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Signaling in leokocyte transendothelial migration: a roadmap for homing of progenitor cells

van Buul, J.D.

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Citation for published version (APA):van Buul, J. D. (2004). Signaling in leokocyte transendothelial migration: a roadmap for homing of progenitorcells.

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Download date: 28 Jun 2020

Chapterr 1 SIGNALLINGG IN LEUKOCYTE

TRANSENDOTHELIALL MIGRATION.

13 3

Signalingg in Leukocyte Transendothelial Migration Jaapp D. van Buul, Peter L. Hordijk

AbstractAbstract—Under—Under a variety of (patho) physiological conditions, leukocytes wil l leave the bloodstream by crossing the endotheliall monolayer that lines the vessels and migrate into the underlying tissues. It is now clear that the process of extravasationn involves a range of adhesion molecules on both leukocytes and endothelial cells, as well as extensive intracellularr signaling that drives adhesion and chemotaxis on the one hand and controls a transient modulation of endotheliall integrity on the other. We review here the current knowledge of the intracellular signaling pathways that are activatedd in the context of transendothelial migration in leukocytes and in endothelial cells. In leukocytes, polarization off receptors and of the signaling machinery is of key importance to drive adhesion and directional migration. Subsequent adhesion-inducedd signaling in endothelial cells, mediated by Rho-like GTPases and reactive oxygen species, induces a transientt and focal loss of endothelial cell-cell adhesion to allow transmigration of the leukocyte. This review underscoress the notion that we have likely just scratched the surface in revealing the complexity of the signaling that controlss leukocyte transendothelial migration. (Arterioscler Thromb Vase Biol. 2004;24:1-11.)

Thee migratory properties of white blood cells are indis-pensablee to drive immune responses throughout the

body.. To ensure migration to the proper locations, the traffickingg of leukocytes is tightly regulated. The migration andd extravasation of leukocytes across the endothelium that liness the vessel walLpccurs inseveral distinct steps, refe|red too as the multi-step.,, paradigm, originally introduced by Butcher11 and extended by Springer.2 The first step comprise* i thee rolling of the jjet}]|pcytes over the endothelial cells, mediatedd by transient weak interactions between adhesion, molecules.. Subsequently, loosely attached leukocytes are in suchh close proximjty of the endothelium that they can be. activatedd by chemè|actic cytokines, ipreslnted on the apical surfacee of the endothelium. .As a consequence, the activated leukocytess will spread and firmly adhere to the endothelium andd finally migrate through the in thee endothelial cells to the undëÉ| levell of complexity has been added by work that showed that inn addition to chemokines, shear stress can also act as a transmigration-promotingg stimulus.3 Figure 1 shows a sche-maticc overview of the different stages that occur during transendotheliall migration (TEM) of leukocytes.

Thee extravasation of leukocytes is essential for many (patho)) physiological processes, including migration of T-lymphocytess for immune surveillance, recruitment of acti-vatedd lymphocytes and granulocytes during acute and chronic inflammatoryy responses, and homing and mobilization of hematopoieticc progenitor cells. In the past decade, knowledge off the molecules and the signaling events, both in the leukocytess and in the endothelial cells, that control transen-

dotheliall migration of leukocytes has increased signifïcanüy andd is discussed here.: We have div|(||fijj|is overview into 3 parts:: the first part deals with chemokine-induced leukocyte activation;; me second part describes the signaling events that occurr in endothelial cells after leukocyte adhesion; and the finall part* discusses our current knowledge about the role of thee endothelial cell-cell junctions in the final stage of the transmigrationn process.

