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Originally published online as doi:10.1189/jlb.0204104 on November 29, 2004

Published online before print November 29, 2004
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(Journal of Leukocyte Biology. 2005;77:287-295.)
© 2005 by Society for Leukocyte Biology

Blockade of PSGL-1 attenuates CD14+ monocytic cell recruitment in intestinal mucosa and ameliorates ileitis in SAMP1/Yit mice

Takuya Inoue*, Yoshikazu Tsuzuki*, Koji Matsuzaki*, Hisayuki Matsunaga*, Junichi Miyazaki*, Ryota Hokari*, Yoshikiyo Okada*, Atsushi Kawaguchi*, Shigeaki Nagao*, Kazuro Itoh*, Satoshi Matsumoto{dagger} and Soichiro Miura*,1

* Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan; and
{dagger} Yakult Central Institute for Microbiological Research, Tokyo, Japan

1 Correspondence: Second Department of Internal Medicine, National Defense Medical College, 2-3 Namiki, Tokorozawa, Saitama, 359-8513, Japan. E-mail: miura{at}me.ndmc.ac.jp


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ABSTRACT
 
The pathogenesis of Crohn’s disease (CD) is not known. However, monocytes and macrophages are thought to play important roles in the development of mucosal inflammation. Therefore, in this study, we examined the role of monocyte-endothelial cell interactions in senescence-accelerated mouse P1 (SAMP1)/Yit mice, a murine model of spontaneous ileitis. Fluorescence-labeled CD14+ monocytic cells isolated from the spleen and mesenteric lymph nodes of AKR/J (control) mice were injected into the tail veins of recipient (AKR/J and SAMP1/Yit) mice, and migration in the postcapillary venules (PCV) of Peyer’s patches, submucosal venules, and villus microvessels of the terminal ileum was monitored by using an intravital microscope. Rolling and adhesion of CD14+ monocytic cells in the PCV of Peyer’s patches and microvessels of the terminal ileum were increased in SAMP1/Yit mice. An imunohistochemical study showed increased expression of P-selectin glycoprotein-1 (PSGL-1), P-selectin, and vascular cell adhesion molecule-1 in the terminal ileum of SAMP1/Yit mice. Antibodies against these three adhesion molecules significantly inhibited adhesion of CD14+ monocytic cells to the PCV of Peyer’s patches and microvessels of the terminal ileum, treatment with an anti-PSGL-1 monoclonal antibody (mAb) showing the strongest suppressive effect. Anti-PSGL-1 mAb also attenuated T cell adhesion in microvessels of intestinal mucosa. In addition, periodical administration of an anti-PSGL-1 mAb for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice. The results suggest that PSGL-1-P-selectin interaction plays an important role in monocyte-endothelial cell interactions and the development of ileitis in a murine model of CD and that the blockade of this adhesion molecule may be a novel strategy for treating CD.

Key Words: SAMP1/Yit • P-selectin • VCAM-1


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INTRODUCTION
 
In Crohn’s disease (CD), transmural inflammation, fistulas, and granuloma, formation can be randomly distributed throughout the gastrointestinal tract from the mouth to the anus [1 ]. The pathogenesis of CD is not known, but CD is, in part, characterized by an accumulation of inflammatory cells in affected regions of the gastrointestinal tract. Among the various types of inflammatory cells, monocytes and macrophages are thought to play an important role in the development of mucosal inflammation and ulceration [2 ]. Transmural perivascular mononuclear cell infiltrates are a feature of CD, and the vast majority of these cells includes CD68+ and CD31+ monocytes/macrophages surrounded by naïve and memory T cells [3 ].

CD14 is the receptor for the lipopolysaccharide (LPS)-LPS-binding protein complex, and its presence on monocytes/macrophages represents cell activation [4 , 5 ]. Moreover, a large proportion of macrophages in inflamed mucosa strongly expresses CD14, which is not usually expressed in normal mucosa, and CD14 expression by macrophages in CD and ulcerative colitis is significantly up-regulated compared with that in noninflamed intestinal mucosa [5 , 6 ]. Therefore, blood CD14+ monocytic cells are thought to be actively recruited to the mucosa of inflamed bowel, and CD14 is a marker of recently recruited monocytes/macrophages in inflammatory bowel disease (IBD) [2 , 5 ].

