscopic damage score. Little or no ulceration of the colonic mucosa were observed in control or vehicle-treated animals. As expected, the administration of TNBS to Balb/c mice resulted in increased colonic damage (ulcer index: 2.2 ± 0.1; Fig. 4A). Moreover, there was no effect on TNBS-induced colonic damage in mice treated with the isotype control mAb (ulcer index: 1.9 ± 0.1). In contrast, TNBS-mediated colonic damage was significantly reduced in animals treated with the MIP-3{alpha} mAb (ulcer index: 1.1 ± 0.2) compared with mice given TNBS alone (P < 0.001).


Figure 4
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  		Fig. 4. Administration of a neutralizing MIP-3{alpha} mAb reduces TNBS-mediated colonic ulceration and histological damage in Balb/c mice. A: colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: Ctl, vehicle (i.e., 50% EtOH), TNBS alone, TNBS + MIP-3{alpha} mAb, and TNBS + isotype control mAb. Animals were killed after 10 days, and the degree of colonic ulceration was determined using a macroscopic damage score. Results are expressed as means ± SE; n = 6–11 mice/group. B: colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: Ctl, vehicle (i.e., 50% EtOH), TNBS alone, TNBS + MIP-3{alpha} mAb, and TNBS + isotype control mAb. Animals were killed after 10 days, and the degree of microscopic colonic injury was determined using a histologic damage score. Results are expressed as means ± SE; n = 6–11 mice/group.

 
Similar to our findings examining macroscopic mucosal damage, MIP-3{alpha} neutralizing mAb treatment also significantly reduced microscopic colonic damage resulting from TNBS administration. As shown in Fig. 4B, TNBS-mediated histological damage was significantly decreased (P < 0.01) in mice given the MIP-3{alpha} mAb (histology score: 2.2 ± 0.3) compared with animals given only TNBS (histology score: 3.5 ± 0.4). Histological damage in mice treated with the isotype control mAb (histology score: 3.1 ± 0.2) was similar to that seen in animals treated with TNBS alone. As expected, no signs of microscopic colonic damage were observed in vehicle-treated or control animals.

To determine whether the reduction in colonic inflammation seen with MIP-3{alpha} mAb therapy was associated with decreased production of proinflammatory mediators, we next measured colonic mucosal levels of MPO, IL-1beta, and TNF-{alpha}. As shown in Fig. 5A, colonic MPO levels in vehicle-treated Balb/c mice were similar to those seen in untreated control animals. Compared with untreated control mice, animals given TNBS had markedly increased colonic MPO levels (~4.5-fold), as expected. In contrast, colonic mucosal levels of MPO were substantially reduced in mice treated with the MIP-3{alpha} mAb compared with animals treated with TNBS alone (P < 0.05). Surprisingly, the beneficial effects of MIP-3{alpha} mAb treatment on TNBS-induced colitis did not appear to be associated with a reduction in mucosal levels of IL-1beta (Fig. 5B) or TNF-{alpha} (data not shown).


Figure 5
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  		Fig. 5. Anti-MIP-3{alpha} mAb therapy reduces colonic MPO, but not IL-1beta, content during TNBS-induced colitis. A: colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: Ctl, vehicle (i.e., 50% EtOH), TNBS alone, TNBS + MIP-3{alpha} mAb, and TNBS + isotype control mAb. Animals were killed after 10 days, and the MPO activity in colonic homogenates was determined. Results are expressed as means ± SE; n = 6–11 mice/group. B: colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: Ctl, vehicle (i.e., 50% EtOH), TNBS alone, TNBS + MIP-3{alpha} mAb, and TNBS + isotype control mAb. Animals were killed after 10 days, and the IL-1beta content in colonic homogenates was determined by ELISA. Results are expressed as means ± SE; n = 6–11 mice/group.