Chemokine-inducedd Leukocyte Activation Thee migration of leukocytes across the endothelium is mainly dfivenn by a large family of extracellular ligands: the chemo-tacticc cytokines, better known as chemokines. Chemokines aree small (8 to 14 kDa) structurally related molecules that

gg G-protein-coupled Jacking.44 Today, >40

differentt chemokines have been identified, and this number is stilll growing. The nomenclature of chemokines is based on thee arrangement of the N-terminal cysteine residues. These residuess can be adjacent (CC chemokines) or have one or moree extra amino acids in between them (CXC or CX3C chemokines).. Two chemokines have only one cysteine at the N-terminus,, ie, lymphotactin/SCM-la and SCM-10. In the currentt nomenclature, an "L" (as in CXCL12) refers to a ligand,, whereas an "R" (as in CXCR4) refers to a chemokine receptor.55 The main topics of this review are discussed with referencee to stromal cell-derived factor-1 (SDF-1 or, accord-ingg to the current nomenclature, CXCL12), because SDF-1/ CXCL122 is involved in the migration of many, if not all,

Receivedd October 28, 2003; revision accepted January 25, 2004. Fromm Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands. Correspondencee to Dr Peter L. Hordijk, Department of Experimental Immunohematology, Sanquin Research at CLB and Landsteiner Laboratory,

Academicc Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. E-mail p.hordijk@sanquin.nl ©© 2004 American Heart Association, Inc.

DOI:: 10.1161/01.ATV.0000122854.76267.5C ArteriosclerArterioscler Thromb Vase Biol is available at http://www.atvbaha.org

14 14

ArteriosclerArterioscler Thromb Vase Bio I. April 2004 15

II Rollin g II Adhesio n and Spreading / activatio n n

IIII Transmigration

Endotheli i

i i L-setec(l n n

Leukocytes :: PSGL-1 Sialy ll Lewi s X

P-,, E-selecti n

Endothelium::CD34 4 MadCAM-1 1

III aJktffM) aHp22 a*-»

afifisunt afifisunt a,, {>,(««>

ICAM-1,-2 2

VCAM-1 1 Ftbronectir t t

III I

PECAM-1 1

LFA-11 COM

II I JAM-11 CD99

PECAM-1 1 VE-Cadhori n n

Figuree 1. Schematic overview of the distinct steps that comprise leukocyte transendothelial migration. The first step (I) represents the rollingg of leukocytes and involves adhesion molecules such as selectins on the leukocytes or endothelial cells and their ligands on the cellularr counterparts. The second step (II) represents firm adhesion, mediated through immobilized chemoattractants on the endotheli-umm and adhesion molecules such as integrins on the leukocytes and CAMs on the endothelium. The third step (III) represents the dia-pedesiss of the leukocytes through the endothelial cell-cell junctions, in which homophilic binding molecules such as the JAMs, PECAM-1,, CD99, and VE-cadherin are involved. ^

i M M ization,, spreading at trie leading edgelïffne cell and contrac-tionn at the back.22-2* Directional migration of leukocytes is accompaniedd by polarization of the cell body, of the actin and

differentt types of leukocytes and various types of tumor cells6-77 and is expressed in virtually all human tissues.8-9

Chemokiness and their receptors are one of the many levels thatt coordinate theAigration of leukocytes and leukocyte tubulin cytoskeleton, and of a wide range of intracellular subsetss at various lèvfc, fps veryy complex system or' havee shown that 'leukocyte n i idoth hal migratie

olledd and

ïuMuii» » erationn is

f Kgg :::: ' -

okinee . itflfenWoIanëë gfadiehts":pérsist in

thee bloodstream, because the gradient will be rapidly washed away.3-133 It is there ort nerallj c pted that leukocytes respondd to chemokines thai are immobilized on the surfaie df diee endothelium,jyJ^jc^jgjjndejïrareol_by detailed studies showingg that ch CXCL12,, are indeed present on the vascular endothelium in vivo.13-166 These immobilized chemokines are presented,t< nearbyy rolling leukocytes by heparin-sulfate proteoglycans highlyy glycosylated proteins that are expressed on the surface off endothelial cells.17-19 Middleton et al have shown that IL-8,, a potent chemokine for neutrophils, is presented on the microvillous-likee extensions on the luminal surface of the endothelium.. Moreover, these investigators showed that after injectionn of IL-8 into the skin of a rabbit, IL-8 is transported throughh the endothelial cells from the basolateral side to the luminall side.13 For SDF-1/CXCL12, it has been shown extensivelyy that immobilization of this chemokine leads to increasedd adhesion and TEM of leukocytes under physiolog-icall flow.3-16-20-21 Chemokinee Receptors Chemokiness transmit their pro-migratory signals through G-protein-coupled,, 7-times-spanning membrane receptors. Thesee receptors initiate adhesion and motility via (pertussis toxin-sensitive)) G-proteins, leading to integrin activation via inside-outt signaling, followed by coordinated actin polymer-