The mechanism of the migration of monocytes, which are the major origin of tissue macrophages, from peripheral blood into the inflamed tissue involves several steps: The first step is rolling of monocytes along the activated endothelium and then reduction in the velocity of rolling; the second step is monocyte adhesion; and the third step is transmigration into the interstitium [7 ]. It has been reported that L-selectin (on leukocytes), P-selectin (on platelets and endothelial cells), and their counterparts, peripheral lymph node addressins (PNAd) and P-selectin glycoprotein ligand-1 (PSGL-1), mediate the first step [8 ] and that the second step is mediated by CD11a/CD18, {alpha}4ß1, and {alpha}4ß7 integrins interacting with their counter-ligands among the immunoglobulin (Ig) superfamily, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) on the endothelium [9 ]. Then monocytes transmigrate through endothelial cells into the tissue. During transmigration, monocyte chemoattractant protein-1 (MCP-1), which is one of the C–C chemokines, is thought to attract and activate monocytic cells, and this chemokine has been found to be up-regulated in IBD [10 11 12 ]. However, it is not clear how CD14+ monocytic cells actually interact with the vascular endothelium in the inflamed intestine of CD and what molecules mediate the interactions during the active recruitment process.

Recently, the senescence-accelerated mouse P1/Yit (SAMP1/Yit) mouse [13 ] has been reported to be a useful model of CD, as the SAMP1/Yit mouse has been shown to have spontaneous small bowel inflammation, especially at the end of ileum, under specific pathogen-free conditions. Moreover, the mucosal inflammation is histologically characterized by a discontinuous and transmural granulomatous inflammation similar to CD in humans. Most previous models do not have these features of CD, indicating that the SAMP1/Yit mouse can yield fundamental insights into the immunopathogenesis of CD [14 , 15 ]. It has also been reported that inflammatory changes, including infiltration of immune cells, start after 10 weeks of age and that ileitis develops in most mice after 20 weeks of age.

Therefore, in this study, we investigated in situ monocyte migration in lymphoid (Peyer’s patches) and nonlymphoid (villus) regions of the intestinal mucosa of SAMP1/Yit mice with spontaneous ileitis and examined the roles of PSGL-1, P-selectin, and VCAM-1 in this recruitment process by using an intravital microscope. We focused on the role of PSGL-1 molecules in the development of spontaneous ileitis and investigated whether periodical administration of an anti-PSGL-1 monoclonal antibody (mAb) attenuates inflammation in SAMP1/Yit mice.


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MATERIALS AND METHODS
 
Immunohistochemistry for adhesion molecules in the intestinal mucosa
Localization and expression of PSGL-1, P-selectin, and VCAM-1 in the intestinal mucosa were assessed by immunohistochemistry using the labeled streptavidin-biotin method. The terminal ileum of each mouse was removed and fixed in periodate lysine paraformaldehyde solution. Then the tissue was washed with phosphate-buffered saline (PBS) and infused with 10%, 15%, and 20% sucrose phosphate buffer. Fixed tissue was embedded in optical cutting temperature compound (Tissue-Tek, Miles Inc., Pittsburgh, PA) and stored at –70°C. Cryostat sections of 8 µm in thickness were transferred to poly-L-lysine-coated slides and air-dried for 1 h at 20°C. After the sections had been washed in PBS (pH 7.4) containing 1% Triton X-100 for 5 min, they were incubated in 5% normal goat serum in PBS. mAb against PSGL-1 (2PH2; 0.5 mg/ml), P-selectin (RB40.34; 0.5 mg/ml), VCAM-1 (429; 0.5 mg/ml), MCP-1 (2H5; 0.5 mg/ml), and CD68 (FA-11; 0.5 mg/ml) were diluted 50 times with PBS and layered onto the sections, and the sections were incubated overnight at 4°C. The sections were then incubated with a secondary antibody, biotinylate anti-rat IgG antibody (Amersham, Little Chalfont, UK), for 1 h at room temperature. Then the sections were incubated with fluorescein isothiocyanate-conjugated streptavidin (Amersham) for 30 min at room temperature. Rinsing with PBS containing 1% bovine serum albumin was performed between each step. A coverslip was applied by using glycerol jelly. The sections were observed under a fluorescent microscope (BX60; Olympus, Tokyo, Japan).

