Mucosal Addressin Cell Adhesion Molecule-1 Expression Research Articles

Blocking MAdCAM-1 in vivo reduces leukocyte extravasation and reverses chronic inflammation in experimental colitis

Leukocyte recruitment to sites of intestinal inflammation is a crucial multi-step process, leading ultimately to the accumulation of cells in the inflamed tissue. These interactions in the gut are critically dependent on the mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is expressed on endothelial cells within the mesenteric lymph nodes and the lamina propria of the intestine. Here, we investigate the pathophysiologic role of MAdCAM-1 in the intestinal microcirculation in vivo. Using a standard mouse model, chronic colitis was established after four cycles of dextran sodium sulfate (DSS) application. MAdCAM-1 expression was investigated by immunohistochemistry and Western blotting, as well as real-time polymerase chain reaction (PCR). Intravital microscopy was used to study the role of MAdCAM-1 on leukocyte-endothelium interactions and leukocyte extravasation. Significant changes in MAdCAM-1 were observed in mice with chronic DSS-induced colitis. Upregulation of MAdCAM-1 expression in chronic colitis was demonstrated on a protein and messenger ribonucleic acid (mRNA) level. Anti-MAdCAM-1 treatment lead to a marked reduction (>60%) of leukocyte sticking and extravasation in vivo, compared to the controls. This was parallelled by a significant reduction (45%) of intestinal inflammation, as measured by the histologic grading score. These in vivo results demonstrate a distinct role of MAdCAM-1 in inflammatory intestinal diseases, and suggest that therapeutic strategies targeting this adhesion molecule could be useful in the treatment of chronic colitis.

Madcam-1 Expression and Regulation in Murine Colonic Endothelial Cells in Vitro

Although the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is associated with the etiology of inflammatory bowel diseases, few studies have directly examined MAdCAM-1 using microvascular endothelium derived from the colon. This study measured the expression of MAdCAM-1 in a novel colon endothelial line MJC-1, as well as MAdCAM-1 regulation and function in vitro. We cloned microvascular endothelial cells from primary colon cultures using ImmortoMice mice (whose cells express a temperature-sensitive SV40 large T antigen, H-2Kb-tsA58 mice). Expression of MAdCAM-1 after stimulation with cytokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, or interferon (IFN)-gamma] was determined by Western blotting. Signal paths regulating MAdCAM-1 expression were examined using pharmacological blockers before cytokines. We also examined lymphocyte adhesion using lymphocytes that constitutively express alpha4beta7 integrin. TNF-alpha induced MAdCAM-1 in a dose-dependent manner by 24 hours. MAdCAM-1 induction was protein kinase C, tyrosine kinase, p38 mitogen activated protein kinase, and nuclear-factor kappa-B/poly adenosine diphosphate ribose polymerase dependent. Lymphocyte adhesion was increased 2.6-fold after TNF-alpha stimulation and was inhibited by anti-MAdCAM-1 antibody before treatment (P < 0.05 control versus TNF-alpha). In vitro, MAdCAM-1 can be induced on colon endothelial cells by TNF-alpha stimulation and may represent a useful model to study microvascular injury in the large intestine.

Mechanisms of MAdCAM-1 gene expression in human intestinal microvascular endothelial cells.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a homing receptor preferentially expressed on gut-associated endothelial cells that plays a central role in leukocyte traffic into the mucosal immune compartment. Although the molecular mechanisms underlying endothelial ICAM-1 or E-selectin expression have been intensively investigated, the mechanisms that regulate human MAdCAM-1 expression have not been defined. We report MAdCAM-1 gene and protein expression in primary cultures of human intestinal microvascular endothelial cells (HIMEC) that was not demonstrated in human umbilical vein endothelial cells. Similar to ICAM-1 and E-selectin expression, MAdCAM-1 gene expression in HIMEC was inducible with TNF-alpha, IL-1beta, or LPS activation. However, in striking contrast to ICAM-1 and E-selectin expression, MAdCAM-1 mRNA and protein expression in HIMEC was heavily dependent on culture duration and/or cellular density, suggesting a prominent role for cell-cell interaction among these endothelial cells in the expression of the mucosal addressin. MAdCAM-1 expression was inhibited by both SN-50 (NF-kappaB inhibitor) and LY-294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor], whereas ICAM-1 and E-selectin expression was inhibited by SN-50 but not by LY-294002. The Akt phosphorylation by TNF-alpha or LPS was greater at higher cell density, demonstrating a pattern similar to that of MAdCAM-1 expression. NF-kappaB activation was not affected by cellular density in HIMEC. MAdCAM-1 expression in human gut endothelial cells is regulated by distinct signaling mechanisms involving both NF-kappaB and PI3-K/Akt. These data also suggest that PI3-K/Akt is involved in the gut-specific differentiation of HIMEC, which results in expression of the mucosal addressin MAdCAM-1.