signalingg proteins, such as PI-3K, PTEN, Rho-like GTPases, andd GjS-y-suburrits. Their differential distribution mediates the amplificationn of the chemokine gradient on the outside into a (sffel)|siWlifJgg gradient inside the cell.24-25 Concomitant pofanzftidhh öfthe chemokine receptors at the cell surface is sibjectt to conflicting reports. Studies with C5a-receptor-GFP fusionn proteins in neutrophils or cAMP-receptor-GFP fusion fwfansHr** Dictyostelium cells showed uniform distribution oXjh^^^cStgrs_,p^Obfi-^toffla... membrane in cells that

rr (chemokine) recep-ors;; such as CCK2 and CCK5 on T-lymphocytes and the

receptorr on neutrophils, are distributed to the leading qjii exposure to their cognate ligands.27-28 In

B-lymphocytes,, SDF-1 induces polarization of CXCR4 to the leadingg edge of the cell.29 In addition, it has been reported thatt SDF-1/CXCL12 can bind to fibronectin and subse-quentlyy induces a polarized distribution of CXCR4 on adher-entt T-lymphocytes.30 We recently showed that immobilized SDF-11 on TNFa-activated endothelium rapidly induces po-larizationn of CXCR4 to the leading edge of the migrating cell andd that CXCR4 subsequenüy co-localizes with cholesterol lipidd rafts (Figure 2A).20 Disruption of lipid rafts by cyclo-dextrinn inhibited SDF-l/CXCL12-induced CXCR4 internal-izationn and cell migration, indicating that internalization and properr SDF-1-mediated signaling requires the presence of lipidd rafts.20-31

Adhesionn Molecules Inn the first phase of extravasation, leukocytes roll over the endothelium,, a process that is mediated by a family of adhesionn molecules called selectins.32-33 Recenüy, it became

vann Buul and Hordijk 16 6

Leadin gg edge

Figur ee 2. A, Schematic overview of the signaling pathways at the leading edge of a migrating cell. A leukocyte adheres through L-selectinn to cellular adhesion molecules (CAM) or glycoproteins such as CD34 on the endothelial cells» L-selectki induces CXCR4 upregulationn from intracellular granules on the leukocyte surface, resulting in increased activation of Rapt. Rapt induces a4-integrin activation,, possibly through phosphorylation. These actions on the leukocyte surface appear to takë'pfacë in al ipa raft. B, Schematic overvieww of the signaling pathways at the trailing edge of a migrating cell. An adhered leukocyte moves forward by leading-edge pro-trusionn and retraction of its tail. Therefore, «Lfe-integrin activates through Vav1, RhoA, which subsequently activates the kinase p160ROCK.. ROCK actjvation leads, through. MLCK activation, to MLC phosphorylation, resulting in retraction of the actin cytoskeleton. Thee actin cytoskeleton is linked via the ERM proteins to the adhesion molecules ICAM-3, PSGL-1, CD43, and CD44, which are expressedd at the rear end of the cell and might serve as an anchor for other migrating cells.

clearr that stimulation of L-selectin by antibody-specific cross-linkingg induced CXCR4 expression in lymphocytes.34

Moreover,, these authors showed that CXCR4 is stored in intracellularr vesitjes "and found that |-selectin, just as CXCR4,, localizes to lipid rafts.355 These findings suggest that selectin-mediatedd rolling of leukocytes over the endothelium, alreadyy primes we leuk^y t ts to^^^ tCXC^to l ip i f l ^ fe Inn addition, Shamri i "-grin (a4/3|)) requires cfiollIterolTSns for adhesive» leukocytee function-associated antigen-1 (LFA-1; aL02) acts independentt from lipid rafts to mediate adhesion.21 These dataa suggest the presence óf independent clusters of adhesion moleculess on the plasma membrane at the leading edge of migratingg leukocytes, before chemokine stimulation (Figure 2A). .

