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. 2004 Feb;135(2):226–232. doi: 10.1111/j.1365-2249.2003.02369.x

Endotoxin stimulates monocyte–endothelial cell interactions in mouse intestinal Peyer's patches and villus mucosa

N ISHII 1, Y TSUZUKI 1, K MATSUZAKI 1, J MIYAZAKI 1, Y OKADA 1, R HOKARI 1, A KAWAGUCHI 1, S NAGAO 1, K ITOH 1, S MIURA 1
PMCID: PMC1808937  PMID: 14738449

Abstract

Although monocyte–endothelial cell interactions represent an initial step in controlling the recruitment of monocytes in inflamed tissues, their dynamic processes in microvessels of lymphoid (Peyer's patches) and non-lymphoid (villus) regions in gut-associated lymphoid tissue remain poorly understood. We monitored the migration of fluorescence-labelled monocytes derived from the spleen in intestinal microvessels with or without lipopolysaccharide (LPS) treatment and investigated the role of adhesion molecules, P-selectin, vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In control mice, there were few interactions between infused monocytes and the endothelium of intestinal microvessels. The monocyte–endothelial interactions (both rolling and adhesion) were significantly increased in intestinal microvessels of LPS-treated mice compared with those in controls. Anti-P-selectin monoclonal antibody (MoAb) significantly suppressed the LPS-induced increase in monocyte rolling in postcapillary venules of Peyer's patches and submucosal venules. Anti-VCAM-1 MoAbs significantly suppressed the LPS-induced increase in monocyte adhesion to postcapillary venules (PCVs) of Peyer's patches, submucosal venules, and villus capillaries. In contrast, anti-ICAM-1 MoAb significantly suppressed the number of adherent monocytes in PCV of Peyer's patches but not in submucosal venules or villus capillaries. These observations demonstrated that LPS treatment resulted in a significant increase in recruitment of monocytes both in microvessels of lymphoid and non-lymphoid regions and that P-selectin, VCAM-1 and ICAM-1 appeared to play important roles in LPS-induced interactions.

Keywords: ICAM-1, LPS, monocyte, P-selectin, VCAM-1

INTRODUCTION

Monocytes/macrophages infiltrate most inflammatory tissues and are crucial contributors to inflammatory reactions because of their ability to secrete cytokines, present antigens, phagocytize foreign particles and release proteolytic enzymes and oxygen radicals [1,2]. Adhesion of monocytes to the vascular endothelium and subsequent diapedesis are important events that occur during chronic inflammation, immune-mediated reactions and atherosclerosis [3–5]. Several monocyte–endothelial adhesion pathways have been described. These pathways include adhesion molecules such as l-selectin-peripheral node addressin (PNAd), β2 integrin-intercellular adhesion molecule-1 (ICAM-1), α4 integrin-vascular cell adhesion molecule (VCAM-1), sialyl LewisX-E-selectin and P-selectin glycoprotein-1 (PSGL-1)-P-selectin [4,6,7]. Monocyte migration into tissues is regulated by at least two mechanisms. One of these is the production of chemotactic factors by inflamed tissue such as interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1) and members of the GRO family of chemoattractants [8–10]. The other mechanism involves the activation of vascular endothelial cells by cytokines such as IL-1, TNF-α, or IFN-γ or by the bacterial product endotoxin [lipopolysaccharide (LPS)], leading to the expression of several proteins on the endothelial surface that facilitate adhesion and migration of monocytes [11–13]. In vitro studies have demonstrated that monocytes migrated in response to chemotactic factors across both resting and IL-1-activated endothelium and utilized adhesion molecules for migration [14,15]. Monocyte–adhesive interactions with TNF-α-activated human umbilical vein endothelial cell (HUVEC) monolayers have been examined using an in vitro flow model [16]. A model of atherogenesis showed that the aortic luminal endothelium of cholesterol-fed rabbits displayed markedly enhanced adhesiveness for monocytes in an ex vivo assay [17]. In synovial tissues from patients with rheumatoid arthritis, attachment of blood monocytes to high endothelial venules was observed under rotating conditions [18].