Isolation of CD14+ monocytic cells and T lymphocytes, labeling with carboxyfluorescein diacetate succinimidyl ester (CFDSE)
Mouse spleen and mesenteric lymph nodes were isolated after a midline incision and crushed with slide glasses. Pellets were incubated with collagenase, hyarulonidase, and DNase for 10 min, washed three times with Dulbecco’s phosphate saline (PBS), and hemolyzed with NH3-Tris buffer. Then monocytic cells were isolated by magnetic cell sorting (MACS; Miltenyi Biotec, Auburn, CA) with a rabbit anti-mouse CD14 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and bead-conjugated anti-rabbit IgG (Miltenyi Biotec). The purity of CD14+ monocytic cells was evaluated by using a fluorescence-activated cell sorter (FACS-440, Becton Dickinson, Mountain View, CA) using a rat anti-mouse F4/80 mAb (Cosmo Bio, Tokyo, Japan). The phenotype of CD14+ cells was evaluated by rat anti-mouse CD62L (MEL-14, PharMingen, San Diego, CA) and rat anti-mouse {alpha}2 integrin (Immunotec, Marseille, France). Conversely, T lymphocytes were isolated by T cell isolation column (CL101, Cedarlane, Ontario, Canada). CFDSE (Molecular Probes, Eugene, OR) was dissolved in dimethyl sulfoxide at 15.6 mM and divided into small aliquots (each 300 µl), and the aliquots were stored in a cuvette sealed with argon gas at –20°C until use in the experiments. CD14+ monocytic cells (5x106) in 1 ml PBS were incubated with CFDSE solution for 30 min at 37°C and washed twice with PBS.

Animal preparation for intravital observation
Male SAMP1/Yit mice were kindly donated by Yakult Central Institute for Microbiological Research (Tokyo, Japan) and were maintained in an animal colony at the National Defense Medical College (NDMC; Saitama, Japan). AKR/J mice (Japan Clea Co., Tokyo), from which SAMP1/Yit mice were derived and do not have ileitis, were purchased and maintained in an animal colony at NDMC. SAMP1/Yit mice and AKR/J mice (as control mice) were used in this study. The care and use of animals were in accordance with the guidelines for laboratory animals in NDMC. For migration studies, mice at 30 weeks of age were anesthetized with 50 mg/kg pentobarbital sodium, and the abdomen of each mouse was opened with a midline incision. An ileal segment of 1–3 cm in length ending at the ileocecal valve was chosen for observation and placed on wet cotton. The intestine was kept warm and moist by continuous superfusion with PBS warmed to 37°C. Each side of a suitable ileal segment was ligated with care taken to prevent disruption to microcirculation, and PBS was injected into the closed segment using a 30-gauge needle. The labeled CD14+ monocytic cells (5x106) were infused from tail vein for 3 min. The behavior of infused cells in postcapillary venules (PCV) of Peyer’s patches and submucosal venules was observed from the serosal side by using an intravital microscope. In the same animals, the migration in mucosal villus microvessels was also observed from the mucosal surface after opening the intestinal lumen along its antimesenteric side. The microcirculation was observed by a fluorescence microscope (BX51WI, Olympus) equipped with a silicon-intensified target tube camera with a contrast-enhancing unit (C-2400-08, Hamamatsu Photonics, Shizuoka, Japan) and a x10 or x20 ultraviolet-fluorite objective lens (Fluor, Nikon, Tokyo, Japan) according to a previously described method [16 ] and was recorded on digital videotape with a high-speed video recording system (WV-DR7, Sony, New York, NY).

In another sets of experiments, the labeled T lymphocytes (1x107) were infused from tail vein for 3 min, and the behavior of infused cells was observed in PCV of Peyer’s patches, submucosal venules, and mucosal villus microvessels under fluorescence microscopy and recorded using the same system as described above.

Administration of mAb at intravital observation
In some experiments, 2 mg/kg rat anti-mouse PSGL-1 (2PH2, PharMingen), P-selectin (2H5, PharMingen), and VCAM-1 mAb (MVCAM429, PharMingen) were administered via a tail vein 30 min prior to CFSE-labeled CD14+ monocytic cell infusion. In addition, effects of anti-PSGL-1 antibody on T cell adhesion were also evaluated. Then the suppressive effects of these antibodies were compared with that of isotype-matched rat IgG (PharMingen).

