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Am J Physiol Cell Physiol 294: C391-C401, 2008. First published October 31, 2007; doi:10.1152/ajpcell.00056.2007
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

MCP-1 targeting inhibits muscularis macrophage recruitment and intestinal smooth muscle dysfunction in colonic inflammation

Masatoshi Hori, Hiromi Nobe, Kazuhide Horiguchi, and Hiroshi Ozaki

Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

Submitted 9 February 2007 ; accepted in final form 26 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Upregulation of muscularis macrophage numbers and activities plays an important role in the intestinal dysmotility associated with intestinal inflammation. The present study aimed to clarify changes in population dynamics of intestinal muscularis macrophages during colonic inflammation and to test possible inhibitory actions of agents targeting monocyte chemoattractant protein-1 (MCP-1) on muscularis macrophage dynamics and motility disorder in the colonic inflammation elicited by 2,4,6-trinitrobenzene sulfonic acid. In the inflamed muscle layer, ED1 antibody-positive monocytes and monocyte-derived macrophages were increased, followed by increasing resident macrophages positively staining for ED2 antibody. Initiation of the ED1-positive macrophage dynamic is associated with MCP-1 mRNA expression. MCP-1 was expressed in both ED1- and ED2-positive macrophages after inflammation. Electromicroscopic analysis revealed that the cell-division phase of muscularis macrophages was seen only in the early stages of inflammation. In addition, ED1 and ED2 double-positive macrophages can be detected during inflammation. Treatment with dominant negative MCP-1 or neutralizing MCP-1 antibodies markedly inhibited numbers of both ED1- and ED2-positive macrophages. Inflammation-mediated dysmotility was partially recovered by treatment with neutralizing MCP-1 antibodies. These results suggest that the inflamed muscle layer is initially infiltrated by monocytes, which then differentiate and develop into muscularis-resident macrophages. These macrophages express MCP-1 for further recruitment of monocytes. MCP-1 may be one potential therapeutic target for inhibiting intestinal motility disorders in gut inflammation.

monocyte chemoattractant protein-1; intestine; 2,4,6-trinitrobenzene sulfonic acid; colitis

THE IMMUNE SYSTEM OF THE GASTROINTESTINAL mucosa represents the first line of defense against foreign pathogens. This first barrier system of the mucosal layer is generally considered to be closely involved in many intestinal diseases, including inflammatory bowel disease (IBD) and intestinal obstruction. Considerable attention has been given to the finding that inflammation at the mucosal region has a severe impact on this first line of defense, subsequently progressing into the muscular layer to induce motility disorder (3, 15–17, 30, 41, 43).

Recent reports have noted that a dense network of resident macrophages populates the intestinal muscularis (13, 27). These macrophages are regularly distributed at the level of the myenteric plexus within the muscle layer (22, 24, 25, 27, 33). Several lines of evidence suggest that these muscularis-resident macrophages appear to be inactive in the basal state but can be activated by endotoxins, intestinal inflammation, and intestinal surgical manipulation, subsequently initiating various inflammatory responses that lead to intestinal dysmotility (6, 12, 19, 41). We recently found that muscularis-resident macrophages upregulate expression of toll-like receptor 4 (TLR-4) as a result of intestinal microfloral overgrowth and that these activated macrophages may produce proinflammatory cytokines to induce a motility disorder in the dilated part of the ileum in a rat model of Hirschsprung's disease (endothelin B receptor-null rat) (37, 44). Interestingly, in the same study (37), the number of macrophages resident within the myenteric plexus in the dilated part of the intestine was significantly increased. A similar phenomenon was found in rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis (16) and in a surgical induction of intestinal obstruction model rat (43).

Monocyte chemoattractant protein-1 (MCP-1) is considered to be one important chemokine regulating migration and infiltration of monocytes/macrophages. MCP-1 belongs to a CC chemokine subfamily, and its effects are mediated through CC chemokine receptor 2 (CCR2) (35). In human inflammatory intestinal diseases such as Crohn's disease and ulcerative colitis, MCP-1 levels are increased within the inflamed mucosal layer (1, 21, 34, 40). In experimental models of intestinal disease, MCP-1 levels are also upregulated in inflamed mucosal and muscle layers (16, 32, 36, 39). In addition, MCP-1 plays a pivotal role in inducing fibrosis and mucosal inflammation in the gut (14, 29). On the other hand, Bauer's group (39) used the endotoxemic ileus model rat to demonstrate that muscularis macrophages produce MCP-1 to recruit more monocytes and neutrophils into the muscle layer. These findings suggest that MCP-1 may be important for inducing intestinal inflammation not only in the mucosal region but also in the muscularis region during inflammation. However, upregulation and activation of muscularis-resident macrophages during inflammation are not understood well.