Integrinss play a major role in TEM of leukocytes and are, inn fact, indispensable for proper extravasation.36 The main integrinss that regulate SDF-1-induced adhesion and spread-ingg of leukocytes are LFA-1 (aJ32), Mac-1 (aMfe), VLA-4 («4/3,),, and VLA-5 (a5/3,).37-3s LFA-1, Mac-1, and VLA-4 bindd to the adhesion molecules intercellular cell adhesion molecule-11 (ICAM-1) and ICAM-2 (for LFA-1 and Mac-1), andd vascular cell adhesion molecule-1 (VCAM-1) (for VLA-4),, present on the apical surface of the endothelium.39-40 For thee integrins VLA-4 and VLA-5, it has been shown that they bindd to fibronectin.41 Studies in mice that lack ft-integrins or studiess that used blocking antibodies against /32-integrins showedd that neutrophil traffic was dependent on this inte-grin.42-433 Similarly, mice that lacked the a4-integrin showed

impairedd T-lynSphocyte homing to Peyer patches, although nott to other secondary lymphoid organs.44 In vivo studies wfc*f»oclfflg*antibodiess to the a4-integrin VLA-4 also slowedd that this integrin was involved in migration of CD34+

Is."" These data point to a prominent role for these •• Tie control of trafficking of leukocytes in

ndss induces multiple signalingg cascades in the leukocytes.46-47 This outside-in signalingg is translated by the leukocyte into cell polarization andd forward movement. This movement is generally assumed too be driven by the combination of leading edge protrusion andd contractility at both the front and the back of the cell, whichh allows displacement of the cell body.48-51 Some of thesee studies have used Dictyostelium discoidea amoebae as aa model, because the ability to sense and to respond to extracellularr signals is remarkably similar to that of mamma­liann leukocytes.49

AA large number of structural and regulatory proteins are locatedd at the leading edge of the migrating cell, including proteinss that regulate the dynamics of the actin cytoskeleton, suchh as the small GTPase Racl, its effector PAK1, and the Arp2/33 complex.52-53 Also, focal adhesion and focal contact proteinss such as adapter proteins, tyrosine kinases, and integrinss are found at the leading edge.52-53 These studies mainlyy focused on fibroblasts; therefore, one should take in accountt that different cell types might use different signaling pathwayss to achieve the same goals.

ArteriosclerArterioscler Thromb Vase Biol.April 2004 17 7

Att the back of the migrating cell, proteins such as ICAM-3, PSGL-1,, CD43, and CD44 are localized.54-55 ERM proteins actt as adapter proteins and link these transmembrane proteins too the actin cytoskeleton.56-57 To move forward, cells have to formm new adhesion sites and break-down old ones, both in the protrudingg and in the retracting part of the cell. The turnover off focal adhesions is very complex and involves integrins58

andd microtubules.59 Because of space limitations, we cannot discusss the complex mechanisms that drive of focal adhesion turnover.600 Recently, it has been shown that the a4-integrin is phosphorylatedd on serine residues at the leading edge of polarizedd cells and is required for lamellipodia stability, althoughh a4 is also present at the trailing edge.61 In addition, thesee authors showed that paxillin, a protein present in focal adhesions,622 co-localizes with a4 at the trailing edge but not at thee leading edge with the phosphorylated a4. Moreover, enforcedd association of paxillin with <*4 inhibits migration, indicatingg that the <*4-paxillin interaction mediates attachment andd detachment of membrane protrusions, which is required forr migrating cells to move forward.