Analysis of fluorescence-labeled cell dynamics
The numbers of rolling and adherent CD14+ monocytic cells and T lymphocytes were determined off-line by digital videotaped images. Infused fluorescence-labeled cells adhering to the vascular walls with occasional movement were defined as rolling. We counted the numbers of labeled cell influx and rolling in a field using a x20 objective lens for 1 min at 10 and 20 min after the injection. The results are expressed as an average of rolling percentage at 10 min and at 20 min, rolling number/total number of cell influx x 100 (%). Labeled cells adhering to the vascular walls without movement and remaining stationary for a period of >30 s were defined as adherence. We counted the numbers of adherent CD14+ monocytic cells and T cells in the observation field using a x20 objective lens at 10-min intervals up to 60 min after the administration.

Effects of periodical administration of anti-PSGL-1 on ileitis of SAMP1/Yit mice
We next examined whether periodical administration of anti-PSGL-1 antibody ameliorates ileitis of SAMP1/Yit mice. Anti-PSGL-1 or isotype-matched IgG (2 mg/kg) was administered intraperitoneally to SAMP1/Yit mice every other day from the 14th to 21st week. Mice were killed in the 21st week, and body weight, small intestinal length, villus height, submucosal thickness, and the number of infiltrating immune cells were determined. The expression of CD4, CD8, PSGL-1, and CD68 was immunohistochemically evaluated using the labeled streptavidin-biotin method with CD4 (L3T4, PharMingen), CD8 (Ly-2, PharMingen), PSGL-1 (2PH2, PharMingen), and CD68 (FA-11, PharMingen) as described above.

Blocking effects of 7-week administration of anti-PSGL-1 antibody on CD14+ monocytic cell adherence
Next, to evaluate whether long-term administration of anti-PSGL-1 antibody still has a blocking effect on CD14+ monocytic cell adherence to intestinal microvessels, we determined how monocytic cell adherence was inhibited in SAMP/Yit mice after a 7-week administration of anti-PSGL-1 antibody. We observed the CD14+ monocytic cell adhesion in the microvessels using the same observation system as described above in SAMP/Yit mice, which had received a 7-week periodical administration of anti-PSGL-1 antibody compared with control, IgG-treated mice.

Statistics
All results are expressed as means ± SE. For comparison of rolling and adherence, the mean value was statistically evaluated by a nonparametric Mann-Whitney U-test. Statistical significance was defined as P < 0.05.


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RESULTS
 
Immunohistochemistry for PSGL-1, P-selectin, VCAM-1, and CD68
SAMP1/Yit mice more than 12 weeks of age showed a discontinuous thickening of the intestinal wall of the terminal ileum. Histologically, the lesions in the ileum were characterized by submucosal thickening and inflammatory cell infiltration as was reported previously [13 ]. These lesions were not observed in control AKR/J mice of the same age. We examined the expression of the mucin-like glycoprotein ligand PSGL-1 and the endothelial adhesion molecule P-selectin and VCAM-1 by immunofluorescence using the labeled streptavidin-biotin method. The numbers of PSGL-1-positive cells in the epithelium and the lamina propria of SAMP1/Yit mice were greater than those in AKR/J mice (Fig. 1 ). The number of P-selectin-positive vessels was also greater in the lamina propria of SAMP1/Yit mice than that in AKR/J mice. Few submucosal vessels were VCAM-1-positive in AKR/J mice, and VCAM-1-positive vessels were significantly increased in the ileal mucosa of SAMP1/Yit mice. The number of CD68-positive cells was also increased in the lamina propria of SAMP1/Yit mice (Fig. 1) .



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  		Figure 1. Immunohistochemistry of PSGL-1, P-selectin, VCAM-1, and CD68 in the intestinal mucosa. Methods used for immunostaining are described in Materials and Methods. PSGL-1- and CD68-positive cells in the epithelium and the lamina propria, P-selectin-positive vessels in the lamina propria, and VCAM-1-positive vessels in the ileal mucosa of SAMP1/Yit mice were increased compared with those in AKR/J mice.

