Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.     

Dynamics of vascular endothelial-cadherin and beta-catenin localization by vascular endothelial growth factor-induced angiogenesis in human umbilical vein cells

The adherens junctional molecule, vascular endothelial cadherin (VE-cadherin), functions to maintain adherens junction stability and to suppress apoptosis of endothelial cells by forming a complex with vascular endothelial growth factor (VEGF) receptor 2 and members of the armadillo family of cytoplasmic proteins. In order to investigate the dynamics of the association of VE-cadherin with adherens junctions during the initial stages of angiogenesis, human umbilical cord endothelial cells (HUVECs) were stimulated with VEGF to undergo angiogenesis in type-I collagen three-dimensional culture. In confluent monolayers of HUVECs, VE-cadherin and its signaling partner, beta-catenin, as well as the paracellular transmembrane adhesion molecule platelet-endothelial cell adhesion molecule (PECAM-1), were all present in regions of cell-cell contact. Within 3 h of stimulation of angiogenesis, VE-cadherin and beta-catenin were lost from these regions. In contrast, the distribution pattern of PECAM-1 did not alter. After 6 h the majority of endothelial cells had migrated to form a network of capillary cords with cell-cell contacts that contained all three molecules. By metabolic labeling of HUVECs it was found that de novo synthesis of VE-cadherin was not essential for the formation of new adherens junctions. Coimmunoprecipitation and immunoblotting experiments showed that the VE-cadherin and beta-catenin remained associated after they were lost from adherens junctions. Detergent extraction of cells with Triton X-100 indicted that the majority of VE-cadherin and beta-catenin was Triton soluble, indicating that they are only weakly associated with the actin-based cytoskeleton.     

Endothelial cell PECAM-1 confers protection against endotoxic shock

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a 130-kDa member of the Ig superfamily that is expressed on platelets and leukocytes and is highly enriched at endothelial cell-cell junctions. Previous studies showed that this vascular cell adhesion and signaling receptor functions to regulate platelet activation and thrombosis, to suppress apoptotic cell death, to mediate transendothelial migration of leukocytes, and to maintain the integrity of the vasculature. Because systemic exposure to the bacterial endotoxin LPS triggers an acute inflammatory response that involves many of these same processes, we compared the pathophysiological responses of wild-type versus PECAM-1-deficient mice to LPS challenge. We found that PECAM-1-deficient mice were significantly more sensitive to systemic LPS administration than their wild-type counterparts and that the lack of PECAM-1 expression at endothelial cell-cell junctions could account for the majority of the increased LPS-induced mortality observed. The diverse functional roles played by PECAM-1 in thrombosis, inflammation, apoptosis, and the immune response may make this molecule an attractive target for the development of novel therapeutics to manage and treat endotoxic shock.     

Platelet-endothelial cell adhesion molecule-1 modulates endothelial migration through its immunoreceptor tyrosine-based inhibitory motif

Coordinated migration of endothelial cells models the remodeling of existing endothelia as well as angiogenesis and vasculogenesis. Platelet-endothelial cell adhesion molecule-1, PECAM-1, a transmembrane endothelial adhesion protein, binds and activates the tyrosine phosphatase SHP-2 via phosphotyrosines 663 and 686. PECAM-1 phosphorylation and recruitment of SHP-2 are regulated by cell-cell and cell-substrate adhesion. We found that PECAM-1 is dephosphorylated on tyrosine 686 during endothelial migration, resulting in diffuse dispersal of PECAM-1 and SHP-2. Overexpression of native PECAM-1 slowed, and nonphosphorylatable PECAM-1 increased, endothelial migration, implying that the SHP-2-regulatory phosphotyrosines negatively regulate migration. Using differentially phosphorylated recombinant proteins we found that phosphotyrosine 686 preferentially mediates binding and 663 mediates activation of SHP-2 by PECAM-1. In PECAM-1-null endothelial cells, SHP-2 bound and dephosphorylated an alternative set of phosphoproteins and its distribution to the cytoskeletal fraction was significantly decreased. Tyrosine phosphorylation of beta-catenin and focal adhesion kinase was increased in endothelial cells overexpressing nonphosphorylatable PECAM-1. Thus homophilically engaged, tyrosine-phosphorylated PECAM-1 locally activates SHP-2 at cell-cell junctions; with disruption of the endothelial monolayer, selective dephosphorylation of PECAM-1 leads to redistribution of SHP-2 and pro-migratory changes in phosphorylation of cytoskeletal and focal contact components.     

Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow

The vascular endothelial cell cadherin complex (VE-cadherin, alpha-, beta-, and gamma-catenin, and p120/p100) localizes to adherens junctions surrounding vascular endothelial cells and may play a critical role in the transendothelial migration of circulating blood leukocytes. Previously, we have reported that neutrophil adhesion to human umbilical vein endothelial cell (HUVEC) monolayers, under static conditions, results in a dramatic loss of the VE-cadherin complex. Subsequent studies by us and others (Moll, T., E. Dejana, and D. Vestweber. 1998. J. Cell Biol. 140:403-407) suggested that this phenomenon might reflect degradation by neutrophil proteases released during specimen preparation. We postulated that some form of disruption of the VE-cadherin complex might, nonetheless, be a physiological process during leukocyte transmigration. In the present study, the findings demonstrate a specific, localized effect of migrating leukocytes on the VE-cadherin complex in cytokine-activated HUVEC monolayers. Monocytes and in vitro differentiated U937 cells induce focal loss in the staining of VE-cadherin, alpha-catenin, beta-catenin, and plakoglobin during transendothelial migration under physiological flow conditions. These events are inhibited by antibodies that prevent transendothelial migration and are reversed following transmigration. Together, these data suggest that an endothelial-dependent step of transient and focal disruption of the VE-cadherin complex occurs during leukocyte transmigration.     

TIMP-1 inhibits microvascular endothelial cell migration by MMP-dependent and MMP-independent mechanisms

It was reported over a decade ago that tissue inhibitor of metalloproteinases-1 (TIMP-1) suppresses angiogenesis in experimental models but the mechanism is still incompletely understood. This in vitro study focused on the molecular basis of TIMP-1-mediated inhibition of endothelial cell (EC) migration, a key step in the angiogenic process. Both recombinant human TIMP-1 and the synthetic MMP inhibitors, GM6001 and MMP-2-MMP-9 Inhibitor III, suppressed migration of human dermal microvascular endothelial cells (HDMVEC) in a dose-dependent fashion. The MMP-dependent inhibition of migration was associated with increased expression of the junctional adhesion proteins, VE-cadherin and PECAM-1, and VE-cadherin accumulation at cell-cell junctions. TIMP-1 also caused MMP-independent dephosphorylation of focal adhesion kinase (FAK) (pY397) and paxillin, which was associated with reduced number of F-actin stress fibers and focal adhesions. Moreover, TIMP-1 stimulated expression of PTEN that has been shown to reduce phosphorylation of FAK and inhibit cell migration. Our data suggest that TIMP-1 inhibits HDMVEC migration through MMP-dependent stimulation of VE-cadherin and MMP-independent stimulation of PTEN with subsequent dephosphorylation of FAK and cytoskeletal remodeling.     

ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.     

VE-Cadherin Mediates Endothelial Cell Capillary Tube Formation in Fibrin and Collagen Gels

Various cell adhesion molecules mediate the diverse functions of the vascular endothelium, such as cell adhesion, neutrophil migration, and angiogenesis. In order to identify cell adhesion molecules important for angiogenesis, we used anin vitromodel (Chalupowicz, Chowdhury, Bach, Barsigian, and Martinez,J. Cell Biol.130, 207–215, 1995) in which human umbilical vein endothelial cell monolayers are induced to form capillary-like tubes when a second gel, composed of either fibrin or collagen, is formed overlying the apical surface. In the present investigation, we observed that a monoclonal antibody directed against the first extracellular domain of human vascular endothelial cadherin (VE-cadherin, cadherin 5) inhibited the formation of capillary tubes formed between either fibrin or collagen gels. Moreover, when added to preformed capillary tubes, this antibody disrupted the capillary network. In contrast, monoclonal antibodies directed against the extracellular domain of N-cadherin, the α_vβ₃integrin, and PECAM-1 failed to inhibit capillary tube formation. During capillary tube formation, Western blot and RT-PCR analysis revealed no marked change in VE-cadherin expression. Immunocytochemical studies demonstrated that VE-cadherin was concentrated at intercellular junctions in multicellular capillary tubes. Thus, VE-cadherin plays a specific role in fibrin-induced or collagen-induced capillary tube formation and is localized at areas of intercellular contact where it functions to maintain the tubular architecture. Moreover, its function at tubular intercellular junctions is distinct from that at intercellular junctions present in confluent monolayers, since only the former was inhibited by monoclonal antibodies.     

Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown

Tumor invasiveness depends on the ability of tumor cells to breach endothelial barriers. In this study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the B-Raf(V600E) mutation in metastatic melanoma cells up-regulated tissue factor (TF) expression on cell membranes and promoted thrombin production. Co-culture of endothelial monolayers with metastatic melanoma cells mediated the opening of inter-endothelial spaces near melanoma cell contact sites in the presence of platelet-free plasma (PFP). By using small interfering RNA (siRNA), we demonstrated that B-Raf(V600E) and TF silencing attenuated the focal disassembly of adherens junction induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on Ser-879 and VE-cadherin on Tyr-658, Tyr-685, and Tyr-731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1, in endothelial cells. We also provided strong evidence that tumor-derived thrombin enhanced melanoma TEM by inducing ubiquitination-coupled VE-cadherin internalization, focal adhesion formation, and actin assembly in endothelium. Confocal microscopic analysis of tumor TEM revealed that junctions transiently opened and resealed as tumor cells accomplished TEM. In addition, in the presence of PFP, tumor cells preferentially transmigrated via paracellular routes. PFP supported melanoma transmigration under shear conditions via a B-Raf(V600E)-thrombin-dependent mechanism. We concluded that the activation of thrombin generation by cancer cells in plasma is an important process regulating melanoma extravasation by disrupting endothelial junction integrity.     

The unfolding tale of PECAM-1

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a member of the immunoglobulin (Ig) superfamily that has distinctive features of an immunoreceptor based upon its genomic structure and the presence of intrinsic immunoreceptor tyrosine inhibitory motifs (ITIMs) in its ligand binding polypeptide. This has lead to its subclassification into the Ig-ITIM superfamily. Its amino-terminal Ig-like domain of PECAM-1 is necessary for its homophilic binding, which plays an important role in cell–cell interactions. Its intracellular ITIMs serve as scaffolds for recruitment of signalling molecules including protein-tyrosine phosphatases to mediate its inhibitory co-receptor activity. Increasing evidence has implicated PECAM-1 in a plethora of biological phenomena, including modulation of integrin-mediated cell adhesion, transendothelial migration, angiogenesis, apoptosis, cell migration, negative regulation of immune cell signalling, autoimmunity, macrophage phagocytosis, IgE-mediated anaphylaxis and thrombosis. In this review, we discuss some of the new developments attributed to this molecule and its unique roles in biology.     

Regulation of Endothelial Cell Barrier Function by Antibody-driven Affinity Modulation of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1)

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane-proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.     

Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration.

Cell migration is an important process in such phenomena as growth, development, and wound healing. The control of cell migration is orchestrated in part by cell surface adhesion molecules. These molecules fall into two major categories: those that bind to extracellular matrix and those that bind to adjacent cells. Here, we report on the role of a cell-cell adhesion molecule, platelet-endothelial cell adhesion molecule-1, (PECAM-1), a member of the lg superfamily, in the modulation of cell migration and cell-cell adhesion. PECAM-1 is a 120-130 kDa integral membrane protein that resides on endothelial cells and localizes at sites of cell-cell contact. Since endothelial cells express PECAM-1 constitutively, we studied the effects of PECAM-1 on cell-cell adhesion and migration in a null-cell population. Specifically, we transfected NIH/3T3 cells with the full length PECAM-1 molecule (two independent clones). Transfected cells containing only the neomycin resistance gene, cells expressing a construct coding for the extracellular domain of the molecule, and cells expressing the neu oncogene were used as controls. The PECAM-1 transfectants appeared smaller and more polygonal and tended to grow in clusters. Indirect immunofluorescence of PECAM-1 transfectants showed peripheral staining at sites of cell-cell contact, while the extracellular domain transfectants and the control cells did not. In two quantitative migration assays, the full-length PECAM-1 transfectants migrated more slowly than control cells. Thus, PECAM-1 transfected into a null cell appears to localize to sites of cell-cell contact, promote cell-cell adhesion, and diminish the rate of migration. These findings suggest a role for this cell-cell adhesion molecule in the process of endothelial cell migration.     

Discontinuous expression of endothelial cell adhesion molecules along initial lymphatic vessels in mesentery: the primary valve structure

BACKGROUND: Understanding lymphatic fluid uptake requires investigation of the primary valve system located at endothelial cell junctions. The objective of this study was to evaluate the expression pattern of adhesion molecules at endothelial cell junctions in an adult initial lymphatic network. METHODS AND RESULTS: Mesenteric tissues from adult male Wistar rats were labeled with antibodies against PECAM-1 and VE-cadherin. Endothelial cells along initial lymphatics and blood microvascular networks expressed both junctional molecules. In contrast to continuous junctional labeling along blood vessels, PECAM and VE-cadherin labeling patterns were discontinuous with gaps along lymphatic endothelial cell junctions. Along larger draining vessels in proximal regions of the initial lymphatic network, the majority of labeling gaps along junctions were less than 1microm. In comparison to draining vessels, terminal lymphatics exhibited a decrease in PECAM staining intensity and a decrease in endothelial cell junctional length defined by positive PECAM and VE-cadherin staining. CONCLUSION: These results suggest that primary valves responsible for unidirectional interstitial fluid uptake along initial lymphatic vessels are associated with discontinuous expression of endothelial junction molecules. This feature may render the ability to separate local membrane regions between neighboring endothelial cells.     

Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34(+) cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34(+) cells in response to stromal cell-derived factor-1alpha. Stromal cell-derived factor-1alpha-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34(+) cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.     

Activated leukocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration

Transendothelial leukocyte migration is a major aspect of the innate immune response. It is essential in repair and regeneration of damaged tissues and is regulated by multiple cell adhesion molecules (CAMs) including members of the immunoglobulin (Ig) superfamily. Activated leukocyte cell adhesion molecule (ALCAM/CD166) is an Ig CAM expressed by activated monocytes and endothelial cells. Hitherto, the functional relevance of ALCAM expression by endothelial cells and activated monocytes remained unknown. In this report, we demonstrate soluble recombinant human ALCAM significantly inhibited the rate of transendothelial migration of monocyte cell lines. Direct involvement of ALCAM in transendothelial migration was evident from the robust inhibition of this process by ALCAM blocking antibodies. However, soluble recombinant ALCAM had no impact on monocyte migration or adhesion to endothelium. Localization of ALCAM specifically at cell-cell junctions in endothelial cells supported its role in transendothelial migration. This study is the first to localize ALCAM to endothelial cell junctions and demonstrate a functional relevance for co-expression of ALCAM by activated monocytes and endothelial cells.     

PECAM-1 isoform-specific activation of MAPK/ERKs and small GTPases: implications in inflammation and angiogenesis

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) and leukocytes. PECAM-1 plays an important role in endothelial-leukocyte and endothelial-endothelial cell-cell interactions. The anti-PECAM-1 antibody-mediated blockade of these interactions inhibits transendothelial migration (TEM) of leukocytes and angiogenesis. PECAM-1 may accommodate these processes through the regulation of cell adhesive and migratory mechanisms. How PECAM-1 regulates these dynamic processes remain unknown. Here we show that PECAM-1 transduces outside-in signals, which activate MAPK/ERKs and small GTPases. This occurs through PECAM-1-mediated formation of intracellular-signaling complexes, Shc/Grb2/SOS1 and/or Crkl/C3G, which is initiated by PECAM-1 engagement on the surface of leukocytes and/or EC. Src, SHP2, and alternative PECAM-1 pre-mRNA splicing play a regulatory role in these signaling events. Our findings reveal that PECAM-1 engagement on the cell surface can transduce "outside-in" signals and activate MAPK/ERKs and small GTPases, impacting both cadherin-mediated cell-cell and integrin-mediated cell-matrix interactions. Thus, we propose PECAM-1 is an important mediator of vascular barrier and regulator of leukocyte and EC adhesion and migration.     

Modulation of VE-cadherin and PECAM-1 mediated cell-cell adhesions by mitogen-activated protein kinases

Endothelial cell transition from a differentiated, quiescent phenotype to a migratory, proliferative phenotype is essential during angiogenesis. This transition is dependent on alterations in the balanced production of stimulatory and inhibitory factors, which normally keep angiogenesis in check. Activation of MAPK/ERKs is essential for endothelial cell migration and proliferation. However, its role in regulation of endothelial cell adhesive mechanisms requires further delineation. Here, we show that sustained activation of MAPK/ERKs results in disruption of cadherin-mediated cell-cell adhesion, down-regulation of PECAM-1 expression, and enhanced cell migration in microvascular endothelial cells. Expression of a constitutively active MEK-1 in mouse brain endothelial (bEND) cells resulted in down-regulation of VE-cadherin and catenins expression concomitant with down-regulation of PECAM-1 expression. In contrast, inhibition of MEK-1 restored parental morphology, cadherin/catenins expression and localization. These data are further supported by our observation that sustained activation of MAPK/ERKs in phorbol myristate acetate incubated HUVEC lead to disruption of cadherin-mediate cell-cell interactions and enhanced capillary formation on Matrigel. Thus, sustained activation of MAPK/ERKs plays an important role in disruption of cell-cell adhesion and migration of endothelial cells.     

Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.