The objective of this study was to characterize time-dependent dynamic changes in monocytes and monocyte-derived macrophages (stained with ED1 antibody) and resident macrophages (stained with ED2 antibody) in the myenteric plexus region during TNBS-induced colonic inflammation. We also examined the possible ameliorative actions of dominant negative MCP-1 (7ND-MCP-1) or neutralizing anti-MCP-1 antibody on ED1- and/or ED2-positive macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of inflammation. Animal experiments and care were performed in strict compliance with the guidelines outlined within the Guide to Animal Use and Care from the University of Tokyo. This research project was approved by the Insitutional Review Board of Graduate School of Agriculture and Life Sciences of the University of Tokyo and permitted by the committee. The notification number is 181T0044. Male Sprague-Dawley rats (160–200 g; Charles River Japan, Yokohama, Japan) were anesthetized with diethyl ether. The abdomen was opened by midline laparotomy, and the proximal colon was gently exteriorized. Colitis was induced by injecting 100 mg/kg TNBS (Tokyo Kasei Kogyo, Tokyo, Japan) into the colonic lumen 2–3 cm distal to the cecal-colonic junction, as previously reported (16–18). In this study, TNBS was not dissolved in 50% ethanol to avoid the acute inflammatory effects of ethanol. Abdominal surgery to inject TNBS through the colonic wall induces colonic inflammation not only by TNBS but also by abdominal surgery alone (41). Indeed, we confirmed that mRNA expressions of inflammatory cytokines were transiently upregulated and then quickly decreased to baseline levels over 24 h in sham-operated rat (18). However, this model has an advantage in that the TNBS injection site in the proximal colon of each animal can be fixed and the degree of inflammation is not variable compared with colitis models using intrarectal injection of TNBS. In this study, we thus considered surgical intestinal manipulation and TNBS as inflammatory stimuli, and nontreatment was used as a comparative reference state, unless otherwise stated.

Whole mount immunohistochemistry. Rats were killed 1, 2, and 7 days after induction of inflammation by TNBS treatment. Abdomens were opened, then the proximal colon was removed and opened along the mesenteric border. Luminal contents were washed away by using normal physiological salt solution. Opened segments were pinned to the silicon base of a dish with the mucosal side facing up, and the mucosal and submucosal layers were gently removed by using microforceps under light microscopy. Whole mount muscularis preparations were fixed in 4% formaldehyde in PBS (0.05 M, pH 7.2) for 30 min. Following fixation, preparations were washed for 1 h in PBS. Nonspecific binding was reduced by incubating tissues in 5% nonfat milk diluted in PBS containing 0.3% (vol/vol) Triton X-100 for 1 h at room temperature. Preparations were then incubated with anti-rat macrophage antibodies (ED1 and ED2; 1:500; BMA Biomedicals, Augst, Switzerland) and anti-rat MCP-1 (1:200; Antigenix America) at 4°C for 12–15 h, rinsed in PBS, and incubated with FITC or Texas red-labeled secondary antibodies (1:200; Vector Laboratories, Burlingame, CA). Labeled preparations were examined under fluorescence microscopy (Eclipse E800; Nikon, Tokyo, Japan) with a cooled charge-coupled device camera (Media Cybernetics, Silver Spring, MD) or LSM510 confocal microscope (Carl Zeiss Japan, Tokyo, Japan) with an excitation wavelength appropriate for FITC or Texas red. Immunopositive cell numbers were then counted and quantified as follows. We injected TNBS at the same site in each animal. Thus we can confirm the injection site in each animal. We prepared whole mount sections of the injection sites and made two preparation sheets (1 cm x 1 cm). We then took representative pictures from each sheet for analysis.

