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HAPTER 6
Vascular Endothelial Cell Adhesion and Signaling During Leukocyte Recruitment

Martin S. Kluger, PhD*
Department of Dermatology and Interdepartmental Program in Vascular Biology and Transplantation, Yale University School of Medicine, New Haven, Connecticut
ABSTRACT During inammation, coordinated expression of cytokine-induced adhesion molecules (CAMs) on postcapillary venular endothelial cells (ECs) regulates leukocyte recruitment. During their recruitment from blood, leukocytes adhere to EC CAMs, activating signaling pathways inside ECs. In a forthcoming paradigm, leukocyte transendothelial migration requires active EC participation, with extracellular adhesive CAM functions mirrored by cytoplasmic domain-dependent intracellular events. These events serve to reorganize the EC actin cytoskeleton. Investigators have visualized this as changes in EC shape, transient opening of EC-EC contacts, and redistribution of CAMs expressed on the luminal EC surface. In this review, we (1) summarize the overlapping extracellular adhesive properties of the 3 EC CAMs most important for leukocyte recruitment during inammation, namely, E-selectin, vascular cell adhesion molecule, and intercellular adhesion molecule-1; (2) explore the role of these 3 CAMs as signal transducers by identifying the intracellular signals (Ca, Rho/Rac, and phosphatidylinositol 4,5-bisphosphate) that upon leukocyte engagement, reorganize the EC cytoskeleton and redistribute these apical CAMs, thereby favoring leukocyte recruitment; and (3) describe how CAM-derived signals lead to ezrin-radixin-moesin complex formation and
*E-mail correspondence: martin-kluger@yale.edu
Advances in Dermatology, vol 20 Copyright 2004, Mosby, Inc. All rights reserved.
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M. S. Kluger how this complex of plasma membranecytoskeleton adapter proteins coordinates CAM-driven intracellular signals with extracellular adhesive CAM functions. This literature review suggests that the cytoplasmic domains of these EC CAMs and their downstream effectors represent new and potentially benecial intracellular therapeutic targets for treating diseases of the skin.
EDITORS COMMENT Endothelial cells (ECs) lining the vascular spaces of postcapillary venules in skin (and other peripheral tissues) are among the rst regulators of inammation. The upregulation of adhesion molecules by endothelial cells, as well as the ability of ECs to present chemoattractant cytokines, leads to the adhesion of leukocytes on the endothelium, a prerequisite step before their transmigration into inamed tissue. In this chapter, Dr Martin Kluger details the adhesion molecules involved in the initial steps of leukocyte trafcking. Moreover, it is clear that ECs do not behave as passive participants in transendothelial migration of inammatory cells. Dr Kluger describes the molecular signaling mechanisms involved in the EC response to leukocyte adhesion and transmigration. The identication of key molecular players in leukocyte-EC interactions may lead to new targets for inhibitors of skin inammation.
Sam Hwang, MD, PhD
nammation governs the complex response of vascularized tissue to cellular injury due to any cause. In skin disease as diverse as psoriasis, allergic contact dermatitis, atopic dermatitis, and cutaneous T-cell lymphoma, inammation involves activation of the vascular endothelium, a monolayer layer of epithelial cells forming the sheet-like inner lining of all blood vessels in the body. Ideally positioned as an anatomic interface between owing blood and vascularized tissue, endothelium regulates access by the cellular components of the immune system to tissue such as skin according to the state of endothelial cell (EC) activation. Although ECs comprise a single cell type, they demonstrate heterogeneity among different vascular beds, with different segments of the vasculature reecting variable barrier properties; for example, in dermis, the interendothelial junctions are less restrictive than in brain and more restrictive than in liver. The site of leukocyte recruitment in skin and in most other tissues is the postcapillary venule.1,2 To mount a successful inammatory response, activation by cytokines such as tumor necrosis factor- (TNF-) must upregulate expression of specic adhe-
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sion molecules on the luminal surface of venular ECs. Identication of the rst cytokine-inducible endothelial adhesion molecule, Eselectin, occurred approximately 20 years ago.3 Since then, much knowledge has accrued on the role of cytokine-induced adhesion molecule (CAM) gene expression on endothelium4-6 and on the specic binding interactions of EC CAMs with cognate ligands expressed on the surface of interactive leukocytes.7 A paradigm shift in the way that we regard EC CAMs shall be forthcoming based on observations that, just before transendothelial migration (TEM), leukocyte adhesion to EC CAMs activates EC intracellular signaling pathways. In this paradigm, leukocyte recruitment requires active EC participation, with EC CAM extracellular adhesive functions mirrored by intracellular events causing reorganization of the actin cytoskeleton. Many investigators have visualized changes in EC shape, transient opening of EC-EC contacts, and redistribution of CAMs expressed on the luminal (in vitro: apical) surface or at EC-EC junctions.8-10 Specically, clustering of CAMs on the apical plasma membrane near points of contact assists leukocytes up to and into junctional openings. Immediately before paracellular transmigration, CAMs engaged by leukocytes become linked to the EC actin cytoskeleton via membranecytoskeleton adapter proteins. Clustering within regions of plasma membrane specialization can potentiate signaling or enable assistance of leukocytes just before they squeeze through the junctional cleft of adjacent ECs. The purpose of this chapter is to (1) summarize the extracellular adhesive properties of the 3 EC CAMs most important for leukocyte recruitment during inammation, namely, E-selectin, vascular cell adhesion molecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1); (2) identify the signaling components active within ECs upon leukocyte engagement that modify the EC cytoskeleton and redistribute these apical CAMs; and (3) describe how CAM-derived signals lead to ezrin-radixin-moesin (ERM) complex formation, and how this complex of plasma membranecytoskeleton adapter proteins coordinates CAM-driven intracellular signals with extracellular adhesive function.
SEQUENTIAL STEPS OF LEUKOCYTE RECRUITMENT INVOLVE OVERLAPPING ADHESIVE FUNCTIONS OF EC CAMs

A progressive sequence of binding events among cognate pairs of EC CAMs and leukocyte ligands serves as a prelude to leukocyte transmigration across endothelium. These steps are usually described as tethering, rolling, leukocyte activation, and rm adhesion (Fig 1).7,11 The nal step of this sequence, TEM, has most often been described
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FIGURE 1. Overlapping roles of cytokine-induced adhesion molecules (CAMs) on the endothelial cell (EC) surface in a multistep model of leukocyte recruitment. Blood ow (arrows) drives leukocyte circulation and inuences leukocyte recruitment by imparting shear stress. The EC CAMs, P- and E-selectin, vascular cell adhesion molecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1), are indicated above the schematic diagram at functionally relevant steps of leukocyte recruitment. Their importance may vary by pathologic condition, and for the sake of clarity, not all EC CAMs are shown. P-selectin is the CAM most procient at leukocyte tethering, whereas P- and E-selectin and VCAM all mediate rolling. During rolling, leukocytes may detach (dotted arrow), or upon encounter with chemokines present on the EC surface, become activated (exclamation point). Chemokine-activated leukocytes atten and rmly adhere to EC immunoglobulin-CAMs, VCAM and ICAM1, triggering other (bidirectional) activation signals in leukocytes and in ECs (lightning symbol; ligand binding to E-selectin may also trigger such signals in EC).Leukocyte ligand binding to EC CAMs also triggers signals in ECs that relax interendothelial junctions (shown by disappearance of the interendothelial junction symbol). Flattened leukocytes may locomote toward relaxed junctions where interactions with colocalized VCAM and ICAM-1 help initiate transendothelial migration (TEM). Other EC molecules situated within the interendothelial cleft, platelet endothelial cell adhesion molecule (PECAM) and CD-99, assist in leukocyte traversal of the EC monolayer. After squeezing through adjacent ECs, extravasated leukocytes must still traverse a layer of basal lamina before embarking toward distal sites of inammation (eg, in epidermis). Abbreviation: ECM, Extracellular matrix.
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as occurring at junctions where adjacent ECs touch.10,12 This review shall refer to lymphocytes, monocytes, and neutrophils as leukocytes, using distinctions only as necessary. Although beyond the scope of this chapter, after TEM, the leukocyte journey continues further into skin with migration across the basal lamina, through the extracellular matrix, and depending on the site of inammatory insult, potentially across the epithelial barrier into epidermis.12-14
CAM EXPRESSION AND ADHESIONS ON THE APICAL EC SURFACE: THE SELECTINS