Intestinal mucosal monocytes play pivotal roles in specific immunological protection against enteric pathogens [19] and in chronic inflammatory changes such as Crohn's disease, and it has also been reported that the vast majority of mononuclear cells infiltrating the inflamed mucosa and submucosa results from a sustained active recruitment of circulatory cells [20,21]. However, it is not clear how monocytes actually migrate to the inflamed intestinal mucosa from systemic circulation, especially in vivo. Moreover, adhesion receptors and regulatory pathways that are important for monocyte interaction with intestinal microvessels and subsequent migration into gut-associated lymphoid tissue (GALT) remain to be identified. In this study, we visualized and analysed the nature and magnitude of monocyte migration into lymphoid (Peyer's patches) and nonlymphoid (villus) regions of intestinal mucosa using an intravital system [22]. In addition, we studied the influence of Gram-negative bacterial LPS on the recruitment of monocytes in different vessels, the contribution of P-selectin to monocyte rolling and the roles of VCAM-1 and ICAM-1 in monocyte-endothelial cell adhesion in inflamed intestinal microvessels.

MATERIALS AND METHODS

Isolation of monocytes and labelling with carboxyfluorescein diacetate succinimidyl ester

The mouse spleen was 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-buffered saline (PBS) and haemolysed with NH3Cl-Tris buffer. Thereafter, monocytes were isolated by magnetic cell sorting (MACS, Miltenvi Biotec, CA, USA) with rabbit antimouse CD14 polyclonal antibody (Santa Crus Biotec, CA, USA) and beads-conjugated antirabbit IgG (MACS, Cruz Biotec, CA, USA). The purity of monocytes was evaluated by a fluorescence-activated cell sorter (FACS-440, Becton-Dickinson, Mountain View, CA, USA) using rat antimouse IgG F4/80 MoAb (Cosmo Bio, Tokyo, Japan).

Carboxyfluorescein diacetate succinimidyl ester (CFDSE: Molecular Probes, Eugene, OR, USA) was dissolved in dimethylsulphoxide at 15·6 mm, divided into small aliquots (each 300 µl), and stored in a cuvette sealed with argon gas at -20°C until use in the experiments. Monocytes (2 × 106) in 1 ml of PBS were incubated with CFDSE solution for 30 min at 37°C and washed twice by PBS.

Animal preparation for intravital observation

Male balb/c mice, weighing 20 g, were maintained on standard laboratory chow (Oriental Yeast, Tokyo, Japan). The care and use of laboratory animals were in accordance with the guidelines of the animal facility in National Defence Medical College. For migration studies, mice were anaesthetized 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 selected 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 to prevent the damage to microcirculation, and PBS was injected into the closed segment using a 30-gauge needle. The behaviour of monocytes in postcapillary venules (PCV) of Peyer's patches and submusal venules was observed from the serosal side using an intravital microscope. In the same animals, the migration in mucosal villus capillaries was also observed from the mucosal surface after opening the intestinal lumen along its antimesenteric side. The behaviour of fluorescence-labelled monocytes in PCV of Peyer's patches, submusal venules and mucosal villus capillaries of the small intestine was visualized on a monitor through a silicon-intensified target image tube (SIT) system, using a previously described method [22], and recorded on digital videotape with a high-speed video recording system (WV-DR7, Sony, Tokyo, Japan). Microcirculation was observed by a fluorescence microscope (BX51WI, Olympus, Tokyo, Japan) equipped with a silicon-intensified target image tube (SIT) camera with a contrast-enhancing unit (C-2400–08, Hamamatsu Photonics, Shizuoka, Japan) and ×10 or ×20 ultraviolet-fluorite objective lens (Fluor, Nikon, Tokyo, Japan).

Preparation of LPS-treated mice

LPS from Escherichia coli O-111 B4 (Sigma Chemical, MO, USA) was dissolved in PBS at a concentration of 1 mg/ml and 0·5 mg of LPS was administered intraperitoneally into host mice 3 h before carboxyfluorescein succinimidyl ester (CFSE) labelled monocytes were injected via a tail vein.

Administration of monoclonal antibodies

In some experiments, 2 mg/kg of rat antimouse P-selectin (RB40·34; BD Pharmingen, San Diego, CA, USA), rat antimouse VCAM-1 (429 MVCAM.A; BD Pharmingen) and/or rat antimouse ICAM-1 MoAbs (3E2; BD Pharmingen) were administered via the tail vein 30 min prior to CFSE-labelled monocyte administration, and their effects were compared with those of isotype-matched control rat IgG1 or IgG2a (BD Pharmingen).

