Introduction

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Lymphocytes always recirculate from blood to lymphoid tissue to maintain immunological surveillance. Lymphocyte homing from blood to lymphoid tissue and inflammatory sites depends on the interaction between lymphocytes and high endothelial venules. This interaction is in multistep theory mediated by selectins, integrins, and Ig superfamily adhesion molecules (Butcher, 1991; Shimizu et al., 1992; Springer, 1994; Bargatze et al., 1995). The homing of lymphocytes appears to be organ specific, and lymphocyte homing to Peyer's patches and mucosal sites is thought to be regulated by 47 integrin and its counter ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (Berlin et al., 1993). MAdCAM-1 is an Ig superfamily adhesion molecule expressed on mucosal endothelium, and blocking monoclonal antibodies for 47 integrin and MAdCAM-1 have been reported to inhibit normal homing of 47-positive lymphocytes to the small intestine, Peyer's patches, and mesenteric lymph nodes (Hamann et al., 1994).

It is becoming increasingly apparent that inflammation of the intestine is associated with enhanced expression of adhesion molecules in both experimental animals and humans (Nakamura et al., 1993; Miura et al., 1996). In human ulcerative colitis and Crohn's disease, the expression of MAdCAM-1 is up-regulated in factor VIII-positive vessels in inflamed colonic mucosa (Briskin et al., 1997). It has been reported that the expression of MAdCAM-1 is increased in some animal models of colitis (Viney et al., 1996; McDonald et al., 1997; Picarella et al., 1997; Connor et al., 1999), in islet vessels of nonobese diabetic mice (Hanninen et al., 1993; Faveeuw et al., 1994), and in spinal cord vessels of experimental allergic encephalomyelitis (O'Neill et al., 1991). However, whether enhanced expression of MAdCAM-1 in inflamed intestinal mucosa plays a significant role in the development of inflammatory bowel diseases has not been clearly determined.

Oral administration of dextran sulfate sodium (DSS) induces acute and chronic colitis in mice (Okayasu et al., 1990; Cooper et al., 1993). The histology of this experimental colitis includes destruction of crypts and infiltration of inflammatory cells, including lymphocytes, macrophages, and granulocytes in the inflamed colon. This model resembles human ulcerative colitis, and has been widely investigated as a form of reproducible mucosal colonic inflammation in mice (Elson et al., 1995). However, whether MAdCAM-1 plays a role in the pathogenesis of DSS-induced colitis remains unknown. The aims of this study were 1) to evaluate the time course changes in expression of MAdCAM-1 and inflammatory cell infiltration in the colonic mucosa of DSS-induced colitis, and 2) to examine whether treatment with anti-MAdCAM-1 antibody has a prophylactic effect on the development of this model.

	Materials and Methods

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Induction of Colitis. Specific pathogen-free female BALB/c mice (18-20 g, 8 weeks old) were housed in wire-mesh-bottom cages in our animal laboratory center. The care and use of laboratory animals were in accordance with the National Institutes of Health guidelines. DSS (mol. wt. 5000; Wako, Osaka, Japan) was dissolved in drinking water to a final concentration of 5%. All mice were allowed free access to animal chow (CE-2; Nippon Clea Inc., Osaka, Japan) and water. Occult blood in the feces was evaluated by the orthotolidine and the guajac methods, and blood clot around the anus was considered as the gross blood per rectum.

Assessment of Histological Damage. Changes over time in colonic damage induced by DSS were determined. To estimate the formation of colitis, mice were sacrificed on days 0, 7, 14, and 21 after the start of induction of colitis. Under pentobarbital anesthesia, the colon was removed and opened longitudinally. The colon was divided into three segments (cecum, proximal and distal colon). Each segment was cut in half longitudinally and one of each segment was fixed in 10% buffered formalin. Tissues were embedded in paraffin and 4-µm longitudinal sections were stained with H&E. Histological damage score was measured by the method of crypt scoring by Cooper et al. (1993): grade 0, intact crypt; grade 1, loss of the basal one-third of the crypt; grade 2, loss of the basal two-thirds of the crypt; grade 3, loss of entire crypt with the surface epithelium remaining intact; and grade 4, loss of both the entire crypt and surface epithelium. Histological damage score was measured in each segment and averaged in proportion with the length of muscularis mucosa. The number of granulocytes in each segment was counted at high-power field (400×) and averaged in proportion with the length of muscularis mucosa.