CD14+ monocytic cell rolling and the effects of antibodies against PSGL-1 and P-selectin in the ileal mucosa of AKR/J and SAMP1/Yit mice
FACS analysis demonstrated that ~90% of CD14+ cells we obtained using MACS expressed F4/80 (data not shown) [17 ], one of the specific markers for monocytes and macrophages. These CD14+ cells express CD62L and {alpha}2 integrin (Fig. 2 ), suggesting that these CD14+ cells may be CX3CR1lowGR1+ monocytes [18 ]. The movements and interactions of CFSE-labeled CD14+ monocytic cells within microvessels were visualized by using a fluorescence microscope. When the total influx of labeled CD14+ monocytic cells were counted in PCV of Peyer’s patches, there was no significant difference in the number of influx between two strains (i.e., AKR/J vs. SAMP1/Yit, 124±27 cells/min vs. 114±32 cells/min at 10 min). The percentage of rolling cells in PCV of Peyer’s patches in SAMP1/Yit mice was significantly higher than that in PCV of Peyer’s patches in AKR/J mice at 10–20 min (25.8±4.3% and 12.1±1.5%, respectively; Fig. 3 ). In the ileal villus microvessels, the ratio of rolling CD14+ monocytic cells was also significantly greater in SAMP1/Yit mice than in AKR/J mice (23.1±4.1% and 12.0±1.7%, respectively). In submucosal microvessels, the ratio of rolling cells was also greater in SAMP1/Yit mice, but there was no significant difference between the two groups.



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  		Figure 2. The phenotype of CD14+ cells was evaluated by rat anti-mouse CD62L (MEL-14, PharMingen) and rat anti-mouse {alpha}2 integrin (Immunotec). R1, Mononuclear cell area. SSC, Side-scatter; FSC, forward-scatter.



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  		Figure 3. Ratios of rolling CD14+ monocytic cells in PCV of Peyer’s patches, submucosal venules, and villus microvessels. Effects of anti-PSGL-1 antibody and anti-P-selectin antibody. The percentage of rolling CD14+ monocytic cells in PCVs of Peyer’s patches and in the ileal villus capillaries in SAMP1/Yit mice was significantly larger than in that of AKR/J mice. *, P < 0.05, versus AKR/J; #, P < 0.05, versus SAMP1/Yit; n = 5 in each group.

We examined the effects of anti-PSGL-1 mAb and anti-P-selectin mAb on monocytic cell rolling in PCV of Peyer’s patches, submucosal venules, and mucosal villus capillaries in SAMP1/Yit mice after infusion of CFSE-labeled cells (Fig. 3) . There was no significant difference in CD14+ monocytic cell behavior after control IgG treatment (data not shown). In these experiments, the ratio of rolling/total number of CD14+ monocytic cells was significantly decreased by anti-PSGL-1 mAb, anti-P-selectin mAb, and a combination of these mAb at 10–20 min compared with control IgG at the three sites. However, the degrees of inhibition by these antibodies at the three sites were not statistically different.

CD14+ monocytic cell adherence and the effects of antibodies against PSGL-1, P-selectin, and VCAM-1 in the ileal mucosa of AKR/J and SAMP1/Yit mice
Figure 4 shows microscopic images of the distribution of fluorescence-labeled, adherent CD14+ monocytic cells in the intestinal mucosa of SAMP1/Yit mice at 60 min after infusion. The number of adhered CD14+ monocytic cells gradually increased up to 60 min after infusion (Fig. 4) . In control AKR/J mice, a few CD14+ monocytic cells adhering to PCV of Peyer’s patches were seen (20.0±4.7/mm2). The number of adhered CD14+ monocytic cells in SAMP1/Yit mice (37.8±3.7/mm2) was significantly (P<0.05) larger than that in AKR/J at 60 min. Significant increases in CD14+ monocytic cell adherence in submucosal venules and villus microvessels of the ileum were also observed in SAMP1/Yit mice compared with those in AKR/J mice (submucosal vessels, 9.4±1.9/mm2 and 15.8±3.4/mm2, respectively; villus microvessels, 15.4±3.1/mm2 and 9.2±2.0/mm2, respectively).



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  		Figure 4. Representative photos of in vivo observation of CD14+ monocytic cell adhesion in the intestinal mucosa of SAMP1/Yit mice at 60 min by using an intravital microscope. The increased number of adhered CD14+ monocytic cells in SAMP1/Yit mice was significantly inhibited by anti-PSGL-1 mAb in PCV of Peyer’s patches, submucosal venules, or villus capillaries.

We next examined the effects of anti-PSGL-1 mAb, anti-P-selectin mAb, and anti-VCAM-1 mAb on CD14+ monocytic cell adherence in PCV of Peyer’s patches, submucosal venules, and mucosal villus microvessels after infusion of CFSE-labeled CD14+ monocytic cells (Figs. 4 and 5 ). These three mAb significantly inhibited adhesion of CD14+ monocytic cells during the observation periods compared with the effect of control IgG treatment. Among the mAb, anti-PSGL-1 markedly reduced the number of adherent CD14+ monocytic cells in PCV of Peyer’s patches compared with the effect of the other antibodies (P<0.01), and anti-VCAM-1 showed a significantly weaker, inhibitory effect compared with the inhibitory effect of the other mAb in the submucosal venules (P<0.05).