For double staining of ED1 and ED2, we directly labeled ED1 antibody with Alexa Fluor 488 by using a direct labeling kit (Zenon Alexa Fluor 488 mouse IgG1 labeling kit; Invitrogen/Molecular Probes, Tokyo, Japan). After staining ED2-positive cells with ED2 antibody and anti-mouse IgG antibody labeled with Alexa Fluor 568 (Invitrogen/Molecular Probes), we stained ED1-positive cells by using the direct-labeled ED1 antibody with Alexa Fluor 488.

Semiquantitative RT-PCR. Total RNA was extracted from mucosa-free muscularis preparations by using the acid guanidinium isothiocyanate-phenol chloroform method, and concentrations of RNA were adjusted to 1 µg/µl with RNase-free distilled water. Semiquantitative RT-PCR was performed as previously reported (12). Briefly, first-strand cDNA was synthesized by using a random 9-mer primer and avian myeloblastosis virus (AMV) Reverse Transcriptase XL at 30°C for 10 min, 55°C for 45 min, 99°C for 5 min, and finally 4°C for 5 min.

Hot-start PCR amplification was performed by using Taq Gold Polymerase (Perkin-Elmer Japan, Yokohama, Japan). PCR product sizes are shown in the online supplemental table, along with the oligonucleotide primer sequences for the following rat genes: MCP-1 (Gene Bank No. M57441 [GenBank] ), macrophage colony-stimulating factor (M-CSF: Gene Bank No. AF515736 [GenBank] ), granulocyte-macrophage colony-stimulating factor (GM-CSF: Gene Bank No. U00620 [GenBank] ), and glyceraldehyde 3-phosphate dehydrogenase (Gene Bank No. XX00972). After initial denaturation at 95°C for 10 min, 28–40 cycles (four-cycle interval) of amplification at 94°C for 40 s, 55°C for 1 min, and 72°C for 1.5 min were performed by using a thermal cycler (PCR Thermal Cycler MP; Takara Biomedicals, Shiga, Japan). PCR products in each cycle were electrophoresed on 2% agarose gel containing 0.1% ethidium bromide. Possible contamination of DNA was confirmed by PCR using total RNA without the reverse transcription step. Detectable fluorescent bands were visualized with an ultraviolet transilluminator by using FAS-III (Toyobo), and density of band areas was measured by using NIH Image software.

Recombinant dominant negative MCP-1 and neutralizing MCP-1 antibody. In the present study, an NH2-terminal deletion dominant negative mutant of rat MCP-1 gene (7ND-MCP-1) was constructed by recombinant PCR using wild-type rat MCP-1 cDNA as a template, as previously described by Zhang et al. (46, 47). The 7ND-MCP-1 construct was cloned into Hind III (5') and Kpn I (3') sites of the pFLAG-CTC expression vector (Sigma, St. Louis, MO) to generate a recombinant 7ND-MCP-1 protein with a COOH-terminal FLAG tag. This 7ND-MCP-1-FLAG construct was then transfected into BL-21(DE3) host cells. Protein induction was performed by adding isopropylthiogaslactoside (IPTG; 1 mM) for 10 h at 37°C. Western blots of cell lysate were performed to confirm production of IPTG-induced 7ND-MCP-1 protein using anti-FLAG antibody (Sigma, Tokyo, Japan) and anti-MCP-1 antibody (Chemicon International, Temecula, CA). After purification using an anti-FLAG antibody affinity column (Sigma Japan), 7ND-MCP-1 protein could be detected as a single band. Either recombinant purified 7ND-MCP-1 (500 µg) or neutralizing anti-rat MCP-1 antibody (100 µg; Chemicon International), dissolved in PBS, was injected intravenously via caudal veins into male Sprague-Dawley rats (160–200 g; Charles River Japan) 2 h before TNBS treatment.

Contraction studies. The colon was cut open along the mesenteric attachment, and the mucosa and submucosa were removed. Circular strips were made and suspended along the circular axis in a tissue bath filled with a normal physiological salt solution of (in mM) 136.9 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 23.8 NaHCO3, 5.5 glucose, and 0.01 EDTA (pH 7.4). Muscle strips were maintained at 37°C in an atmosphere of 95% O2-5% CO2. Responses of strips were measured isometrically under a resting tension of 10 mN and were recorded on a multipen recorder (Yokogawa, Tokyo, Japan) and on a computer with the PowerLab system (ADI Instruments, Colorado Springs, CO). Calculations were performed with PowerLab playback software.