Nextt to the a4-integrin, LFA-1 (aLft) signaling is required forr proper leukocyte migration. Whereas the a4-integrin uses paxillinn to regulate the attachment and detachment of the cells'' leading edge, LFA-1 seems to signal through myosin lightt chain kinase and through pl60ROCK, which regulates celll contraction and detachment from the matrix (Figure 2B).633 Moreover, LFA-1 engagement induces actin polymer­izationn at the leading edge of the cell,64 possibly through the Rhoo family exchange factor \?avl.65 Thus, bothjWl Integrins andd LFA-1 are required for leukocyte migration, acting throughh distinct signaling cascades, to drive efficient,.., chemotaxis.. i I IS l ^ S 1 %

JLL JL MM, % „ # . § • MA,. •• • Smalll GTPasesf J^ ,m.. _ 1 f - J Ass is well known from studies in fibroblasts as well as in leukocytes,, Rho-like small GTPases (in particular Rho, Rac, andd Cdc42) are crucial for coordinated cell adhesion, spread- ,„ ing,, and migration.66 Racl, being a critical regulator of cell spreading,, actin polymerization, and membrane ruffling in anyy type of cell, is found throughout the cytoplasm under restingg conditions. However, in its active, GTP-bound form, Racll localizes to growth factor-induced membrane ruffles andd to the leading edge of migrating cells.67 These authors showedd additionally that /3,-integrins induce the interaction of activee Racl with its downstream effectors, such as PAK1.67-68

Usingg C-terminal cell-permeable peptides as selective inhib­itors,, we recently showed that Racl (and not Rac2 or CDC42) playss an important role in SDF-1-induced actin polymeriza­tionn in HL-60 cells.69

Nextt to the involvement of Racl in driving membrane protrusion,, the small GTPase Rap 1 was shown to be involved inn the control of the adhesive capacities of LFA-1 and VLA-4.70-711 Moreover, Rapl can be activated by SDF-1, and inn its active form induces cell polarization. In addition, active Rapll promotes transendothelial migration of T-lymphocytes.72

Recently,, RAPL, a Rapl-binding protein, was shown to associatee with Rapl after SDF-1 stimulation. RAPL induces polarizationn of T-lymphocytes and distribution of LFA-1 to thee leading edge of the cell.73 These data suggest that Rapl

cann signal between chemokine-activated G-protein-coupled receptors,, such as CXCR4, and integrin activation (Figure 2A). .

Ass described, lamellipodia and membrane protrusions at thee front of the cell are required to move the cell forward. Consequently,, the rear of the cell has to contract and to detach fromm the underlying matrix, ie, from the endothelial mono­layer.. RhoA and one of its effectors, the serine/threonine kinasee plöOROCK, have been found to mediate tail retraction off migrating leukocytes.7475 Inhibition of RhoA activity by C33 transferase in monocytes prevents migration through an endoüieliall monolayer, although overall attachment of mono­cytess to activated endothelial monolayers was not affected. Interestingly,, the adhesion to either ICAM-1 or VCAM-1 was largelyy promoted on inhibition of pl60ROCK.75 In addition, thee /32-integrin mislocalized in these cells from the leading edgee to the trailing edge, indicating that RhoA and plóOROCKK signaling mediates integrin localization at the leadingg edge. The results suggest a model in which RhoA activityy is inhibited at the front of the cell to allow the inductionn of protrusions, in concert with enhanced Racl activityy at the leading edge. Conversely, RhoA is activated at thee rear of the cell to retract and detach the uropod and Rac 1 mayy become inhibited at sites of tail retraction. The events thatt occur in chemokine-mediated leukocytes and are in­volvedd in migration are summarized in Figure 2A and 2B.

