icam vcam

8/13/2019 icam vcam

1/40

CHAPTER 6Vascular EndothelialCell Adhesion andSignaling During

Leukocyte RecruitmentMartin S. Kluger, PhD*Department of Dermatology and Interdepartmental Program in Vascular

Biology and Transplantation, Yale University School of Medicine,

New Haven, Connecticut

ABSTRACT

During inflammation, coordinated expression of cytokine-induced adhesionmolecules (CAMs) on postcapillary venular endothelial cells (ECs) regulatesleukocyte recruitment. During their recruitment from blood, leukocytes adhere

to EC CAMs, activating signaling pathways inside ECs. In a forthcomingparadigm, leukocyte transendothelial migration requires active EC participa-tion, with extracellular adhesive CAM functions mirrored by cytoplasmic do-main-dependent intracellular events. These events serve to reorganize the ECactin cytoskeleton. Investigators have visualized this as changes in EC shape,transient opening of EC-EC contacts, and redistribution of CAMs expressedon the luminal EC surface. In this review, we (1) summarize the overlappingextracellular adhesive properties of the 3 EC CAMs most important for leuko-cyte recruitment during inflammation, namely, E-selectin, vascular cell adhe-sion molecule, and intercellular adhesion molecule-1; (2) explore the role ofthese 3 CAMs as signal transducers by identifying the intracellular signals(Ca, Rho/Rac, and phosphatidylinositol 4,5-bisphosphate) that upon leu-kocyte engagement, reorganize the EC cytoskeleton and redistribute theseapical CAMs, thereby favoring leukocyte recruitment; and (3) describe how

CAM-derived signals lead to ezrin-radixin-moesin complex formation and

*E-mail correspondence: [email protected]

Advances in Dermatology, vol 20 163Copyright 2004, Mosby, Inc. All rights reserved.

8/13/2019 icam vcam

2/40

how this complex of plasma membranecytoskeleton adapter proteins coordi-

nates CAM-driven intracellular signals with extracellular adhesive CAM func-tions. This literature review suggests that the cytoplasmic domains of these ECCAMs and their downstream effectors represent new and potentially benefi-cial intracellular therapeutic targets for treating diseases of the skin.

EDITORS COMMENT

Endothelial cells (ECs) lining the vascular spaces of postcapillary venules inskin (and other peripheral tissues) are among the first regulators ofinflammation. The upregulation of adhesion molecules by endothelial cells,as well as the ability of ECs to present chemoattractant cytokines, leads to theadhesion of leukocytes on the endothelium, a prerequisite step before theirtransmigration into inflamed tissue. In this chapter, Dr Martin Klugerdetails the adhesion molecules involved in the initial steps of leukocytetrafficking. Moreover, it is clear that ECs do not behave as passiveparticipants in transendothelial migration of inflammatory cells. Dr Klugerdescribes the molecular signaling mechanisms involved in the EC response toleukocyte adhesion and transmigration. The identification of key molecularplayers in leukocyte-EC interactions may lead to new targets for inhibitors ofskin inflammation.

Sam Hwang, MD, PhD

Inflammation governs the complex response of vascularized tissueto cellular injury due to any cause. In skin disease as diverse as

psoriasis, allergic contact dermatitis, atopic dermatitis, and cutane-ous T-cell lymphoma, inflammation involves activation of the vas-cular endothelium, a monolayer layer of epithelial cells forming thesheet-like inner lining of all blood vessels in the body. Ideally posi-tioned as an anatomic interface between flowing blood and vascu-larized tissue, endothelium regulates access by the cellular compo-nents of the immune system to tissue such as skin according to thestate of endothelial cell (EC) activation. Although ECs comprise asingle cell type, they demonstrate heterogeneity among differentvascular beds, with different segments of the vasculature reflectingvariable barrier properties; for example, in dermis, the interendothe-

lial junctions are less restrictive than in brain and more restrictivethan in liver. The site of leukocyte recruitment in skin and in mostother tissues is the postcapillary venule.1,2 To mount a successfulinflammatory response, activation by cytokines such as tumor ne-crosis factor- (TNF-) must upregulate expression of specific adhe-

164 M. S. Kluger

8/13/2019 icam vcam

3/40

sion molecules on the luminal surface of venular ECs. Identification

of the first cytokine-inducible endothelial adhesion molecule, E-selectin, occurred approximately 20 years ago.3 Since then, muchknowledge has accrued on the role of cytokine-induced adhesionmolecule (CAM) gene expression on endothelium4-6 and on the spe-cific binding interactions of EC CAMs with cognate ligands ex-pressed on the surface of interactive leukocytes.7 A paradigm shiftin the way that we regard EC CAMs shall be forthcoming based onobservations that, just before transendothelial migration (TEM), leu-kocyte adhesion to EC CAMs activates EC intracellular signalingpathways. In this paradigm, leukocyte recruitment requires activeEC participation, with EC CAM extracellular adhesive functionsmirrored by intracellular events causing reorganization of the actin

cytoskeleton. Many investigators have visualized changes in ECshape, transient opening of EC-EC contacts, and redistribution ofCAMs expressed on the luminal (in vitro: apical) surface or at EC-ECjunctions.8-10 Specifically, clustering of CAMs on the apical plasmamembrane near points of contact assists leukocytes up to and intojunctional openings. Immediately before paracellular transmigra-tion, CAMs engaged by leukocytes become linked to the EC actincytoskeleton via membranecytoskeleton adapter proteins. Cluster-ing within regions of plasma membrane specialization can potenti-ate signaling or enable assistance of leukocytes just before theysqueeze through the junctional cleft of adjacent ECs. The purpose ofthis chapter is to (1) summarize the extracellular adhesive proper-ties of the 3 EC CAMs most important for leukocyte recruitment dur-ing inflammation, namely, E-selectin, vascular cell adhesion mol-ecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1); (2)identify the signaling components active within ECs upon leuko-cyte engagement that modify the EC cytoskeleton and redistributethese apical CAMs; and (3) describe how CAM-derived signals leadto ezrin-radixin-moesin (ERM) complex formation, and how thiscomplex of plasma membranecytoskeleton adapter proteins coor-dinates CAM-driven intracellular signals with extracellular adhe-sive function.

SEQUENTIAL STEPS OF LEUKOCYTE RECRUITMENT INVOLVEOVERLAPPING ADHESIVE FUNCTIONS OF EC CAMs

A progressive sequence of binding events among cognate pairs of ECCAMs and leukocyte ligands serves as a prelude to leukocyte trans-migration across endothelium. These steps are usually described astethering, rolling, leukocyte activation, and firm adhesion (Fig 1).7,11

The final step of this sequence, TEM, has most often been described

Vascular Endothelial Cell and Leukocyte Recruitment 165

8/13/2019 icam vcam

4/40

FIGURE 1.

Overlapping roles of cytokine-induced adhesion molecules (CAMs) on the endothe-lial cell (EC)surface in a multistep model of leukocyte recruitment. Blood flow(arrows)drives leukocyte circulation and influences leukocyte recruitment by im-

parting shear stress. The EC CAMs, P- and E-selectin, vascular cell adhesion mol-ecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1), are indicatedabove the schematic diagram at functionally relevant steps of leukocyte recruit-ment. Their importance may vary by pathologic condition, and for the sake of clar-ity, not all EC CAMs are shown. P-selectin is the CAM most proficient at leukocytetethering, whereas P- and E-selectin and VCAM all mediate rolling. During rolling,leukocytes may detach (dotted arrow), or upon encounter with chemokines presenton the EC surface, become activated (exclamationpoint). Chemokine-activated leu-kocytes flatten and firmly adhere to EC immunoglobulin-CAMs, VCAM and ICAM-1, triggering other (bidirectional) activation signals in leukocytes and in ECs (light-ning symbol; ligand binding to E-selectin may also trigger such signals inEC).Leukocyte ligand binding to EC CAMs also triggers signals in ECs that relaxinterendothelial junctions (shown by disappearance of theinterendothelial junc-tion symbol). Flattened leukocytes may locomote toward relaxed junctions whereinteractions with colocalized VCAM and ICAM-1 help initiate transendothelial mi-

gration (TEM). Other EC molecules situated within the interendothelial cleft, plate-let endothelial cell adhesion molecule (PECAM) and CD-99, assist in leukocyte tra-versal of the EC monolayer. After squeezing through adjacent ECs, extravasatedleukocytes must still traverse a layer of basal lamina before embarking toward dis-tal sites of inflammation (eg, in epidermis). Abbreviation:ECM, Extracellular ma-trix.

166 M. S. Kluger

8/13/2019 icam vcam

5/40

as occurring at junctions where adjacent ECs touch.10,12 This review

shall refer to lymphocytes, monocytes, and neutrophils as leuko-cytes, using distinctions only as necessary. Although beyond thescope of this chapter, after TEM, the leukocyte journey continuesfurther into skin with migration across the basal lamina, through theextracellular matrix, and depending on the site of inflammatory in-sult, 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 2different families of apical surface EC adhesion molecules respon-sible for leukocyte recruitment from blood into skin. The selectinfamily consists of E-selectin (CD62E; relative mass [Mr] of 97 and

107-115 kd, whose expression is unique to activated ECs), P-selec-tin (CD62P; Mrof 120-140 kd, expressed by activated ECs and byactivated platelets), and L-selectin (CD62L; Mrof 74 or 95 kd, ex-pressed by neutrophils, monocytes, macrophage, eosinophils, naveand some memory lymphocytes). Selectins are single transmem-

brane glycoproteins with homologous extracellular domains thatvary in length, depending on the number of complement regulatoryprotein repeats present (there are 6, 9, and 2 of these for E-, P-, andL-selectin, respectively). In contrast, the selectin intracellular cyto-plasmic domains lack sequence homology. Nevertheless, E-selectinis conserved across different species, suggesting an adaptation forintracellular signaling, control of surface expression, or both. P-selectin surface protein reaches the surface within minutes of ECstimulation with histamine or thrombin before recycling back intoEC Weibel-Palade storage granules.15,16 In contrast, E-selectin sur-face expression requires de novo synthesis and is strictly limited bycytoplasmic tail interactions leading to internalization and lysoso-mal degradation, which is more rapid among ECs derived from largevessels (eg, human umbilical vein ECs [HUVECs]) than from mi-crovessels (eg, from skin).15,17,18 The E-selectin carboxyl-terminuscytoplasmic tail consists of just 32 amino acids and contains a phos-phorylable serine residue19 that, in association with a di-leucinetype internalization motif,20 mediates constitutive internalization.Binding of HL-60 leukocytes (a cell line from a patient with promy-elocytic leukemia that produces subcutaneous tumors in mice) trig-

gers E-selectindependent intracellular signaling but does not affectthe cytoplasmic taildependent downregulation of E-selectin sur-face expression.21 This differs from ICAM-1 (see below). It suggeststhat different interactions regulate E-selectin internalization and sig-naling.