The selectins and the immunoglobulin (Ig-) superfamily are 2 different families of apical surface EC adhesion molecules responsible for leukocyte recruitment from blood into skin. The selectin family consists of E-selectin (CD62E; relative mass [Mr] of 97 and 107-115 kd, whose expression is unique to activated ECs), P-selectin (CD62P; Mr of 120-140 kd, expressed by activated ECs and by activated platelets), and L-selectin (CD62L; Mr of 74 or 95 kd, expressed by neutrophils, monocytes, macrophage, eosinophils, nave and some memory lymphocytes). Selectins are single transmembrane glycoproteins with homologous extracellular domains that vary in length, depending on the number of complement regulatory protein repeats present (there are 6, 9, and 2 of these for E-, P-, and L-selectin, respectively). In contrast, the selectin intracellular cytoplasmic domains lack sequence homology. Nevertheless, E-selectin is conserved across different species, suggesting an adaptation for intracellular signaling, control of surface expression, or both. Pselectin surface protein reaches the surface within minutes of EC stimulation with histamine or thrombin before recycling back into EC Weibel-Palade storage granules.15,16 In contrast, E-selectin surface expression requires de novo synthesis and is strictly limited by cytoplasmic tail interactions leading to internalization and lysosomal degradation, which is more rapid among ECs derived from large vessels (eg, human umbilical vein ECs [HUVECs]) than from microvessels (eg, from skin).15,17,18 The E-selectin carboxyl-terminus cytoplasmic tail consists of just 32 amino acids and contains a phosphorylable serine residue19 that, in association with a di-leucine type internalization motif,20 mediates constitutive internalization. Binding of HL-60 leukocytes (a cell line from a patient with promyelocytic leukemia that produces subcutaneous tumors in mice) triggers E-selectindependent intracellular signaling but does not affect the cytoplasmic taildependent downregulation of E-selectin surface expression.21 This differs from ICAM-1 (see below). It suggests that different interactions regulate E-selectin internalization and signaling.
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E-selectin has a special signicance to inammatory skin disease. As originally noted in 16 of 18 cases of patch/plaque stage cutaneous T-cell lymphoma but not at all in noncutaneous lymphoma,22 most CD45RO memory T cells at cutaneous sites were positive for the HECA452 antigen (representing the E-selectin ligand cutaneous lymphocyteassociated antigen CLA), an observation conrmed by others.23 A comparative immunohistochemical analysis of skin biopsy specimens (from patients with psoriasis, allergic contact dermatitis, and other chronic active dermatides) with biopsy specimens from other inamed tissues led to the hypothesis that during chronic inammation, skin is unique in favoring persistent expression of E-selectin, and that HECA452 immunoreactivity represents E-selectin ligand expression on a distinct subset of skinhoming memory T cells.24 This is now thought to be an oversimplication. During chronic inammation, E-selectin expression persists in a variety of vascular beds other than skin,5,25-29 and T cells recruited into skin represent several different subsets that vary by chemokine receptor expression.30-32 Chemokines found on dermal ECs help specify recruitment of different T-cell subsets into skin (reviewed here recently).33 The importance of selectin adhesive functions during inammation is strikingly illustrated by leukocyte adhesion deciency type II (LAD II), in which there is an absence of fucosylated glycan structures normally present on selectin ligands. LAD II neutrophils have diminished ability to roll on endothelium, and LAD II disorder is characterized by recurrent bacterial infections, persistent leukocytosis, and severe mental and growth retardation.34,35 The recruitment capabilities of E- and P-selectin differ. Both mediate the initial tethering step but by virtue of an elongated extracellular structure, P-selectin is more efcient. P-selectin mediates faster rolling (12-20 m/s by mouse neutrophils in vivo) than E-selectin (rolling speed, 5 m/s).36-38 During slow rolling on E-selectin, extended exposure to the chemokine-laden EC surface promotes integrin activation, the subsequent step of leukocyte recruitment. Migration into inamed skin of normal mice by antigen-activated, radioactively labeled CD4 T cells expressing ligands for both E- and P-selectin was inhibited by either antiP or by antiE-selectin antibody (Ab), and use of both Abs together was additive, resulting in a nearly complete reduction of recruitment.39 In E-selectindecient mice, Ab blockade of P-selectin was still required to deter neutrophil immigration,40 emphasizing overlapping (adhesive) function of the selectins. Moreover, double knockout mice (lacking E- and P-selectin genes) but not mice lacking just a single selectin gene exhibit a phe-
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notype similar to patients with LAD IIthat is, a virtual lack of leukocyte rolling, low extravasation, and ulcerative dermatitis. Rolling in E-selectin-/- mice after TNF treatment was not reduced, but double mutants showed a 46-fold reduction compared with normal littermates, versus only a 20-fold reduction found in P-selectin-/- mice.41 This is also indicative of an overlap in the adhesive functions of EC selectins.
ICAM EXPRESSION AND ADHESION

ICAM-1 (CD54) was identied as an aggregate-forming adhesion molecule expressed on lymphocytes.42 Isolation of a complementary DNA clone of ICAM-1 revealed a type I integral membrane glycoprotein with repeating Ig domains in the extracellular region, a structural signature of the Ig superfamily.43 Heterogeneity among different cell types gives rise to Mr for ICAM-1 of 97 to 114 kd, most likely resulting from differential patterns of glycosylation, since the nonN-glycosylated form resulting from tunicamycin treatment has an Mr of just 55,000.44 Low constitutive expression of E-selectin and ICAM-1 on dermal venules contributes to homeostatic T-cell immunosurveillance.45 On cultured ECs, ICAM-1 (but not E-selectin or VCAM) is constitutively expressed at low levels. ICAM-1 expression levels are upregulated up to 40-fold by TNF endothelial activation, reaching peak levels after 24 hours of TNF, a time point corresponding to downregulation of E-selectin expression.18,46 A second ICAM isoform, ICAM-2 (CD102; Mr 55-65 kd), is partially homologous to ICAM-1 but has only 2 Ig-like extracellular domains compared with 5 such domains for ICAM-1. Constitutive expression on ECs is high but is downregulated by TNF treatment.47 ICAM-1 dimerizes on the EC surface, but dimerization does not increase ligand binding.48,49 It is unknown whether ICAM-1derived signals require dimerization, or whether ICAM-1 dimerization is necessary for colocalization with an ERM complex. Unlike Eselectin, there is little or no constitutive internalization of ICAM-1 surface protein, which is relatively long-lived on ECs.17,18,50 Instead, ligand engagement may accelerate internalization of ICAM1.51 ICAM-1 clusters induced by antiICAM-1coated microspheres, but not monomeric ICAM-1, are rapidly internalized from the surface of TNF-treated HUVECs, suggesting that interactions of the ICAM-1 cytoplasmic tail important for internalization are promoted by clustering.52 Cross-linking of ICAM-1 upregulates gene expression of ICAM-1 and VCAM (but not of E-selectin) a positive feedback mechanism,53,54 which would be expected to replace ICAM-1 protein cleared by internalization. The intracellular trafcking path-
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ways of ICAM-1 protein in connection with leukocyte TEM warrants further investigation as has recently been done for a related EC CAM, platelet endothelial cell adhesion molecule (PECAM/ CD31).55 ICAM-1 is believed important for leukocyte recruitment during a wide range of inammatory and noninammatory circumstances. In humans with leukocyte deciency I (LAD I), CD18 integrin deciency reduces levels of ICAM-1 ligands on leukocytes, resulting in recurrent bacterial infections and poor wound healing. In LFA-1 decient mice, neutrophils and activated T cells are unable to cross EC monolayers in response to a chemokine gradient.56 In mouse skin inamed by a delayed-type hypersensitivity response, lymphocyte inltration is prevented by ICAM-1 (but not ICAM-2) Ab blockade.57 ICAM-1/ mice exhibit leukocytosis, lymphocytosis, and diminished tissue inltration by neutrophils despite normal production of proinammatory cytokines.58,59 Together, these data suggest a critical role for ICAM-1 in the recruitment of circulating cells from blood during skin inammation.
VCAM EXPRESSION AND ADHESION