 
Anti-MIP-3{alpha} mAb therapy significantly reduces the number of lamina propria CD4+ and CD8+ T cells in TNBS-treated mice. Since the beneficial effect of anti-MIP-3{alpha} mAb therapy on TNBS-induced colitis did not appear to be related to reduced mucosal proinflammatory cytokine production, we next examined whether antibody treatment was associated with decreased numbers of colonic CCR6+ T cells. For these experiments, lamina propria mononuclear cells isolated from control animals, animals given TNBS alone, animals given TNBS together with the MIP-3{alpha} mAb, or animals given TNBS together with the isotype control mAb were analyzed by three-color flow cytometry. As expected, increased numbers of CCR6-bearing lamina propria CD4+ and CD8+ T cells were observed in mice treated with TNBS for 7 days compared with untreated control mice or mice given vehicle alone (Fig. 6). In contrast, the numbers of CCR6-bearing lamina propria CD4+ and CD8+ T cells were reduced (by ~70% and ~60%, respectively) in mice treated with TNBS plus the MIP-3{alpha} mAb compared with mice given TNBS alone or mice given TNBS plus the isotype control mAb.


Figure 6
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  		Fig. 6. Anti-MIP-3{alpha} mAb therapy significantly reduces the number of CCR6-bearing lamina propria CD4+ and CD8+ T cells in TNBS-treated mice. Colitis was induced in male Balb/c mice using TNBS as described in MATERIALS AND METHODS. Animals were divided into 5 experimental groups: Ctl, vehicle (i.e., 50% EtOH), TNBS (7 days), TNBS + anti-MIP-3{alpha} mAb, and TNBS + isotype control mAb. At the end of the experiment, animals were killed, and lamina propria lymphocytes were isolated from each colon as described in MATERIALS AND METHODS. Flow cytometric analysis then performed as described in Fig. 2. A: numbers of CD4+CCR6+ lamina propria lymphocytes were determined by flow cytometry and expressed as percentages of the total numbers of CD3{epsilon}+ cells. Results are expressed as means ± SE; n = 3–4 mice/group. B: numbers of CD8+CCR6+ lamina propria lymphocytes were determined by flow cytometry and expressed as percentages of the total numbers of CD3{epsilon}+ cells. Results are expressed as means ± SE; n = 3–4 mice/group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we showed that colonic production of the C-C chemokine MIP-3{alpha} is upregulated in Balb/c mice following treatment with the haptenizing agent TNBS and that TNBS-mediated increases in colonic MIP-3{alpha} are associated with a marked increase in the number of CCR6-expressing T lymphocytes in the lamina propria of these animals. We also showed that treatment of Balb/c mice with a neutralizing mAb directed against murine MIP-3{alpha} is capable of significantly reducing colonic inflammation and injury induced by TNBS. We found that anti-MIP-3{alpha} mAb therapy significantly reduced TNBS-induced increases in colonic weight-to-length ratio, mucosal ulceration, histological damage, and mucosal MPO activity. In addition, we found that the increase in CCR6-bearing lamina propria T cells induced by TNBS administration could be substantially reduced by anti-MIP-3{alpha} mAb treatment. Taken together, these findings suggest that MIP-3{alpha} participates in intestinal inflammation induced by TNBS and may have an important functional role in the pathogenesis of colonic inflammatory responses.

Previous studies from our group and others (15, 16, 20) have clearly demonstrated that enterocytes are a major site of MIP-3{alpha} production in the human colon and that epithelial MIP-3{alpha} mRNA and protein levels are elevated in active IBD. Despite these important observations, however, the functional relevance of increased colonic MIP-3{alpha} production during intestinal inflammation remains poorly understood. In this study, we found that colonic MIP-3{alpha} production was upregulated following TNBS administration and that this correlated with an increase in the number of CCR6-bearing CD4+ and CD8+ T lymphocytes in the lamina propria of Balb/c mice. Furthermore, anti-MIP-3{alpha} mAb therapy significantly reduced the number of CCR6-bearing lamina propria CD4+ and CD8+ T cells in TNBS-treated mice. Tanaka et al. (33) have reported that MIP-3{alpha} production in normal mice is mainly localized to epithelial cells in the colon. In keeping with the findings of this study, these authors found that colonic MIP-3{alpha} production was markedly enhanced in mice treated with the proinflammatory mediator LPS. Similarly, a recent study by Scheerens et al. (30) has shown that the production of MIP-3{alpha} is also significantly elevated during chronic murine colitis. More specifically, these investigators found that MIP-3{alpha} (and CCR6) mRNA expression was markedly upregulated in inflamed colons of Rag2-deficient mice reconstituted with pathogenic CD45RBhigh CD4+ T cells and in inflamed colons of IL-10-deficient mice. Interestingly, inhibition of colonic inflammation in the IL-10 knockout model by treatment with anti-IL-12 mAb resulted in downregulation of MIP-3{alpha} mRNA levels. Since active Crohn's disease, TNBS-induced colitis, IL-10 knockout colitis, and T cell transfer colitis are each characterized by infiltration of the colonic mucosa by CD4+ T lymphocytes, these data suggest that MIP-3{alpha} may play an especially important role in the recruitment of these cells to the epithelial layer during intestinal inflammation.