8/13/2019 icam vcam

6/40

8/13/2019 icam vcam

7/40

notype similar to patients with LAD IIthat is, a virtual lack of leu-

kocyte rolling, low extravasation, and ulcerative dermatitis. Rollingin E-selectin-/- mice after TNF treatment was not reduced, but doublemutants showed a 46-fold reduction compared with normal litter-mates, versus only a 20-fold reduction found in P-selectin -/- mice.41

This is also indicative of an overlap in the adhesive functions of ECselectins.

ICAM EXPRESSION AND ADHESIONICAM-1 (CD54) was identified as an aggregate-forming adhesionmolecule expressed on lymphocytes.42 Isolation of a complemen-tary DNA clone of ICAM-1 revealed a type I integral membrane gly-coprotein with repeating Ig domains in the extracellular region, astructural signature of the Ig superfamily.43 Heterogeneity among

different cell types gives rise to Mrfor ICAM-1 of 97 to 114 kd, mostlikely resulting from differential patterns of glycosylation, since thenonN-glycosylated form resulting from tunicamycin treatment hasan Mrof just 55,000.

44 Low constitutive expression of E-selectin andICAM-1 on dermal venules contributes to homeostatic T-cell immu-nosurveillance.45 On cultured ECs, ICAM-1 (but not E-selectin orVCAM) is constitutively expressed at low levels. ICAM-1 expres-sion levels are upregulated up to 40-fold by TNF endothelial activa-tion, reaching peak levels after 24 hours of TNF, a time point corre-sponding to downregulation of E-selectin expression.18,46 A secondICAM isoform, ICAM-2 (CD102; Mr 55-65 kd), is partially homolo-gous to ICAM-1 but has only 2 Ig-like extracellular domains com-pared with 5 such domains for ICAM-1. Constitutive expression onECs is high but is downregulated by TNF treatment.47

ICAM-1 dimerizes on the EC surface, but dimerization does notincrease ligand binding.48,49 It is unknown whether ICAM-1de-rived signals require dimerization, or whether ICAM-1 dimerizationis necessary for colocalization with an ERM complex. Unlike E-selectin, there is little or no constitutive internalization of ICAM-1surface protein, which is relatively long-lived on ECs.17,18,50 In-stead, ligand engagement may accelerate internalization of ICAM-1.51 ICAM-1 clusters induced by antiICAM-1coated micro-spheres, but not monomeric ICAM-1, are rapidly internalized fromthe surface of TNF-treated HUVECs, suggesting that interactions ofthe ICAM-1 cytoplasmic tail important for internalization are pro-

moted by clustering.52

Cross-linking of ICAM-1 upregulates gene ex-pression of ICAM-1 and VCAM (but not of E-selectin) a positive feed-back mechanism,53,54 which would be expected to replace ICAM-1protein cleared by internalization. The intracellular trafficking path-


8/13/2019 icam vcam

8/40

ways of ICAM-1 protein in connection with leukocyte TEM war-

rants further investigation as has recently been done for a related ECCAM, platelet endothelial cell adhesion molecule (PECAM/CD31).55

ICAM-1 is believed important for leukocyte recruitment duringa wide range of inflammatory and noninflammatory circumstances.In humans with leukocyte deficiency I (LAD I), CD18 integrin defi-ciency reduces levels of ICAM-1 ligands on leukocytes, resulting inrecurrent bacterial infections and poor wound healing. In LFA-1deficient mice, neutrophils and activated T cells are unable to crossEC monolayers in response to a chemokine gradient.56 In mouse skininflamed by a delayed-type hypersensitivity response, lymphocyteinfiltration is prevented by ICAM-1 (but not ICAM-2) Ab blockade.57

ICAM-1/ mice exhibit leukocytosis, lymphocytosis, and dimin-

ished tissue infiltration by neutrophils despite normal productionof proinflammatory cytokines.58,59 Together, these data suggest acritical role for ICAM-1 in the recruitment of circulating cells from

blood during skin inflammation.

VCAM EXPRESSION AND ADHESIONVCAM (CD106, Mr100-110 kd), discovered in activated HUVECs

by expression cloning with monoclonal Ab (mAb) E 1/6,60 is ex-pressed by activated ECs and follicular dendritic cells. VCAM is in-volved in disease pathogenesis during inflammatory bowel disease,atheroscelerosis, and the asthmatic response, and in the skin duringerythroderma (exfoliative dermatitis), urticaria, allograft rejection,and infection.61 The VCAM extracellular domain also contains re-peating Ig domains, but because of alternate posttranscriptionalsplicing there are 2 VCAM messenger RNAs, a more abundant full-length transcript and a variant that lacks exon 5. The VCAM variantmaintains the same cytoplasmic domain but has a shorter extracel-lular domain.62 As for E-selectin, the cytoplasmic tail of humanVCAM 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 HUVECsappears to be short-lived and internalized rapidly, similar to E-selectin.50,63

ICAM-1, ICAM -2, and VCAM each promote firm adhesion toECs. Moreover, VCAM and to a greater extent ICAM-1 probably as-

sist leukocyte entry into the interendothelial junction (detailed be-low). These Ig-CAMs interact with different integrin heterodimersexpressed on the leukocyte surface: ICAM-1 and ICAM-2 interactwith leukocyte function-associated antigen-1 (LFA-1) expressed onall leukocytes, ICAM-1 (but not ICAM-2) interacts with macrophage

170 M. S. Kluger

8/13/2019 icam vcam

9/40

receptor-1 (Mac-1) expressed on neutrophils, and VCAM interacts

with very late antigen-4 (VLA-4) on lymphocytes, monocytes, andeosinophils, but only rarely expressed on neutrophils.* Using an invivo migration assay, investigators demonstrated that antiVLA-4(but not anti-LFA-1) blockade of resting T-cell migration into rat skinwas further blocked by antibodies to P- and E-selectin, suggestingoverlapping functions of VCAM (but not of ICAMs) with the endo-thelial selectins.64 Indeed, because of multiple VLA-4 activationstates, VCAM/VLA-4 binding is highly versatile and can mediatetethering, rolling, and firm arrest under flow conditions in vitro.65,66

The VCAM knockout mouse dies early during embryonic develop-ment, revealing little about VCAM function,67but in triple-selectinnull mice, the small amount of residual rolling is dependent on

VCAM interactions.

68

In summary, the adhesive functions of E-selectin, VCAM, andICAM-1 are sequential and redundant. The overlapping functions ofthese EC CAMs may prevent functional inadequacy, ensuring suc-cessful recruitment of different leukocytes under varied pathologicstates. Conversely, in the absence of inflammation, strict control ofEC CAM gene expression normally protects vascularized tissue fromunwarranted and potentially harmful contact with bloodborne leu-kocytes. Before discussing how EC CAMs activate intracellular sig-naling pathways, we will outline similar signals present in ECs dur-ing vascular leak and consider the concurrent activation ofleukocytes.

LEUKOCYTE ACTIVATION IN RESPONSE TO EC CONTACTIt has been known for some time that the ability of T cells to leave thecirculation depends on their activation state.69 Activation occursduring rolling, when leukocytes encounter chemoattractants (C5a,platelet activating factor, leukotriene B4, formyl peptides) and che-moattractant cytokines (chemokines; short, 70- to 120amino acidsingle-chain peptides) that attach to and oligomerize on heparan sul-fate proteoglycans of the luminal EC surface.7,70,71Chemokines bindto specific leukocyte receptors that trigger heterotrimeric G-proteindependent leukocyte signaling.72,73 Such signals lead to clustering,and greater affinity/avidity of the integrins LFA-1, Mac-1, and VLA-4for their cognate Ig-superfamily EC-CAMs.74-76 Other intracellularevents signaling activation are induced in leukocytes during E-

*There are several integrin nomenclatures. LFA-1 is also CD11a/CD18 or 12, andMac-1 is also CD11b/CD18 or M2. The VCAM ligand VLA-4 is also CD49d/CD29 or

41.


8/13/2019 icam vcam

10/40

selectin tethering,36 VLA-4 cross-linking,77,78 or LFA-1 binding to

ICAM-1.79

In sum, leukocyte-EC encounters generate bidirectionalactivation signals, but until the advent of recent EC CAM studies,chemokine and integrin-activated pathways in leukocytes have re-ceived far greater attention.

In vivo, TEM occurs under flow conditions characterized bymeasurable levels of fluid shear stress. Shear stress is likely to influ-ence control of leukocyte TEM by ECs, since within 5 minutes itgenerates EC signals that causes EC elongation.80 A new in vitromodel to study leukocyte transmigration under flow was estab-lished recently.81 Using a similar flow model, our group preparedretrovirally transduced HUVECs that constitutively (in the absenceof cytokine) express E-selectin, ICAM-1, and VCAM-1, either in pair-

wise combinations or in triple combination. We find that pairwiseexpression increases TEM and that triple adhesion moleculeex-pressing cells are able to support CD4 T-cell TEM equally well asTNF-treated ECs. These unpublished observations suggest that ad-hesion molecule expression can alone account for the proinflamma-tory effects of TNF on recruitment of chemokine-activated T cells byECs, 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 LEUKOCYTETRANSMIGRATION

Vascular leak refers to the transendothelial passage of blood macro-molecules into extravascular tissue, commonly seen as edema. Inter-EC contacts normally act as a barrier to leukocyte transmigration andto vascular leak. It is a current working hypothesis that during vas-cular leak and leukocyte TEM, interendothelial contacts are re-duced by different processes of EC activation (Fig 2).8,82,83 Briefly,chemical mediators such as thrombin or histamine induce vascularleak. For example, histamine released by degranulation of residentmast cells is taken up by receptors located on postcapillary venularECs.84,85 Blood flow increases with nitric oxidemediated vessel di-lation, and permeability to macromolecules increases as gaps form

between EC neighbors, but only in a transient reversible manner (15-30 minutes). This coincides with escape ofexudate, a protein-richfluid containing blood proteins such as fibronectin and fibrin, from

blood into the interstitium. These integrin ligand proteins form aprovisional extravascular matrix allowing for the subsequent effi-cient migration of extravasated leukocytes toward the site of injuryin dermis or beyond.

172 M. S. Kluger

8/13/2019 icam vcam

11/40

FIGURE 2.

Gap formation between adjacentendothelial cells (ECs) occurs during vascular leakand leukocyte transendothelial migration (TEM).A, The integrity of EC junctionscontrols the barrier function of vascular endothelium, separating macromoleculesand circulating leukocytes in blood from the underlying tissue.B, During early in-

flammation, histamine from mast cells, or later in inflammation, tumor necrosisfactor(TNF)from macrophage or T cells, is taken up by specific receptors locatedon ECs. These inflammatory mediators trigger EC signals that lead to gap formationand leakage of blood macromolecules into the extravascular space. Vascular endo-thelial (VE)-cadherin expression on the plasma membrane surface of adjoining ECsnormally maintains barrier function, but is reduced during vascular leak. C, Leuko-cyte ligand engagement of cytokine-induced adhesion molecules (CAMs) ex-pressed on the luminal EC surface triggers EC signal pathways similar to those oc-curring in vascular leak that also lead to gap formation, but only at ECscontacted bycaptured leukocytes. The short cytoplasmic tails of E-selectin, vascular cell adhe-sion molecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1) are re-quired for these signals to occur. Cytoskeletal reorganization, redistribution of VE-cadherin and induction of matrix metalloproteinases (MMPs) are believed tocoordinate EC gap formation with initiation of TEM by captured leukocytes local-ized to the interendothelial junction.