Internal biliary drainage improves decreased number of gut mucosal T lymphocytes and MAdCAM-1 expression in jaundiced rats

BackgroundAlthough the effect of preoperative biliary drainage in patients with obstructive jaundice is controversial, bacterial or endotoxin translocation is one of the main postoperative problem in jaundiced patients. Failure in gut barrier functions causes bacterial translocation; homing and distribution of T lymphocytes in the intestinal lamina propria are important for gut mucosal immune defense. This study was performed to examine whether bile regulates the numbers of T lymphocyte subsets or the expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in experimental jaundice in rats with and without external and internal biliary drainage. MethodsFour groups of Wistar rats were used: those that received a sham operation (SHAM), common bile duct ligation (CBDL), CBDL followed by external drainage (ED), and CBDL followed by internal drainage (ID). Numbers of CD4+ and CD8+ T lymphocytes and MAdCAM-1–positive cells in the lamina propria were counted immunohistochemically in the specimens of jejunum and ileum of each group. Bacterial translocation was examined by culturing from the mesenteric lymph node complex isolated from rats in each group. ResultsA significant decrease in numbers of CD4+ and CD8+ T lymphocytes and MAdCAM-1–positive cells in the lamina propria was seen in obstructive jaundice, although numbers of peripheral blood lymphocytes increased in comparison with the sham-operated control. The numbers of CD4+ and CD8+ T lymphocytes and MAdCAM-1 expression in the lamina propria did not recover to a normal level after external drainage, but did so after internal drainage. Frequencies of bacterial translocation were high in the CBDL and ED group. In contrast, bacterial translocation was not present in any animals in the SHAM group and was at a low percentage in the ID group. ConclusionsChanges in the number of T lymphocytes and MAdCAM-1 expression are associated with the presence of bile in the gastrointestinal tract and are inversely correlated with the frequency of bacterial translocation induced by CBD ligation. MAdCAM-1 expression maintained by the presence of bile may regulate T-lymphocyte homing to the lamina propria in obstructive jaundice.

CYP450 dietary inhibitors attenuate TNF-alpha-stimulated endothelial molecule expression and leukocyte adhesion.

Enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and other endothelial cell adhesion molecules (ECAMs) are associated with the onset and progression of inflammatory bowel disease (IBD). We show in this study that two cytochrome p-450 (CYP450) inhibitors from Citrus paradis (grapefruit), bergamottin, and 6',7'-dihydroxybergamottin (DHB) block tumor necrosis factor (TNF)-alpha-stimulated expression of MAdCAM-1 in cultured endothelial cells and also reduce alpha(4)beta(7)-dependent lymphocyte adhesion. Bergamottin (20-50 microM) or DHB (10-30 microM) pretreatment dose-dependently reduced TNF-alpha-mediated expression of MAdCAM-1 and lymphocyte adhesion. Bergamottin and DHB also prevented expression of two other ECAMs, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (but not E-selectin). SKF-525a, a specific CYP450 inhibitor, also blocked the expression of MAdCAM-1 mediated by TNF-alpha. Similar to SKF-525a (20 microM), bergamottin (20 microM) and DHB (20 microM) directly inhibited the activity of CYP450 3A4. These results suggest that natural CYP450 inhibitors may be effective in reducing ECAM expression and leukocyte adhesion and therefore be useful in the clinical treatment of inflammatory states like IBD.