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  		Figure 5. Time courses of adhesion of CD14+ monocytic cells in the intestinal mucosa in PCV of Peyer’s patches (a), in submucosal venules (b), and in villus microvessels (c). Effects of anti-PSGL-1, anti-P-selectin, and VCAM-1 mAb on adhesion in SAMP/Yit mice. *, P < 0.05, versus AKR/J; #, P < 0.05, versus SAMP1/Yit; {dagger}, P < 0.05, versus anti-VCAM-1; , P < 0.05, versus anti-P-selectin; n = 5 in each group.

T lymphocyte adherence and the effects of antibodies against PSGL-1 in the ileal mucosa of AKR/J and SAMP1/Yit mice
To evaluate the effects of anti-PSGL-1 antibody on T cell adherence, we performed a blocking study. In AKR/J mice, a few T lymphocytes adhering to PCV of Peyer’s patches were seen (3.5±1.0/mm2). The number of adhered T lymphocytes in SAMP1/Yit mice (11.4±2.7/mm2) was significantly (P<0.05) larger than that in AKR/J at 60 min. Significant increases in T cell adherence in Peyer’s patches, submucosal venules, and villus microvessels of the ileum were also observed in SAMP1/Yit mice compared with those in AKR/J mice; however, the inhibitory effects of anti-PSGL-1 at 60 min were only seen in villus microvessels (P<0.01; Fig. 6 ).



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  		Figure 6. The inhibitory effects of anti-PSGL-1 antibody on T cell adherence in microvessels of intestinal mucosa. Isolated T cells from AKR/J mice spleen were fluorescent-labeled and injected via tail vein. The inhibitory effects of anti-PSGL-1 antibody on T cell adherence in PCV of Peyer’s patches (PP), in submucosal venules (SV), and in villus microvessels (VM) were evaluated at 60 min after T cell administration. *, P < 0.05, versus SAMP1/Yit; n = 5 in each group.

Effect of periodical administration of anti-PSGL-1 on ileitis
Monocyte rolling and adherence in the intestinal mucosa of SAMP1/Yit mice were significantly increased compared with that in AKR/J mice and were significantly attenuated by anti-PSGL-1 mAb, anti-P-selectin mAb, and anti-VCAM-1 mAb. Among the three antibodies, anti-PSGL-1 showed the strongest inhibitory effect on monocyte rolling and adherence. We therefore examined whether periodical administration of anti-PSGL-1 mAb ameliorates ileitis of SAMP1/Yit mice. As shown in Figure 7 , characteristic features of SAMP1/Yit mice at 21 weeks included submucosal thickening with decreased villus height and significant infiltration of CD4-, CD8-, and CD68-positive cells in the ileal mucosa compared with that in control AKR/J mice. Anti-PSGL-1 mAb treatment for 7 weeks significantly suppressed villus atrophy and infiltration of CD4-, CD8-, and CD68-positive cells in the ileal mucosa compared with those in mice treated with the control IgG. Although infiltration of cells in the ileal mucosa was attenuated to the levels of AKR/J mice, submucosal thickening was not remarkably attenuated by anti-PSGL-1 mAb treatment.



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  		Figure 7. (A) Effects of the administration of anti-PSGL-1 mAb on chronic inflammation (hematoxylin and eosin staining). (B) Effects of the administration of anti-PSGL-1 mAb on villus height and (C) submucosal thickness. *, P < 0.05, versus AKR/J; #, P < 0.05, versus IgG. (D) Number of CD4-positive cells in the intestinal mucosa. *, P < 0.05, versus AKR/J; #, P < 0.05, versus IgG. (E) Number of CD8-positive cells in the intestinal mucosa. *, P < 0.05, versus AKR/J; #, P < 0.05, versus IgG. (F) Number of CD68-positive cells in the intestinal mucosa: effects of anti-PSGL-1 mAb. *, P < 0.05, versus AKR/J; #, P < 0.05, versus IgG; n = 4 in each group.