8/13/2019 icam vcam

12/40

The scientific community is still divided about whether the

most important regulation of vascular leak and transmigration oc-curs at EC contacts. An alternative explanation proposed is transcel-lular passage of macromolecules through a vesiculo-vaculoar or-ganelle network within ECs.86 Similarly,neutrophiltransmigrationhas been observed (primarily in vivo) to occur via a transcellularroute.87 Data gathered by electron microscopy include detailed de-scriptions of transmigration directly through thinned regions of in-dividual ECs.88 But different observations made in vitro show thatneutrophil TEM also occurs through junctions, often at tricellularjunctions that may differ in their cell-cell contacts.12,14,89 Despitethese distinct recruitment patterns, signaling by EC CAMs and con-trol of EC junctions remain important mechanistic components evenfor neutrophils.90-92

One signal pathway leading to EC gap formation common to his-tamine-induced vascular leak and CAM signaling during leukocyterecruitment is the rapid and transient elevation of intracellular freecalcium concentration (Ca

i).93-95 Another form of vascular leak

involves the Rho/Rac signaling pathways and occurs later in inflam-mation after EC activation by cytokines (TNF, interleukin-1 [IL-1], orIL-2). Increasing Ca

iand activating Rho/Rac can lead to reorgani-

zation of the actin cytoskeleton, contraction, and elongation, caus-ing ECs to take on a fibroblast-like appearance.10,80,95-97 In quiescentECs, the actin cytoskeleton appears as a meshwork of dense periph-eral bands outlining individual ECs. Treatment with TNF-causesthe actin-based cytoskeleton to polymerize, cross-link, and reorga-nize into a configuration referred to as stress fiber formation.96-98

Stress fiber formations also occur after EC activation upon ligandengagement of VCAM and ICAM (see below).

The third major mechanism common to vascular leak and CAMsignaling during leukocyte recruitment consists of adhesive interac-tions across EC-EC junctions. Vascular endothelial (VE)-cadherin(CD144), expressed at adherens junctions only by ECs, is of centralimportance to junctional integrity during both vascular leak and leu-kocyte transmigration.8,99,100 VE-cadherin is a transmembrane gly-coprotein linked to - and -catenin that associates with the actincytoskeleton through-catenin.83,100 In vivo evidence for the impor-tance of VE-cadherin as a primary mediator of EC junctional contactis that mAb against VE-cadherin accelerates neutrophil recruitment

into the inflamed peritoneum.101

Reports that neutrophil adhesionsto HUVECs disrupt the EC junction through a VE-cadherindepen-dent mechanism102,103 were disputed by a study showing that thesedata derived from a postfixation artifact caused by detergent lysis

174 M. S. Kluger

8/13/2019 icam vcam

13/40

release of a neutrophil protease.104 Fixation artifacts were circum-

vented by using real-time imaging under flow to show that humanneutrophils and monocytes transmigrate through transient gapsformed upon redistribution of a VE-cadheringreen fluorescenceprotein (GFP) construct.14 The same group later showed that (unex-pectedly) there was no defect in transmigration under flow with neu-trophils derived from mice deficient in neutrophil elastase and ma-trix metalloproteinase9.105 These studies bolster the concept thatcontrol of EC junctional integrity by VE-cadherin is important dur-ing TEM. Two other junctional molecules that appear to functionsequentially during TEM as leukocytes pass down through adjoin-ing 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 cy-

tokine- and neutrophil-induced vascular leak.106-109 For example,no permeability increase of EC monolayers was observed as a resultof neutrophil TEM.110-112These investigators observed transient for-mation of inter-EC gaps that were restricted just to those ECs partici-pating in TEM. Therefore, activation of the mechanism of EC gapformation during TEM must be similarly restricted. Leukocyte en-gagement of E-selectin, VCAM, and ICAM-1 occurs specifically inthose ECs participatory to recruitment, suggesting an elegant solu-tion to this problem. EC CAM engagement by leukocyte ligand (orAb cross-linking) induces CAM signals that reduce barrier functionat interendothelial junctions by removal of junctional VE-cadherinand by actin cytoskeleton reorganization, processes believed to con-trol EC shape changes pertinent to TEM.

SIGNALING MECHANISMS OF ENDOTHELIAL CAMs: OVERVIEWCoordination of extracellular CAM adhesions with intracellular ac-tin cytoskeleton organization requires transmission of inbound sig-nals. E-selectin, VCAM, and ICAM-1 each have short intracellulardomains (tails) to assist this communication. Mutation/deletionstudies of E-selectin or ICAM-1 have shown cytoplasmic taildepen-dent activation of EC signal pathways,21,49,113 and the cytoplasmictail of VCAM mediates interactions with effectors of actin reorgani-zation.114 These EC CAMs produce 3 signals critical for cytoskeletalreorganization: transient elevation of intracellular Ca levels, acti-vation of Rho/Rac signaling, and phosphoinositide messengers.

Increase in Ca

iand activation of Rho or Rac by EC CAMs canlead to reorganization of the actin cytoskeleton and endothelial gapformation. EC CAMinitiated of Rho/Rac activation also can gener-ate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2), an addi-


8/13/2019 icam vcam

14/40

tional mediator of actin reorganization. Finally, Rho, Rac, and phos-

phoinositide signals can each activate the ERM family of cyto-skeletal adaptor proteins to form a plasma membrane complexlinking clustered EC CAMs to the actin cytoskeleton. The many sig-naling events stemming from the cytoplasmic tails of E-selectin,VCAM, and ICAM discussed below are summarized in Table 1 andFig 3.

TABLE 1.Signals and Events After Ligand Engagement or Ab Cross-linking of EC CAMs

EC CAM Signal or Event References and Comments

E-selectin Cytoplasmic taildependent ECactivation and cytoskeletal

linkage

21

Increased [Cai] 121, 122. Also invascular leak: 93-95

EC shape change and stress

fiber formation

121, 122

E-selectin redistribution tospecialized plasma membrane

regions enriched for caveolin-1

and PLC activation

21, 125, 126

VCAM Cytoplasmic taildependent EC

activation and actin reorganization

114, 130

Increased [Cai] 122, 123, 130

ROS formation 130, 139

MMP production 135, 136

Rac activation 97, 123, 137.

Also in vascularleak: 97

VCAM- and/or Rac-derived VE-

cadherin redistribution awayfrom interendothelial junctions

137, 139, 140.

Also in vascularleak: 99, 100

Rac-induced ERM complex

formation

165

VCAM interaction or

colocalization with ERMcomplexes in a specialized

plasma membrane structure

114

EC shape change and transient

gap formation assistingleukocyte entry

14, 137, 139, 140

(continued)

176 M. S. Kluger

8/13/2019 icam vcam

15/40

CALCIUM SIGNALING IN ECs DURING LEUKOCYTE RECRUITMENT

Calcium, a key second messenger in many cell types, is a criticalregulator of EC junctional integrity. In quiescent ECs, a transient risein Ca

ifrom less than 100 nmol/L to approximately 5- to 10-fold

higher decreases EC barrier function.95 The level of Caiincreases

during leukocyte ligand adhesion, transmigration, or both, and neu-trophil TEM is inhibited by a cell permeant Cabuffer.111 Further,neutrophils induce phosphorylation of specific serine/threonineresidues on EC myosin light-chain kinase (MLCK), suggesting thatCa

i flux can lead to actin-induced cytoskeletal contractility

through phosphorylation of myosin light chains.115 (The myosinlight chain is the regulatory part of the myosin molecule.Upon phos-phorylation, it induces actin contractility through myosin confor-

mation and sliding along filamentous [F-] actin, a flexible helicalpolymer composed of 5- to 9-nm-diameter globular [G-] actin mono-mers.)116 Two different teams used inhibitors of endothelial MLCKto assess this pathway. Neutrophil TEM across bovine pulmonary

ICAM-1 Cytoplasmic taildependent EC

activation and actin reorganization

49, 113

Increased [Cai] 9, 53, 98, 124

ICAM-1 dimerization 48, 49

ROS formation 90, 91

Rho activation, EC shape change,and stress fiber formation

49, 142, 145.Also in vascular

leak: 80, 96, 97

Rho-induced ERM complexformation

163, 164

ICAM-1 interaction or

colocalization with ERMcomplexes in specialized

plasma membrane structures

114, 125,

128, 160,181, 186

Transient gap formation

and/or assisting leukocyteentry to EC junctions

14, 124,

189, 190

ICAM-1 signals leading to

changes in gene expression

53, 54

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 metalloprotein-

ase;VE-cadherin, vascular endothelial cadherin; ERM, ezrin, radixin, and moesin; ICAM-1, intercel-

lular adhesion molecule-1.


8/13/2019 icam vcam

16/40

FIGURE 3.

Integration of endothelialcell (EC) cytokine-induced adhesion molecule (CAM) ad-hesion and signaling. Intercellular adhesion molecule-1 (ICAM-1) expressed on theluminal surface of cytokine-activated ECs mediates the firm binding of leukocytesvia cognate leukocyte ligands, triggering cytoplasmic domain-dependent intracel-lular signals in ECs. Left, Rho activation and a transient increase in intracellularfree calcium concentration (Ca

i) lead to actin reorganization. Downstream of

Rho, Rho kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2

)inter-

act with folded, unassembled monomers of ezrin, radixin, and moesin (ERM), caus-ing their phosphorylation, unfolding, and head-to-tail assembly into a complex thatrelocates to the inner leaflet of the plasma membrane. Once relocated, the ERMcomplex supports further actin reorganization. Right, Actin reorganization leads toEC shape change and EC gap formation that eases leukocyte transendothelial mi-gration (TEM) at interendothelial junctions. ICAM-1 redistributes, colocalizingwith ERM complexes in cup-shaped regions of plasma membrane that serve asdocking sites for adherent leukocytes en route to transmigration. ICAM-1 ho-modimers form upon ligand engagement, possibly enhancing cytoplasmic domain-dependent signaling. Positive feedback signal pathways include ongoing Rho-mediated actin reorganization through inhibition of Rho guanine dissociationinhibitor (GDI) and auto-upregulation of ICAM-1 gene expression. Different contri-butions by vascular cell adhesion molecule (VCAM), E-selectin, and other ECCAMs to EC regulation of leukocyte TEM also occur (see text) but are omitted forclarity. Molecular interactions shown are either direct (solid arrows)or omit inter-mediary steps(dashed arrows).Abbreviation:MLCK, Myosin light-chain kinase.