VCAM (CD106, Mr 100-110 kd), discovered in activated HUVECs by expression cloning with monoclonal Ab (mAb) E 1/6,60 is expressed by activated ECs and follicular dendritic cells. VCAM is involved in disease pathogenesis during inammatory bowel disease, atheroscelerosis, and the asthmatic response, and in the skin during erythroderma (exfoliative dermatitis), urticaria, allograft rejection, and infection.61 The VCAM extracellular domain also contains repeating Ig domains, but because of alternate posttranscriptional splicing there are 2 VCAM messenger RNAs, a more abundant fulllength transcript and a variant that lacks exon 5. The VCAM variant maintains the same cytoplasmic domain but has a shorter extracellular domain.62 As for E-selectin, the cytoplasmic tail of human VCAM shows substantial homology across species (human, mouse, rat, and pig), with conservation at 17 of 19 amino acid residues.19 Surface protein expression of VCAM by cytokine-activated HUVECs appears to be short-lived and internalized rapidly, similar to Eselectin.50,63 ICAM-1, ICAM -2, and VCAM each promote rm adhesion to ECs. Moreover, VCAM and to a greater extent ICAM-1 probably assist leukocyte entry into the interendothelial junction (detailed below). These Ig-CAMs interact with different integrin heterodimers expressed on the leukocyte surface: ICAM-1 and ICAM-2 interact with leukocyte function-associated antigen-1 (LFA-1) expressed on all leukocytes, ICAM-1 (but not ICAM-2) interacts with macrophage
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receptor-1 (Mac-1) expressed on neutrophils, and VCAM interacts with very late antigen-4 (VLA-4) on lymphocytes, monocytes, and eosinophils, but only rarely expressed on neutrophils.* Using an in vivo migration assay, investigators demonstrated that antiVLA-4 (but not anti-LFA-1) blockade of resting T-cell migration into rat skin was further blocked by antibodies to P- and E-selectin, suggesting overlapping functions of VCAM (but not of ICAMs) with the endothelial selectins.64 Indeed, because of multiple VLA-4 activation states, VCAM/VLA-4 binding is highly versatile and can mediate tethering, rolling, and rm arrest under ow conditions in vitro.65,66 The VCAM knockout mouse dies early during embryonic development, revealing little about VCAM function,67 but in triple-selectin null mice, the small amount of residual rolling is dependent on VCAM interactions.68 In summary, the adhesive functions of E-selectin, VCAM, and ICAM-1 are sequential and redundant. The overlapping functions of these EC CAMs may prevent functional inadequacy, ensuring successful recruitment of different leukocytes under varied pathologic states. Conversely, in the absence of inammation, strict control of EC CAM gene expression normally protects vascularized tissue from unwarranted and potentially harmful contact with bloodborne leukocytes. Before discussing how EC CAMs activate intracellular signaling pathways, we will outline similar signals present in ECs during vascular leak and consider the concurrent activation of leukocytes.
LEUKOCYTE ACTIVATION IN RESPONSE TO EC CONTACT

It has been known for some time that the ability of T cells to leave the circulation depends on their activation state.69 Activation occurs during rolling, when leukocytes encounter chemoattractants (C5a, platelet activating factor, leukotriene B4, formyl peptides) and chemoattractant cytokines (chemokines; short, 70- to 120amino acid single-chain peptides) that attach to and oligomerize on heparan sulfate proteoglycans of the luminal EC surface.7,70,71 Chemokines bind to specic leukocyte receptors that trigger heterotrimeric G-protein dependent leukocyte signaling.72,73 Such signals lead to clustering, and greater afnity/avidity of the integrins LFA-1, Mac-1, and VLA-4 for their cognate Ig-superfamily EC-CAMs.74-76 Other intracellular events signaling activation are induced in leukocytes during E*There are several integrin nomenclatures. LFA-1 is also CD11a/CD18 or 12, and Mac-1 is also CD11b/CD18 or M2. The VCAM ligand VLA-4 is also CD49d/CD29 or 41.
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selectin tethering,36 VLA-4 cross-linking,77,78 or LFA-1 binding to ICAM-1.79 In sum, leukocyte-EC encounters generate bidirectional activation signals, but until the advent of recent EC CAM studies, chemokine and integrin-activated pathways in leukocytes have received far greater attention. In vivo, TEM occurs under ow conditions characterized by measurable levels of uid shear stress. Shear stress is likely to inuence control of leukocyte TEM by ECs, since within 5 minutes it generates EC signals that causes EC elongation.80 A new in vitro model to study leukocyte transmigration under ow was established recently.81 Using a similar ow model, our group prepared retrovirally transduced HUVECs that constitutively (in the absence of cytokine) express E-selectin, ICAM-1, and VCAM-1, either in pairwise combinations or in triple combination. We nd that pairwise expression increases TEM and that triple adhesion moleculeexpressing cells are able to support CD4 T-cell TEM equally well as TNF-treated ECs. These unpublished observations suggest that adhesion molecule expression can alone account for the proinammatory effects of TNF on recruitment of chemokine-activated T cells by ECs, and that there is overlap in the functions of E-selectin, ICAM-1, and VCAM-1 in the recruitment of CD4 T cells.
RELATIONSHIP OF VASCULAR LEAK AND LEUKOCYTE TRANSMIGRATION