Semiquantative analysis of expression of mucosal addressin cell adhesion molecule-1 during small bowel graft rejection in rats

Aim Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated lymphoid tissues (GALT). We performed a semiquantative analysis of MAdCAM-1 expression during small bowel graft rejection. Methods Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Animals were sacrificed on days 3, 4, 5, 6, and 7 after SBT. Cryostat sections were prepared from grafts, including Peyer's patches (PPs). Indirect immunoperoxidase staining was performed using mAbs against MAdCAM-1. The degree of vascular endothelial staining on high endothelial venules (HEV) in the PPs was graded from 1 (low levels) to 5 (high levels), and in the vessels of the lamina propria from 1 (faint), 2 (low at the base of villi), 3 (low to the middle of villi), 4 (high to the middle of villi), to 5 (high to villus tip). Results MAdCAM-1 expression on HEVs in PPs was down-regulated during rejection. In contrast its expression on endothelial cells of vessels in the lamina propria was up-regulated during rejection. Conclusion Alteration in MAdCAM-1 expression may be associated with the development of SB graft rejection. The vessels at the base of villi, which are associated with lymphocyte recruitment, may become sites of intense immune reactivity during the early phase of small bowel allograft rejection.

Mucosal vaccination increases endothelial expression of mucosal addressin cell adhesion molecule 1 in the human gastrointestinal tract.

Homing of leukocytes to various tissues is dependent on the interaction between homing receptors on leukocytes and their ligands, addressins, on endothelial cells. Mucosal immunization results in homing of antigen-specific lymphocytes back to the mucosa where they first encountered the antigen. However, it is unknown whether this homing of antigen-specific cells is mediated by an altered endothelial addressin expression after vaccination. Using different immunization routes with an oral cholera vaccine, we show that the endothelial expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is increased in the gastric and upper small intestinal mucosae after immunization through various local routes in the upper gastrointestinal tract. In contrast, rectal immunization did not influence the levels of MAdCAM-1 in the gastric or duodenal mucosa. Furthermore, we show that MAdCAM-1 can be induced on human endothelial cells by tumor necrosis factor alpha (TNF-alpha) and gamma interferon. The vaccine component cholera toxin B subunit (CTB) increased MAdCAM-1 expression on endothelial cells in cultured human gastric explants, an effect that seemed to be mediated by TNF-alpha. In conclusion, MAdCAM-1 expression is increased in the upper gastrointestinal tract after local immunizations with a vaccine containing CTB. This strongly suggests the involvement of MAdCAM-1 in the preferential homing of mucosal lymphocytes to their original site of activation.

Trefoil peptide TFF2 treatment reduces VCAM-1 expression and leukocyte recruitment in experimental intestinal inflammation.

There is evidence for a beneficial effect of trefoil peptides in animal models of gastric damage and intestinal inflammation, but the optimal treatment strategy and the mechanistic basis have not been explored thoroughly. It has been suggested that these proteins may modulate the inflammatory response. The aims of this study were to compare the protective and curative value of systemic and topical trefoil factor family (TFF)2 administration in dextran sulfate sodium-induced experimental colitis and to investigate the relationship between the therapeutic effects of TFF2 and modulation of leukocyte recruitment and expression of cell adhesion molecules. Clinical and morphologic severity of colitis was evaluated at the end of the study (Day 10). Leukocyte-endothelial cell interactions were determined in colonic venules by fluorescence intravital microscopy. The expression of cell adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was measured by the dual radiolabeled monoclonal antibody technique. Pretreatment with TFF2 by subcutaneous or intracolonic (ic) route ameliorated the clinical course of colitis, and the luminal route had a significantly superior effect. This beneficial effect was correlated with significant reductions in endothelial VCAM-1 but not MAdCAM-1 expression and leukocyte adhesion to intestinal venules, which returned to levels similar to those of controls. In established colitis, ic TFF2 treatment did not modify the severity of colonic lesions. In conclusion, TFF2 is useful in the treatment of colitis, and topical administration is superior to the systemic route. Reduction in adhesion molecule expression and leukocyte recruitment into the inflamed intestine contributes to the beneficial effect of this treatment.