178 M. S. Kluger

8/13/2019 icam vcam

17/40

arterial ECs was reduced by specific inhibitors of MLCK and pro-

moted by inhibitors of a myosin-associated phosphatase.92

Pretreat-ment with a different MLCK inhibitor reduced F-actin formation,MLC phosphorylation, and neutrophil TEM across HUVECs cul-tured on an amniotic membrane substrate.117 Whether initiated byneutrophil products, EC CAMs, or both, it is noteworthy that el-evated Ca

iwas seen to occur in ECs making direct contact with

transmigrating neutrophils.118 Mechanisms of actin reorganizationmay be EC-type specific. During neutrophil adherence to TNF-activated human pulmonary microvascular ECs, changes observedin the F-actin cytoskeleton were phosphoinositide dependent butCa

iindependent.90,119 This discrepant result may be explained

by the use of microvascular ECs (with no chemoattractant), which

differs from studies finding Ca

-dependent transmigration that in-volved large vesselderived HUVECs (which were exposed to che-moattractant).

CALCIUM SIGNALING BY E-SELECTIN, VCAM, AND ICAM-1E-selectindependent leukocyte adhesion does not require cytoskel-etal interaction.120 Hence, the evolutionary conservation of the shortE-selectin cytoplasmic tail19 likely relates to control of surface ex-pression by endocytosis, intracellular signaling, or both. Cross-linking E-selectin expressed on IL-1treated ECs increases Ca

i

and causes EC shape change.121 Using Fura-2loaded ECs, investi-gators found that cross-linking of HUVEC VCAM and E-selectin (butnot ICAM-1 or PECAM) raised Ca

iand caused stress fiber forma-

tion.122 Neutrophil and monocyte adhesion also induced thesechanges, which were inhibited by mAb blockade of E-selectin li-gand binding. VCAM- and ICAM-1induced Ca signaling in ECshas been described by others.98,123 For example, Ab blockade wasused to show that an increase in HUVEC Ca

iwas derived from

lymphocyte adhesion to ICAM-1.98 Cross-linking of ICAM-1 onHUVECs and on mouse brain ECs each induces a rapid increase ofCa concentration.9,53 ICAM-induced Ca signaling appearsimportant for TEM but not for leukocyte adhesion, since calciumchelator pretreatment of 2 different rat brain EC lines reducesICAM-1dependent TEM but not adhesion of lymphocytes.124 Inthis system, cross-linking of ICAM-1 resulted in tyrosine phosphor-

ylation activation of phosphatidylinositol-phospholipase C (PLC)-1, which mediated release of Castores via inositol 1,4,5-triphos-phate (IP3).


8/13/2019 icam vcam

18/40

REDISTRIBUTION AND CLUSTERING OF E-SELECTIN

During leukocyte recruitment, there is cytoplasmic taildependentredistribution of CAMs expressed on the apical EC surface. Wild-type (but not a cytoplasmic deletion) E-selectin construct clusters atthe site of HL-60 leukocyte adhesion and links to the actin cytoskel-eton.21 These clusters assemble in cholesterol-containing lipidrafts,125,126 a type of cell surface microenvironment that favors cyto-skeletal interactions and signal cascade activation.127 More pre-cisely, ligand-induced clustering redistributes E-selectin to caveo-lin-1containing rafts where it associates with and activates PLC.Since PLC hydolyzes PtdIns[4,5]P2, resulting in IP3 production andsubsequent release of stored Ca

i, the observed E-selectinin-

duced rise in Cai

121,122 may derive from the E-selectin subpopu-

lation localized in caveolin

lipid rafts. Consequently, it will be im-portant to determine whether the E-selectin cytoplasmic tailsequence contains a binding site for PtdIns[4,5]P2 as already de-scribed for ICAM-1.128

SIGNALING BY VCAM

In a VCAM-dependent static transmigration model, resting (ie, notstimulated by antigen recognition) mouse splenic lymphocytes re-quire VLA-4 interaction with VCAM to spontaneously bind andtransmigrate across lymph nodederived mouse EC lines.129 Minuscytokine activation, these lines (mHEVa and mHEVc) constitutivelyexpress VCAM, but not other EC CAMs (ie, P- and E-selectin, ICAM-1, mucosal vascular addressin cell adhesion molecule [MAdCAM-1;an EC receptor for 4 integrin], and PECAM-1). Lymphocyte TEMmediated by VCAM was dependent on EC calcium flux and reactiveoxygen species (ROS) production, but not on tyrosine kinase orphosphatidylinositol-3-kinase (PI3K) activity, based on EC pretreat-ment with either herbimycin A (a tyrosine kinase inhibitor) or wort-manin (a PI3K inhibitor).130

ROS production indicates EC activation. NADPH oxidase is anenzyme oxidizer of the reduced form of the electron carrier nico-tinamide adenine dinucleotide phosphate (NADPH) and of othersubstrates. In professional phagocytes it catalyzes the production ofthe ROS superoxide, which when dismutated becomes hydrogenperoxide.131,132 ECs may express NADPH oxidases similar to those

in phagocytes.133

In general, cross talk between the cellular redoxstate and other signaling pathways can impair EC barrier function,and dihydrorhodamine-123, a membrane-permeable peroxide indi-cator, has been used to show that VCAM cross-linking leads to EC

180 M. S. Kluger

8/13/2019 icam vcam

19/40

ROS production, and that ROS production is critical for VCAM-

mediated TEM. ROS can relax constraints on EC positioning by theactivation of EC matrix metalloproteinases (MMPs).132,134 MMPsfunction as important effectors for degradation of basement mem-

brane during TEM.135,136

Just below the site of lymphocyte binding there appears to be acoalescence of endothelial actin indicative of the VCAM cytoskele-ton interaction.130 VCAM-dependent ROS production may promotethis 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 ofactin, dispersion of cadherin from intercellular junctions, and gapformation.97 Of interest is that the Rho/Rac ratio appears to be anespecially sensitive barometer for actin contractility and junctional

integrity; the activation of one GTPase can lead to the inactivation ofthe other, and dual activation may have opposing effects on barrierfunction.85,138 VCAM-derived Rac signaling induced by cross-link-ing, or by the cell permeant constitutively active Tat-RacV12 chi-meric peptide, each lead to HUVEC gap formation secondary to lossof junctional VE-cadherin localization.137,139,140 In an experimentcomparing constitutively active peptides, Tat-RacV12 uptake in-duced stress fiber formation, VE-cadherin redistribution, and EC gapformation, but a comparable Rho-based peptide (Tat-RhoV14) didnot induce gap formation, only stress fibers without redistributionof VE-cadherin.139 VE-cadherin redistribution initiated by Rac pep-tide was shown to be ROS dependent (through use of the oxygenscavenger N-acetylcysteine). In keratinocytes, Rac activation suf-ficed to disassemble cadherin-mediated contacts.141 Rho-based pep-tide actin stress fiber formation without VE-cadherin redistributionimplies that during HUVEC gap formation, distinct signals controlactin contractility and VE-cadherin redistribution.

During leukocyte TEM, transientinterendothelial junctionalgapformation is hypothesized to require release and redistribution ofVE-cadherin.8,12,99,102 Based on these observations, VCAM-depen-dent ROS and MMP production may coordinate EC gap formationwith adhesion and TEM by VLA-4T cells/monocytes, which showa greater preference for transmigration at the site of de novo gapsthan neutrophils.14 EC gap formation may be less important for VLA-4independent neutrophil TEM, which may not require VCAM-

induced Rac signaling or EC-derived ROS (although ICAM-1 on ECscan also generate ROS).90 Further study is warranted on how VCAM-induced Rac activation leads to VE-cadherin redistribution and ECgap formation.


8/13/2019 icam vcam

20/40

RHO SIGNALING BY ICAM-1

Neutrophil TEM, but not fibrinogen-dependent adhesion, is abol-ished by deletion of the ICAM-1 cytoplasmic region from ICAM-1transfected Chinese hamster ovary cells.142 The Rho inhibitor C3was used in this study to show that neutrophil TEM required Rhoactivation, and to implicate Rho as a downstream effector of theICAM-1 cytoplasmic domain in ECs. Rho refers to 3 Rho isoforms,Rho A,B, and C,and the specificityof C3for Rho isat least 100 timesmore efficient that that for Rac or Cdc42, other small GTPases (C3 isa bacterial toxin from Clostridium botulinum that inactivates Rho byadenosine diphosphate ribosylation at amino acid 41).143,144 C3 in-hibition of Rho signaling can block lymphocyte TEM and formationof actin stress fibers initiated by mAb cross-linking of ICAM-1.145

This group also showed that transfection of rat brain microvas-cular ECs with a full-length, but not a cytoplasmic-deleted ICAM-1construct, confers TEM. Results were consistent in ICAM-1/ICAM-2-double-deficient mouse brain endothelioma cells.49,113 Un-expectedly, the lone cytoplasmic tyrosine in ICAM-1 remainsunphosphorylated during ICAM cluster formation.49,145 In sum-mary, the ICAM-1 cytoplasmic domain is essential for EC regulationof TEM, Rho is a key downstream mediator of ICAM-1, and tyrosinephosphorylation of ICAM-1 does not occur. So how does Rho for-ward ICAM-derived signals to effector molecules, and what down-stream events are initiated?

Rho is a molecular switch, an evolutionarily conserved mem-ber of the Ras superfamily of small (20-25 kd) monomeric cyto-plasmic GTPases. Twenty different Rho-related related GTPaseshave been identified that are referred to as either Rho-, Rac-, orCdc42-GTPases, each of which regulates different signal transduc-tion pathways linking cell surface protein receptors to the assemblystate of a filamentous actin cytoskeleton. When bound to GTP, Rho isin an active state, and when bound to guanosine diphosphate(GDP), Rho is in an inactive state. Rho turns off by GTP hydroly-sis, which generates the inactive Rho-GDP form. Rho GTPase activ-ity is regulated by several other molecules: guanine nucleotide ex-change factors activate by catalyzing the exchange of GDP to GTP,GTPase-activating proteins inactivate by stimulating GTP hydroly-sis, and guanine dissociation inhibitors (GDIs) sustain inactivation

by sequestering inactive Rho-GDP away from the plasma mem-brane.144,146

Rho-binding proteins identified by rigorous biochemical meth-ods include the serine/threoninedirected Rho kinases (Rho kinase/

182 M. S. Kluger

8/13/2019 icam vcam

21/40

ROCKII and p160ROCK/ROCK), together referred to as Rho ki-

nase.144

Rho kinase functions immediately downstream of Rho and(like the calcium-calmodulin pathway) mediates MLC phosphoryla-tion via phosphorylation/activation of MLCK.85 Rho/Rho kinase canalso reorganize actin along a parallel route by threonine/serine phos-phorylating (inactivating) myosin light-chain phophatase, whichserves to inhibit dephosphorylation of MLC.147,148

Pharmaceutical inhibitors of the Rho pathway can be used toreduce TEM. MLC phosphorylation, actin polymerization, and neu-trophil TEM were reduced by treatment of ECs with C3 or withY-27632.149 Used to dissect ICAM-1 signaling pathways, the pyri-dine derivative Y-27632 is a Rho kinase inhibitor 200 times moreselective for inhibition of Rho kinase than for protein kinase C orMLCK.150,151 Inhibitors of MLCK diminished neutrophil migration

across a transwell of confluent bovine pulmonary ECs induced bythe chemoattractant leukotriene B4 by 30% to 70%.92 In the samestudy, an inhibitor of myosin-associated phosphatase (calyculin)that increased phosphorylation of myosin light chains, caused ECcontraction and enhanced neutrophil migration. In response to TNF,the effects of Rho activation on actin reorganization include gap for-mation at EC junctions,97 and it is tempting to speculate thatICAM-induced Rho signaling, necessary for ICAM-1dependent TEM, maysimilarly induce gap formation. However, Rho inhibition can, undercertain circumstances, lead to Rac activation.139,152 So further sort-ing out of the relationship of these molecular switch molecules Rhoand Rac, which lie downstream of ICAM-1 and VCAM, will be nec-essary.