Vascular leak refers to the transendothelial passage of blood macromolecules into extravascular tissue, commonly seen as edema. InterEC contacts normally act as a barrier to leukocyte transmigration and to vascular leak. It is a current working hypothesis that during vascular leak and leukocyte TEM, interendothelial contacts are reduced by different processes of EC activation (Fig 2).8,82,83 Briey, chemical mediators such as thrombin or histamine induce vascular leak. For example, histamine released by degranulation of resident mast cells is taken up by receptors located on postcapillary venular ECs.84,85 Blood ow increases with nitric oxidemediated vessel dilation, and permeability to macromolecules increases as gaps form between EC neighbors, but only in a transient reversible manner (1530 minutes). This coincides with escape of exudate, a protein-rich uid containing blood proteins such as bronectin and brin, from blood into the interstitium. These integrin ligand proteins form a provisional extravascular matrix allowing for the subsequent efcient migration of extravasated leukocytes toward the site of injury in dermis or beyond.
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FIGURE 2. Gap formation between adjacent endothelial cells (ECs) occurs during vascular leak and leukocyte transendothelial migration (TEM). A, The integrity of EC junctions controls the barrier function of vascular endothelium, separating macromolecules and circulating leukocytes in blood from the underlying tissue. B, During early inammation, histamine from mast cells, or later in inammation, tumor necrosis factor (TNF) from macrophage or T cells, is taken up by specic receptors located on ECs. These inammatory mediators trigger EC signals that lead to gap formation and leakage of blood macromolecules into the extravascular space. Vascular endothelial (VE)-cadherin expression on the plasma membrane surface of adjoining ECs normally maintains barrier function, but is reduced during vascular leak. C, Leukocyte ligand engagement of cytokine-induced adhesion molecules (CAMs) expressed on the luminal EC surface triggers EC signal pathways similar to those occurring in vascular leak that also lead to gap formation, but only at ECs contacted by captured leukocytes. The short cytoplasmic tails of E-selectin, vascular cell adhesion molecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1) are required for these signals to occur. Cytoskeletal reorganization, redistribution of VEcadherin and induction of matrix metalloproteinases (MMPs) are believed to coordinate EC gap formation with initiation of TEM by captured leukocytes localized to the interendothelial junction.
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The scientic community is still divided about whether the most important regulation of vascular leak and transmigration occurs at EC contacts. An alternative explanation proposed is transcellular passage of macromolecules through a vesiculo-vaculoar organelle network within ECs.86 Similarly, neutrophil transmigration has been observed (primarily in vivo) to occur via a transcellular route.87 Data gathered by electron microscopy include detailed descriptions of transmigration directly through thinned regions of individual ECs.88 But different observations made in vitro show that neutrophil TEM also occurs through junctions, often at tricellular junctions that may differ in their cell-cell contacts.12,14,89 Despite these distinct recruitment patterns, signaling by EC CAMs and control of EC junctions remain important mechanistic components even for neutrophils.90-92 One signal pathway leading to EC gap formation common to histamine-induced vascular leak and CAM signaling during leukocyte recruitment is the rapid and transient elevation of intracellular free calcium concentration (Cai).93-95 Another form of vascular leak involves the Rho/Rac signaling pathways and occurs later in inammation after EC activation by cytokines (TNF, interleukin-1 [IL-1], or IL-2). Increasing Cai and activating Rho/Rac can lead to reorganization of the actin cytoskeleton, contraction, and elongation, causing ECs to take on a broblast-like appearance.10,80,95-97 In quiescent ECs, the actin cytoskeleton appears as a meshwork of dense peripheral bands outlining individual ECs. Treatment with TNF- causes the actin-based cytoskeleton to polymerize, cross-link, and reorganize into a conguration referred to as stress ber formation.96-98 Stress ber formations also occur after EC activation upon ligand engagement of VCAM and ICAM (see below). The third major mechanism common to vascular leak and CAM signaling during leukocyte recruitment consists of adhesive interactions across EC-EC junctions. Vascular endothelial (VE)-cadherin (CD144), expressed at adherens junctions only by ECs, is of central importance to junctional integrity during both vascular leak and leukocyte transmigration.8,99,100 VE-cadherin is a transmembrane glycoprotein linked to - and -catenin that associates with the actin cytoskeleton through -catenin.83,100 In vivo evidence for the importance of VE-cadherin as a primary mediator of EC junctional contact is that mAb against VE-cadherin accelerates neutrophil recruitment into the inamed peritoneum.101 Reports that neutrophil adhesions to HUVECs disrupt the EC junction through a VE-cadherindependent mechanism102,103 were disputed by a study showing that these data derived from a postxation artifact caused by detergent lysis
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release of a neutrophil protease.104 Fixation artifacts were circumvented by using real-time imaging under ow to show that human neutrophils and monocytes transmigrate through transient gaps formed upon redistribution of a VE-cadheringreen uorescence protein (GFP) construct.14 The same group later showed that (unexpectedly) there was no defect in transmigration under ow with neutrophils derived from mice decient in neutrophil elastase and matrix metalloproteinase9.105 These studies bolster the concept that control of EC junctional integrity by VE-cadherin is important during TEM. Two other junctional molecules that appear to function sequentially during TEM as leukocytes pass down through adjoining ECs are PECAM-1 and CD99, reviewed elsewhere.10,11 Leukocyte TEM is not believed to cause vascular leak. Instead, mechanisms distinct from TEM have been proposed to explain cytokine- and neutrophil-induced vascular leak.106-109 For example, no permeability increase of EC monolayers was observed as a result of neutrophil TEM.110-112 These investigators observed transient formation of inter-EC gaps that were restricted just to those ECs participating in TEM. Therefore, activation of the mechanism of EC gap formation during TEM must be similarly restricted. Leukocyte engagement of E-selectin, VCAM, and ICAM-1 occurs specically in those ECs participatory to recruitment, suggesting an elegant solution to this problem. EC CAM engagement by leukocyte ligand (or Ab cross-linking) induces CAM signals that reduce barrier function at interendothelial junctions by removal of junctional VE-cadherin and by actin cytoskeleton reorganization, processes believed to control EC shape changes pertinent to TEM.
SIGNALING MECHANISMS OF ENDOTHELIAL CAMs: OVERVIEW

Coordination of extracellular CAM adhesions with intracellular actin cytoskeleton organization requires transmission of inbound signals. E-selectin, VCAM, and ICAM-1 each have short intracellular domains (tails) to assist this communication. Mutation/deletion studies of E-selectin or ICAM-1 have shown cytoplasmic taildependent activation of EC signal pathways,21,49,113 and the cytoplasmic tail of VCAM mediates interactions with effectors of actin reorganization.114 These EC CAMs produce 3 signals critical for cytoskeletal reorganization: transient elevation of intracellular Ca levels, activation of Rho/Rac signaling, and phosphoinositide messengers. Increase in Cai and activation of Rho or Rac by EC CAMs can lead to reorganization of the actin cytoskeleton and endothelial gap formation. EC CAMinitiated of Rho/Rac activation also can generate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2), an addi-
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tional mediator of actin reorganization. Finally, Rho, Rac, and phosphoinositide signals can each activate the ERM family of cytoskeletal adaptor proteins to form a plasma membrane complex linking clustered EC CAMs to the actin cytoskeleton. The many signaling events stemming from the cytoplasmic tails of E-selectin, VCAM, and ICAM discussed below are summarized in Table 1 and Fig 3.
TABLE 1. Signals and Events After Ligand Engagement or Ab Cross-linking of EC CAMs EC CAM E-selectin Signal or Event Cytoplasmic taildependent EC activation and cytoskeletal linkage Increased [Cai] EC shape change and stress ber formation E-selectin redistribution to specialized plasma membrane regions enriched for caveolin-1 and PLC activation VCAM Cytoplasmic taildependent EC activation and actin reorganization Increased [Cai] ROS formation MMP production Rac activation References and Comments 21
121, 122. Also in vascular leak: 93-95 121, 122 21, 125, 126
114, 130 122, 123, 130 130, 139 135, 136 97, 123, 137. Also in vascular leak: 97 137, 139, 140. Also in vascular leak: 99, 100 165 114
VCAM- and/or Rac-derived VEcadherin redistribution away from interendothelial junctions Rac-induced ERM complex formation VCAM interaction or colocalization with ERM complexes in a specialized plasma membrane structure EC shape change and transient gap formation assisting leukocyte entry
14, 137, 139, 140
(continued)