Analysis of monocyte dynamics

The number of adherent monocytes was determined off-line by digital videotaped images. Monocytes adhering to vascular walls with occasional movements were defined as rolling monocytes. We counted the numbers of total influx and rolling monocytes using a ×20 objective lens over a period of 1 min at 10 and 20 min after the injection, and the results are expressed as the ratio of number of rolling monocytes/total monocytes in the observation field. Monocytes adhering to vascular walls without movement and remaining stationary for a period of more than 30 s were defined as adherent monocytes. We counted the numbers of adherent monocytes in the field using a × 20 objective lens at 10-min intervals up to 60 min after monocyte administration.

Immunohistochemistry for VCAM-1 in intestinal mucosa

The expression of VCAM-1 was assessed by immunohistochemistry using the labelled streptavidin biotin method. Small intestines were isolated and fixed in periodate, lysine-paraformaldehyde solution, and tissues were embedded in an optimum cutting temperature compound (Miles Inc., Elkhart, IN, USA) before being frozen in dry ice and acetone. Cryostat sections, 7 µm in thickness, were transferred to poly l-lysine (PLL)-coated slides and air-dried for 1 h at 20°C. After they had been washed for 5 min in phosphate-buffered saline (PBS; pH 7·4) containing 1% Triton X, the sections were incubated in 5% normal goat serum in PBS. Monoclonal antibody against mouse VCAM-1 (429 MVCAM.A; BD PharMingen) was diluted 50 times in PBS and layered on the sections overnight at 4°C. After treatment with anti-VCAM-1 MoAb, the sections were incubated with a secondary antibody, a biotinylated antirat IgG-class antibody (Amersham International plc, Buckinghamshire, UK), for 1 h at room temperature. The sections that had been treated with the secondary antibody were incubated with fluorescein isothiocyanate (FITC)-conjugated streptavidin (Amersham International plc) for 30 min at room temperature. After each step, the sections were rinsed in PBS containing 1% bovine serum albumin. A cover slip was applied using glycerol jelly, and the sections were observed under a fluorescence microscope (BX60; Olympus, Tokyo, Japan).

Statistics

All results are expressed as means ± SEs of 5–8 mice. For comparison of rolling and adhesion, the mean values were statistically evaluated by a non-parametric Mann–Whitney U-test. Statistical significance was defined as P < 0·05.

RESULTS

Effects of LPS on monocyte rolling and adhesion

FACS analysis demonstrated that 89·6% of the monocyte population we obtained as CD14+ cells using MACS expressed F4/80 [23], one of the specific markers for mouse monocytes (data not shown). After the infusion of fluorescence-labelled cells, some monocytes showed a characteristic rolling behaviour on the surface of the endothelial wall in the PCVs of Peyer's patches, although the percentage of rolling cells during the period from 10 to 20 min under the control condition was small (about 16%). In submucosal venules of the villus mucosa, there was also a rolling behaviour in some (about 17%) of the monocytes under the control condition (Table 1). In contrast to these sites, there was no obvious rolling behaviour in the monocyte fraction in mucosal microvessels of the intestinal villus. On the other hand, LPS treatment significantly increased the percentage of rolling monocytes in PCVs of Peyer's patches from 16 ± 8% to 44 ± 7% (Table 1). Similarly, the percentage of rolling monocytes was increased by LPS treatment in submucosal venules from 17 ± 5% to 31 ± 7%. The total number of monocytes that had entered PCVs of Peyer's patches or submucosal venules between 10 and 20 min of being infused into the tail vein did not differ between control and LPS-treated animals.

Table 1.

Effect of LPS treatment on the percentage of rolling monocytes in PCV of Peyer's patches and submucosal venules of ileal mucosa after infusion of monocytes; effects of antiadhesion molecules

PCV of Peyer’s patches (%) Submucosal venules (%)
Control 16 ± 8 17 ± 5
Control + Anti-VCAM-1 17 ± 4 18 ± 3
LPS 44 ± 7* 31 ± 7*
LPS + Anti-VCAM-1 34 ± 9 n.s. 21 ± 8 n.s.
LPS + Anti-ICAM-1 30 ± 7 n.s. 22 ± 5 n.s.
LPS + Anti-VCAM-1 + anti-ICAM-1 28 ± 9 n.s. 23 ± 9 n.s.
LPS + anti-P-selectin 10 ± 1† 11 ± 2†
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Rolling monocytes were counted between 10 and 20 min after infusion of CFSE-labelled monocytes and expressed the percentages of total number of appearing monocytes. Values are means ± SE of 5–8 animal experiments.

*

P < 0·05 versus control.

†

P < 0·05 versus LPS. n.s.: not significant against LPS.