RHO/RAC SIGNALING THROUGH PtdIns[4,5]P2Along with calcium, Rho, and Rac, PtdIns[4,5]P2is now recognizedas a major regulator of the actin cytoskeleton.153,154 Although tradi-tionally PtdIns[4,5]P2 has been viewed as a substrate for synthesis ofIP3 and diacylglycerol, PtdIns[4,5]P2 is also a downstream effector ofCa flux or Rho/Rac cytoskeletal reorganization.154-156 Rac inter-acts with type I phosphatidyl-4-phosphate 5-kinase (PtdIns[4]P 5-ki-nase), and PtdIns[4]P 5-kinase (acting through PtdIns[4,5]P2) in-duces actin filament uncapping and assembly in permeabilizedplatelets.157 Conversely, in mouse-derived, immortalized NIH 3T3fibroblasts, dominant negative Rac1 decreases PtdIns[4,5]P2 levels

by 50% (mimicking the effect of fibroblast detachment from sub-strate) because of a reduction in the enzymatic activity of PtdIns[4]P5-kinase.158 Like Rac, Rho has also been reported to regulatePtdIns[4,5]P2via PtdIns[4]P 5-kinase.

155


8/13/2019 icam vcam

22/40

ERM PROTEIN ACTIVATION AND ERM COMPLEX FORMATION

OCCURS VIA RHO/RAC AND PtdIns[4,5]P2The ERM family of homologous, interactive proteins regulates cellshape and cell adhesion by linkage of membrane adhesion receptorsto the actin cytoskeleton. ERM protein cytoskeletal linkages occur asa complex on the cytoplasmic side of the plasma membrane wherethey 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 C-terminal ends of their roughly 600 amino acids are 2 different inter-active ERM association domains (known as N- and C-ERMADs). The

bulk cytoplasmic pool of ERM protein appears as folded, nonphos-phorylated monomers resulting from intramolecular association of

N- and C-ERMADs on each ezrin, radixin, or moesin molecule.Structure/function studies indicate that folding normally masksERMAD sites, shielding them from dimerization or interaction withtransmembrane adhesion molecules.161,162 Rho/Rho kinase can ini-tiate unfolding and head-to-tail association by ERM protein phos-phorylation at a COOH-terminal threonine conserved in all 3 ERMproteins (T558 in moesin);163,164 Rac can activate moesin in a similarmanner.165 Once unfolded, they are unmasked and active,166-168 canform homo- or hetero-oligomers,169 and can translocate to the cyto-plasmic side of the plasma membrane as an ERMcomplex.164,170Un-folding also renders accessible a 35amino acid C-terminal regioncontaining a functional F-actin binding site.171-173

Like Rho/Rho kinase signaling, PtdIns[4,5]P2also unmasks anN-ERMAD domain membrane binding site (in ezrin). Direct interac-tion of PtdIns[4,5]P2 with ezrin was shown by mutagenesis of thePtdIns[4,5]P2binding site in the ezrin NH2-terminal domain. By de-letion and terminal truncation mutagenesis, the ezrin/PtdIns[4,5]P2

binding sites were localized to regions containing KK(X)(n)K/RKamino acid motifs, and amino acid mutagenesis was found to altercellular localization.174

ERM proteins are a control point for cell shape and actin con-tractility. ERM proteins bind F-actin in vitro and colocalize with ac-tin at the cytoplasmic surface of the plasma membrane in many celltypes.116,175,176 A clever experiment was performed to identifymoesin as an effector of actin cytoskeleton reorganization. Constitu-

tively active Rac and Rho proteins were known to cause actin reor-ganization, stress fiber and lamellipodia formation in digitonin-permeabilized, serum-starved Swiss 3T3 cells. This ability was lostafter cell permeabilization with digitonin but was restored by recon-

184 M. S. Kluger

8/13/2019 icam vcam

23/40

stitution with cytosolic extract, the active component of which,

when biochemically isolated, was found to be moesin.165

In sum-mary, Rho/Rho kinase and PtdIns[4,5]P2 each promote unfolding,complex formation, and plasma membrane translocation of ERMproteins to sites of plasma membrane adhesion molecule expressionwhere they can interact with the actin cytoskeleton to affect changesin cell shape.

OTHER FUNCTIONS OF THE ERM COMPLEX

ERM proteins can sequester Rho GDI, thereby shifting Rho equilib-rium toward an active state by reducing Rho GDI-mediated inhibi-tion of the conversion of Rho-GDP to Rho-GTP.170,177 This suggeststhat the assembled ERM complex can amplify Rho/Rac signals via a

positive feedback loop. Redundancy of individual ERM proteinfunction is suggested by moesin-knockout mice, which show nor-mal development and are unimpaired in certain ERM functions.178

Hence, to genetically test the individual role in integration of CAMadhesions and cytoskeletal contractility by ERM proteins, it may benecessary to produce an ERM triple knockout mouse.

VCAM AND ICAM CLUSTERING AND ERM COMPLEX ASSOCIATIONIn general, studies involving interactions between cytoskeletal fila-ments and transmembrane glycoproteins have shown that physicalassociations do not occur directly but instead involveadaptor mol-eculeslinking transmembrane proteins to the cytoskeleton.179 Onesuch adaptor protein is-actinin, a homodimer equipped with 2 do-mains per subunit: a globular actin-binding region and a rod-shapeddomain that binds to the cytoplasmic tails of transmembrane pro-teins.180 ICAM-1surfacedistributionmediatedby-actinincytoskel-etal association was demonstrated first in transfected COS cells byimmunofluorescence. A wild-type ICAM construct was found local-ized to specialized membrane regions of microvilli, whereas a gly-cophosphatidyl-anchored ICAM-1 construct, without any cytoplas-mic domain, showed a uniform surface distribution.181 Microvillilocalization of ICAM-1 required an intact actin cytoskeleton (shown

by disruption of the actin cytoskeleton with cytochalasin B). Directassociation of purified -actinin with ICAM-1 cytoplasmic tail pep-tides and full-length ICAM-1 suggested that juxtamembrane posi-

tively charged amino acid residues of the ICAM-1 cytoplasmic tailwere interactive.ERM adaptor protein binding to transmembrane molecules was

established by finding that in vitro, moesin bound to the cytoplas-


8/13/2019 icam vcam

24/40

mic domain of CD43, ICAM-2, and CD44,182 and by mapping the

ezrin-binding site to a membrane-proximal 9amino acid regionwithin the CD44 cytoplasmic domain.183 A PtdIns[4,5]P2-depen-dent association of ezrin with ICAM-1 was convincingly demon-strated by affinity precipitation of ezrin with an ICAM-1 cytoplas-mic domain peptide and by direct interaction using surface plasmonresonance. Specific amino acid residues were not identified, but ba-sic and hydrophobic residues resembling the PtdIns[4,5]P2consen-sus binding sequences of K/RXKX(K/R)(K/R) or KX(3)KXKK184 werenoted as juxtamembrane in the ICAM-1 cytoplasmic tail sequence.In the proposed model, ERM-induced ICAM-1 clustering (observedon the tips of transfected COS cell microvilli) might magnify ICAM-1adhesiveness, thereby increasing the efficiency of lymphocyte re-cruitment by ECs during inflammation.128 Direct interaction of

VCAM with moesin and ezrin (the predominate ERM proteins foundin ECs) was shown by a glutathione-S-transferase-VCAM cytoplas-mic tail construct pull down of moesin/ezrin and by coimmunopre-cipitation of VCAM with moesin or ezrin from protein lysates of ac-tivated HUVECs.114 VCAM/ERM binding potentially is mediated bya serine-rich portion of the VCAM cytoplasmic domain (S317through S326; SYSLVEAQKS) that resembles a serine-rich cytoplas-mic motif in ICAM-3, critical for ERM interaction.185 Indirect inter-actions with either ICAM-1 or VCAM may also occur through inter-mediaries such as EBP50 (ezrin-binding phosphoprotein 50) or thesimilar E3KARP.186-188

Just as ligand-induced E-selectin clustering affects redistribu-tion to specialized membrane compartments enriched for caveolin-1, ligand-induced clustering of ICAM-1 and VCAM has been ob-served to coincide with localization to other specialized ECmembrane structures (eg, microspikes, microvilli). Lymphocyte ad-hesion to HUVECs induces VCAM and ICAM-1 colocalization withintracellular moesin and ezrin in a cup-shaped region of membranespecialization that reaches upwards along the z-dimension in 3-di-mensional confocal images, and functions as a lymphocyte dockingstructure.114 Formation of this structure relies on Rho/Rho kinaseand PtdIns[4,5]P2signal pathways. A similar endothelial structureencircling leukocytes was visualized on microvilli, except that thiscup-like region of membrane specialization formed via ICAMLFA-1 interactions, independently of VCAMVLA-4.160 Depen-

dence on Ca

i (rather than on Rho/Rho kinase) suggests thatICAM-1 localization to EC microvilli may entail an ICAM-1depen-dent calcium/PLCpathway.124