Transfection of IL-10 expression vectors into endothelial cultures attenuates alpha4beta7-dependent lymphocyte adhesion mediated by MAdCAM-1.

BackgroundEnhanced expression of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is associated with the onset and progression of inflammatory bowel disease. The clinical significance of elevated MAdCAM-1 expression is supported by studies showing that immunoneutralization of MAdCAM-1, or its ligands reduce inflammation and mucosal damage in models of colitis. Interleukin-10 (IL-10) is an endogenous anti-inflammatory and immunomodulatory cytokine that has been shown to prevent inflammation and injury in several animal studies, however clinical IL-10 treatment remains insufficient because of difficulties in the route of IL-10 administration and its biological half-life. Here, we examined the ability of introducing an IL-10 expression vector into endothelial cultures to reduce responses to a proinflammatory cytokine, TNF-αMethodsA human IL-10 expression vector was transfected into high endothelial venular ('HEV') cells (SVEC4-10); we then examined TNF-α induced lymphocyte adhesion to lymphatic endothelial cells and TNF-α induced expression of MAdCAM-1 and compared these responses to control monolayers.ResultsTransfection of the IL-10 vector into endothelial cultures significantly reduced TNF-α induced, MAdCAM-1 dependent lymphocyte adhesion (compared to non-transfected cells). IL-10 transfected endothelial cells expressed less than half (46 ± 6.6%) of the MAdCAM-1 induced by TNF-α (set as 100%) in non-transfected (control) cells.ConclusionOur results suggest that gene therapy of the gut microvasculature with IL-10 vectors may be useful in the clinical treatment of IBD.

Expression of Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) During Small-Bowel Graft Rejection in Rats

Background: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a critical endothelial adhesion molecule for lymphocyte trafficking to gut-associated lymphoid tissues (GALT) under physiological conditions. It is expressed on special postcapillary venules, that is the high endothelial venules (HEV) in GALT. In this study, we investigated changes in MAdCAM-1 expression during small-bowel graft rejection. Methods: Orthotopic small-bowel transplantation (SBT) with portacaval drainage was performed from brown Norway (BN) rats to Lewis (LEW) rats. Isografted (LEW → LEW) and untransplanted animals served as controls. Animals were killed on days 3, 4, 5, 6 and 7 after SBT. Cryostat sections (6 μm thick) were prepared from normal small-bowel tissues and small-bowel grafts, including Peyer patches (PPs). Indirect immunoperoxidase staining was performed using monoclonal antibodies (mAbs) against rat MAdCAM-1 (OST12). Results: In the PPs of controls, MAdCAM-1 antibodies specifically stained the endothelial cells of HEV, which were predominantly located in the interfollicular areas. In the allografts on day 4 after SBT, the MAdCAM-1 expression was weaker on the HEV in the PPs than in the controls. In the lamina propria of controls, a faint expression of MAdCAM-1 on vessels was observed. On day 4 after allogeneic SBT, the MAdCAM-1 was more strongly expressed on the endothelial cells of the vessels at the base of the villi. As rejection developed, the MAdCAM-1 expression on the vessels progressed toward the villus tip. Conclusions: The change of MAdCAM-1 expression may be involved in the development of small-bowel graft rejection. The vessels at the base of villi, which is associated with lymphocyte recruitment, may become a site of intense immune reactivity in the early phase of small-bowel allograft rejection.

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland.