ICAM-1 Cytoplasmic taildependent EC activation and actin reorganization Increased [Cai] ICAM-1 dimerization ROS formation Rho activation, EC shape change, and stress ber formation Rho-induced ERM complex formation ICAM-1 interaction or colocalization with ERM complexes in specialized plasma membrane structures Transient gap formation and/or assisting leukocyte entry to EC junctions ICAM-1 signals leading to changes in gene expression 49, 113 9, 53, 98, 124 48, 49 90, 91 49, 142, 145. Also in vascular leak: 80, 96, 97 163, 164 114, 125, 128, 160, 181, 186 14, 124, 189, 190 53, 54
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Abbreviations: Ab, Antibody; EC, endothelial cell; CAM, cytokine-induced adhesion molecule; VCAM, vascular cell adhesion molecule; [Cai], intracellular free calcium concentration; PLC, phosphatidylinositol-phospholipase C; ROS, reactive oxygen species; MMP, matrix metalloproteinase; VE-cadherin, vascular endothelial cadherin; ERM, ezrin, radixin, and moesin; ICAM-1, intercellular adhesion molecule-1.
CALCIUM SIGNALING IN ECs DURING LEUKOCYTE RECRUITMENT

Calcium, a key second messenger in many cell types, is a critical regulator of EC junctional integrity. In quiescent ECs, a transient rise in Cai from less than 100 nmol/L to approximately 5- to 10-fold higher decreases EC barrier function.95 The level of Cai increases during leukocyte ligand adhesion, transmigration, or both, and neutrophil TEM is inhibited by a cell permeant Ca buffer.111 Further, neutrophils induce phosphorylation of specic serine/threonine residues on EC myosin light-chain kinase (MLCK), suggesting that Cai ux can lead to actin-induced cytoskeletal contractility through phosphorylation of myosin light chains.115 (The myosin light chain is the regulatory part of the myosin molecule. Upon phosphorylation, it induces actin contractility through myosin conformation and sliding along lamentous [F-] actin, a exible helical polymer composed of 5- to 9-nm-diameter globular [G-] actin monomers.)116 Two different teams used inhibitors of endothelial MLCK to assess this pathway. Neutrophil TEM across bovine pulmonary
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FIGURE 3. Integration of endothelial cell (EC) cytokine-induced adhesion molecule (CAM) adhesion and signaling. Intercellular adhesion molecule-1 (ICAM-1) expressed on the luminal surface of cytokine-activated ECs mediates the rm binding of leukocytes via cognate leukocyte ligands, triggering cytoplasmic domain-dependent intracellular signals in ECs. Left, Rho activation and a transient increase in intracellular free calcium concentration (Cai) lead to actin reorganization. Downstream of Rho, Rho kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) interact with folded, unassembled monomers of ezrin, radixin, and moesin (ERM), causing their phosphorylation, unfolding, and head-to-tail assembly into a complex that relocates to the inner leaet of the plasma membrane. Once relocated, the ERM complex supports further actin reorganization. Right, Actin reorganization leads to EC shape change and EC gap formation that eases leukocyte transendothelial migration (TEM) at interendothelial junctions. ICAM-1 redistributes, colocalizing with ERM complexes in cup-shaped regions of plasma membrane that serve as docking sites for adherent leukocytes en route to transmigration. ICAM-1 homodimers form upon ligand engagement, possibly enhancing cytoplasmic domaindependent signaling. Positive feedback signal pathways include ongoing Rhomediated actin reorganization through inhibition of Rho guanine dissociation inhibitor (GDI) and auto-upregulation of ICAM-1 gene expression. Different contributions by vascular cell adhesion molecule (VCAM), E-selectin, and other EC CAMs to EC regulation of leukocyte TEM also occur (see text) but are omitted for clarity. Molecular interactions shown are either direct (solid arrows) or omit intermediary steps (dashed arrows). Abbreviation: MLCK, Myosin light-chain kinase.
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arterial ECs was reduced by specic inhibitors of MLCK and promoted by inhibitors of a myosin-associated phosphatase.92 Pretreatment with a different MLCK inhibitor reduced F-actin formation, MLC phosphorylation, and neutrophil TEM across HUVECs cultured on an amniotic membrane substrate.117 Whether initiated by neutrophil products, EC CAMs, or both, it is noteworthy that elevated Cai was seen to occur in ECs making direct contact with transmigrating neutrophils.118 Mechanisms of actin reorganization may be EC-type specic. During neutrophil adherence to TNFactivated human pulmonary microvascular ECs, changes observed in the F-actin cytoskeleton were phosphoinositide dependent but Cai independent.90,119 This discrepant result may be explained by the use of microvascular ECs (with no chemoattractant), which differs from studies nding Ca-dependent transmigration that involved large vesselderived HUVECs (which were exposed to chemoattractant).
CALCIUM SIGNALING BY E-SELECTIN, VCAM, AND ICAM-1

E-selectindependent leukocyte adhesion does not require cytoskeletal interaction.120 Hence, the evolutionary conservation of the short E-selectin cytoplasmic tail19 likely relates to control of surface expression by endocytosis, intracellular signaling, or both. Crosslinking E-selectin expressed on IL-1treated ECs increases Cai and causes EC shape change.121 Using Fura-2loaded ECs, investigators found that cross-linking of HUVEC VCAM and E-selectin (but not ICAM-1 or PECAM) raised Cai and caused stress ber formation.122 Neutrophil and monocyte adhesion also induced these changes, which were inhibited by mAb blockade of E-selectin ligand binding. VCAM- and ICAM-1induced Ca signaling in ECs has been described by others.98,123 For example, Ab blockade was used to show that an increase in HUVEC Cai was derived from lymphocyte adhesion to ICAM-1.98 Cross-linking of ICAM-1 on HUVECs and on mouse brain ECs each induces a rapid increase of Ca concentration.9,53 ICAM-induced Ca signaling appears important for TEM but not for leukocyte adhesion, since calcium chelator pretreatment of 2 different rat brain EC lines reduces ICAM-1dependent TEM but not adhesion of lymphocytes.124 In this system, cross-linking of ICAM-1 resulted in tyrosine phosphorylation activation of phosphatidylinositol-phospholipase C (PLC)1, which mediated release of Ca stores via inositol 1,4,5-triphosphate (IP3).
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REDISTRIBUTION AND CLUSTERING OF E-SELECTIN