Immunohistochemistry. Another part of removed colon was fixed for 12 h at 4°C in periodate-lysine-paraformaldehyde. Subsequently, these specimens were washed and dehydrated for 12 h with PBS containing 10, 15, and 20% sucrose. After fixation, they were embedded in Tissue-Tek O.C.T. compound (Sakura Fineteck Inc., Tokyo, Japan) and frozen in liquid nitrogen. Cryostat sections of frozen tissue were cut at 7 µm. Immunohistochemistry was performed by the labeled streptavidin biotin technique. Primary antibodies used in the immunostaining were monoclonal antibodies (mAbs) that react to MAdCAM-1 (MECA367, rat IgG2a) (Streeter et al., 1988), intercellular adhesion molecule (ICAM)-1 (3-E-2, hamster IgG1), vascular cell adhesion molecule (VCAM)-1 (429, rat IgG2a), 7-integrin (M293, rat IgG2a), CD4 (RM4-5, rat IgG2a), CD8 (53-6.7, rat IgG2a), and CD45R/B220 (RA3-6B2, rat IgG2a) for B-cell marker and MOMA-2 (rat IgG2b) for macrophage marker. These antibodies were obtained from PharMingen (San Diego, CA). The tissue sections were incubated with primary antibodies for overnight at 4°C, and treated with biotinylated goat anti-rat IgG (Southern Biotechnology Associates, Inc., Birmingham, AL) or anti-hamster IgG (KPL, Guilford, UK) for 1 h at room temperature. They were visualized by streptavidin-fluorescein isothiocyanate (FITC) for 30 min. The MAdCAM-1-positive vessels in lamina propria were quantified as area of positively stained vessels per millimeter of muscularis mucosa. The infiltrating cells were expressed as the number of CD4-, CD8-, B220-, and 7-integrin-positive cells per millimeter of muscularis mucosa.

Inhibitory Effect of Anti-MAdCAM-1 mAb on DSS-Induced Colitis. For the study assessing the effect of anti-MAdCAM-1 mAb on DSS-induced colitis, mice were randomized into four groups. Anti-MAdCAM-1 antibody or isotype and species-matched Ig (rat IgG; Chemicon Intern. Inc., Temecula, CA) was administered i.p. at the dose of 200 µg/mouse. For the daily administration group, DSS-treated mice were administered mAb everyday from day 0 to day 13. As positive control group, DSS-treated mice were administered isotype-matched control IgG everyday from day 0 to day 13. For the early-phase treatment group, DSS-treated mice were administered mAb every day from day 0 to day 6, and for the late-phase treatment group, DSS-treated mice were administered mAb everyday from day 7 to day 13. Histological damage score and immunohistochemical evaluation also were determined in these four groups at day 14. To evaluate the binding of MAdCAM-1 mAb to the colonic mucosa, the localization of infused mAb was examined by immunohistochemistry. Cryostat sections of treated groups were incubated only with a secondary antibody and visualized with streptavidin-FITC.

Statistical Analysis. Results are expressed as mean ± S.E. The parametric data were statistically analyzed by one-way ANOVA and Fisher's protected least-significant difference test (number of infiltrate cells, area of MAdCAM-1-positive vessels). The nonparametric data were statistically analyzed by Kruskal-Wallis test and Scheffé F test (histological damage score). P values of .05 or less were considered to be statistically significant.

	Results

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Characterization of DSS-Induced Colitis. Diarrhea was observed after day 14 and bloody stool was observed on day 21 in DSS-treated mice. The earliest histological changes were observed on day 14. The lesions induced by DSS were characterized by slowly progressive colitis with infiltration by a large number of lymphocytes, lymphocyte aggregation in the lamina propria, and crypt distortion on day 14. Loss of entire crypts and erosions were observed on day 21. Histological changes were most common in the distal colon. Histological damage scores were therefore determined for the distal colon. The changes over time in score are shown in Fig. 1. The score was significantly increased after day 14 and further increased on day 21. 