In the present study, we used neutralizing mAbs to elucidate the functional relevance of MIP-3{alpha} production in TNBS-induced colitis. Using this approach, we found that MIP-3{alpha} mAb therapy could significantly ameliorate lamina propria T cell accumulation and colonic inflammation in TNBS-treated Balb/c mice. An alternative model with which to study the role of MIP-3{alpha} in intestinal inflammatory responses is to use CCR6-deficient mice, which lack the only known receptor for this chemokine. Interestingly, in CCR6-deficient mice, dendritic cells expressing CD11c+ and CD11b+ are absent from the subepithelial dome of Payer's patches while the number of T lymphocytes in the intestinal mucosa is increased (6, 36). These animals also fail to generate a normal humoral immune response to orally administered antigens or rotavirus infection (6). Recently, Varona et al. (35) reported, in contrast to the findings of this study, that colonic inflammation is exacerbated in C57BL/6 CCR6-deficient mice treated with TNBS compared with wild-type control animals. One possible explanation for the discrepancy between our findings and those of Varona et al. may relate to differences between the experimental protocols and mouse strains used in each study. In this study, we examined acute TNBS-mediated colonic inflammation (at 10 days) in susceptible Balb/c mice, whereas Varona et al. studied TNBS-induced intestinal injury in a more chronic setting (at 21 days) using resistant C57BL/6 mice. A previous study (32) has shown that CD4 T cell responses following TNBS treatment are variable and depend upon the mouse strain used. More specifically, at early time points, similar to those used in our study, the administration of TNBS to Balb/c mice has been shown to elicit an immune reaction that is characterized by Th1 cytokine production (10). In contrast, during the later stages of colitis, as studied by Varona et al., C57BL/6 mice appear to develop a prominent T helper type 2 (Th2) cytokine response following TNBS treatment (9). These observations raise the intriguing possibility that blockade of MIP-3{alpha}/CCR6 may have differential effects on TNBS-mediated colitis depending on whether Th1 or Th2 responses are dominant. Another potential explanation for the differences between the two studies is that the increased numbers of lymphocytes already present in the intestines of CCR6-deficient mice may render these animals more susceptible to TNBS-mediated colitis. Consistent with this, Varona et al. found that colonic mucosal mRNA levels for CD4, CD8, IFN-{gamma}, IL-10, IL-4, and IL-13 were two- to fourfold higher in CCR6-deficient C57BL/6 mice compared with wild-type control animals. Moreover, after TNBS treatment, the T cell cytokine profile of CCR6-deficient C57BL/6 mice, but not wild-type C57BL/6 mice, was similar to that observed in colitis-susceptible Balb/c mice.

Treatment of mice with TNBS induces a colitis that mimics some features of human Crohn's disease (11, 32). Inflammation in this model is thought to involve an acute phase in which there is necrosis and focal basal cryptitis, followed by a chronic phase involving transmural inflammation mediated by CD4+ T lymphocyte recognition of hapten-modified self-proteins. In the present study, treatment of Balb/c mice with TNBS led to a dramatic increase in the numbers of lamina propria CD4+ T lymphocytes bearing CCR6, the specific receptor for MIP-3{alpha}, which could be markedly reduced by the administration of anti-MIP-3{alpha} mAb. These findings suggest that upregulation of mucosal MIP-3{alpha} production directly induces the recruitment of CCR6-bearing T cells to the colon or, alternatively, stimulates the expansion of CCR6-expressing T cells in the lamina propria. In keeping with our findings, previous studies (5, 12, 22) have shown that MIP-3{alpha} can directly regulate the migration of T cells (especially those in the memory subset) and induce T cell adhesion to gastrointestinal-specific vascular addressin MAd-CAM-1. Moreover, a recent report (34) utilizing intravital microscopy has demonstrated that T and B cell adhesion to colonic microvessels during dextran sodium sulfate-induced colitis can be markedly reduced by pretreatment of mice with a MIP-3{alpha} neutralizing mAb or by desensitization of CCR6 with excess MIP-3{alpha}. Thus, the beneficial effect of MIP-3{alpha} mAb therapy on TNBS-mediated colonic inflammation may reflect blockade of T lymphocyte homing to the intestine.