Consequencess of Leukocyte^Endotheliumm Interactions Huangg ef al were the first to show that intracellular calcium levelss in endothelial cells were increased on adhesion and transmigrationn bf neutrophils.4 Moreover, they showed that thisthis was afsó' required for efficient TEM. In addition, Hixen-b|ughh et al showed that chemoattractant-stimulated neutro­philss induce an increase in the phosphorylation of myosin lightt chain (MLC) in endothelial cells.76 Increased MLC phosphorylationn accompanies RhoA activation, subsequent celll contraction, and a loss of endothelial cell-cell junctions, whichh tfien leads to increased permeability of the endothelial barrier.7'-788 Using an inhibitor to ML,C kinase (MLCK) on endotheliall cells, Saito et al showed that transmigration of neutrophilss was blocked and MLC phosphorylation was inhibited.799 Adhesion molecules such as P-selectin and E-selectinn and VCAM-1 on the endothelium might be respon­siblee for transmitting the signals, induced by adhered neutro­philss into the endothelium, because activation of these adhe­sionn molecules through antibody-specific cross-linking resultss in a transient increase of intracellular calcium.80 These studiess put forward the notion that leukocyte adhesion affects thee endothelial cells in a specific fashion and that endothelial celll function contributes importantly to TEM. Today, the involvementt of endothelial signaling in transmigration of leukocytess is an accepted phenomenon, and various signaling eventss in endothelial cells have been implicated in the process off leukocyte TEM, although the exact mechanisms by which thee endothelial cells facilitate the passage of leukocytes is still nott entirely clear.81-82

Ass noted, leukocytes use mainly /3, (VLA-4/a4j3,) and /32

(LFA-l/aL/32)) integrins to firmly adhere to the endothelium.

vann Buul and Hordijk 18 8

Thee ligands for these integrins on the endothelium are VCAM-11 and ICAM-1, respectively. More recently, a novel familyy of integrin ligands, called junctional adhesion mole­culess (JAM), has been discovered.83 The role of JAMs in transendotheliall migration will be discussed. Signaling throughh ICAM-1, induced by antibody-mediated cross-linking,, activates RhoA.84 Moreover, other proteins that regulatee the actin cytoskeleton, such as FAK, Cas, and cortactin,, are phosphorylated on tyrosine on antibody-inducedd cross-linking of ICAM-1.85 Thus, engagement of ICAM-11 induces signaling in endothelial cells. However, one shouldd take into account that receptor cross-linking is a rather globall stimulus compared with leukocyte binding to the endothelium.. The short (28 amino acids) intracellular tail of ICAM-11 interacts with ezrin, an adapter protein that links ICAM-11 with the actin cytoskeleton.86 Ezrin is a member of thee ERM protein family and plays an important role in lamellipodiaa induction at the leading edge of fibroblasts.87-88

Moreover,, it appears that ezrin links the proteoglycan syndecan-2,, which might possibly bind SDF-1, to the actin cytoskeleton.899 Detailed analysis of monocytes adherent to activatedd endothelium or cross-linking of the adhesion recep­torss ICAM-1 and VCAM-1 showed clustering of the ERM proteinss near those sites of activation.90 Next to its function as ann adapter protein, there is evidence that shows ezrin to act as aa signaling molecule, because active ezrin is reported to be responsiblee for the formation of lamellipodia.91 In addition, thesee authors showed^that Racl, but nat.RhoA or,Cdc42, is activatedd in cells that express the active form of ezrin. Other previouss work suggested that the ÈRM proteins are rapidly phosphorylatedd in a Rho>dependent manner.9? I3arreiro et al recentlyy showed that the endothelial cells form a so-called dockingg structure to "catch"" the migrating leukocyte, and theyy subsequently showed that moesin and ezrin localized to thosee docking structures, together with ICAM-1 and VCAM-1.93 3

Wee recendy showed that clustering of VCAM-1 activates Racl.944 Inhibition of downstream signaling by Racl with an inhibitoryy peptide, encoding the effector loop of Racl (aal 7-32;95),, reduced the migration of leukocytes across endothelial monolayers.944 Because transduction of an active mutant of Racl,, RacV12, reduces endothelial cell-cell contacts,96-98

blockingg Rac activity might prevent opening of endothelial cell-celll contacts, thereby preventing leukocytes from mi­grating.. Moreover, ligation of VCAM-1 activates phosphati-dylinositoll 3-kinase (PI3-K), implicated as an activator of R a c ll 99.100