Blocking effects of 7-week administration of anti-PSGL-1 antibody on CD14+ monocytic cell adherence
To assess whether anti-PSGL-1 antibody treatment still maintains its effective blocking function after long-term administration, we performed an in vivo observation of CD14+ monocytic cell adherence to intestinal microvessels after a 7-week periodical administration of this antidody. As shown in Figure 8 , there was a significant decrease in monocytic cell adherence to PCV of Payer’s patches, submucosal venules, or microvessels in the villi after a 7-week periodical administration of anti-PSGL-1 antibody compared with control antibody, suggesting that the blocking ability of anti-PSGL-1 antibody was well maintained at least for 7 weeks.



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  		Figure 8. Blocking effects of a 7-week administration of anti-PSGL-1 antibody on CD14+ monocytic cell adherence. To assess whether anti-PSGL-1 antibody treatment still maintains its effective blocking function after long-term administration, we performed in vivo observation of CD14+ monocytic cell adherence to intestinal microvessels after a 7-week periodical administration of this antidody. PP, PCV of Peyer’s patches; SV, submucosal venules; VM, villus microvessels. *, P < 0.05, versus SAMP1/Yit + IgG; n = 5 in each group.


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DISCUSSION
 
In the present study, we demonstrated that the expression levels of PSGL-1, P-selectin, and VCAM-1 were increased in SAMP1/Yit mice with spontaneous ileitis and that antibodies against PSGL-1, P-selectin, and VCAM-1 significantly attenuated monocyte rolling and adherence in microvessels of the ileal mucosa. Moreover, it should be noted that periodical administration of anti-PSGL-1 ameliorated ileitis of SAMP1/Yit mice. It has been reported that a part of the pathogenesis of CD may be abnormal infiltration of monocytes/macrophages and the immune responses of these cells to luminal nutritional or bacterial antigens. Our findings showed over-infiltration of not only lymphocytes but also CD14+ monocytic cells/macrophages in ileitis in accordance with previously reported results and indicated that the monocytic cells-endothelial cell interactions may play an important role in the development of ileitis [2 , 3 ]. Although there have been several studies on lymphocyte migration in vivo and on interaction of lymphocytes with endothelial cells of venules in the intestinal mucosa [16 , 19 20 21 ], only a few reports have described direct observations of trafficking of monocytic cells/macrophages in the murine small intestinal mucosa and its role in the development of intestinal inflammation [22 ].

Therefore, in this study, we examined the precise interaction of CD14+ monocytic cells, i.e., CD14+ cells, and endothelial cells in the ileum of SAMP1/Yit mice, a model of CD [13 ]. We found that the numbers of rolling and adherent CD14+ monocytic cells in PCV of Peyer’s patches, submucosal venules, and mucosal villus microvessels of the small intestine were increased in SAMP1/Yit mice compared with those in control AKR/J mice. Moreover, an immunohistochemical study using CD68 showed an increase in the number of macrophages as a result of increased accumulation of CD14+ monocytic cells in the lamina propria in the ileum of SAMP1/Yit mice compared with that in control mice. Macrophages identified in IBD have been shown to express an activated phenotype (CD14-positive), which is not normally expressed in the normal intestine and contributes to the inflammatory cell-mediated damage in IBD [5 ]. Conversely, it has been suggested that the expression of chemokines MCP-1, -2, and -3 and macrophage inflammatory protein-1{alpha} and -1ß is up-regulated in IBD [12 , 23 ]. MCP-1 has been shown to exhibit a higher expression level than the level of other chemokines [12 ], and its expression was also found to be up-regulated in the ileal mucosa of SAMP1/Yit mice in our preliminary study (data not shown). Therefore, monocyte accumulation, increased level of tissue macrophages, and chemokine MCP-1 may play important roles in mucosal inflammation in SAMP1/Yit mice.