Some infused monocytes adhered to the endothelial walls of PCVs of Peyer's patches after rolling. Figure 1a,b shows photomicrographs of adherent monocytes in Peyer's patches at about 50 min after the infusion of monocytes. Figure 2a shows the time-course of the number of adherent cells to PCVs of Peyer's patches after the administration of monocytes in control and LPS-treatment conditions (expressed as number of adherent cells in a 1 mm2 field via a ×20 objective lens). In control animals, only a few monocytes (1–3 per field) were observed in the field during the observation period (Figs 1a, 2a). However, LPS treatment induced a significant increase in the number of monocytes that adhered selectively to PCVs in Peyer's patches (Fig. 1b). Under the LPS-treatment conditions, the number of adherent monocytes increased in a time-dependent manner within 30 min, and this increase reached a plateau at around 40 min (Fig. 2a).

Fig. 1.

Fig. 1

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Photographs of adherence of CFSE-labelled monocytes in mouse ileal Peyer's patches (a) and (b) and submucosal venules (c) and (d) observed from the serosal side at about 50 min after infusion of CFSE-labelled monocytes under a fluorescent microscope. (a,c) Control mice; (b,d) LPS-treated mice. The number of monocytes that adhered to the venular endothelium was increased significantly in both Peyer's patches (b) and submucosal venules (d). Bar: 100 µm (objective lens ×10).

Fig. 2.

Fig. 2

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Effects of LPS treatment on time-courses of adherence of monocytes to microvessels in three different regions, PCVs of Peyer's patches (a), submucosal venules (b) and villus microvessels of ileal mucosa (c). Monocytes located inside microvesels were counted in a 1-mm2 observation field. (□), control mice; (▴), LPS-treated mice. Values are means ± SE of five animal experiments. *P < 0·05 compared with values of control mice.

Figure 1c,d shows the photomicrographs of CFSE-labelled monocytes in submucosal venules observed from the serosal side at about 50 min after monocyte administration under a fluorescent microscope. In the control condition, there were few monocytes that adhered to the submucosal venules during the observation period (Fig. 1c). On the other hand, LPS treatment induced a significant increase in the number of adherent monocytes in submucosal venules after rolling (Fig. 1d). In the LPS-treated group, the number was remarkably increased within 10 min after the infusion of monocytes, and this increase reached a plateau at about 20 min (Fig. 2b).

Figure 2c shows the time-course of the number of monocytes adhered to villus microvessels of the ileal mucosa in control and LPS-treatment conditions. In the control conditions, only a small number of CFSE-labelled monocytes adhered to the mucosal microvessels of the ileal mucosa (2–3/mm2 field) during the observation period when observed from the mucosal side under a fluorescent microscope. In the LPS-treatment conditions, the number of adherent monocytes gradually and significantly increased in villus microvessels. However, the rate of increase (140%) was smaller than that in Peyer's patches (310%; P < 0·05) or submucosa (680%; P < 0·01) at 60 min.

Effects of pretreatment with anti-adhesion molecules on LPS-induced monocyte interactions

The effects of pretreatment with anti-P-selectin MoAb, anti-VCAM-1 MoAb, anti-ICAM-1 MoAb and a combination of anti-VCAM-1 MoAb and anti-ICAM-1 MoAb on monocyte rolling in LPS-treated animals were determined in PCVs of Peyer's patches and submucosal venules in the early phase (between 10 and 20 min) after infusion of CFSE-labelled cells. The effects of anti-VCAM-1 on monocyte rolling in control animals were also examined (Table 1). The percentage of monocyte rolling was not affected in control animals by anti-VCAM-1 in either Peyer's patches or submucosal venules, and the percentage of rolling monocytes in LPS-treated animals was only slightly reduced by treatment with anti-VCAM-1 MoAb, anti-ICAM-1 MoAb or a combination of these two MoAbs in either PCVs of Peyer's patches or submucosal venules. In contrast, anti-P-selectin attenuated LPS-induced monocyte rolling from 44 ± 7% to 10 ± 1%, below the control level in PCVs of Peyer's patches. Anti-P-selectin also attenuated the LPS-induced monocyte rolling from 31 ± 7% to 11 ± 2% in submucosal venules.