The mammary gland performs a variety of immunological functions, including protecting itself from mastitis and protecting neonates from infectious agents. Several molecules that mediate lymphocyte trafficking in the immune system are also expressed in the mammary gland. This review is focused on the immunological function of these molecules, especially glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the mammary gland. GlyCAM-1 is expressed in the lactating mouse mammary gland. Endothelial cells produce this protein and secrete it into milk. The glycosylated modification of mammary gland GlyCAM-1 is different from that of the lymph nodes, and lacks the binding ability for L-selectin on lymphocytes. GlyCAM-1 in the mammary gland is not involved in lymphocyte migration, and probably has another function besides that of the lymph nodes. MAdCAM-1 is expressed on endothelial cells of small venules around mouse mammary lobules during lactation. This molecule has the ability to interact with alpha4beta7 integrin on lymphocytes and mediates lymphocyte recruitment to the mammary gland. The density of beta7+/CD3+ T-cells is correlated with the density of the MAdCAM-1-stained area, suggesting that MAdCAM-1 may mediate the migration of these cells. In contrast, there is no relationship between MAdCAM-1 expression and the number of beta7+/c-IgA+ B-cells, implying that some other factor is involved in lymphocyte migration to the mammary gland. Chemokines, such as IL-8, GRO-alpha, MCP-1, RANTES and MEC, have been detected in human and mouse mammary glands. Although little information is available, these molecules may contribute to lymphocyte migration to the mammary gland.

Expression of mucosal addressin cell adhesion molecule 1 on vessel endothelium of gastric mucosa in patients with nodular gastritis

The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin alpha4beta7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT). Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center, strongly associated with H pylori infection. The purpose of this study was to address the implication of the MAdCAM-1/integrin beta7 pathway in NG. We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls. A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM-1 and integrin beta7. In simultaneous viewing of serial sections, the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was calculated. We also performed immunostaining with anti-CD20, CD4, CD8 and CD68 antibodies to determine the lymphocyte subsets co-expressing integrin beta7. Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori infection. Of note, the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive controls. Strong expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid aggregates. Integrin beta7-expressing mononuclear cells, mainly composed of CD20 and CD4 lymphocytes, were associated with vessels lined with MAdCAM-1-expressing endothelium. Our results suggest that the MAdCAM-1/integrin alpha4beta7 homing system may participate in gastric inflammation in response to H pylori-infection and contributes to MALT formation, typically leading to the development of NG.

Increased expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and lymphocyte recruitment in murine gastritis induced by Helicobacter pylori.

Although T cell involvement in Helicobactor pylori-induced gastritis is known, mechanism about T cell recruitment is not understood. In this study we examined how mucosal addressin cell adhesion -molecule-1 (MAdCAM-1) is involved in lymphocyte recruitment in murine chronic gastritis induced by H. pylori. C57 BL/6 mice were infected with Sydney strain (SS1). Six months after infection, the stomach was removed. The expression of adhesion molecules, MAdCAM-1, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and the cell surface antigens CD4, CD8, CD45R/B220 or beta7-integrin were determined by immunohistochemistry. A significant increase in CD4 lymphocytes was observed in the body portion of stomach in SS1-infected mice and most of these CD4 cells express beta7-integrin, a known counter ligand for MAdCAM-1 molecule. Strong MAdCAM-1 expression was observed adjacent to these cells in the lamina propria as well as in the submucosa of SS1-infected stomach. Quantitative analysis showed that the area of MAdCAM-1 expression well correlated with the infiltration of beta7-integrin positive lymphocytes. On the other hand, expression of ICAM-1 or VCAM-1 in the lamina propria was few even in the SS1-infected stomach. Increased expression of MAdCAM-1 was well correlated to the location of lymphocytes, which express CD4 and beta7-integrin. These results suggest the possibility that MAdCAM-1 may be largely involved in the lymphocyte recruitment in the gastritis mucosa with H. pylori.

Mucosal addressin expression and binding-interactions with naive lymphocytes vary among the cranial, oral, and nasal-associated lymphoid tissues.