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Changes over time in histological damage score of distal colon in DSS-treated mice. BALB/c mice were fed orally 5% DSS (mol. wt. 5000) in drinking water. Mice were sacrificed on day 0, 7, 14, and 21. The tissue sections examined under a light microscope were given a score on a 0 to 4 scale based on the criteria as described under Materials and Methods. Results are expressed as the mean ± S.E. of six animals. *P < .05 compared with day 0 (controls).

View larger version (68K): [in this window] [in a new window]   Fig. 2.   Immunohistochemical study of MAdCAM-1 expression in the colonic mucosa of DSS-treated mice. A, MAdCAM-1 expression of nontreated mice (day 0) was weak and sporadic on the vessels in the lamina propria (200×). B, MAdCAM-1 expression of DSS-treated mice on day 7, demonstrating the increased MAdCAM-1 on the vessels in the lamina propria (200×). C, MAdCAM-1 expression of DSS-treated mice on day 14, demonstrating the increased MAdCAM-1 on the vessels in the lamina propria as well as in the submucosal layer (200×).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Changes over time in MAdCAM-1 expression in the lamina propria of distal colon in DSS-treated mice. MAdCAM-1-positive vessels in the lamina propria were determined as area of positively stained vessels per millimeter of muscularis mucosa on days 0, 7, 14, and 21. Results are expressed as the mean ± S.E. of six animals. *P < .05 compared with day 0 (controls).

View larger version (127K): [in this window] [in a new window]   Fig. 4.   Immunohistochemical staining of 7-integrin-positive cells in the colonic mucosa of DSS-treated mice. A, 7-integrin-positive cells of DSS-treated mice on day 0, showing only a few cells infiltrating the lamina propria (200×). B, 7-integrin-positive cells in DSS-treated mice on day 14, showing a significant cell infiltration in the lamina propria (200×).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Changes over time in 7-integrin-, CD4-, CD8-, and B220-positive cells in the lamina propria of distal colon in DSS-treated mice. The number of these cells was determined and expressed as cells per millimeter of muscularis mucosa on days 0, 7, 14, and 21. A, 7-integrin-positive cells (). B, CD4 ()- and CD8 ()-positive cells. C, B220-positive cells (). Results are expressed as the mean ± S.E. of six animals. *P < .05 compared with day 0 (controls).

View larger version (89K): [in this window] [in a new window]   Fig. 6.   Immunohistochemical study of ICAM-1 and VCAM-1 expression in the colonic mucosa of DSS-treated mice. A, strong expression of ICAM-1 demonstrated in vascular endothelium of the submucosa on day 21 (400×). B, strong expression of VCAM-1 demonstrated in vascular endothelium of the submucosa on day 21 (400×).

Effect of Anti-MAdCAM-1 mAb on DSS-Induced Colitis. The attenuating effect of anti-MAdCAM-1 mAb on chronic DSS-induced colitis was investigated. Because the expression of MAdCAM-1 was significantly increased on day 7, we decided to consider early-phase and late-phase treatments as those before and after day 7, respectively. Figure 7, A and B, compares H&E-stained sections of colonic tissues of control IgG-treated and anti-MAdCAM-1-mAb-treated groups on day 14. As shown in this figure, the degree of inflammation and the extent of mucosal damage were significantly attenuated by daily administration of mAb compared with the positive control group (DSS + control IgG). To confirm the binding of administered MAdCAM-1 mAb to the colonic mucosa, the localization of infused mAb was determined by immunohistochemistry by using only a secondary antibody with streptavidin-FITC. As Fig. 7C demonstrates, MAdCAM-1 mAb was shown to bind to the endothelium of vessels in lamina propria and submucosa. Figure 8 compares the histological damage scores of mAb treatment groups and the positive control group on day 14. The early-phase treatment group exhibited no significant difference from the positive control group in histological damage score, although late-phase treatment and daily administration group significantly prevented histological damage. There was no significant difference between the late-phase group and the daily administration group in degree of decrease in histological damage score.