Studiess performed by our group already showed that inhibitionn of endothelial RhoA by C3 transferase decreased monocytee migration across human umbilical vein endothelial cells,, although the C3 pretreatment had little or no effect on thee migration of primary CD34+ cells across bone manow endodieliall cells.12-94 Also, inhibition of plóOROCK signal­ingg in endothelial cells by Y-27632 decreases not only migrationn of lymphocytes across endothelium but also adhe­sion.933 The latter results indicate that RhoA activity is linked too adhesion molecules on the endothelium and that blockade off RhoA activity or downstream effectors prevents leukocyte adhesionn as well as endothelial cell contraction and subse­

quentt loss of cell-cell contacts. Thus, blocking signaling throughh Racl or RhoA in endothelial cells inhibits transen­dotheliall migration of monocytes. It is therefore likely that bothh RhoA and Racl in endothelial cells are involved in leukocyte-inducedd endothelial signaling: RhoA through liga­tiontion of ICAM-1 and Racl through the ligation of VCAM-1.

Forr many years, it has been known that reactive oxygen speciess (ROS) play an important role in the maintenance of thee endothelial monolayer integrity.101102 The generated ROS aree spontaneously or enzymatically converted to hydrogen peroxidee (H202), which may disrupt the integrity of die endotheliall monolayer, possibly through inhibition of ty­rosinee phosphatases.103104 Some studies already reported the presencee of components of the neutrophil-NADPH-oxidase (gp91)) complex in endothelial cells.105106 Other studies suggestedd the expression of various NADPH-oxidase homo-loguess in endothelial cells.107 Upregulation of Nox-based NADPH-oxidasess after vascular damage have been report­ed.108-1099 Nevertheless, how the activity or intracellular local­izationn of these protein complexes is regulated remains unclearr and warrants further study. Our group has previously shownn that active Racl induces ROS production in endothe­liall cells and that endothelial, ROS are actively involved in TEM,, because leukocyte migration across endothelium is inhibitedd when die endothelial monolayer is pretreated with oxygen-radicall scavengers.97 Similar findings have been re­portedd by Wang and Doerschuk, who showed that engage­mentt of ICAM-1 induces ROS production in endothelial cells,1100 In line with these findings, we and others have found thatt activationrof VCAM-1 and PECAM-1, by antibody-mediatedd crosSflinking, results in increased levels of ROS predEcuon.12-,1,-n22 These findings suggest that leukocyte adhesionn to endothelium induces ROS production, which in turnn might transmit signals into the endothelium to facilitate leukocytee passjge.

Activatiorroff ICAM-1, by cross-linking, remodels the actin cytoskeletonn through activation of p38 mitogen-activated proteinn kinase.1" Also, VCAM-1 is able to activate p38 mitogen-activatedd protein kinase in a ROS-dependent man­ner.9** Extracellular signal-regulated kinase-2, which is also involvedd in motility and in proliferation, is also activated by VCAM-1.999 Lorenzon et al showed previously that VCAM-1 cann act as a signaling receptor.80 In their studies, VCAM-1 cross-linkingg was found to induce a transient increase in endotheliall calcium concentration, which is required for efficientt TEM.

Thesee data suggest that engagement of ICAM-1 and VCAM-1,, through the integrin-mediated adhesion of leuko­cytes,, induces intracellular signaling in the endothelial cells thatt finally results in the opening of the endothelial cell-to-celll junctions. This hypothesis is supported by studies that showedd that active Racl induces loss of cell-to-cell adhesion inn endothelial cells.96-98 This contrastss with epithelial cells, in whichh active Racl promotes cadherin-based cell-to-cell ad­hesion.1144 Although a lot is known about epithelial regulation off cell-to-cell junctions, relatively little is known about the controll of endothelial cell-to-cell junctions with respect to the openingg and resealing of these junctions during the passage of

ArteriosclerArterioscler Thromb Vase Biol. April 2004 19 9

leukocytes.. The last part of this review discusses our current knowledgee about this topic.