Muscularis inflammation induced by TNBS is accompanied by a decrease in muscle contractility and hypertrophy of the muscle layer. Discriminating between changes in contractility due to functional or trophic changes is thus crucial (28). To discriminate between functional and trophic changes in the present study, we used a normalization procedure as described before (15). In brief, we exenterated the colonic inflammatory site in situ to fix the length at 15 mm. The muscle layer of the exenterated colitis tissue was isolated, and width was fixed at 8 mm. In these preparations, changes in wet weight of each strip reflect trophic changes. After experiments, wet weight of each strip was measured and the magnitude of absolute force was normalized for wet weight of the strip.

Transmission electron microscopy. Short segments of proximal colon were removed from rats, under ether anesthesia, 2 or 7 days after injection of TNBS and were placed in fixative containing 3% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 2 h at room temperature. After being rinsed in the same buffer, specimens were postfixed in 1% osmium tetroxide for 2 h at 4°C, rinsed in distilled water, block-stained with saturated aqueous uranyl acetate solution for 3 h, dehydrated in a graded series of ethyl alcohol, and embedded in Epon epoxy resin. Ultrathin sections were cut by using a Reichert microtome (Reichert Japan, Nishinomiya, Japan), double-stained with uranyl acetate and lead citrate, and examined with a JEOL JEM 1200 EX II electron microscope (JEOL, Tokyo, Japan).

Statistics. Results are expressed as means ± SE. Statistical evaluation of data was performed by using unpaired Student's t-tests for comparisons between two groups and by one-way analysis of variance (ANOVA) followed by Dunnett's test for comparisons among three or more groups. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Time-dependent increment of ED1- or ED2-positive macrophages in myenteric plexus. We used immunohistochemistry to characterize changes in number and morphology of muscularis macrophages within the rat colonic myenteric plexus region during TNBS-induced colonic inflammation. Figure 1 shows the effects of TNBS treatment for 1, 2, and 7 days on the distribution of both ED1-stained monocytes/monocyte-derived macrophages and ED2-stained muscularis-resident macrophages. Position of the myenteric plexus region was confirmed by double staining with ED2 and protein gene product 9.5, which detects myenteric nerve plexus (16, 22). In control rats, there were very few ED1-positive cells in the myenteric plexus region (Fig. 1A, top). In some staining conditions, we could detect weakly stained cells with ED1. These cells were ED2-positive resident macrophages, because resident macrophages are well known to also weakly express ED1 antigen. In contrast, in the TNBS-injected colitis model rat at 1 day, numerous ED1-positive, small, round-shaped cells were seen. Increased ED1-positive cells were maintained over 7 days after TNBS injection (Fig. 1A, top, and Fig. 1B). Conversely, numerous ED2-stained muscularis-resident macrophages were detected in the colonic myenteric plexus region from control rats. All ED2-positive cells displayed a ramified shape and were regularly arranged (Fig. 1A, bottom). In TNBS-treated rat colon, the number of muscularis-resident macrophages increased gradually beginning 2 days after inflammation, and arrangement seemed to become irregular, particularly at 7 days after induced inflammation (Fig. 1A, bottom, and Fig. 1C). Furthermore, some ED2-positive cells at 2 days after TNBS treatment were small and displayed a round shape. Figure 1, B and C, shows that ED1-positive monocyte-derived macrophages increased in number much faster than ED2-positive resident macrophages during inflammation. No fluorescence-positive cells were identified when whole mount preparation was stained with the secondary antibody alone (data not shown).


Figure 1
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  		Fig. 1. Time-dependent changes in monocyte/monocyte-derived macrophages (ED1-positive) or resident macrophages (ED2-positive) in the myenteric plexus region during 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colonic inflammation. A: myenteric plexus region whole mount immunohistochemistry showing macrophages stained with ED1 (top) or ED2 (bottom) at 0 (control), 1, 2, and 7 days after TNBS injection. Results typical of 6–8 experiments are shown. Scale bar, 15 µm. B and C: quantification of ED1- (B) or ED2-positive (C) cells in the myenteric plexus region. **P < 0.01 compared with control (day 0).

