Mucosal Addressin Cell Adhesion Molecule-1 Research Articles

The Binding Specificity and Selective Antagonism of Vedolizumab, an Anti-α4β7Integrin Therapeutic Antibody in Development for Inflammatory Bowel Diseases

Vedolizumab is a humanized monoclonal antibody that targets the alpha(4)beta(7) integrin exclusively, and modulates inflammation in the gastrointestinal tract without inducing the systemic immunosuppression that characterizes anti-alpha(4) chain monoclonal antibodies, such as natalizumab. This unique pharmacologic profile is largely attributable to four determinants. The first determinant is the restriction of the expression of the alpha(4)beta(7) integrin to subsets of leukocytes. Vedolizumab does not bind to the majority of memory CD4(+) T lymphocytes (60%), neutrophils, and most monocytes. The highest level of vedolizumab binding is to a subset (approximately 25%) of human peripheral blood memory CD4(+) T lymphocytes that include gut-homing interleukin 17 T-helper lymphocytes. Vedolizumab also binds to eosinophils at high levels, and to naive T-helper lymphocytes, naive and memory cytotoxic T lymphocytes, B lymphocytes, natural killer cells, and basophils at lower levels; vedolizumab binds to memory CD4(+) T and B lymphocytes with subnanomolar potency (EC(50) = 0.3-0.4 nM). The second determinant is binding specificity; vedolizumab binds exclusively to the alpha(4)beta(7) integrin, and not to the alpha(4)beta(1) and alpha(E)beta(7) integrins. The third determinant is selective antagonism; vedolizumab selectively inhibits adhesion of alpha(4)beta(7)-expressing cells to mucosal addressin cell adhesion molecule 1 (median inhibition concentration [IC(50)] = 0.02-0.06 microg/ml) and fibronectin (IC(50) = 0.02 microg/ml), but not vascular cell adhesion molecule 1. The fourth determinant is the gastrointestinal-specific tropism of the alpha(4)beta(7) integrin function. These pharmacologic properties of vedolizumab, in conjunction with the gastrointestinal tropism of alpha(4)beta(7) integrin function, may ultimately confer an improved risk-to-benefit profile for patients with inflammatory bowel diseases.

Mucosal Addressin Cell-Adhesion Molecule-1 Controls Plasma-Cell Migration and Function in the Small Intestine of Mice

Immunoglobulin (Ig) A secretion into the intestinal lumen is an important immune mechanism of the gastrointestinal (GI) tract. B cells proliferate and differentiate into IgA-secreting plasma cells (PC) within lymphoid organs then migrate directly into the intestinal lamina propria. We aimed to elucidate the in vivo role of the mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which is constitutively expressed in the GI-associated lymphoid tissue, in B-cell migration. We generated MAdCAM-1-deficient mice (MAdCAM(Delta)) and evaluated the B-cell compartment of the GI-associated lymphoid tissue. We also assessed PC migration to the small intestine and the intestinal immune response after oral immunization. In MAdCAM(Delta) mice, the size of Peyer's patches was drastically reduced, compared with that of wild-type mice; this difference was detectable by 3 days after birth, indicating that MAdCAM-1 is dispensable for embryonic Peyer's patch development but mediates recruitment of lymphocytes into this lymphoid organ at later stages. Moreover, antigen-specific, IgA-positive PC were severely compromised in their migration to the small intestine; accordingly, there was a reduced number of IgA-secreting PC in the lamina propria of the small intestine. The MAdCAM(Delta) mice were unable to mount a normal intestinal IgA response after oral immunization with cholera toxin. These data provide in vivo evidence that MAdCAM-1 is required for the localization and function of IgA-secreting PC in the intestine.