The head and neck lymph nodes (LN)--or cranial, oral, and nasal-associated lymphoid tissue (CONALT)--help disseminate activated lymphocytes to produce salivary immune responses, especially after intranasal immunization. To elucidate the mechanisms that induce immunity at these sites, we investigated the interactions between addressins and homing-receptors that allow for lymphocyte binding to high endothelial venules (HEV) of CONALT. In vivo lymphocyte trafficking to CONALT was mediated primarily through interactions between peripheral node addressin (PNAd) and L-selectin, whereas interactions between mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and alpha 4 beta 7 played a role in retention in cervical LN (CLN). Upon immunofluorescent staining for PNAd and MAdCAM-1, nearly all HEV in CONALT expressed PNAd, with varying MAdCAM-1 expression among these LN. The parotid gland LN (PRLN) and submaxillary gland LN (SMLN) rely exclusively upon PNAd-L-selectin interactions for naive-lymphocyte binding, whereas the CLN utilize PNAd-L-selectin interactions and MAdCAM-1-alpha 4 beta 7 interactions for binding. Intense staining of non-HEV-expressed vascular cell adhesion molecule-1 (VCAM-1) was observed in PRLN, whereas SMLN and CLN displayed less VCAM-1 but showed intense staining for diffuse MAdCAM-1. This study suggests that though PNAd-L-selectin interactions play an important role in the trafficking of lymphocytes throughout CONALT, varying MAdCAM-1 and VCAM-1 addressin expression and usage impart important differences among the PRLN, SMLN, and CLN.

Mucosal immunity preservation with bombesin or glutamine is not dependent on mucosal addressin cell adhesion molecule-1 expression.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is an adhesion molecule that directs naive T and B cells into Peyer's patches for sensitization and distribution to intestinal and extraintestinal sites. With no enteral stimulation, its expression drops rapidly in association with reduced Peyer's patch cell populations and increases rapidly with reinstitution of enteral feeding. Because both glutamine (GLN) and bombesin (BBS) preserve mucosal immunity, this study examined whether they preserve MAdCAM-1 expression. In 2 separate experiments, animals were randomized to IV cannulation with chow, total parenteral nutrition (TPN), and (experiment 1) 15 microg/kg BBS 3 times per day or (experiment 2) an isocaloric, isonitrogenous 2% GLN-supplemented solution. After 5 days of feeding, MAdCAM-1 expression in Peyer's patches, spleen, and intestine was measured using a dual radiolabeled monoclonal antibody technique. MAdCAM-1 expression was not significantly improved from TPN levels either with BBS or GLN supplementation. Levels of MAdCAM-1 expression remained unchanged in non-Peyer's patch sites. Although MAdCAM-1 is considered the gateway molecule for cell entry into mucosal immunity, this does not seem to be the mechanism for mucosal immunity preservation in nonenterally fed mice receiving bombesin or glutamine.

Melatonin reduces TNF-a induced expression of MAdCAM-1 via inhibition of NF-kappaB.

BackgroundEndothelial MAdCAM-1 (mucosal addressin cell adhesion molecule-1) expression is associated with the oxidant-dependent induction and progress of inflammatory bowel disease (IBD). Melatonin, a relatively safe, potent antioxidant, has shown efficacy in several chronic injury models may limit MAdCAM-1 expression and therefore have a therapeutic use in IBD.MethodsWe examined how different doses of melatonin reduced endothelial MAdCAM-1 induced by TNF-a in an in vitro model of lymphatic endothelium. Endothelial monolayers were pretreated with melatonin prior to, and during an exposure, to TNF-a (1 ng/ml, 24 h), and MAdCAM-1 expression measured by immunoblotting.ResultsMAdCAM-1 was induced by TNF-a. Melatonin at concentrations over 100 μm (10-4 M) significantly attenuated MAdCAM-1 expression and was maximal at 1 mM.ConclusionsOur data indicate that melatonin may exert therapeutic activity in IBD through its ability to inhibit NF-kB dependent induction of MAdCAM-1.

Differential expression of cell adhesion molecules within inflamed ileal pouch mucosa: relationship to recruited cell subtypes.