Although blockade of T cell recruitment and/or expansion may explain the protective effect of anti-MIP-3{alpha} mAb therapy seen in this study, we cannot rule out the possibility that the reduced colonic injury seen in antibody-treated animals is mediated via mechanisms involving other CCR6-bearing cell types. For example, previous studies (7, 8, 19) have suggested that MIP-3{alpha} likely plays a key role in the migration of CCR6-bearing immature myeloid dendritic cells. In the intestine, these important antigen-presenting cells have been shown to reside in a subepithelial position where, via direct interactions with epithelial cells, they help to maintain gut immune homeostasis as well as screen the gut lumen for potential pathogens (26, 27). In addition, activated neutrophils have been reported to express CCR6 and MIP-3{alpha} (1, 29, 37). Consistent with this, we found that anti-MIP-3{alpha} therapy could markedly reduce TNBS-mediated increases in mucosal MPO activity, suggesting that MIP-3{alpha} may also regulate, at least in part, the activity of neutrophils during colon inflammation. Finally, previous studies from our laboratory and others (4, 14, 38) have shown that colonic epithelial cells express CCR6 in vivo and that activation of this receptor by MIP-3{alpha} in vitro can induce a variety of cellular responses including cell proliferation, cell migration, and electrogenic ion secretion. Which, if any, of these potential non-T cell mechanisms may account for the ameliorating effect of anti-MIP-3{alpha} therapy on TNBS-induced colitis is the subject of current studies in our laboratory.

In summary, in this study, we showed that antibodies directed against the C-C chemokine MIP-3{alpha} can substantially reduce colonic injury and inflammation induced by TNBS in mice. To our knowledge, this is the first evidence indicating that MIP-3{alpha} plays a functional role in the pathophysiology of intestinal inflammation and suggests that anti-MIP-3{alpha} therapy may be effective in the treatment of IBD.