W1209 Mucosal Addressin Cell Adhesion Molecule (Madcam) Is Not Expressed in Normal and MS Brain Tissue

W1209 Mucosal Addressin Cell Adhesion Molecule (Madcam) Is Not Expressed in Normal and MS Brain Tissue

Pharmacological characterization of PF‐00547659, an anti‐human MAdCAM monoclonal antibody

The adhesion molecule mucosal addressin cell adhesion molecule (MAdCAM) plays an essential role in the recruitment of lymphocytes to specialized high endothelial venules of the gastrointestinal tract and in their excessive tissue extravasation observed in inflammatory conditions, such as Crohn's disease. We have characterized the in vitro pharmacological properties of two monoclonal antibodies blocking MAdCAM, MECA-367 and PF-00547659, and determined their pharmacokinetic/pharmacodynamic profiles in vivo. Functional adhesion assays and surface plasmon resonance were used to characterize, in vitro, the pharmacological properties of MECA-367 and PF-00547659. The in vivo effects of MECA-367 and PF-00547659 on restriction of beta(7) (+) memory T lymphocytes were determined in mice and macaques, respectively, over the pharmacological dose range to confirm pharmacokinetic/pharmacodynamic relationships. MECA-367 and PF-00547659 bound with high affinity to mouse and human MAdCAM with K(d) values of 5.1 and 16.1 pmol.L(-1) respectively and blocked the adhesion of alpha(4)beta(7) (+) leukocytes to MAdCAM with similar potency. MECA-367 and PF-00547659 induced a similar, dose-dependent two- to threefold increase in circulating populations of beta(7) (+) memory T-cells in the mouse and macaque; without affecting the beta(7) (-) populations. PF-00547659 has potential utility in the treatment of inflammatory conditions by blocking tissue homing of activated alpha(4)beta(7) (+) leukocytes. The characterization of a rodent cross-reacting antibody as a surrogate for PF-00547659 in the search for potential pharmacological biomarkers and the determination of efficacious doses was effective in addressing the restricted orthologous cross-reactivity of PF-00547659 and the challenges this poses with respect to efficacy and safety testing.

Possible impact of MADCAM1 gene single nucleotide polymorphisms to the outcome of allogeneic hematopoietic stem cell transplantation

Mucosal addressin cell adhesion molecule–1 (MAdCAM-1) contributes to the recruitment of donor T cells into the mucosal tissues of the recipient after allogeneic hematopoietic stem cell transplantation (aHSCT). The aim of our study was to determine whether selected single nucleotide polymorphisms (SNPs) of the MADCAM1 gene are associated with development of serious complications after aHSCT. Three MADCAM1 gene single nucleotide polymorphisms (rs758502 C/T, rs2302217 A/G, rs3745925 G/T) were genotyped by polymerase chain reaction with sequence-specific primers in 87 Czech, HLA-identical donor-recipient aHSCT pairs. MADCAM1 rs2302217 AA homozygous recipients developed chronic GVHD more frequently than patients with other genotypes (65% vs. 34%; p = 0.025). Furthermore, multivariate analysis revealed the MADCAM1 rs2302217 AA genotype in recipient being also an independent factor associated with development of acute GVHD ( p = 0.036) and decreased overall survival ( p = 0.001). These data suggest that MADCAM1 gene polymorphisms may be associated with the risk of chronic GVHD and may, also, affect mortality related to aHSCT.

Gut Homing Receptors on CD8 T Cells Are Retinoic Acid Dependent and Not Maintained by Liver Dendritic or Stellate Cells

Lymphocytes primed by intestinal dendritic cells (DC) express the gut-homing receptors CCR9 and alpha4beta7, which recognize CCL25 and mucosal addressin cell-adhesion molecule-1 in the intestine promoting the development of regional immunity. In mice, imprinting of CCR9 and alpha4beta7 is dependent on retinoic acid during T-cell activation. Tissue specificity is lost in primary sclerosing cholangitis (PSC), an extraintestinal manifestation of inflammatory bowel disease, when ectopic expression of mucosal addressin cell-adhesion molecule-1 and CCL25 in the liver promotes recruitment of CCR9+alpha4beta7+ T cells to the liver. We investigated the processes that control enterohepatic T-cell migration and whether the ability to imprint CCR9 and alpha4beta7 is restricted to intestinal DCs or can under some circumstances be acquired by hepatic DCs in diseases such as PSC. Human and murine DCs from gut, liver, or portal lymph nodes and hepatic stellate cells were used to activate CD8 T cells. Imprinting of CCR9 and alpha4beta7 and functional migration responses were determined. Crossover activation protocols assessed plasticity of gut homing. Activation by gut DCs imprinted high levels of functional CCR9 and alpha4beta7 on naïve CD8 T cells, whereas hepatic DCs and stellate cells proved inferior. Imprinting was RA dependent and demonstrated plasticity. Imprinting and plasticity of gut-homing human CD8 T cells requires primary activation or reactivation by gut DCs and is retinoic acid dependent. The inability of liver DCs to imprint gut tropism implies that alpha4beta7+CCR9+ T cell that infiltrate the liver in PSC are primed in the gut.