 
MCP-1 mRNA expression. We next investigated mRNA expression of MCP-1 in the muscularis layer during the course of TNBS-induced inflammation. From control muscle tissues, only trace amounts of MCP-1 mRNA were detected at 32 cycles of amplification (day 0 in Fig. 2A). In contrast, MCP-1 mRNA expression was dramatically upregulated at 1 day after TNBS injection and then gradually decreased toward resting levels by 7 days after inflammation.


Figure 2
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  		Fig. 2. Changes in monocyte chemoattractant protein (MCP)-1, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression in the muscularis region during colonic inflammation. Top: typical results of MCP-1 (A), M-CSF (B), GM-CSF (C), and GAPDH RT-PCR products. Bottom: level of each gene mRNA expression is expressed as a ratio of GAPDH mRNA expression. Each column shows quantified results (mean ± SE) of 4 independent experiments. *P < 0.05, **P < 0.01 vs. control.

 
We also examined mRNA expressions of macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the muscularis layer during the course of TNBS-induced inflammation. M-CSF and GM-CSF mRNAs were expressed in muscularis preparations from control rat (0.75 ± 0.02 in M-CSF and 0.28 ± 0.05 in GM-CSF vs. GAPDH mRNA, respectively, n = 4 each; day 0 in Fig. 2, B and C). After TNBS injection, M-CSF and GM-CSF mRNA expressions gradually increased, peaking 7 days after inflammation [1.25 ± 0.08 (P < 0.05) in M-CSF and 0.68 ± 0.07 (P < 0.01) in GM-CSF mRNA expression vs. GAPDH mRNA, n = 4 each; Fig. 2, B and C].

Immunohistochemistry of MCP-1. To determine the cellular origin of MCP-1 within the myenteric region, we performed double immunostaining of MCP-1-positive cells with ED1 or ED2 (Fig. 3; n = 6 each). In control rat colon, MCP-1 antibody-positive cells were scarcely detected. At 1 day after TNBS injection, more cells, relatively small and round-shaped, were positively stained with MCP-1. MCP-1-positive cells were also detected at the myenteric plexus region at 2 and 7 days after inflammation. Almost all MCP-1-positive cells were also ED1- or ED2-positive, indicating that they were macrophages. MCP-1-immunoreactive ED2-positive macrophages formed relatively small, round shapes.


Figure 3
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  		Fig. 3. Double staining of MCP-1 and ED1 or ED2 during colonic inflammation in the myenteric region. Typical representative fields of the myenteric plexus region stained with both MCP-1 and ED1 (A) or both MCP-1 and ED2 (B) at 0, 1, 2, and 7 days after TNBS injection. Each picture is representative of results seen in 4–6 independent experiments. Arrowheads indicate merged cells. Scale bar, 20 µm.

 
Transmission electron microscopy of macrophages in the myenteric region. We examined myenteric plexus region tissues from control (day 0) and TNBS-induced inflamed colons under electron microscopy. In control rat colon, some resident macrophages were associated with myenteric ganglia (Fig. 4A). Macrophages were identified by well-developed cell processes, lysosomes, vacuoles, and coated vesicles (Fig. 4C) as previously reported (16, 38). Macrophages possessed electron-lucent cytoplasm and several short processes (Fig. 4, B and C). The processes of interstitial cells of Cajal (ICC), which were characterized by electron-dense cytoplasm, numerous mitochondria, and caveolae, were closely associated with resident macrophages (Fig. 4B).


Figure 4
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  		Fig. 4. Transmission electron micrography of myenteric plexus region in control rat colon. Myenteric ganglia in the myenteric plexus region in representative control colon (A). Left and right flames in A are shown in B and C, respectively. G, myenteric ganglia; LM, longitudinal smooth muscle cells; CM, circular smooth muscle cells; MP, macrophages; IC, interstitial cell of Cajal; Go, Golgi body; arrow, lysosome. Scale bar in A, 2 µm. Scale bars in B and C, 1 µm.