During leukocyte recruitment, there is cytoplasmic taildependent redistribution of CAMs expressed on the apical EC surface. Wildtype (but not a cytoplasmic deletion) E-selectin construct clusters at the site of HL-60 leukocyte adhesion and links to the actin cytoskeleton.21 These clusters assemble in cholesterol-containing lipid rafts,125,126 a type of cell surface microenvironment that favors cytoskeletal interactions and signal cascade activation.127 More precisely, ligand-induced clustering redistributes E-selectin to caveolin-1containing rafts where it associates with and activates PLC. Since PLC hydolyzes PtdIns[4,5]P2, resulting in IP3 production and subsequent release of stored Cai, the observed E-selectininduced rise in Cai121,122 may derive from the E-selectin subpopulation localized in caveolin lipid rafts. Consequently, it will be important to determine whether the E-selectin cytoplasmic tail sequence contains a binding site for PtdIns[4,5]P2 as already described for ICAM-1.128
SIGNALING BY VCAM
In a VCAM-dependent static transmigration model, resting (ie, not stimulated by antigen recognition) mouse splenic lymphocytes require VLA-4 interaction with VCAM to spontaneously bind and transmigrate across lymph nodederived mouse EC lines.129 Minus cytokine activation, these lines (mHEVa and mHEVc) constitutively express VCAM, but not other EC CAMs (ie, P- and E-selectin, ICAM1, mucosal vascular addressin cell adhesion molecule [MAdCAM-1; an EC receptor for 4 integrin], and PECAM-1). Lymphocyte TEM mediated by VCAM was dependent on EC calcium ux and reactive oxygen species (ROS) production, but not on tyrosine kinase or phosphatidylinositol-3-kinase (PI3K) activity, based on EC pretreatment with either herbimycin A (a tyrosine kinase inhibitor) or wortmanin (a PI3K inhibitor).130 ROS production indicates EC activation. NADPH oxidase is an enzyme oxidizer of the reduced form of the electron carrier nicotinamide adenine dinucleotide phosphate (NADPH) and of other substrates. In professional phagocytes it catalyzes the production of the ROS superoxide, which when dismutated becomes hydrogen peroxide.131,132 ECs may express NADPH oxidases similar to those in phagocytes.133 In general, cross talk between the cellular redox state and other signaling pathways can impair EC barrier function, and dihydrorhodamine-123, a membrane-permeable peroxide indicator, has been used to show that VCAM cross-linking leads to EC
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ROS production, and that ROS production is critical for VCAMmediated TEM. ROS can relax constraints on EC positioning by the activation of EC matrix metalloproteinases (MMPs).132,134 MMPs function as important effectors for degradation of basement membrane during TEM.135,136 Just below the site of lymphocyte binding there appears to be a coalescence of endothelial actin indicative of the VCAM cytoskeleton interaction.130 VCAM-dependent ROS production may promote this coalescence through the Rho-related guanosine triphosphatase (GTPase), Rac.123,137 Like the related small GTPase Rho (discussed below), Rac is a molecular switch associated with reorganization of actin, dispersion of cadherin from intercellular junctions, and gap formation.97 Of interest is that the Rho/Rac ratio appears to be an especially sensitive barometer for actin contractility and junctional integrity; the activation of one GTPase can lead to the inactivation of the other, and dual activation may have opposing effects on barrier function.85,138 VCAM-derived Rac signaling induced by cross-linking, or by the cell permeant constitutively active Tat-RacV12 chimeric peptide, each lead to HUVEC gap formation secondary to loss of junctional VE-cadherin localization.137,139,140 In an experiment comparing constitutively active peptides, Tat-RacV12 uptake induced stress ber formation, VE-cadherin redistribution, and EC gap formation, but a comparable Rho-based peptide (Tat-RhoV14) did not induce gap formation, only stress bers without redistribution of VE-cadherin.139 VE-cadherin redistribution initiated by Rac peptide was shown to be ROS dependent (through use of the oxygen scavenger N-acetylcysteine). In keratinocytes, Rac activation sufced to disassemble cadherin-mediated contacts.141 Rho-based peptide actin stress ber formation without VE-cadherin redistribution implies that during HUVEC gap formation, distinct signals control actin contractility and VE-cadherin redistribution. During leukocyte TEM, transient interendothelial junctional gap formation is hypothesized to require release and redistribution of VE-cadherin.8,12,99,102 Based on these observations, VCAM-dependent ROS and MMP production may coordinate EC gap formation with adhesion and TEM by VLA-4 T cells/monocytes, which show a greater preference for transmigration at the site of de novo gaps than neutrophils.14 EC gap formation may be less important for VLA4independent neutrophil TEM, which may not require VCAMinduced Rac signaling or EC-derived ROS (although ICAM-1 on ECs can also generate ROS).90 Further study is warranted on how VCAMinduced Rac activation leads to VE-cadherin redistribution and EC gap formation.
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RHO SIGNALING BY ICAM-1

Neutrophil TEM, but not brinogen-dependent adhesion, is abolished by deletion of the ICAM-1 cytoplasmic region from ICAM-1 transfected Chinese hamster ovary cells.142 The Rho inhibitor C3 was used in this study to show that neutrophil TEM required Rho activation, and to implicate Rho as a downstream effector of the ICAM-1 cytoplasmic domain in ECs. Rho refers to 3 Rho isoforms, Rho A, B, and C, and the specicity of C3 for Rho is at least 100 times more efcient that that for Rac or Cdc42, other small GTPases (C3 is a bacterial toxin from Clostridium botulinum that inactivates Rho by adenosine diphosphate ribosylation at amino acid 41).143,144 C3 inhibition of Rho signaling can block lymphocyte TEM and formation of actin stress bers initiated by mAb cross-linking of ICAM-1.145 This group also showed that transfection of rat brain microvascular ECs with a full-length, but not a cytoplasmic-deleted ICAM-1 construct, confers TEM. Results were consistent in ICAM-1/ICAM2-double-decient mouse brain endothelioma cells.49,113 Unexpectedly, the lone cytoplasmic tyrosine in ICAM-1 remains unphosphorylated during ICAM cluster formation.49,145 In summary, the ICAM-1 cytoplasmic domain is essential for EC regulation of TEM, Rho is a key downstream mediator of ICAM-1, and tyrosine phosphorylation of ICAM-1 does not occur. So how does Rho forward ICAM-derived signals to effector molecules, and what downstream events are initiated? Rho is a molecular switch, an evolutionarily conserved member of the Ras superfamily of small (20-25 kd) monomeric cytoplasmic GTPases. Twenty different Rho-related related GTPases have been identied that are referred to as either Rho-, Rac-, or Cdc42-GTPases, each of which regulates different signal transduction pathways linking cell surface protein receptors to the assembly state of a lamentous actin cytoskeleton. When bound to GTP, Rho is in an active state, and when bound to guanosine diphosphate (GDP), Rho is in an inactive state. Rho turns off by GTP hydrolysis, which generates the inactive Rho-GDP form. Rho GTPase activity is regulated by several other molecules: guanine nucleotide exchange factors activate by catalyzing the exchange of GDP to GTP, GTPase-activating proteins inactivate by stimulating GTP hydrolysis, and guanine dissociation inhibitors (GDIs) sustain inactivation by sequestering inactive Rho-GDP away from the plasma membrane.144,146 Rho-binding proteins identied by rigorous biochemical methods include the serine/threoninedirected Rho kinases (Rho kinase/
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ROCKII and p160ROCK/ROCK), together referred to as Rho kinase.144 Rho kinase functions immediately downstream of Rho and (like the calcium-calmodulin pathway) mediates MLC phosphorylation via phosphorylation/activation of MLCK.85 Rho/Rho kinase can also reorganize actin along a parallel route by threonine/serine phosphorylating (inactivating) myosin light-chain phophatase, which serves to inhibit dephosphorylation of MLC.147,148 Pharmaceutical inhibitors of the Rho pathway can be used to reduce TEM. MLC phosphorylation, actin polymerization, and neutrophil TEM were reduced by treatment of ECs with C3 or with Y-27632.149 Used to dissect ICAM-1 signaling pathways, the pyridine derivative Y-27632 is a Rho kinase inhibitor 200 times more selective for inhibition of Rho kinase than for protein kinase C or MLCK.150,151 Inhibitors of MLCK diminished neutrophil migration across a transwell of conuent bovine pulmonary ECs induced by the chemoattractant leukotriene B4 by 30% to 70%.92 In the same study, an inhibitor of myosin-associated phosphatase (calyculin) that increased phosphorylation of myosin light chains, caused EC contraction and enhanced neutrophil migration. In response to TNF, the effects of Rho activation on actin reorganization include gap formation at EC junctions,97 and it is tempting to speculate that ICAMinduced Rho signaling, necessary for ICAM-1dependent TEM, may similarly induce gap formation. However, Rho inhibition can, under certain circumstances, lead to Rac activation.139,152 So further sorting out of the relationship of these molecular switch molecules Rho and Rac, which lie downstream of ICAM-1 and VCAM, will be necessary.
RHO/RAC SIGNALING THROUGH PtdIns[4,5]P2