View larger version (136K): [in this window] [in a new window]   Fig. 7.   A, H&E-staining of distal colon 14 days after the treatment with IgG, demonstrating loss of the two-thirds of crypts with the surface epithelium remaining intact (grade 2) (200×). B, H&E staining of distal colon of DSS-treated mice 14 days after the continuous treatment with anti-MAdCAM-1 mAb (200 µg/day), demonstrating the almost intact crypt (grade 0) (200×). C, immunohistochemical localization of infused anti-MAdCAM-1 antibody in the colonic mucosa 14 days after the continuous administration to DSS-treated mice. Incubation with a secondary antibody demonstrates clearly that the antibody firmly binds to the endothelium of vessels in the lamina propria and in the submucosa (200×).

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Effect of administration of anti-MAdCAM-1 antibody on histological damage score of distal colon of DSS-treated mice on day 14. Mice were treated with anti-MAdCAM-1 antibody (200 µg/day) or isotype-matched control antibody. Positive control: treatment with control antibody for 14 days in DSS-treated mice. Early phase: early-phase treatment group, treated with anti-MAdCAM-1 antibody from day 0 to 6. Late phase: late-phase treatment group, treated with anti-MAdCAM-1 antibody from day 7 to 13. Daily administration: daily administration group, treated with anti-MAdCAM-1 antibody from day 0 to 13. Results are expressed as the mean ± S.E. of six animals. *P < .05 compared with the positive control.

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Effect of administration of anti-MAdCAM-1 antibody on number of (A) 7-integrin-positive cells () and (B) CD4 ()-, CD8 ()-, and B220 ()-positive cells in the lamina propria of distal colon in DSS-treated mice on day 14. Mice were treated with anti-MAdCAM-1 antibody (200 µg/day) or isotype-matched control antibody. Positive control: treatment with control antibody for 14 days in DSS-treated mice. Early phase: early-phase treatment group, treated with anti-MAdCAM-1 antibody from day 0 to 6. Late phase: late-phase treatment group, treated with anti-MAdCAM-1 antibody from day 7 to 13. Daily administration: daily administration group, treated with anti-MAdCAM-1 antibody from day 0 to 13. Results are expressed as the mean ± S.E. of six animals. *P < .05 compared with the positive control. P < .05 compared with the early-phase treatment.

	Discussion

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In this study we induced a slowly progressive colitis in BALB/c mice with 5% DSS (mol. wt. 5000). The severity of colitis was highest on the left side of colon, as Cooper et al. (1993) demonstrated; however, in our model, shortening of crypts and increase in number of infiltrating lymphocytes were recognized only on day 14. An important finding of our study is that expression of MAdCAM-1 was already significantly increased in the vessels of lamina propria on day 7, when histological change was not yet observed. The accumulation of 7-integrin-positive T cells as well as B cells was observed in sites of inflammation after day 14 following significant increase in MAdCAM-1 expression, suggesting that MAdCAM-1 is a major inducer of lymphocyte migration into inflamed colonic mucosa.

Increased expression of MAdCAM-1 has been recognized in other animal models, such as interleukin (IL)-10-deficient mice (Connor et al., 1999), IL-2 knockout mice (McDonald et al., 1997), SCID mice with CD45 RB^high CD4+ T cells (Picarella et al., 1997) and trinitrobenzene sulfonic acid colitis (Viney et al., 1996). In these models, expression of MAdCAM-1 was increased on the vessels in the lamina propria and the submucosal layer. In this study, we also demonstrated that in DSS-induced colitis there was strong expression of MAdCAM-1 in inflamed colon. Although expression of MAdCAM-1 was increased at sites of inflammation in inflammatory bowel diseases, human MAdCAM-1 was recognized in endothelium in gut and lymphoid tissues (Briskin et al., 1997; Souza et al., 1999). In this study, expression of MAdCAM-1 was slight on the small vessels at the bases of crypts in nontreated mice. As for other animal models of experimental colitis, we speculate that expression of MAdCAM-1 was up-regulated on the endothelium of mucosal and submucosal vessels where MAdCAM-1 had already been present.