 

Figure 6
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  		Fig. 6. Double staining of ED1 and ED2 during colonic inflammation in the myenteric region. Typical representative fields of the myenteric plexus region stained with both ED1 and ED2 at 0, 1, 2, and 7 days after TNBS injection. Each picture is representative of results seen in 4–6 independent experiments. Scale bar, 10 µm.

 
At 2 or 7 days after inflammation, contours of the myenteric ganglia became irregular in TNBS-treated rat colons, and numerous macrophages were observed around ganglia (Fig. 5D). These macrophages were closely associated with nerve elements and were often engaged with the irregular outlines of ganglia. In addition, we often found macrophages undergoing cell mitosis only 2 days after inflammation (Fig. 5, B and C).


Figure 5
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  		Fig. 5. Transmission electron micrography of myenteric plexus region in inflamed colon. Mitotic division of macrophages in the colonic myenteric region were found at 2 days after TNBS-induced inflammation in B and C. Nuclear envelope is no longer evident, but agglutinated chromatin can be seen (arrow). Scale bars in A, B, and C, 1 µm. D: myenteric ganglia in myenteric plexus region 7 days after TNBS treatment. Scale bar, 2 µm. F, fibroblast cells.

 
Double staining of ED1 and ED2. We next performed double staining of ED1 and ED2 by using whole-mount immunohistochemistry (Fig. 6). In control rats, only ED2-positive ramified resident macrophages were detected. In contrast, at 2 or 7 days after inflammation, we were able to detect ED1 and ED2 double-positive macrophages in the inflamed myenteric plexus region. At 7 days after inflammation, ED1 and ED2 double-positive cells varied from small and round to ramified forms.

Effects of neutralizing MCP-1 antibody and 7ND-MCP-1 on ED1- and ED2-positive cell numbers during inflammation. Seven amino acids within the NH2 terminal of human MCP-1 are essential for inducing chemoattraction (48), and an NH2-terminal deletion mutant of the human MCP-1 gene (7ND-MCP-1) has a dominant negative effect by inhibiting wild-type MCP-1 activity via interference at the CCR2 receptor (47, 48). In addition, 7ND-MCP-1 dramatically attenuates atherosclerosis in apolipoprotein E-knockout mice (31). Moreover, 7ND-MCP-1 reduces vascular remodeling by inhibiting endothelial nitric oxide synthesis in rats (5), suggesting that human MCP-1 functionally cross-reacts with rat MCP-1. We thus investigated the effects of neutralizing anti-MCP-1 antibody and 7ND-MCP-1 on macrophage population in the TNBS-induced colitis model rat. Numbers of ED1- or ED2-positive cells in the myenteric plexus region were increased at 2 days after inflammation (Fig. 7). Treatment with neutralizing anti-MCP-1 antibody or 7ND-MCP-1 dramatically decreased numbers of ED1- or ED2-positive cells. Neutralizing anti-MCP-1 antibody and 7ND-MCP-1 treatments significantly inhibited numbers of ED1- or ED2-positive cells (Fig. 7). Neutralizing anti-MCP-1 antibody and 7ND-MCP-1 treatments had no effect on populations of ED1- or ED2-positive cells in control animals (ED1, 20.4 ± 6.9 cells/x200 magnification field; ED2, 39.4 ± 6.9 cells/x200 magnification field; n = 3 each). Heat-denatured 7ND-MCP-1 and neutralizing anti-MCP-1 administration did not affect TNBS-induced ED1- and ED2-positive cell infiltration (n = 2, data not shown).


Figure 7
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  		Fig. 7. Effects of neutralizing anti-MCP-1 antibody (MCP-1 Ab) and dominant negative MCP-1 (7ND-MCP-1) on the ED1-or ED2-positive cell populations in the myenteric plexus region at 2 days after inflammation. Each picture is representative of results from 3 independent experiments. A: MCP-1 Ab treatment and 7ND-MCP-1 treatment had no effect on cell populations of ED1- and ED2-positive cells in control colon. Scale bar, 20 µm. B: quantification of ED1-or ED2-positive cell numbers the myenteric plexus region at 2 days after inflammation with or without MCP-1 Ab or 7ND-MCP-1 treatments. C and D: each column indicates mean ± SE of 3 independent experiments. *P < 0.05, **P < 0.01 vs. TNBS. #P < 0.05, ##P < 0.01 vs. control.