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This work was supported by National Institutes of Health (NIH) Grants DK-075942, DK-58858, AIO-53069 and PO1-DK-033506, by the Massachusetts General Hospital/New England Regional Primate Research Center for the Study of Inflammatory Bowel Diseases (NIH Grant DK-43551), by a First Award from the Crohn's and Colitis Foundation of America and the William and Shelby Modell Family Foundation (to A. C. Keates), and by an American Gastroenterological Association/TAP Endowed Research Award in Acid Related Diseases (to S. Keates).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. C. Keates, Div. of Gastroenterology, Dana 501, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (e-mail: akeates{at}bidmc.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Akahoshi T, Sasahara T, Namai R, Matsui T, Watabe H, Kitasato H, Inoue M, Kondo H. Production of macrophage inflammatory protein 3alpha (MIP-3alpha) (CCL20) and MIP-3beta (CCL19) by human peripheral blood neutrophils in response to microbial pathogens. Infect Immun 71: 524–526, 2003.[Abstract/Free Full Text]
  2. Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ. A new class of membrane-bound chemokine with a CX 3 C motif. Nature 385: 640–644, 1997.[CrossRef][Medline]
  3. Ben-Baruch A, Michiel DF, Oppenheim JJ. Signals and receptors involved in recruitment of inflammatory cells. J Biol Chem 270: 11703–11706, 1996.[Web of Science]
  4. Brand S, Olszak T, Beigel F, Diebold J, Otte JM, Eichhorst ST, Goke B, Dambacher J. Cell differentiation dependent expressed CCR6 mediates ERK-1/2, SAPK/JNK, and Akt signaling resulting in proliferation and migration of colorectal cancer cells. J Cell Biochem 97: 709–723, 2006.[CrossRef][Web of Science][Medline]
  5. Campbell JJ, Hedrick J, Zlotnik A, Siani MA, Thompson DA, Butcher EC. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279: 381–384, 1998.[Abstract/Free Full Text]
  6. Cook DN, Prosser DM, Forster R, Zhang J, Kuklin NA, Abbondanzo SJ, Niu XD, Chen SC, Manfra DJ, Wiekowski MT, Sullivan LM, Smith SR, Greenberg HB, Narula SK, Lipp M, Lira SA. CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue. Immunity 12: 495–503, 2000.[CrossRef][Web of Science][Medline]
  7. Dieu-Nosjean MC, Massacrier C, Homey B, Vanbervliet B, Pin JJ, Vicari A, Lebecque S, Dezutter-Dambuyant C, Schmitt D, Zlotnik A, Caux C. Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors. J Exp Med 192: 705–718, 2000.[Abstract/Free Full Text]
  8. Dieu MC, Vanbervliet B, Vicari A, Bridon JM, Oldham E, Ait-Yahia S, Briere F, Zlotnik A, Lebecque S, Caux C. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 188: 373–386, 1998.[Abstract/Free Full Text]
  9. Dohi T, Fujihashi K, Kiyono H, Elson CO, McGhee JR. Mice deficient in Th1- and Th2-type cytokines develop distinct forms of hapten-induced colitis. Gastroenterology 119: 724–733, 2000.[CrossRef][Web of Science][Medline]
  10. Elson CO, Beagley KW, Sharmanov AT, Fujihashi K, Kiyono H, Tennyson GS, Cong Y, Black CA, Ridwan BW, McGhee JR. Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance. J Immunol 157: 2174–2185, 1996.[Abstract]
  11. Elson CO, Sartor RB, Tennyson GS, Riddell RH. Experimental models of inflammatory bowel disease. Gastroenterology 109: 1344–1367, 1995.[CrossRef][Web of Science][Medline]
  12. Fitzhugh DJ, Naik S, Caughman SW, Hwang ST. Cutting edge: C-C chemokine receptor 6 is essential for arrest of a subset of memory T cells on activated dermal microvascular endothelial cells under physiologic flow conditions in vitro. J Immunol 165: 6677–6681, 2000.[Abstract/Free Full Text]
  13. Gurtner GJ, Newberry RD, Schloemann SR, McDonald KG, Stenson WF. Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic acid colitis in mice. Gastroenterology 125: 1762–1773, 2003.[CrossRef][Web of Science]
  14. Han X, Keates S, Kelly C, Keates AC. MIP-3alpha mediates EGF receptor transactivation and Erk MAP kinase signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin (Abstract). Gastroenterology 128: W1653, 2005.
  15. Izadpanah A, Dwinell MB, Eckmann L, Varki NM, Kagnoff MF. Regulated MIP-3{alpha}/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity. Am J Physiol Gastrointest Liver Physiol 280: G710–G719, 2001.[Abstract/Free Full Text]
  16. Kaser A, Ludwiczek O, Holzmann S, Moschen AR, Weiss G, Enrich B, Graziadei I, Dunzendorfer S, Wiedermann CJ, Murzl E, Grasl E, Jasarevic Z, Romani N, Offner FA, Tilg H. Increased expression of CCL20 in human inflammatory bowel disease. J Clin Immunol 24: 74–85, 2004.[CrossRef][Web of Science][Medline]