Endothelial-bound cell adhesion molecules are important in recruiting inflammatory cells to the mucosa in inflammatory bowel disease (IBD). Little is known of the expression of these molecules in relation to the recruitment of particular cell subtypes in the early course of mucosal inflammation. We therefore studied the expression of several adhesion molecules to examine their potential correlation with the cellular infiltrate in the inflamed ileal pouch, a possible disease model for ulcerative colitis. Eleven patients (group 1) with familial adenomatous polyposis (FAP) with no evidence of ileal pouch inflammation and 14 patients (group 2) with ileal pouch inflammation (all with a prior diagnosis of ulcerative colitis) underwent pouch endoscopy with biopsy. Cryostat sections of biopsies were immunostained using a three-stage immunoperoxidase method for the adhesion molecules intercellular adhesion molecule (ICAM-1), vascular cellular adhesion molecule (VCAM-1), E-selectin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). These results were correlated with immunostaining for the cell markers CD3, CD4, CD8, CD45RO, CD14 and CD15, which were quantified by computer image analysis. MAdCAM-1, ICAM-1 and VCAM-1 were expressed to similar degrees on the endothelia of groups 1 and 2. In contrast, E-selectin was significantly increased in group 2 (P = 0.003) and correlated with immunostaining for CD15 (r = 0.72), CD4 (r = 0.55) and CD14 (r = 0.53). MAdCAM-1 expression did not correlate with any cell subset. CD15 was the only cell marker to be altered significantly, being increased in group 2 (P = 0.002). The inflammatory process seen in ileal pouch inflammation is characterized by up-regulation of E-selectin and recruitment of CD15-positive cells, emphasizing the role of neutrophil recruitment and migration to the epithelium in the pathogenesis of this condition.

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-alpha using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-alpha induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappa B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-alpha within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-alpha activated NF-kappa B through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-alpha stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-kappa B/PARP. MAdCAM-1 expression requires NF-kappa B translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

Adhesion molecules in lymphocyte trafficking and colitis

Background & Aims: In adults, binding of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) to lymphocyte α4β7 integrin directs cell trafficking to gut, whereas interaction of peripheral node addressins (PNAd) with lymphocyte L-selectin targets immune cells to peripheral lymph nodes (PLNs). Because nothing is known about these addressins during human development, we studied the expression and function of MAdCAM-1 (and PNAd for comparison) in fetuses and children. Methods: Series of human tissue samples obtained from fetuses (7-40 weeks), children (2 months-7 years), and adults were immunostained with monoclonal antibodies. The function of the addressins and their lymphocyte counter-receptors was tested in in vitro binding assays on fetal and adult tissues. Results: Unlike in adults, MAdCAM-1 is widely expressed from embryonic week 7 onwards, and it only gradually becomes polarized to mucosal vessels after birth. In utero MAdCAM-1 functionally governs lymphocyte adhesion to vessels both in the gut and PLNs by binding to α4β7 integrin. The later induction of PNAd gradually starts to dominate the binding of lymphocytes to PLNs during childhood. Conclusions: There are striking age-dependent switches and species-specific variation in the molecular mechanisms of lymphocyte migration. In utero and during early childhood, the mucosal addressin MAdCAM-1 plays a dominant role in lymphocyte-endothelial cell adhesion at mucosal and nonmucosal sites.

Immune cell trafficking in uterus and early life is dominated by the mucosal addressin MAdCAM-1 in humans

Background & Aims: In adults, binding of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) to lymphocyte α4β7 integrin directs cell trafficking to gut, whereas interaction of peripheral node addressins (PNAd) with lymphocyte L-selectin targets immune cells to peripheral lymph nodes (PLNs). Because nothing is known about these addressins during human development, we studied the expression and function of MAdCAM-1 (and PNAd for comparison) in fetuses and children. Methods: Series of human tissue samples obtained from fetuses (7–40 weeks), children (2 months–7 years), and adults were immunostained with monoclonal antibodies. The function of the addressins and their lymphocyte counter-receptors was tested in in vitro binding assays on fetal and adult tissues. Results: Unlike in adults, MAdCAM-1 is widely expressed from embryonic week 7 onwards, and it only gradually becomes polarized to mucosal vessels after birth. In utero MAdCAM-1 functionally governs lymphocyte adhesion to vessels both in the gut and PLNs by binding to α4β7 integrin. The later induction of PNAd gradually starts to dominate the binding of lymphocytes to PLNs during childhood. Conclusions: There are striking age-dependent switches and species-specific variation in the molecular mechanisms of lymphocyte migration. In utero and during early childhood, the mucosal addressin MAdCAM-1 plays a dominant role in lymphocyte-endothelial cell adhesion at mucosal and nonmucosal sites.GASTROENTEROLOGY 2001;121:853-864