MAdCAM-1 is induced on a murine endothelial cell line, bEND.3, by tumor necrosis factor (TNF)- and IL-1-mediated nuclear factor-B protein in vitro (Sikorski et al., 1993; Takeuchi and Baichwal, 1995). It was previously reported that expression of MAdCAM-1 was time-dependently increased in the colon of wild-type C57BL/6 mice by systemic administration of TNF- in vivo (Connor et al., 1999). TNF- and IL-1 are thought to be produced chiefly by activated macrophages (Sartor, 1994), and it was reported that DSS was observed in macrophages in mesenteric lymph nodes and colonic mucosa (Kitajima et al., 1999). Actually, it has been reported that i.p.-administered anti-TNF antibody and anti-IL-1-antibody ameliorated DSS-induced colitis (Kojouharoff et al., 1997; Arai Y et al., 19981). Based on these observations, we speculate that the time-dependent increase in MAdCAM-1 expression in DSS-induced colitis is induced by enhanced TNF- and IL-1 production from activated macrophages in inflamed mucosa. IL-1 also can stimulate production of IL-2 and interferon- by T cells. Subsequent interaction between macrophages and lymphocytes augments inflammation of colonic mucosa with crossregulation of Th1/Th2 responses (Mosmann and Moore, 1991).

We examined the prophylactic effect of anti-MAdCAM-1 antibody treatment on DSS-induced colitis. We found that daily administration of mAb significantly inhibited the progression of colitis. However, we also found that early-phase treatment with mAb did not significantly attenuate histological damage, although it did decrease the number of infiltrating lymphocytes. These results suggest the possibility that MAdCAM-1 plays a key role in the development of DSS-induced colitis by inhibiting lymphocyte migration, but not in the initiation phase of DSS-colitis before up-regulation of MAdCAM-1.

Prophylactic effects of administration of antiadhesion molecules were previously reported for other experimental animal models. Immunoneutralizaton of MAdCAM-1 and anti-7-integrin antibody reduced the severity of SCID mice treated with CD45 RB^high CD4+ T cells (Picarella et al., 1997). ICAM-1 and VCAM-1 are other adhesion molecules induced in sites inflammation in colon. Anti-ICAM-1 mAb or anti-ICAM-1 antisense oligonucleotide has been used to prevent acute DSS-induced colitis in rats and mice (Bennett et al., 1997; Taniguchi et al., 1998). Anti-VCAM-1 antibody also was reported to abrogate increased leukocyte adhesion in colonic venules in trinitrobenzene sulfonic acid colitis in rats (Sans et al., 1999). More recently, inhibition of DSS-induced colitis in mice by intracolonically administered antibodies against endothelial leukocyte adhesion molecule-1 or ICAM-1 was reported (Hamamoto et al., 1999). However, in our model expression of neither ICAM-1 nor VCAM-1 was recognized in colonic mucosa of normal mice by immunohistochemistry. Moreover, in the present study, significant expression of ICAM-1 and VCAM-1 was only demonstrated at day 21 of DSS colitis in the vessels of submucosal layer where significant infiltration of PMNLs was seen and significant enhancement of MAdCAM-1 had already occurred. These results suggest that MAdCAM-1 plays a more important role in the induction of DSS-induced colitis than ICAM-1 and VCAM-1, which may play roles mainly in the neutrophil-mediated tissue injury in the later phase of this type of colitis. In clinical studies, levels of ICAM-1, VCAM-1, and MAdCAM-1 were increased in the intestinal mucosa in active ulcerative colitis and Crohn's disease (Nakamura et al., 1993; Jones et al., 1995). Some clinical trials with antiadhesion molecules were previously performed. For example, ICAM-1 antisense oligonucleotide therapy was used to prevent Crohn's disease (Yacyshyn et al., 1998). Because ICAM-1 is generally expressed on the outside of gastrointestinal tract as well, and because MAdCAM-1 is an adhesion molecule more unique to the gut mucosa than ICAM-1, attenuation of MAdCAM-1 may be more specific therapy for colitis. The findings of our present study suggest that anti-MAdCAM-1 therapy may be an effective and organ-specific strategy for treating human inflammatory bowel diseases.