 
Effects of neutralizing MCP-1 antibody on carbachol-induced contractions in TNBS-induced inflamed colon. Finally, we investigated the effect of neutralizing MCP-1 antibody administration on smooth muscle dysfunction induced by TNBS. Carbachol (0.1–10 µM) induced contractions in a concentration-dependent manner (Fig. 8). In the inflamed colonic smooth muscle of TNBS-induced colitis model rat, carbachol-mediated contraction was significantly inhibited. Treatment with neutralized MCP-1 antibody significantly recovered contractions. Conversely, heat-denatured neutralized MCP-1 antibody administration did not recover contractility (n = 2, data not shown).


Figure 8
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  		Fig. 8. Effect of neutralized anti-MCP-1 antibody administration on smooth muscle dysfunction in the TNBS-induced colitis model rat. Carbachol-induced contractility at 2 days after TNBS induction with or without neutralized anti-MCP-1 antibody was measured as described in MATERIALS AND METHODS. Each plot indicates a mean ± SE of 8 samples from 3 independent experiments. bullet, Control; {circ}, TNBS alone; {blacktriangleup}, TNBS with anti-MCP-1 antibody treatment. *P < 0.05, **P < 0.01 vs. TNBS. #P < 0.05, ##P < 0.01 vs. control. n.s., Not significant.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we found that the number of ED1-positive monocyte-derived macrophages increased in the myenteric plexus 1 day after TNBS injection. These changes coincided with increased expression of MCP-1 mRNA levels. We also observed that numbers of ED2-positive resident macrophages increased, although this upregulation lagged behind the altered dynamics of ED1-positive cells. Muscularis-resident macrophages are morphologically ramified in normal intestine, but the round and swollen shape of ED2-positive macrophages (immature type) was often observed at 2 or 7 days after inflammation. These results indicate that infiltrating ED1-positive monocytes/monocyte-derived macrophages may differentiate and/or mature into ED2-positive muscularis-resident macrophages. To clarify this point, we performed double staining for both ED1 and ED2 in inflamed whole mount preparations. We detected numerous ED1 and ED2 double-positive cells at 2 and 7 days after inflammation. In addition, at 7 days after inflammation, ED1 and ED2 double-positive cells tended to become ramified. These results strongly support our suggestion. On the other hand, transmission electron microscopy indicated increased mitotic activity of muscularis macrophages at 2 days after inflammation, but not later at 7 days after inflammation. These results suggest that, at an early stage of inflammation, increases in the muscularis macrophage population in the myenteric plexus may also result from increased proliferative activity. At present, whether mitotic macrophages originated from ED1-positive exudative macrophages or ED2-positive resident macrophages remains unclear. Flores-Langarica et al. (8) recently suggested that immature and mature dendritic cells account for 20–30% of total major histocompatibility complex (MHC) class II-positive cells in the myenteric plexus region. Further examination will be necessary to clarify pathophysiological functions of dendritic cells in the inflamed myenteric plexus.

In patients with IBD, MCP-1 expression is increased in the inflamed colonic mucosal region. In addition, MCP-1 mRNA is upregulated not only in the mucosal region, but also in cells of the myenteric plexus of IBD patients (10, 20). In the present study and our previous investigation (16), MCP-1 mRNA production could be detected in mucosa-free muscularis preparations in the TNBS-induced colitis rat colon, and MCP-1 protein was immunohistochemically expressed in both ED1- and ED2-positive cells in the myenteric plexus, implying that dynamic changes in muscularis macrophages for this rat model are similar to those in IBD patients.