Influence of glucagon-like peptide-2 on intestinal lymphocyte homing in mice with acute pancreatitis

To investigate the influence of glucagon-like peptide-2 (GLP-2) on intestinal lymphocyte homing receptor-integrin alpha 4 beta 7 and homing ligand-intestinal mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in mice with acute pancreatitis (AP). A total of 96 mice were divided into three groups randomly (n=32 in each group): AP group, GLP-2 group and control group. Murine AP model was reproduced by intraperitoneal injection of caerulein and lipopolysaccharides (LPS). GLP-2 (250 microg/kg) was injected intraperitoneally at 15 minutes after the establishment of model, then it was injected twice a day for 3 days in GLP-2 group, while the mice in control group received normal saline instead. Mice were sacrificed at 6, 12, 24 and 48 hours after reproduction of AP, and tissue specimens were harvested. Integrin alpha 4 beta 7 positive peripheral blood lymphocytes were determined by flow cytometry. The expression of MAdCAM-1 in the terminal ileum mucosa and Peyer patch was measured by immunohistochemistry. Same observations were also done in the control and GLP-2 groups. Compared with control group, integrin alpha 4 beta 7 positive lymphocyte in peripheral blood and the expression of MAdCAM-1 in the terminal ileum mucosa and Peyer patch were significantly reduced at 6, 12, 24 and 48 hours in AP mice (all P<0.05). Integrin alpha 4 beta 7 positive lymphocyte and the expression of MAdCAM-1 were markedly higher in GLP-2 group than those in AP group (all P<0.05), but were lower in GLP-2 group than those in control group (all P>0.05). Administration of GLP-2 may restore expression of integrin alpha 4 beta 7 and MAdCAM-1, promote lymphocyte homing to intestine, thus improve the immunological function of intestine.

Increased expression of mucosal addressin cell adhesion molecule 1 in the duodenum of patients with active celiac disease is associated with depletion of integrin α4β7-positive T cells in blood

Mucosal addressin cell adhesion molecule 1, expressed on gut endothelial cells, in conjunction with integrin alpha(4)beta(7), expressed on lymphocytes, is critical in lymphocyte homing to the gut. The mucosal addressin cell adhesion molecule 1/integrin alpha(4)beta(7) pathway is involved in the pathogenesis of chronic intestinal inflammation by recruiting lymphocytes into inflamed gut. We explored the duodenal expression of mucosal addressin cell adhesion molecule 1 and the peripheral T-cell expression of integrin alpha(4)beta(7) in patients with celiac disease. Duodenal biopsies and a peripheral blood sample were collected from 15 celiac patients, before and after 12 months of gluten-free diet, and from 12 control subjects. Treated celiac biopsies were cultured with peptic-tryptic digest of gliadin and/or an anti-interferon alpha neutralizing antibody. Mucosal addressin cell adhesion molecule 1 was determined by confocal immunofluorescence microscopy and immunoblotting. Integrin beta(7)-positive T cells were analyzed by flow cytometry. Mucosal addressin cell adhesion molecule 1 expression was significantly higher in active celiac disease than in normal mucosa. After gluten-free diet, a dramatic reduction of mucosal addressin cell adhesion molecule 1 was also observed. No difference was seen between patients with celiac disease after treatment and controls. Ex vivo peptic-tryptic digest of gliadin challenge induced a marked increase of mucosal addressin cell adhesion molecule 1 expression. Blocking interferon alpha inhibited the peptic-tryptic digest of gliadin-induced mucosal addressin cell adhesion molecule 1 overexpression. The percentage of circulating beta(7)-positive T cells was significantly lower in untreated celiac disease in comparison to controls but normalized after gluten-free diet. Mucosal addressin cell adhesion molecule 1 is strongly up-regulated in active celiac disease dependent on interferon alpha and is associated with peripheral depletion of integrin alpha(4)beta(7)-expressing T cells. We conclude that mucosal addressin cell adhesion molecule 1 may represent an important determinant for the generation of mucosal damage in celiac disease.

GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitis

A diffuse lymphocyte infiltrate is 1 of the characteristic features of ulcerative colitis (UC). Such lymphocyte recruitment requires lymphocyte rolling mediated by L-selectin ligand carbohydrates (6-sulfo sialyl Lewis X-capped O-glycans) and/or mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venule (HEV)-like vessels. The present study was undertaken to elucidate the role of MAdCAM-1 posttranslationally modified ("decorated") with L-selectin ligand carbohydrates in UC pathogenesis and consequent clinical outcomes. Biopsy specimens composed of active and remission phases of UC as well as normal colonic mucosa were immunostained for CD34, MAdCAM-1, and MECA-79, and the immunostained sections were quantitatively analyzed. Reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out to evaluate transcripts of MAdCAM-1 and N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6STs). CHO and Lec2 cells transfected with CD34 and MAdCAM-1 together with enzymes involved in L-selectin ligand carbohydrate biosynthesis were analyzed by immunofluorescence, FACS, and Western blotting to characterize the biochemical properties of GlcNAc6STs. The number of MAdCAM-1(+) vessels was increased in UC, with no significant difference between active and remission phases. An increased ratio of MECA-79(+) to MAdCAM-1(+) vessels with preferential GlcNAc6ST-1 transcripts was observed in the active phase of UC compared to the remission phase. MAdCAM-1 protein was colocalized with L-selectin ligand carbohydrates at the luminal surface of HEV-like vessels in situ. GlcNAc6ST-1 preferentially utilizes MAdCAM-1 as a scaffold protein for GlcNAc-6-O-sulfation in L-selectin ligand carbohydrate biosynthesis. UC disease activity is not regulated by expression of MAdCAM-1 protein itself, but rather by GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates.

α4β7/MAdCAM-1 Interactions Play an Essential Role in Transitioning Cryptopatches into Isolated Lymphoid Follicles and a Nonessential Role in Cryptopatch Formation

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.

CD46-Induced Immunomodulatory CD4+ T Cells Express the Adhesion Molecule and Chemokine Receptor Pattern of Intestinal T Cells

Tissue homing of activated T cells is typically mediated through their specific integrin and chemokine receptor repertoire. Activation of human primary CD4(+) T cells in the presence of CD46 cross-linking induces the development of a distinct immunomodulatory T cell population characterized by high IL-10/granzyme B production. How these regulatory T cells (Tregs) migrate/home to specific tissue sites is not understood. In this study, we determined the adhesion protein and chemokine receptor expression pattern on human CD3/CD46-activated peripheral blood CD4(+) T cells. CD3/CD46-activated, but not CD3/CD28-activated, T cells up-regulate the integrin alpha(4)beta(7). The interaction of alpha(4)beta(7) with its ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) mediates homing or retention of T cells to the intestine. CD3/CD46-activated Tregs adhere to/roll on MAdCAM-1-expressing HeLa cells, similar to T cells isolated from the human lamina propria (LP). This interaction is inhibited by silencing MAdCAM-1 expression in HeLa cells or by the addition of blocking Abs to beta(7). CD46 activation of T cells also induced the expression of the surface-bound cytokine LIGHT and the chemokine receptor CCR9, both marker constitutively expressed by gut LP-resident T cells. In addition, we found that approximately 10% of the CD4(+) T lymphocytes isolated from the LP of patients undergoing bariatric surgery contain T cells that spontaneously secrete a cytokine pattern consistent with that from CD46-activated T cells. These data suggest that CD46-induced Tregs might play a role in intestinal immune homeostasis where they could dampen unwanted effector T cell responses through local IL-10/granzyme B production.

Stromal Cells Confer Lymph Node-Specific Properties by Shaping a Unique Microenvironment Influencing Local Immune Responses

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103(+) dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.

Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats.