186 M. S. Kluger

8/13/2019 icam vcam

25/40

EC CAMs WITHIN THE ERM COMPLEX GUIDE ADHERENT LEUKOCYTES

The ICAM-1/LFA-1 interaction seems to follow the leukocyte up tothe moment of transmigration when it squeezes through adjacentECs.189,190 This was first suggested by immunoelectron microscopicanalysis in which VCAM-1 was localized on the apical EC surface

but absent from the precise EC region in which leukocyte transmi-gration was in progress.189 In contrast, ICAM-1 showed continuouscontact with T cells on the EC membrane regions where T-cell mi-gration progressed into EC-EC junctions. Recently, these observa-tions have been extended to include VCAM and ICAM-1 distributedwithin the ERM complex. During firm adhesion of VLA-4/LFA-1

lymphoblasts to HUVECs, VCAM and ICAM-1 were found clusteredwith intracellular moesin and ezrin; VCAM participated in the cup-

shaped docking structure elevated over the apical surface thatstrengthened over time, but only ICAM stayed bound to the lympho-cyte and followed moesin into the cleft. Further, with the use ofHUVECs transfected with either VCAM-GFP or ICAM-GFP, it wasshown by time-lapse confocal microscopy and real-time video mi-croscopy that only ICAM followed the lymphoblast downward intothe interendothelial cleft, and that VCAM did not participate in thetransmigration act per se.114 Overall, these real-time observations ofdynamic lymphoblast associations by VCAM and ICAM from withinan ERM docking structure support earlier impressions that VCAMand ICAM-1 each mediate leukocyte firm adhesion and redistributein response to EC cytoskeletal remodeling before TEM, but that con-tact with ICAM-1 and not VCAM, continues as the leukocyte entersinto 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 ANDSIGNALING FUNCTIONS OF VCAM AND ICAM-1

This chapter highlights a less explored facet of the EC CAMs E-selectin, VCAM, and ICAM-1: their role as intracellular signal trans-ducers during leukocyte recruitment. EC CAM signal pathwayscome full circle by influencing their own distribution through in-duction of the ERM complex. Specifically, formation of the ERMcomplex is a consequence of leukocyte ligand binding that occursdownstream within the Rho/Rho kinase, PtdIns[4,5]P2, and Ca

pathways. Complex formation serves to usher in transmigrating leu-kocytes by clustering of ICAM and VCAM into a dynamic membranedocking structure. CAM-induced Rho/Rac and Casignals directly


8/13/2019 icam vcam

26/40

regulate actin contractility, and EC-CAM/ERM association superim-

poses extra layers of control; the ERM complex serves as a bridgefrom EC CAMs to the cytoskeleton and secondly, amplifies Rho ac-tivation through sequestering of Rho GDI. In sum, the ERM mem-

branecytoskeletal adaptor proteins integrate timing of dynamic EC-leukocyte docking adhesions with cytoskeletal remodeling toinitiate TEM. Other important EC molecules are also likely to be im-portant but were not cited because of space limitations.

CONCLUSIONSWhat has all this got to do with the world of dermatology? Thesedata offer insight as to how vascular ECs regulate leukocyte entryinto skin from blood. The logical connection is that solving themechanisms of vascular EC biology is central to increasing under-

standing of inflammation, and that inflammation forms the basis formuch of skin disease. In a new paradigm, ECs are active participantsduring leukocyte TEM, and the cytoplasmic tails of E-selectin,VCAM, and ICAM-1 activate intracellular signaling pathways. Justas the new biologic drug for psoriasis, efalizumab, targets extracel-lular CAM adhesions by blocking leukocyte LFA-1 interaction withICAM-1 on ECs,191,192 the cytoplasmic domains of EC CAMs andtheir downstream effectors represent new and potentially beneficialintracellular therapeutic targets for treating diseases of the skin.

ACKNOWLEDGMENTSThe author thanks Brad Rosenberg and Jeff Schechner for criticalreading of the manuscript and David Ennis and Jaehyuk Choi for

helpful comments. Miriam Kluger offered her support and encour-agement, an important factor leading to completion of this project.

REFERENCES

1. Marchesi VT: The site of leucocyte emigration during inflammation. Q JExp Physiol Cogn Med Sci46:115-118, 1961.

2. Marchesi VT, Florey HW: Electron micrographic observations on theemigration of leucocytes.Q J Exp Physiol Cogn Med Sci45:343-348,1960.

3. Pober JS, Bevilacqua MP, Mendrick DL, et al: Two distinct monokines,interleukin 1 and tumor necrosis factor, each independently inducebiosynthesis and transient expression of the same antigen on the sur-face of cultured human vascular endothelialcells.J Immunol136:1680-

1687, 1986.4. Caughman SW, Li LJ, Degitz K: Human intercellular adhesion mol-

ecule-1 gene and its expression in the skin.J Invest Dermatol98:61S-65S, 1992.

188 M. S. Kluger

8/13/2019 icam vcam

27/40

5. Tedder TF, Steeber DA, Chen A, et al: The selectins: Vascular adhesion

molecules.FASEB J9:866-873, 1995.6. de Boer OJ,Das PK:Adhesion molecules in the immunopathogenesis of

skin diseases, in Paul LC, Issekutz TB (eds): Adhesion Molecules in

Health and Disease. New York, Marcel Dekker, 1997, pp 589-617.

7. von Andrian UH, Mackay CR: T-cell function and migration: Two sides

of the same coin.N Engl J Med343:1020-1034, 2000.

8. Vestweber D: Regulation of endothelial cell contacts during leukocyte

extravasation.Curr Opin Cell Biol14:587-593, 2002.

9. Greenwood J, Etienne-Manneville S, Adamson P, et al: Lymphocyte mi-

gration into the central nervous system: Implication of ICAM-1 signal-

ing at the blood-brain barrier.Vasc Pharmacol38:315-322, 2002.

10. Muller WA: Leukocyte-endothelial-cell interactions in leukocyte trans-

migration and the inflammatory response. Trends Immunol24:326-

333, 2003.

11. Muller WA: Leukocyte-endothelial cell interactions in the inflamma-

tory response.Lab Invest82:521-533, 2002.

12. Liu Y, Shaw SK, Ma S, et al: Regulation of leukocyte transmigration:

Cell surface interactions and signaling events. J Immunol 172:7-13,

2004.

13. Schon MP, ZollnerTM, Boehncke W-H: The molecular basis of lympho-

cyte recruitment to the skin: Clues for pathogenesis and selective thera-

pies of inflammatory disorders.J Invest Dermatol121:951-962, 2003.14. Shaw SK, Bamba PS, Perkins BN, et al: Real-time imaging of vascular

endothelial-cadherin during leukocyte transmigration across endothe-lium.J Immunol167:2323-2330, 2001.

15. Subramaniam M, Koedam JA, Wagner DD: Divergent fates of P- and E-selectins after their expression on the plasma membrane. Mol Biol Cell

4:791-801, 1993.16. Takano M, Meneshian A, Sheikh E, et al: Rapid upregulation of endo-

thelial P-selectin expression via reactive oxygen species generation.Am J Physiol Heart Circ Physiol283:H2054-H2061, 2002.

17. von Asmuth EJU, Smeets EF, Ginsel LA, et al: Evidence for endocytosisof E-selectin in human endothelial cells. Eur J Immunol22:2519-2526,1992.

18. Kluger MS, Johnson DR, Pober JS: Mechanism of sustained E-selectinexpression in cultured human dermal microvascular endothelial cells.J Immunol158:887-896, 1997.

19. Chuang PI, Young BA, Thiagarajan RR, et al: Cytoplasmic domain ofE-selectin contains a non-tyrosine endocytosis signal.J Biol Chem272:24813-24818, 1997.

20. Kluger MS, Shiao SL, Bothwell ALM, et al: Cutting edge: Internaliza-

tion of transduced E-selectin by cultured human endothelial cells:Comparison of dermal microvascular and umbilical vein cells andidentification of a phosphoserine-type di-leucine motif.J Immunol168:2091-2095, 2002.


8/13/2019 icam vcam

28/40

21. Yoshida M, Westlin WF, Wang N, et al: Leukocyte adhesion to vascular

endothelium induces E-selectin linkage to the actin cytoskeleton.J CellBiol133:445-455, 1996.

22. Picker LJ, Michie SA, Rott LS, et al: A unique phenotype of skin-

associated lymphocytes in humans. Preferential expression of the

HECA-452 epitope by benign and malignant T cells at cutaneous sites.

Am J Pathol136:1053-1068, 1990.

23. Heald P, Yan, S-L, Edelson R, et al: Skin-selective lymphocyte homing

mechanisms in the pathogenesis of leukemic cutaneous T-cell lym-

phoma.J Invest Dermatol101:222-226, 1993.

24. Picker LJ, Kishimoto TK, Smith CW, et al: ELAM-1 is an adhesion mol-

ecule for skin-homing T cells.Nature349:796-799, 1991.

25. Cotran RS, Gimbrone MA Jr, Bevilacqua MP, et al: Induction and detec-

tion of a human endothelial activation antigen in vivo. J Exp Med164:

661-666, 1986.

26. Ruco LP, Pomponi D, Pigott R, et al: Cytokine production (IL-1 alpha,

IL-1 beta, and TNF alpha) and endothelial cell activation (ELAM-1 and

HLA-DR) in reactive lymphadenitis, Hodgkins disease, and in non-

Hodgkins lymphomas. An immunocytochemical study.Am J Pathol137:1163-1171, 1990.

27. Koch AE, Burrows JC, Haines GK, et al: Immunolocalization of endo-

thelial and leukocyte adhesion molecules in human rheumatoid and

osteoarthritic synovial tissues.Lab Invest64:313-320, 1991.

28. Hebbar M, Lassalle P, Janin A, et al: E-selectin expression in salivary

endothelial cells and sera from patients with systemic sclerosis. Role of

resident mast cell-derived tumor necrosis factor alpha. Arthritis Rheum38:406-412, 1995.

29. Di Stefano A, Maestrelli P, Roggeri A, et al: Upregulation of adhesion

molecules in the bronchial mucosa of subjects with chronic obstructivebronchitis.Am J Respir Crit Care Med149:803-810, 1994.

30. Kunkel EJ, Boisvert J, Murphy K, et al: Expression of the chemokine

receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lym-

phocytes.Am J Pathol160:347-355, 2002.

31. Reiss Y, Proudfoot AE, Power CA, et al: CC chemokine receptor (CCR)4

and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK)

in lymphocyte trafficking to inflamed skin. J Exp Med194:1541-1547,

2001.

32. Fitzhugh DJ, Naik S, Caughman SW, et al: Cutting edge: C-C chemokine

receptor 6 is essential for arrest of a subset of memory T cells on acti-

vated dermal microvascular endothelial cells under physiologic flow

conditions in vitro.J Immunol165:6677-6681, 2000.

33. Hwang ST: Mechanisms of T-cell homing to skin. Adv Dermatol17:211-241, 2001.

34. Becker DJ, Lowe JB: Leukocyte adhesion deficiency type II. Biochim

Biophys Acta1455:193-204, 1999.

190 M. S. Kluger

8/13/2019 icam vcam

29/40

35. Luhn K, Wild MK, Eckhardt M, et al: The gene defective in leukocyte

adhesion deficiency II encodes a putative GDP-fucose transporter. NatGenet28:69-72, 2001.

36. Simon SI, Hu Y, Vestweber D, et al: Neutrophil tethering on E-selectin

activates beta 2 integrin binding to ICAM-1 through a mitogen-acti-

vated protein kinase signal transduction pathway.J Immunol164:4348-

4358, 2000.