Only a few studies have shown roles of adhesion molecules in monocytic cells and their corresponding intestinal endothelial ligands in vivo. Using functional blocking mAb and in vitro frozen section adhesion assays, Salmi and Jalkanen [24 ] found that PSGL-1-P-selectin interaction predominantly mediates synovial adherence of macrophages from IBD gut compared with other multiple homing receptors and their corresponding endothelial ligands (CD11a/CD18– ICAM-1, {alpha}4ß7/{alpha}4ß1 integrin–VCAM-1, L-selectin–PNAd, and CD44). PSGL-1 is an extended homodimeric sialomucin and has a clearly documented function in mediating selectin-dependent cell adhesion under flow conditions [25 ]. In the present study, although pretreatments with anti-PSGL-1 mAb, anti-P-selectin mAb, and anti-VCAM-1 mAb resulted in significant suppression of monocyte adhesion to each site during the observation period, anti-PSGL-1 mAb showed the strongest inhibitory effect, consistent with the findings by Salmi and Jalkanen [24 ]. However, anti-VCAM-1 mAb showed a significantly weak effect compared with the effect of other mAbs. Yago et al. [26 ] examined the adhesive interaction of peripheral blood monocytes with VCAM-1 and analyzed the effect of P-selectin binding to monocytes on the adhesive interaction with VCAM-1 under flow conditions. It is interesting that they found that P-selectin binding to monocytes enhances the adhesive interaction of monocytes with VCAM-1 [26 ]. They suggested that PSGL-1-P-selectin interaction and {alpha}4 integrin-VCAM-1 interaction were able to act sequentially in the adhesion cascade that regulates trafficking of monocytes to an inflammatory lesion. These mechanisms may explain our finding that blockade of PSGL-1 and P-selectin attenuates ileitis stronger than blockade of VCAM-1.

A chemical irritant-induced colitis model has shown that blockade of ICAM-1 or VCAM-1 can dramtically reduce the leukocyte adherence and disease activity index in the inflamed colon [27 28 29 30 ]. Yoshida et al. [31 ] demonstrated that administration of an anti-P-selectin mAb or an anti-ICAM-1 mAb was effective against trinitrobenzenesulfonic acid-induced colitis in rats, although they investigated acute, inflamed colitis mainly associated with neutrophil-endothelial cell interaction. In SAMP1/Yit mice, however, Burns et al. [32 ] demonstrated that blockade of ICAM-1, VCAM-1, or {alpha}4 integrins for three days had no significant beneficial effects and that blockade of ICAM-1 and {alpha}4 integrins or blockade of ICAM-1 and VCAM-1 resulted in a 70% reduction of active inflammation but not chronic inflammation. Therefore, we selected anti-PSGL-1 mAb and investigated whether this mAb ameliorates ileitis as chronic inflammation in this model. Our results showed that periodical administration of anti-PSGL-1 mAb resulted in attenuation of the spontaneously occurring mucosal inflammatory changes. This may be partly a result of the inhibition of monocyte recruitment, as the increase in CD68 cells was significantly blocked by anti-PSGL-1 treatment. However, it should be noted that PSGL-1 is expressed not only in CD14+ monocytic cells but also in lymphocytes and neutrophils and mediates adhesion of these cells to the endothelium through PSGL-1-E-selectin or PSGL-1-L-selectin interaction [33 34 35 36 ]. Therefore, anti-PSGL-1 antibody may inhibit these interactions between CD14+ monocytic-endothelial cells and lymphocytes-endothelial cells, although in the present results, anti-PSGL-1 antibody significantly inhibited T cell adhesion only in the villus microvessels. Recently, Haddad et al. [37 ] demonstrated that P-selectin and PSGL-1 are major determinants for T helper cell type 1 (Th1) CD4+ cell recruitment to nonlymphoid effector sites in the intestinal lamina propria and that T cell activation in the presence of interleukin-12 would promote Th1 recruitment to these sites through selective expression of functional PSGL-1. We also found significant decreases in the numbers of CD4 and CD8 cells in the lamina propria of the ileal mucosa after anti-PSGL-1 treatment. Taken together, the results suggest that not only inhibition of monocyte recruitment but also that of lymphocytes is largely involved in the attenuation of inflammatory, histological damage by anti-PSGL-1 in SAMP1/Yit mice. Conversely, the reason why anti-PSGL-1 did not significantly reduce the submucosal thickness in the ileum of SAMP1/Yit mice is not known. We speculate that cells that do not express PSGL-1, such as fibroblasts, also play an important role in the histological changes in the intestinal mucosa.

The present study provided in vivo evidence of interaction of CD14+ monocytic cells with microvessels in lymphoid (Peyer’s patches) and nonlymphoid (villus) regions of the small intestine of SAMP1/Yit mice. This study also demonstrated for the first time that anti-PSGL-1 mAb treatment ameliorates intestinal inflammation in SAMP1/Yit mice. It has been reported that administration of an anti-{alpha}4 integrin mAb ameliorates CD in clinical trials [38 ]. In addition to this therapy, targeting PSGL-1 and P-selectin may be other effective options for the treatment of IBD.


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ACKNOWLEDGEMENTS
 
T. I. and Y. T. contributed equally to this work.

Received February 23, 2004; revised September 27, 2004; accepted October 26, 2004.


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