Although increased expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) has been demonstrated in inflammatory sites of various diseases, its role in colitis remains unknown. In this study, we examined whether MAdCAM-1 is involved in the pathogenesis of granulomatous colitis induced by peptidoglycan-polysaccharide (PG-PS). Experimental colitis was induced by intramural injection of PG-PS to rat colon. After 3 weeks the colon was removed and the mucosal inflammation was assessed. The area of MAdCAM-1-positive venules and the subsets of infiltrating cells were determined in colonic mucosa by immunohistochemistry. In another experiment, monoclonal antibody against MAdCAM-1 was administered intraperitoneally to examine its attenuating effect on colitis. The intramural injection of PG-PS induced significant colonic inflammation with granuloma formation. The submucosa was drastically thickened with the infiltration of CD4 positive lymphocytes and ED-1 positive macrophages. Intense MAdCAM-1 expression was observed on endothelium of the submucosal venules in inflamed mucosa. Administration of anti-MAdCAM-1 antibody significantly attenuated the PG-PS-induced colonic damage and cell infiltration. Enhanced expression of MAdCAM-1 was demonstrated in venular endothelium of the inflamed colon in PG-PS-induced colitis. The attenuating effect of anti-MAdCAM-1 suggests the importance of the MAdCAM-1-dependent process in the formation of chronic granulomatous colitis.

Nonmucosal Alphavirus Vaccination Stimulates a Mucosal Inductive Environment in the Peripheral Draining Lymph Node

The strongest mucosal immune responses are induced following mucosal Ag delivery and processing in the mucosal lymphoid tissues, and much is known regarding the immunological parameters which regulate immune induction via this pathway. Recently, experimental systems have been identified in which mucosal immune responses are induced following nonmucosal Ag delivery. One such system, footpad delivery of Venezuelan equine encephalitis virus replicon particles (VRP), led to the local production of IgA Abs directed against both expressed and codelivered Ags at multiple mucosal surfaces in mice. In contrast to the mucosal delivery pathway, little is known regarding the lymphoid structures and immunological components that are responsible for mucosal immune induction following nonmucosal delivery. In this study, we have used footpad delivery of VRP to probe the constituents of this alternative pathway for mucosal immune induction. Following nonmucosal VRP delivery, J chain-containing, polymeric IgA Abs were detected in the peripheral draining lymph node (DLN), at a time before IgA detection at mucosal surfaces. Further analysis of the VRP DLN revealed up-regulated alpha4beta7 integrin expression on DLN B cells, expression of mucosal addressin cell adhesion molecule 1 on the DLN high endothelia venules, and production of IL-6 and CC chemokines, all characteristics of mucosal lymphoid tissues. Taken together, these results implicate the peripheral DLN as an integral component of an alternative pathway for mucosal immune induction. A further understanding of the critical immunological and viral components of this pathway may significantly improve both our knowledge of viral-induced immunity and the efficacy of viral-based vaccines.

A4b7/MAdCAM‐1 Interactions Play an Essential Role in Transitioning Cryptopatches into Isolated Lymphoid Follicles, but not in Cryptopatch Formation

α4β7 α4β1 and their ligands, mucosal vascular addressin cell adhesion molecule (MAdCAM‐1) and vascular cell adhesion molecule (VCAM‐1), have prominent roles in the formation of secondary lymphoid tissues at early and later time points. In this study, we evaluated the role of α4 integrins in the development of inducible mucosal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse cellular populations express α4β7 and α4β1, including the LTi‐like cells and lymphocytes, while ILF stromal cells and high endothelial venules express VCAM‐1 and MAdCAM‐1 respectively. Evaluation of β7 deficient mice and mice given blocking antibodies to α4β7, MAdCAM‐1, or VCAM‐1 did not identify a role for α4 integrins in the localization or clustering of the LTi‐like cells, or cryptopatch (CP) development. However, these studies demonstrated that α4α7 and MAdCAM‐1 were absolutely required for the transitioning of CP into ILFs. Bone marrow chimeric mouse studies confirmed that β7 expression by lymphocytes was essential for their entry into the developing ILF. Collectively these findings demonstrate a role for α4β7/MAdCAM‐1 interactions at late stages in ILF development corresponding to the influx of mature β7 expressing lymphocytes, and implicate distinctions between the role of early β7 events leading to ILF and secondary lymphoid tissue development.