Along with calcium, Rho, and Rac, PtdIns[4,5]P2 is now recognized as a major regulator of the actin cytoskeleton.153,154 Although traditionally PtdIns[4,5]P2 has been viewed as a substrate for synthesis of IP3 and diacylglycerol, PtdIns[4,5]P2 is also a downstream effector of Ca ux or Rho/Rac cytoskeletal reorganization.154-156 Rac interacts with type I phosphatidyl-4-phosphate 5-kinase (PtdIns[4]P 5-kinase), and PtdIns[4]P 5-kinase (acting through PtdIns[4,5]P2) induces actin lament uncapping and assembly in permeabilized platelets.157 Conversely, in mouse-derived, immortalized NIH 3T3 broblasts, dominant negative Rac1 decreases PtdIns[4,5]P2 levels by 50% (mimicking the effect of broblast detachment from substrate) because of a reduction in the enzymatic activity of PtdIns[4]P 5-kinase.158 Like Rac, Rho has also been reported to regulate PtdIns[4,5]P2 via PtdIns[4]P 5-kinase.155
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ERM PROTEIN ACTIVATION AND ERM COMPLEX FORMATION OCCURS VIA RHO/RAC AND PtdIns[4,5]P2
The ERM family of homologous, interactive proteins regulates cell shape and cell adhesion by linkage of membrane adhesion receptors to the actin cytoskeleton. ERM protein cytoskeletal linkages occur as a complex on the cytoplasmic side of the plasma membrane where they localize in specialized EC-protrusive membrane structures (eg, microvilli or docking structures) that can encompass ICAM-1, ICAM-2, and VCAM (Fig 3, right panel).114,159,160 At the N- and Cterminal ends of their roughly 600 amino acids are 2 different interactive ERM association domains (known as N- and C-ERMADs). The bulk cytoplasmic pool of ERM protein appears as folded, nonphosphorylated monomers resulting from intramolecular association of N- and C-ERMADs on each ezrin, radixin, or moesin molecule. Structure/function studies indicate that folding normally masks ERMAD sites, shielding them from dimerization or interaction with transmembrane adhesion molecules.161,162 Rho/Rho kinase can initiate unfolding and head-to-tail association by ERM protein phosphorylation at a COOH-terminal threonine conserved in all 3 ERM proteins (T558 in moesin);163,164 Rac can activate moesin in a similar manner.165 Once unfolded, they are unmasked and active,166-168 can form homo- or hetero-oligomers,169 and can translocate to the cytoplasmic side of the plasma membrane as an ERM complex.164,170 Unfolding also renders accessible a 35amino acid C-terminal region containing a functional F-actin binding site.171-173 Like Rho/Rho kinase signaling, PtdIns[4,5]P2 also unmasks an N-ERMAD domain membrane binding site (in ezrin). Direct interaction of PtdIns[4,5]P2 with ezrin was shown by mutagenesis of the PtdIns[4,5]P2 binding site in the ezrin NH2-terminal domain. By deletion and terminal truncation mutagenesis, the ezrin/PtdIns[4,5]P2 binding sites were localized to regions containing KK(X)(n)K/RK amino acid motifs, and amino acid mutagenesis was found to alter cellular localization.174 ERM proteins are a control point for cell shape and actin contractility. ERM proteins bind F-actin in vitro and colocalize with actin at the cytoplasmic surface of the plasma membrane in many cell types.116,175,176 A clever experiment was performed to identify moesin as an effector of actin cytoskeleton reorganization. Constitutively active Rac and Rho proteins were known to cause actin reorganization, stress ber and lamellipodia formation in digitoninpermeabilized, serum-starved Swiss 3T3 cells. This ability was lost after cell permeabilization with digitonin but was restored by recon-
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stitution with cytosolic extract, the active component of which, when biochemically isolated, was found to be moesin.165 In summary, Rho/Rho kinase and PtdIns[4,5]P2 each promote unfolding, complex formation, and plasma membrane translocation of ERM proteins to sites of plasma membrane adhesion molecule expression where they can interact with the actin cytoskeleton to affect changes in cell shape.
OTHER FUNCTIONS OF THE ERM COMPLEX

ERM proteins can sequester Rho GDI, thereby shifting Rho equilibrium toward an active state by reducing Rho GDI-mediated inhibition of the conversion of Rho-GDP to Rho-GTP.170,177 This suggests that the assembled ERM complex can amplify Rho/Rac signals via a positive feedback loop. Redundancy of individual ERM protein function is suggested by moesin-knockout mice, which show normal development and are unimpaired in certain ERM functions.178 Hence, to genetically test the individual role in integration of CAM adhesions and cytoskeletal contractility by ERM proteins, it may be necessary to produce an ERM triple knockout mouse.
VCAM AND ICAM CLUSTERING AND ERM COMPLEX ASSOCIATION

In general, studies involving interactions between cytoskeletal laments and transmembrane glycoproteins have shown that physical associations do not occur directly but instead involve adaptor molecules linking transmembrane proteins to the cytoskeleton.179 One such adaptor protein is -actinin, a homodimer equipped with 2 domains per subunit: a globular actin-binding region and a rod-shaped domain that binds to the cytoplasmic tails of transmembrane proteins.180 ICAM-1 surface distribution mediated by -actinin cytoskeletal association was demonstrated rst in transfected COS cells by immunouorescence. A wild-type ICAM construct was found localized to specialized membrane regions of microvilli, whereas a glycophosphatidyl-anchored ICAM-1 construct, without any cytoplasmic domain, showed a uniform surface distribution.181 Microvilli localization of ICAM-1 required an intact actin cytoskeleton (shown by disruption of the actin cytoskeleton with cytochalasin B). Direct association of puried -actinin with ICAM-1 cytoplasmic tail peptides and full-length ICAM-1 suggested that juxtamembrane positively charged amino acid residues of the ICAM-1 cytoplasmic tail were interactive. ERM adaptor protein binding to transmembrane molecules was established by nding that in vitro, moesin bound to the cytoplas-
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mic domain of CD43, ICAM-2, and CD44,182 and by mapping the ezrin-binding site to a membrane-proximal 9amino acid region within the CD44 cytoplasmic domain.183 A PtdIns[4,5]P2-dependent association of ezrin with ICAM-1 was convincingly demonstrated by afnity precipitation of ezrin with an ICAM-1 cytoplasmic domain peptide and by direct interaction using surface plasmon resonance. Specic amino acid residues were not identied, but basic and hydrophobic residues resembling the PtdIns[4,5]P2 consensus binding sequences of K/RXKX(K/R)(K/R) or KX(3)KXKK184 were noted as juxtamembrane in the ICAM-1 cytoplasmic tail sequence. In the proposed model, ERM-induced ICAM-1 clustering (observed on the tips of transfected COS cell microvilli) might magnify ICAM-1 adhesiveness, thereby increasing the efciency of lymphocyte recruitment by ECs during inammation.128 Direct interaction of VCAM with moesin and ezrin (the predominate ERM proteins found in ECs) was shown by a glutathione-S-transferase-VCAM cytoplasmic tail construct pull down of moesin/ezrin and by coimmunoprecipitation of VCAM with moesin or ezrin from protein lysates of activated HUVECs.114 VCAM/ERM binding potentially is mediated by a serine-rich portion of the VCAM cytoplasmic domain (S317 through S326; SYSLVEAQKS) that resembles a serine-rich cytoplasmic motif in ICAM-3, critical for ERM interaction.185 Indirect interactions with either ICAM-1 or VCAM may also occur through intermediaries such as EBP50 (ezrin-binding phosphoprotein 50) or the similar E3KARP.186-188 Just as ligand-induced E-selectin clustering affects redistribution to specialized membrane compartments enriched for caveolin1, ligand-induced clustering of ICAM-1 and VCAM has been observed to coincide with localization to other specialized EC membrane structures (eg, microspikes, microvilli). Lymphocyte adhesion to HUVECs induces VCAM and ICAM-1 colocalization with intracellular moesin and ezrin in a cup-shaped region of membrane specialization that reaches upwards along the z-dimension in 3-dimensional confocal images, and functions as a lymphocyte docking structure.114 Formation of this structure relies on Rho/Rho kinase and PtdIns[4,5]P2 signal pathways. A similar endothelial structure encircling leukocytes was visualized on microvilli, except that this cup-like region of membrane specialization formed via ICAM LFA-1 interactions, independently of VCAMVLA-4.160 Dependence on Cai (rather than on Rho/Rho kinase) suggests that ICAM-1 localization to EC microvilli may entail an ICAM-1dependent calcium/PLC pathway.124
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EC CAMs WITHIN THE ERM COMPLEX GUIDE ADHERENT LEUKOCYTES