37. Kunkel EJ, Ley K: Distinct phenotype of E-selectin deficient mice: E-

selectin is required for slow leukocyte rolling in vivo. Circ Res 79:1196-

1204, 1996.

38. oude Egbrink MG, Janssen GH, Ookawa K, et al: Especially polymor-

phonuclear leukocytes, but also monomorphonuclear leukocytes, roll

spontaneously in venules of intact rat skin: Involvement of E-selectin.J

Invest Dermatol118:323-326, 2002.

39. Tietz W, Allemand Y, Borges E, et al: CD4 T cells migrate into in-

flamed skin only if they expressligands for E- andP-selectin.J Immunol161:963-970, 1998.

40. Labow MA, Norton CR, Rumberger JM, et al: Characterization of E-

selectin-deficient mice: Demonstration of overlapping function of the

endothelial selectins.Immunity1:709-720, 1994.

41. Frenette PS, Mayadas TN, Rayburn H, et al: Susceptibility to infection

and altered hematopoiesis in mice deficient in both P- and E-selectins.

Cell84:563-574, 1996.

42. Rothlein R, Dustin ML, Marlin SD, et al: A human intercellular adhe-

sion molecule (ICAM-1) distinct from LFA-1. J Immunol137:1270-

1274, 1986.

43. Simmons D, Makgoba MW, Seed B: ICAM,an adhesion ligand of LFA-1,

is homologous to the neural cell adhesion molecule NCAM. Nature 331:

624-627, 1988.44. Dustin ML,Rothlein R, Bhan AK, et al: Induction by IL 1 andinterferon-

gamma: Tissue distribution, biochemistry, and function of a natural ad-

herence molecule (ICAM-1).J Immunol137:245-254, 1986.

45. Chong BF, Murphy J-E, Kupper TS, et al: E-selectin, thymus- and acti-

vation-regulated chemokine/ccl17, and intercellular adhesion mol-

ecule-1 are constitutively coexpressed in dermal microvessels: A foun-

dation for a cutaneous immunosurveillance system. J Immunol172:

1575-1581, 2004.

46. Haraldsen G, Kvale D, Lien B, et al: Cytokine-regulated expression of

E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular

cell adhesion molecule-1 (VCAM-1) in human microvascular endothe-

lial cells.J Immunol156:2558-2565, 1996.

47. McLaughlin F, Hayes BP, Horgan CM, et al: Tumor necrosis factor(TNF)-alpha and interleukin (IL)-1beta down-regulate intercellular ad-

hesion molecule (ICAM)-2 expression on the endothelium. Cell Adhes

Commun6:381-400, 1998.


8/13/2019 icam vcam

30/40

48. Jun C-D, Shimaoka M, Carman CV, et al: Dimerization and the effective-

ness of ICAM-1 in mediating LFA-1-dependent adhesion. Proc NatlAcad Sci U S A98:6830-6835, 2001.49. Greenwood J, Amos CL, Walters CE, et al: Intracellular domain of brain

endothelial intercellular adhesion molecule-1 is essential for T lym-phocyte-mediated signaling and migration. J Immunol171:2099-3008,2003.

50. Nakada MT, Tam SH, Woulfe DS, et al: Neutralization of TNF by theantibody cA2 reveals differential regulation of adhesion molecule ex-pression on TNF-activated endothelial cells. Cell Adhes Commun5:491-503, 1998.

51. Almenar-Queralt A, Duperray A, Miles LA, et al: Apical topographyand modulation of ICAM-1 expression on activated endothelium. Am JPathol147:1278-1288, 1995.

52. Muro S, Wiewrodt R, Thomas A, et al: A novel endocytic pathway in-

duced by clustering endothelial ICAM-1 or PECAM-1. J Cell Sci116:1599-1609, 2003.

53. Clayton A, Evans RA, Pettit E, et al: Cellular activation through the li-gation of intercellular adhesion molecule-1. J Cell Sci 111:443-453,1998.

54. Lawson C, Ainsworth M, Yacoub M, et al: Ligation of ICAM-1 on endo-thelial cells leads to expression of VCAM-1 via a nuclear factor-kappaB-independent mechanism.J Immunol162:2990-2996, 1999.

55. Mamdouh Z, Chen X, Pierini LM, et al: Targeted recycling of PECAMfrom endothelial surface-connected compartments during diapedesis.Nature421:748-753, 2003.

56. Andrew DP, Spellberg JP, Takimoto H, et al: Transendothelial migrationand trafficking of leukocytes in LFA-1-deficient mice. Eur J Immunol28:1959-1969, 1998.

57. Lehmann JCU,Jablonski-Westrich D, Haubold U, et al: Overlapping andselective roles of endothelial intercellularadhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking. J Immunol171:2588-2593,2003.

58. Sligh J Jr, Ballantyne C, Rich S, et al: Inflammatory and immune re-sponses are impaired in mice deficient in intercellular adhesion mol-ecule 1.Proc Natl Acad Sci U S A 90:8529-8533, 1993.

59. Xu H, Gonzalo JA, St Pierre Y, et al: Leukocytosis and resistance to sep-tic shock in intercellular adhesion molecule 1-deficient mice. J ExpMed180:95-109, 1994.

60. Osborn L, Hession C, Tizard R, et al: Direct expression cloning of vas-cular cell adhesion molecule 1, a cytokine-induced endothelial proteinthat binds to lymphocytes.Cell59:1203-1211, 1989.

61. Freedberg IM, Eisen AZ, Wolff K, et al (eds):Fitzpatricks Dermatology

in General Medicine, ed 6. New York, McGraw-Hill, 2003.62. Cybulsky MI, Fries JW, Williams AJ, et al: Gene structure, chromosomal

location, and basis foralternative mRNA splicingof the human VCAM1gene.Proc Natl Acad Sci U S A 88:7859-7863, 1991.

192 M. S. Kluger

8/13/2019 icam vcam

31/40

63. Ricard I, Payet MD, Dupuis G: VCAM-1 is internalized by a clathrin-

related pathway in human endothelial cells but its alpha 4 beta 1 inte-grin counter-receptor remains associated with the plasma membrane in

human T lymphocytes.Eur J Immunol28:1708-1718, 1998.

64. Issekutz AC, Issekutz TB: The role of E-selectin, P-selectin, and very

late activation antigen-4 in T lymphocyte migration to dermal inflam-

mation.J Immunol168:1934-1939, 2002.

65. Alon R, Kassner PD, Carr MW, et al: The integrin VLA-4 supports teth-

ering and rolling in flow on VCAM-1.J Cell Biol128:1243-1253, 1995.

66. Chen C, Mobley JL, Dwir O, et al: High affinity very late antigen-4 sub-

sets expressed on T cells are mandatory for spontaneous adhesion

strengthening but not for rolling on VCAM-1 in shear flow.J Immunol162:1084-1095, 1999.

67. Gurtner GC, Davis V, Li H, et al: Targeted disruption of the murine

VCAM1 gene: Essential role of VCAM-1 in chorioallantoic fusion and

placentation.Genes Dev9:1-14, 1995.

68. Collins RG, Jung U, Ramirez M, et al: Dermal and pulmonary inflamma-

tory disease in E-selectin and P-selectin double-null mice is reduced in

triple-selectin-null mice.Blood98:727-735, 2001.

69. Oppenheimer-Marks N, Davis LS, Lipsky PE: Human T lymphocyte ad-

hesion to endothelial cells and transendothelial migration. Alteration

of receptor use relates to the activation status of both the T cell and the

endothelial cell.J Immunol145:140-148, 1990.70. Hoogewerf AJ, Kuschert GS, Proudfoot AE, et al: Glycosaminoglycans

mediate cell surface oligomerization of chemokines. Biochemistry36:13570-13578, 1997.

71. Weber KS, von Hundelshausen P, Clark-Lewis I, et al: Differential im-mobilization and hierarchical involvement of chemokines in monocyte

arrest and transmigration on inflamed endothelium in shear flow. Eur JImmunol29:700-712, 1999.

72. Murdoch C, Finn A: Chemokine receptors and their role in inflamma-tion and infectious diseases.Blood95:3032-3043, 2000.

73. Zlotnik A, Morales J, Hedrick JA: Recent advances in chemokines andchemokine receptors.Crit Rev Immunol19:1-47, 1999.

74. DiVietro JA, Smith MJ, Smith BR, et al: Immobilized IL-8 triggers pro-gressive activation of neutrophils rolling in vitro on P-selectin and in-tercellular adhesion molecule-1.J Immunol167:4017-4025, 2001.

75. Grabovsky V, Feigelson S, Chen C, et al: Subsecond induction of alpha4integrin clustering by immobilized chemokines stimulates leukocytetethering and rolling on endothelial vascular cell adhesion molecule 1under flow conditions.J Exp Med192:495-506, 2000.

76. Constantin G, Majeed M, Giagulli C, et al: Chemokines trigger immedi-

ate beta2 integrin affinity and mobility changes: Differential regulationand roles in lymphocyte arrest under flow. Immunity13:759-769, 2000.

77. Ricard I, Payet MD, Dupuis G: Clustering the adhesion moleculesVLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothe-


8/13/2019 icam vcam

32/40

lial (ECV 304) cells activates the phosphoinositide pathway and trig-

gers Ca2

mobilization.Eur J Immunol27:1530-1538, 1997.78. McGilvray ID, Lu Z, Bitar R, et al: VLA-4 integrin cross-linking on hu-

man monocytic THP-1 cells induces tissue factor expression by a

mechanism involving mitogen-activated protein kinase.J Biol Chem272:10287-10294, 1997.

79. Kanner SB, Grosmaire LS, Ledbetter JA, et al: Beta 2-integrin LFA-1sig-

naling through phospholipase C-gamma 1 activation.Proc Natl Acad

Sci U S A90:7099-7103, 1993.

80. Wojciak-Stothard B, Ridley AJ: Shear stress-induced endothelial cell

polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

J Cell Biol161:429-439, 2003.

81. Cinamon G, Shinder V, Alon R: Shear forces promote lymphocyte mi-

gration across vascular endothelium bearing apical chemokines. Nat

Immunol2:515-522, 2001.

82. Dejana E, Spagnuolo R, Bazzoni G: Interendothelial junctions and their

role in the control of angiogenesis, vascular permeability and leukocyte

transmigration.Thromb Haemost86:308-315, 2001.

83. Bazzoni G, Dejana E: Pores in the sieve and channels in the wall: Con-

trol of paracellular permeability by junctional proteins in endothelial

cells.Microcirculation8:143-152, 2001.