CCL25 and CCL28 promote α4β7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger alpha(4)beta(1)- and alpha(4)beta(7)-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was alpha(4)beta(7) dependent, with no contribution from alpha(4)beta(1), whereas alpha(4)beta(1) mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger alpha(4)beta(7)-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger alpha(4)beta(7)-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus alpha(4)beta(1)/VCAM-1 and alpha(4)beta(7)/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.

4. Gastrointestinal mucosal immunity

Mucosal surfaces constitute a large host-environmental interface that must be protected from pathogenic organisms. The mucosal immune system has evolved as a distinct immune organ functioning independently from its systemic counterpart. The mucosal immune system has the difficult task of mounting protective responses to invading microorganisms while maintaining a state of nonresponsiveness to commensal bacteria and food antigens. The system has unique cellular components and functional aspects that permit it to carry out this dual role.

TNF-α Regulated MAdCAM-1 Expression in Pancreatic Microvessel Endothelium: A Possible Role for MAdCAM-1 in Pancreatitis?

The Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) is associated with active inflammatory bowel disease; however MAdCAM-1 expression in the pancreas has not been previously documented. This study investi- gated MAdCAM-1 expression during cerulein-induced pancreatitis, and examined MAdCAM-1 expression, regulation and function in cytokine-treated pancreatic endothelial cells. Acute pancreatitis was induced by serial injections of ce- rulein over 10h, at which point tissue samples were collected. Pancreas endothelial cells (PMEC) were prepared from H- 2Kb-tsA58 (Immortomouse) mice. MAdCAM-1 expression was examined by immunoblotting after cytokine (TNF-� , IL-� or IFN-� ) stimulation. 2 nd messages regulating MAdCAM-1 were studied by adding pharmacological blockers prior to cy- tokines. MAdCAM-1 dependent lymphocyte adhesion was monitored using � 4� 7-integrin expressing lymphocytes. In ce- rulein-pancreatitis, MAdCAM-1 was upregulated by 10h, and closely correlated with histopathology. In PMEC, only TNF-� induced MAdCAM-1 dose (0-20ng/ml) and time (>12h-48h) dependently. MAdCAM-1 expression was PKC, ty- rosine kinase, p38 MAPK, and NF-� B-PARP dependent. PMEC-lymphocyte adhesion was increased by TNF-� , and sig- nificantly reduced by MAdCAM-1 antibody. MAdCAM-1 is induced in the inflamed pancreas, and in TNF-� stimulated PMEC, and may represent a novel determinant of pancreatic-lymphocyte recruitment in inflammation. PMEC cells might provide a useful system to study mechanisms related to both acute and chronic pancreatitis.

DEC‐205/CD205+ dendritic cells are abundant in the white pulp of the human spleen, including the border region between the red and white pulp

The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.

Effect of FTY720 and Ex Vivo Graft Irradiation in Rat Small Bowel Transplantation: Expression of Mucosal Addressin Cell Adhesion Molecule-1

We performed a semiquantitative analysis of the expression of Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and gut-associated tissues (GALT) during small bowel graft rejection in the rat to confirm the effect of FTY720 and ex vivo graft irradiation. Small bowel transplantations (SBT) were performed from BN rats to LEW rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations (untreated, FTY720, ex vivo graft irradiation, FTY720+ex vivo graft irradiation). Indirect immunoperoxidase staining was performed against CD4 and MAdCAM-1. The number of CD4-positive cells in the allografts was also analyzed by flow cytometry. The graft survival was prolonged only in the FTY720-treated groups. The SBT allografts treated by FTY720 demonstrated less infiltration, but the ex vivo graft irradiation group did not show any effect on its expression. In the FTY720-treated groups, MAdCAM-1 expression on high endothelial venules (HEVs) in Peyer's patches (PPs) was upregulated and its expression on endothelial cells of vessels in the lamina propria was downregulated in comparison with the allograft group without FTY720. It is important to prevent the infiltration of CD4-positive cells, the downregulation of MAdCAM-1 expression on HEVs in PPs and the upregulation of MAdCAM-1 expression on endothelial cells of vessels in the lamina propria for the prolongation of graft survival.