  17. Keates AC, Castagliuolo I, Cruickshank WW, Qiu B, Arseneau KO, Brazer W, Kelly CP. Interleukin 16 is up-regulated in Crohn's disease and participates in TNBS colitis in mice. Gastroenterology 119: 972–982, 2000.[CrossRef][Web of Science][Medline]
  18. Kelner GS, Kennedy J, Bacon KB, Kleyensteuber S, Largaespada DA, Jenkins NA, Copeland NG, Bazan JF, Moore KW, Schall TJ. Lymphotactin: a cytokine that represents a new class of chemokine. Science 266: 1395–1399, 1994.[Abstract/Free Full Text]
  19. Kucharzik T, Hudson JT, 3rd Waikel RL, Martin WD, Williams IR. CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock-in mouse. Eur J Immunol 32: 104–112, 2002.[CrossRef][Web of Science][Medline]
  20. Kwon JH, Keates S, Bassani L, Mayer LF, Keates AC. Colonic epithelial cells are a major site of macrophage inflammatory protein 3alpha (MIP-3alpha) production in normal colon and inflammatory bowel disease. Gut 51: 818–826, 2002.[Abstract/Free Full Text]
  21. LeFrancois L, Lycke N. Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch and lamina propria cells. In: Current Protocols in Immunology. New York: Wiley, 1996, p. 3.19.11–13.19.16.
  22. Liao F, Rabin RL, Smith CS, Sharma G, Nutman TB, Farber JM. CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3 alpha. J Immunol 162: 186–194, 1999.[Abstract/Free Full Text]
  23. Pan Y, Lloyd C, Zhou H, Dolich S, Deeds J, Gonzalo JA, Vath J, Gosselin M, Ma J, Dussault B, Woolf E, Alperin G, Culpepper J, Gutierrez-Ramos JC, Gearing D. Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature 387: 611–617, 1997.[CrossRef][Medline]
  24. Panja A, Barone A, Mayer L. Stimulation of lamina propria lymphocytes by intestinal epithelial cells: evidence for recognition of nonclassical restriction elements. J Exp Med 179: 943–950, 1994.[Abstract/Free Full Text]
  25. Power CA, Church DJ, Meyer A, Alouani S, Proudfoot AE, Clark-Lewis I, Sozzani S, Mantovani A, Wells TN. Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells. J Exp Med 186: 825–835, 1997.[Abstract/Free Full Text]
  26. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immun 2: 361–367, 2001.[CrossRef][Web of Science]
  27. Rimoldi M, Chieppa M, Salucci V, Avogadri F, Sonzogni A, Sampietro GM, Nespoli A, Viale G, Allavena P, Rescigno M. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat Immun 6: 507–514, 2005.[CrossRef][Web of Science]
  28. Salmi M, Jalkanen S. Lymphocyte homing to the gut: attraction, adhesion, and commitment. Immunol Rev 206: 100–113, 2005.[CrossRef][Web of Science][Medline]
  29. Scapini P, Laudanna C, Pinardi C, Allavena P, Mantovani A, Sozzani S, Cassatella MA. Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19. Eur J Immunol 31: 1981–1988, 2001.[CrossRef][Web of Science][Medline]
  30. Scheerens H, Hessel E, Waal-Malefyt R, Leach MW, Rennick D. Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10–/– mice and Rag-2–/– mice reconstituted with CD4+CD45RBhigh T cells. Eur J Immunol 31: 1465–1474, 2001.[CrossRef][Web of Science][Medline]
  31. Schutyser E, Struyf S, Van Damme J. The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 14: 409–426, 2003.[CrossRef][Web of Science][Medline]
  32. Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inflammation. Annu Rev Immunol 20: 495–549, 2002.[CrossRef][Web of Science][Medline]
  33. Tanaka Y, Imai T, Baba M, Ishikawa I, Uehira M, Nomiyama H, Yoshie O. Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans. Eur J Immunol 29: 633–642, 1999.[CrossRef][Web of Science][Medline]
  34. Teramoto K, Miura S, Tsuzuki Y, Hokari R, Watanabe C, Inamura T, Ogawa T, Hosoe N, Nagata H, Ishii H, Hibi T. Increased lymphocyte trafficking to colonic microvessels is dependent on MAdCAM-1 and C-C chemokine mLARC/CCL20 in DSS-induced mice colitis. Clin Exp Immunol 139: 421–428, 2005.[CrossRef][Web of Science][Medline]
  35. Varona R, Cadenas V, Flores J, Martinez A, Marquez G. CCR6 has a non-redundant role in the development of inflammatory bowel disease. Eur J Immunol 33: 2937–2946, 2003.[CrossRef][Web of Science][Medline]
  36. Varona R, Villares R, Carramolino L, Goya I, Zaballos A, Gutierrez J, Torres M, Martinez A, Marquez G. CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses. J Clin Invest 107: R37–R45, 2001.[Medline]
  37. Yamashiro S, Wang JM, Yang D, Gong WH, Kamohara H, Yoshimura T. Expression of CCR6 and CD83 by cytokine-activated human neutrophils. Blood 96: 3958–3963, 2000.[Abstract/Free Full Text]
  38. Yang CC, Ogawa H, Dwinell MB, McCole DF, Eckmann L, Kagnoff MF. Chemokine receptor CCR6 transduces signals that activate p130Cas and alter cAMP-stimulated ion transport in human intestinal epithelial cells. Am J Physiol Cell Physiol 288: C321–C328, 2005.[Abstract/Free Full Text]




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