84. Cotran RS, Kumar V, Robbins SL:Robbins Pathologic Basis of Disease,

ed 5. Philadelphia, WB Saunders, 1994.85. Wojciak-Stothard B, Ridley AJU: Rho GTPases and theregulation of en-

dothelial permeability.Vasc Pharmacol39:187-199, 2002.86. Feng D, Nagy JA, Hipp J, et al: Vesiculo-vacuolar organelles and the

regulation of venule permeability to macromolecules by vascular per-meability factor, histamine, and serotonin.J Exp Med183:1981-1986,

1996.87. Bamforth SD, Lightman SL, Greenwood J: Ultrastructural analysis of

interleukin-1 beta-induced leukocyte recruitment to the rat retina. In-vest Ophthalmol Vis Sci38:25-35, 1997.

88. Feng D, Nagy JA, Pyne K, et al: Neutrophils emigrate from venules by atransendothelial cell pathway in response to FMLP.J Exp Med187:903-915, 1998.

89. Burns AR, Walker DC, Brown ES, et al: Neutrophil transendothelial mi-gration is independent of tight junctions and occurs preferentially attricellular corners.J Immunol159:2893-2903, 1997.

90. Wang Q, Doerschuk CM: Neutrophil-induced changes in the biome-chanical properties of endothelial cells: Roles of ICAM-1 and reactiveoxygen species.J Immunol164:6487-6494, 2000.

91. Wang Q, Doerschuk CM: The signaling pathways induced by neutro-

phil-endothelial cell adhesion.Antioxid Redox Signal4:39-47, 2002.92. Garcia JG, Verin AD, Herenyiova M, et al: Adherentneutrophils activate

endothelial myosin light chain kinase: Role in transendothelial migra-tion.J Appl Physiol84:1817-1821, 1998.

194 M. S. Kluger

8/13/2019 icam vcam

33/40

8/13/2019 icam vcam

34/40

108. Gautam N, Olofsson AM, Herwald H, et al: Heparin-binding protein

(HBP/CAP37): A missing link in neutrophil-evoked alteration of vascu-lar permeability.Nat Med7:1123-1127, 2001.109. Gautam N, Herwald H, Hedqvist P, et al: Signaling via beta integrins

triggers neutrophil-dependent alteration in endothelial barrier func-tion.J Exp Med191:1829-1839, 2000.

110. Huang AJ, Furie MB, Nicholson SC, et al: Effects of human neutrophilchemotaxis across human endothelial cell monolayers on the perme-ability of these monolayers to ions and macromolecules. J Cell Physiol135:355-366, 1988.

111. Huang AJ,Manning JE,Bandak TM,et al: Endothelial cell cytosolic freecalcium regulates neutrophil migration across monolayers of endothe-lial cells.J Cell Biol120:1371-1380, 1993.

112. Allport JR, Muller WA, Luscinskas FW: Monocytes induce reversiblefocal changesin vascular endothelialcadherin complexduringtransen-

dothelial migration under flow.J Cell Biol148:203-216, 2000.113. Lyck R, Reiss Y, Gerwin N, et al: T cell interaction with ICAM-1/ICAM-

2-double-deficient brain endothelium in vitro: The cytoplasmic tail ofendothelial ICAM-1 is necessary for transendothelial migration of Tcells.Blood102:3675-3683, 2003.

114. Barreiro O, Yanez-Mo M, Serrador JM, et al: Dynamic interaction ofVCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelialdocking structure for adherent leukocytes.J Cell Biol157:1233-1245,2002.

115. Hixenbaugh EA, Goeckeler ZM, Papaiya NN, et al: Stimulated neutro-phils induce myosin light chain phosphorylation and isometric ten-sion in endothelial cells.Am J Physiol273:H981-H988, 1997.

116. Bretscher A, Edwards K, Fehon RG: ERM proteins and merlin: Integra-tors at the cell cortex.Nat Rev Mol Cell Biol3:586-599, 2002.

117. Saito H, Minamiya Y, Kitamura M, et al: Endothelial myosin light chainkinase regulates neutrophil migration across human umbilical vein en-dothelial cell monolayer.J Immunol161:1533-1540, 1998.

118. Su WH, Chen HI, Huang JP, et al: Endothelial [Ca(2)](i) signaling dur-ing transmigration of polymorphonuclear leukocytes. Blood96:3816-3822, 2000.

119. Wang Q, Chiang ET, Lim M, et al: Changes in the biomechanical prop-erties of neutrophils and endothelial cells during adhesion.Blood97:660-668, 2001.

120. Kansas G, Pavalko F: The cytoplasmic domains of E- and P-selectin donot constitutively interact with alpha-actinin and are not essential forleukocyte adhesion.J Immunol157:321-325, 1996.

121. Kaplanski G, Farnarier C, Benoliel AM, et al: A novel role for E- andP-selectins: Shape control of endothelial cell monolayers.J Cell Sci107:

2449-2457, 1994.122. Lorenzon P, Vecile E, Nardon E, et al: Endothelial cell E- and P-selectin

and vascular cell adhesion molecule-1 function as signaling receptors.JCell Biol142:1381-1391, 1998.

196 M. S. Kluger

8/13/2019 icam vcam

35/40

123. Cook-Mills JM, Johnson JD, Deem TL, et al: Calcium mobilization and

Rac1 activation are required for VCAM-1 (vascular cell adhesion mol-ecule-1) stimulation of NADPH oxidase activity. Biochem J378:539-547, 2004.

124. Etienne-Manneville S, Manneville JB, Adamson P, et al: ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocytemigration involve intracellular calcium signaling in brain endothelialcell lines.J Immunol165:3375-3383, 2000.

125. Tilghman RW, Hoover RL: E-selectin and ICAM-1 are incorporated intodetergent-insoluble membrane domains following clustering in endo-thelial cells.FEBS Lett525:83-87, 2002.

126. Kiely JM, Hu Y, Garcia-Cardena G, et al: Lipid raft localization of cellsurface E-selectin is required for ligation-induced activation of phos-pholipase C gamma.J Immunol171:3216-3224, 2003.

127. Simons K, Toomre D: Lipid rafts and signal transduction.Nat Rev Mol

Cell Biol1:31-39, 2000.128. Heiska L, Alfthan K, Gronholm M, et al: Association of ezrin with inter-

cellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulationby phosphatidylinositol 4, 5-bisphosphate. J Biol Chem 273:21893-21900, 1998.

129. Cook-Mills JM, Gallagher JS, Feldbush TL: Isolation and characteriza-tion of high endothelial cell lines derived from mouse lymph nodes. InVitro Cell Dev Biol Anim32:167-177, 1996.

130. Matheny HE, Deem TL, Cook-Mills JM: Lymphocyte migration throughmonolayers of endothelial cell lines involves VCAM-1 signaling via en-dothelial cell NADPH oxidase.J Immunol164:6550-6559, 2000.

131. Abo A, Pick E, Hall A, et al: Activation of the NADPH oxidase involvesthe small GTP-binding protein p21rac1.Nature353:668-670, 1991.

132. Kamata H, Hirata H: Redox regulation of cellular signaling.Cell Signal

11:1-14, 1999.133. Jones SA, ODonnell VB, Wood JD, et al: Expression of phagocyte

NADPH oxidase components in human endothelial cells.Am J Physiol271:H1626-H1634, 1996.

134. Lum H, Roebuck KA: Oxidant stress and endothelial cell dysfunction.Am J Physiol Cell Physiol280:C719-C741, 2001.

135. Madri JA, Graesser D, Haas T: The roles of adhesion molecules and pro-teinases in lymphocyte transendothelial migration. Biochem Cell Biol74:749-757, 1996.

136. Madri JA, Graesser D: Cell migration in the immune system: The evolv-ing inter-related roles of adhesion molecules and proteinases. Dev Im-munol7:103-116, 2000.

137. van Wetering S, van den Berk N, van Buul JD, et al: VCAM-1-mediatedRac signaling controls endothelial cell-cell contacts and leukocyte

transmigration.Am J Physiol Cell Physiol285:C343-C352, 2003.138. Sander EE, ten Klooster JP, van Delft S, et al: Rac downregulates Rho

activity: Reciprocal balance between both GTPases determines cellularmorphology and migratory behavior.J Cell Biol147:1009-1022, 1999.


8/13/2019 icam vcam

36/40

139. van Wetering S, van Buul JD, Quik S, et al: Reactive oxygen species

mediate Rac-induced loss of cell-cell adhesion in primary human en-dothelial cells.J Cell Sci115:1837-1846, 2002.

140. van Buul JD, Voermans C, van den Berg V, et al: Migration of human

hematopoietic progenitor cells across bone marrow endothelium is

regulated by vascular endothelial cadherin. J Immunol168:588-596,

2002.

141. Braga VM, Betson M, Li X, et al: Activation of the small GTPase Rac is

sufficient to disrupt cadherin-dependent cell-cell adhesion in normal

human keratinocytes.Mol Biol Cell11:3703-3721, 2000.

142. Sans E, Delachanal E, Duperray A: Analysis of the roles of ICAM-1 in

neutrophil transmigration using a reconstituted mammalian cell ex-

pression model: Implication of ICAM-1 cytoplasmic domain and Rho-

dependent signaling pathway.J Immunol166:544-551, 2001.

143. Ridley AJ, Hall A: The small GTP-binding protein rho regulates the as-

sembly of focal adhesions and actin stress fibers in response to growth

factors.Cell70:389-399, 1992.

144. Sah VP, Seasholtz TM, Sagi SA, et al: The role of Rho in G protein-

coupled receptor signal transduction. Annu Rev Pharmacol Toxicol40:

459-489, 2000.

145. Adamson P, Etienne S, Couraud PO, et al: Lymphocyte migration

through brain endothelial cell monolayers involves signaling through

endothelial ICAM-1 via a rho-dependent pathway. J Immunol162:2964-2973, 1999.

146. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 420:629-635, 2002.

147. van Hinsbergh VW, van Nieuw Amerongen GP: Intracellular signalinginvolved in modulating human endothelial barrier function.J Anat200:

549-560, 2002.148. Kawano Y, Fukata Y, Oshiro N, et al: Phosphorylation of myosin-

binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.JCell Biol147:1023-1038, 1999.

149. Saito H, Minamiya Y, Saito S, et al: Endothelial Rho and Rho kinaseregulate neutrophil migration via endothelial myosin light chain phos-phorylation.J Leukoc Biol72:829-836, 2002.

150. Sahai E, Ishizaki T, Narumiya S, et al: Transformation mediated byRhoA requires activity of ROCK kinases.Curr Biol9:136-145, 1999.

151. Uehata M, Ishizaki T, Satoh H, et al: Calcium sensitization of smoothmuscle mediated by a Rho-associated protein kinase in hypertension.Nature389:990-994, 1997.

152. Rottner K, Hall A, Small JV: Interplay between Rac and Rho in the con-trol of substrate contact dynamics.Curr Biol9:640-648, 1999.

153. Janmey PA: Phosphoinositides and calcium as regulators of cellular ac-tin assembly and disassembly.Annu Rev Physiol56:169-191, 1994.

154. Ma

icam vcam

Documents