Figure 3 shows the effects of pretreatment of anti-VCAM-1 MoAb, anti-ICAM-1 MoAb or a combination of two MoAbs on LPS-induced monocyte adherence to PCVs of Peyer's patches, submucosal venules and microvessels of the villus mucosa at 60 min after monocyte infusion. All results are expressed as percentage of that in LPS-treated animals. In PCVs of Peyer's patches, pretreatment with anti-VCAM-1 MoAb and anti-ICAM-1 MoAb significantly decreased the number of adhered monocytes and the combination of two MoAbs remarkably attenuated LPS-induced adherence. In contrast to the effect in Peyer's patches, pretreatment with anti-ICAM-1 MoAb did not suppress the number of adherent monocytes in submucosal venules, although anti-VCAM-1 MoAb or the combination of two MoAbs significantly attenuated the adherence during the observation period. Similar to the effect in the submucosal venules, pretreatment with anti-ICAM-1 MoAb did not decrease the number of adherent monocytes in the villus mucosa, whereas pretreatment with anti-VCAM-1 MoAb or the combination of two MoAbs constantly reduced the number of adherent cells during the observation period (to 68·6% and 57·6% of those in LPS-treated animals at 60 min, respectively). However, these rates of reduction in number of adherent cells induced by pretreatment with anti-VCAM-1 MoAb and the combination of two MoAbs in the villus mucosa (anti-VCAM-1, 31 ± 6%; combination, 42 ± 5%) were significantly smaller than those in the submucosal venules (anti-VCAM-1, 51 ± 4%; combination, 75 ± 5%; both P < 0·05).

Fig. 3.

Fig. 3

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Effects of pretreatment with anti-VCAM-1 MoAb, anti-ICAM-1 MoAb or a combination of two MoAbs on LPS-induced adherence of monocytes to PCVs of Peyer's patches, submucosal venules and microvessels of villus mucosa at 60 min after infusion of CFSE-labelled monocytes. All results are expressed as percentages of those in LPS-treated mice. Values are means ± SE of five animal experiments. *P < 0·05 compared with values of LPS-treated mice without MoAb.

Effects of LPS on expression of VCAM-1 in intestinal mucosa

As shown in Fig. 4, the degree of up-regulation of VCAM-1 by LPS was larger in submucosal microvessels than in villus mucosa. This may account for the greater inhibition of monocyte adhesion in submucosal microvessels (approximately 55%) than in villus mucosa (approximately 30%).

Fig. 4.

Fig. 4

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Effects of LPS on the expression of VCAM-1 in intestinal mucosa. The method used for immunohistochemistory is described in Materials and methods. (a) VCAM-1 expression in ileal mucosa of control mice. (b) VCAM-1 expression in ileal mucosa of LPS-treated mice. (objective lens ×20).

DISCUSSION

This study has provided in vivo evidence of monocyte interactions with microvessels in lymphoid (Peyer's patches) and non-lymphoid (villus) regions of the small intestine. This study has also shown the influence of LPS on monocyte–endothelial cell interactions in different microvessels and the contribution of adhesion molecules, P-selectin, VCAM-1 and ICAM-1 to the recruitment of monocytes in inflamed microvessels of the gut.

There have been several studies that showed lymphocyte migration in vivo and in which the interactions of lymphocytes with endothelial cells of venules in lymphoid (Peyer's patches) and non-lymphoid (villus) regions of the intestinal mucosa were compared [18,23]. T lymphocytes adhere characteristically to the high endothelial venules (HEV) in Peyer's patches, which sustain intense lymphocyte traffic under physiological conditions [23,24], and the density of adherent T lymphocytes in HEV was shown to be more prominent than that in submucosal venules and villus capillaries. In contrast, our study demonstrated that there were only a few interactions between monocytes and the endothelium of intestinal microvessels under physiological conditions. Moreover, unlike T lymphocyte migration, there was no apparent heterogeneity in monocyte adherence among PCVs of Peyer's patches, submucosal venules and mucosal microvessels of the small intestine under normal conditions.

Stamper-Woodruff assays showed that lymphocytes adhered to HEV in frozen sections of the mouse lymph node but that there was little monocyte adhesion [25,26]. This finding of little monocyte adhesion is intriguing, as monocytes express constitutively l-selectin and β2-integrin receptors for PNAd and ICAM-1 and -2 ligands that are expressed constitutively by HEV, similar to lymphocytes. Thus, although l-selectin and β2-integrin are available to support monocyte–HEV interactions, these molecules alone are not sufficient for monocyte binding, resulting in only a few monocytes adhering to PCVs under control conditions. Alternatively, cytokines or chemokines are essential for the firm adhesion of monocytes, such as in an inflammatory condition [27–29]. Indeed, inflammatory stimuli induced monocyte recruitment via HEV in complete Freund's adjuvant-inflamed lymph nodes in vivo and in the Stamper-Woodruff frozen section assays ex vivo, in accordance with our results [26,27,30].