The main finding of this study was that dominant negative MCP-1 (7ND-MCP-1) or neutralizing anti-MCP-1 antibody prevented population increases for both ED1-positive monocytes/monocyte-derived macrophages and ED2-positive muscularis-resident macrophages. This result also supports the conclusion that ED2-positive resident macrophages may, at least in part, originate from ED1-positive monocyte-derived macrophages in the inflammatory situation. In inflammatory responses to arterial injury, continuous recruitment of monocytes via MCP-1-mediated signaling plays a crucial role in restenosis and atherogenesis. Indeed, mice lacking MCP-1 or the MCP-1 receptor, CCR2, display reduced initial formation of atheroma (2, 11). In these vascular inflammatory diseases, anti-MCP-1 gene therapy using 7ND-MCP-1 is considered as a new therapeutic approach for antirestenosis and antiatherosclerosis paradigms (4). Repeated injection of neutralizing anti-MCP-1 antibody also reduced neointimal formation in a rat model of carotid artery balloon injury (9). These therapeutic trials targeting MCP-1 in vascular remodeling, together with the present results in this rat colitis model, imply that targeting MCP-1 either with gene therapy and/or antibody therapy could offer a novel approach to IBD treatment. Most recently, overexpression of MCP-1 has been reported to induce fibrogenic responses in mouse colon through interactions between T cells and fibroblasts/myofibroblasts (29). In addition, MCP-1 induces a critical role in the development of colonic inflammation in the context of immune and enteric endocrine cells in the colitis model using MCP-1-deficient mice (14). These reports also support the therapeutic benefit of targeting MCP-1 for IBD. However, targeting MCP-1 in the present study was able to ameliorate macrophage infiltration and motility disorder by only 50%, suggesting that another cytokine and/or chemokine plays an important role in inducing macrophage movement and motility disorder during muscularis inflammation.

In M-CSF point-mutated mice (op/op mice) (42, 45), F4/80-positive muscularis-resident macrophages are absent from the small intestine (26). Conversely, F4/80-positive muscularis-resident macrophages can be detected in germ-free adult mice and even in the embryonic intestinal muscle layer of 15 days postcoitum mice (23). These results suggest that M-CSF may be essential for the development and maturation of muscularis-resident macrophage even in the absence of foreign antigens (23). Our findings that GM-CSF and M-CSF mRNA expressions in the muscularis layer were also increased at 7 days after inflammation support the view that infiltrated ED1-positive macrophages may differentiate and/or maturate into ED2-positive resident macrophages under inflammatory conditions.

Bauer's group demonstrated that exogenous lipopolysaccharide causes the extravasation of leukocytes (mainly ED1-positive monocytes) into the intestinal muscularis and that resident muscularis macrophage-derived MCP-1 expression results in the recruitment of monocytes during endotoxemia (39). Muscularis-resident macrophages can easily react with lipopolysaccharide to induce nitric oxide synthase via cyclooxygenase-2 expression, resulting in the induction of a motility disorder (6, 7, 12). We have previously found that TNBS-induced inflammation causes morphological damage of ICC and myenteric nerve networks, resulting in inducing impairment of colonic peristalsis with increases in ED2-positive muscularis macrophages (16). The present study found that neutralized anti-MCP-1 antibody treatment inhibited cell numbers of ED1- and ED2-positive cells and recovered the reduced smooth muscle contractility after TNBS-induced inflammation, suggesting that infiltrated monocytes and muscularis-resident macrophages may play a crucial role in inducing motility disorder. However, we did not observe MCP-1 expression in ED2-positive muscularis-resident macrophages in the early stages of inflammation. These findings suggest that the processes of muscularis macrophage recruitment by MCP-1 may differ between systemic endotoxemia and local intestinal inflammation. It will be necessary to investigate inflammatory events at earlier stage of inflammation by using another kind of colitis model without surgical artifact.

In conclusion, ED1-positive monocytes in this TNBS-induced colitis model initially infiltrated into the inflamed muscle layer and expressed MCP-1 for further recruitment of monocytes. These infiltrated monocytes may differentiate into ED2-positive resident macrophages that also expressed MCP-1 to recruit even more monocytes. In addition, the number of muscularis-resident macrophages may increase via a self-multiplication mechanism in the early stages of inflammation. Another interesting finding of this study was that treatment with dominant negative MCP-1 or neutralizing anti-MCP-1 antibody significantly inhibits monocyte/macrophage infiltration, suggesting that MCP-1 could offer a potential therapeutic target for inhibiting muscularis inflammation in IBD. However, it will be necessary to investigate effect of targeting of MCP-1 on a chronic colitis model, such as an IL-10-deficient colitis model mice, to clarify this point.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the Yakult Bioscience Foundation and a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, and Science.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Hori, Dept. of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The Univ. of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan (e-mail: ahori{at}mail.ecc.u-tokyo.ac.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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