The ICAM-1/LFA-1 interaction seems to follow the leukocyte up to the moment of transmigration when it squeezes through adjacent ECs.189,190 This was rst suggested by immunoelectron microscopic analysis in which VCAM-1 was localized on the apical EC surface but absent from the precise EC region in which leukocyte transmigration was in progress.189 In contrast, ICAM-1 showed continuous contact with T cells on the EC membrane regions where T-cell migration progressed into EC-EC junctions. Recently, these observations have been extended to include VCAM and ICAM-1 distributed within the ERM complex. During rm adhesion of VLA-4/LFA-1 lymphoblasts to HUVECs, VCAM and ICAM-1 were found clustered with intracellular moesin and ezrin; VCAM participated in the cupshaped docking structure elevated over the apical surface that strengthened over time, but only ICAM stayed bound to the lymphocyte and followed moesin into the cleft. Further, with the use of HUVECs transfected with either VCAM-GFP or ICAM-GFP, it was shown by time-lapse confocal microscopy and real-time video microscopy that only ICAM followed the lymphoblast downward into the interendothelial cleft, and that VCAM did not participate in the transmigration act per se.114 Overall, these real-time observations of dynamic lymphoblast associations by VCAM and ICAM from within an ERM docking structure support earlier impressions that VCAM and ICAM-1 each mediate leukocyte rm adhesion and redistribute in response to EC cytoskeletal remodeling before TEM, but that contact with ICAM-1 and not VCAM, continues as the leukocyte enters into the junctional cleft (see the online video clip at http://www .jcb.org/cgi/content/full/jcb.200112126/DC1).
SUMMARY: THE ERM COMPLEX INTEGRATES ADHESIVE AND SIGNALING FUNCTIONS OF VCAM AND ICAM-1
This chapter highlights a less explored facet of the EC CAMs Eselectin, VCAM, and ICAM-1: their role as intracellular signal transducers during leukocyte recruitment. EC CAM signal pathways come full circle by inuencing their own distribution through induction of the ERM complex. Specically, formation of the ERM complex is a consequence of leukocyte ligand binding that occurs downstream within the Rho/Rho kinase, PtdIns[4,5]P2, and Ca pathways. Complex formation serves to usher in transmigrating leukocytes by clustering of ICAM and VCAM into a dynamic membrane docking structure. CAM-induced Rho/Rac and Ca signals directly
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regulate actin contractility, and EC-CAM/ERM association superimposes extra layers of control; the ERM complex serves as a bridge from EC CAMs to the cytoskeleton and secondly, amplies Rho activation through sequestering of Rho GDI. In sum, the ERM membranecytoskeletal adaptor proteins integrate timing of dynamic ECleukocyte docking adhesions with cytoskeletal remodeling to initiate TEM. Other important EC molecules are also likely to be important but were not cited because of space limitations.
CONCLUSIONS
What has all this got to do with the world of dermatology? These data offer insight as to how vascular ECs regulate leukocyte entry into skin from blood. The logical connection is that solving the mechanisms of vascular EC biology is central to increasing understanding of inammation, and that inammation forms the basis for much of skin disease. In a new paradigm, ECs are active participants during leukocyte TEM, and the cytoplasmic tails of E-selectin, VCAM, and ICAM-1 activate intracellular signaling pathways. Just as the new biologic drug for psoriasis, efalizumab, targets extracellular CAM adhesions by blocking leukocyte LFA-1 interaction with ICAM-1 on ECs,191,192 the cytoplasmic domains of EC CAMs and their downstream effectors represent new and potentially benecial intracellular therapeutic targets for treating diseases of the skin. ACKNOWLEDGMENTS The author thanks Brad Rosenberg and Jeff Schechner for critical reading of the manuscript and David Ennis and Jaehyuk Choi for helpful comments. Miriam Kluger offered her support and encouragement, an important factor leading to completion of this project.
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Vascular Endothelial Cell Adhesion and Signaling During Leukocyte Recruitment

Vascular Endothelial Cell and Leukocyte Recruitment

SEQUENTIAL STEPS OF LEUKOCYTE RECRUITMENT INVOLVE OVERLAPPING ADHESIVE FUNCTIONS OF EC CAMs

Vascular Endothelial Cell and Leukocyte Recruitment

CAM EXPRESSION AND ADHESIONS ON THE APICAL EC SURFACE: THE SELECTINS

Vascular Endothelial Cell and Leukocyte Recruitment

ICAM EXPRESSION AND ADHESION

VCAM EXPRESSION AND ADHESION

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LEUKOCYTE ACTIVATION IN RESPONSE TO EC CONTACT

RELATIONSHIP OF VASCULAR LEAK AND LEUKOCYTE TRANSMIGRATION

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SIGNALING MECHANISMS OF ENDOTHELIAL CAMs: OVERVIEW

14, 137, 139, 140

Vascular Endothelial Cell and Leukocyte Recruitment

CALCIUM SIGNALING IN ECs DURING LEUKOCYTE RECRUITMENT

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CALCIUM SIGNALING BY E-SELECTIN, VCAM, AND ICAM-1

REDISTRIBUTION AND CLUSTERING OF E-SELECTIN

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RHO SIGNALING BY ICAM-1

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RHO/RAC SIGNALING THROUGH PtdIns[4,5]P2

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VCAM AND ICAM CLUSTERING AND ERM COMPLEX ASSOCIATION

Vascular Endothelial Cell and Leukocyte Recruitment

EC CAMs WITHIN THE ERM COMPLEX GUIDE ADHERENT LEUKOCYTES

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84. 85. 86.

Vascular Endothelial Cell and Leukocyte Recruitment

Vascular Endothelial Cell and Leukocyte Recruitment

Vascular Endothelial Cell and Leukocyte Recruitment

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