In the present study, the number of adherent monocytes in PCVs of Peyer's patches, submucosal venules and mucosal villus capillaries of the small intestine was increased by LPS treatment compared with that in the control condition during the observation period. However, the magnitude of increase was most prominent in submucosal venules, followed by PCVs of Peyer's patches, while the magnitude of increase in villus capillaries was smallest. These results are consistent with results of studies on chronic inflammatory changes in Crohn's disease showing that focal perivascular infiltrates mainly made up of lymphocytes with monocytes and/or macrophages were located predominantly in the submucosa [20,21]. In Crohn's disease, dilated and highly activated small vessels were observed in the submucosa containing mononuclear cells in the lumen, either close to or adherent to endothelial cells. The increase in adhesion of monocytes to endothelial cells in an inflamed condition could be due to induction of a monocyte-specific activator or chemoattractant, a monocyte-specific adhesion molecule or both [8–13,31] in all sites of the intestine. It has been reported that activation of endothelial cells by agents such as TNF-α, IL-1β or LPS increases the level of ICAM-1 and VCAM-1 expression on endothelial cells [32,33], in accordance with our results. Therefore, these molecules could be candidates for the increase in monocyte adhesion in vivo.

In the present study, pretreatment with anti-VCAM-1 MoAb reduced significantly the number of monocytes that adhered to each site. Pretreatment with anti-ICAM-1 MoAb also suppressed significantly this number in PCVs of Peyer's patches, but not in the submucosal venules and villus microvessels. These results suggest that there is a significant spatial difference in adhesion molecule usage between lymphoid (Peyer's patches) and non-lymphoid (submucosal venules and villus mucosa) regions under inflammatory conditions; namely, the latter appears to be mainly dependent on VCAM-1. It has been reported that the magnitude of up-regulation of VCAM-1 was greater than that of ICAM-1 in the mouse small intestine following systemic administration of LPS [32]. This finding of LPS-induced preferential up-regulation of VCAM-1 is in accordance with our results. Our histological observation that VCAM-1 was mainly up-regulated in the submucosal venules may explain the dominant dependency of monocyte adherence on VCAM-1 in this area. On the other hand, a combination of two MoAbs could suppress the increase in number of adherent monocytes induced by LPS to a number between those in control and LPS treatment conditions at any sites. Although other undefined chemotactic factors or adhesion molecules other than VCAM-1 and ICAM-1 can affect monocyte interactions, our results suggest that VCAM-1 and ICAM-1-dependent processes may be the main mechanisms responsible for the accumulation of monocytes in inflamed intestinal microvessels.

In the present study, LPS treatment increased the percentage of rolling monocytes in intestinal venules. However, the LPS-induced rolling was reduced only slightly by either anti-VCAM-1 or anti-ICAM-1MoAb in both sites. The reason for this may be that monocytes initially attach to activated endothelium via P-selectin and l-selectin-dependent mechanisms, with a smaller contribution by CD49d [34–36]. Once attached initially, subsequent slow rolling was mediated via an overlapping adhesion cascade with CD49d or β2 integrins through an activation-dependent event in inflammation. Issekutz investigated in vitro adhesion and in vivo migration of monocytes to cutaneous inflammation and integrin usage and reported that LFA-1, Mac-1 and VLA-4 are involved in this phenomenon [37]. However, l-selectin and P-selectin, but not VLA-4, are involved in initial attachment of monocytes to TNF-alpha-stimulated endothelium [34]. Taken together with our results, it appears that although CD49d or β2 integrins–VCAM-1 and –ICAM-1 interactions may not play important roles, at least in the initial attachment of monocytes to the endothelium, these molecules play important roles in the subsequent process such as firm adhesion to endothelium.

In this study, we demonstrated the characteristics and magnitude of monocyte migration into lymphoid (Peyer's patches) and non-lymphoid (villus) regions of the small intestine under both physiological and LPS-treatment conditions in vivo. Our results indicated that P-selectin appeared to play important roles in monocyte rolling on the vascular endothelium. Moreover, VCAM-1 and ICAM-1 appeared to play important roles in LPS-induced monocyte adhesion to the vascular endothelium under the condition of LPS stimulation with greater dominancy of VCAM-1.

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