Endotheliall Cell-to-Cell Junctions in Leukocyte Transendotheliall Migration Leukocytess need to migrate through the endothelial cell-cell junctionss to cross the endothelial monolayer in the final stage off extravasation.2 To successfully cross the junctions, the leukocytess have to bypass or block the adhesion, which is mediatedd by junctional proteins. The endothelial cell-to-cell junctionss contain a large number of (adhesion) proteins, includingg vascular endothelial (VE)-cadherin (CD144, cadherin-5),, a homophilic calcium-dependent transmembrane adhesionn molecule, which is localized at the adherens junc­tions.. In addition, the endothelial junctions comprise tight junctionn proteins, eg, occludin, JAM family members, and claudins,, as well as proteins such as CD99 and PECAM-1 (CD31),, which are more diffusely distributed at the cell-cell junctions. .

Manyy studies have described PECAM-1 as an important playerr in the process of transendothelial migration based on thee effects of blocking PECAM-1 in vitro115"118 and in vivo.119-1277 However, PECAM-1 knockout mice do not show aa significant loss of inflammatory response, demonstrating thatt PECAM-1 is not indispensable for transmigration.128 The mechanismss that compensate for the loss of PECAM-1 are nott clear; however, Mamdouh et al recently found that in the absencee of leukocytes, PLC AM-1 is present in a conipartifen directlyy under the' junctional border membr; investigatorss suggest a new mechanism in wh leukocytess trigger these compartments to ] junctionall border membrane. Because PEC* onn the leukocyte as well as on the endothelium, this rriedif-" nismm might facilitate leukocytes to cr(f|s the endothe'i ! cell-to-celll junction i imophilic PECAM-I ing.1299 This mechanism also implies that leukocytes o engulfedd by endo^l^BïcellsJt_fcr_Jnj aforementioned d describedd that neutrophils are also able liall layer by a transcellular route, parti. thiss process is controlled is unknown

PECAM-11 is phosphorylated on tyrosine residues atter endotheliall cell stimulation by mechanical force and shear stresss by means of the Fer tyrosine kinase, which is localized onn microtubules.131132 Moreover, Ilan et al showed that PECAM-11 can function as a reservoir for tyrosine-phosphor-ylatedd /3-catenin.133 These data suggest that PECAM-1 bind­ingg to /3-catenin prevents cytosolic /3-catenin from degrada­tionn by the proteosomes. Subsequently, PECAM-1 engagementt induces SHP-2 phosphorylation through Fer, whichh then dephosphorylates PECAM-1-bound /3-catenin. It hass been shown that increased tyrosine phosphorylation of junctionall proteins leads to loss of cell-to-cell adhesion and thatt subconfluent endothelial monolayers have increased levelss of tyrosine phosphorylation of the junctional proteins VE-cadherin,, /3-catenin, and -y-catenin.134135 Moreover, SHP-22 and /3-catenin are both present in the VE-cadherin complexx at cell-to-cell junctions.136 In addition, inhibition of proteinn tyrosine phosphatase activity promotes the flux of

albuminn across an endothelial monolayer and the extravasa­tionn of neutrophils, indicating that phosphorylation plays an essentiall role in the control of endothelial integrity and of the processs of TEM.137 Phosphatase activity can be inhibited by H202,, because H202 mediates the oxidation of critical cys­teinee residues in tyrosine phosphatases.138 As a result, ty­rosinee phosphorylation levels are increased, triggering loss of endotheliall cell-to-cell junctions. This suggestion links VCAM-1-inducedd ROS production and phosphatase/kinase activityy to the transendothelial migration of leukocytes.

Anotherr family of junctional proteins that has attracted a lott of attention are the JAMs. Recently, new nomenclature for thee JAM family has been put forward by Muller and is used inn this review.139 JAM-A has been cloned and identified recentlyy and appears to mediate homophilic and heterophilic interactions.1400 JAM localizes at the apical regions of cell-to-celll junctions, not only of endothelial cells but also of epitheliall cells, and regulates the electrical resistance across monolayerss of these cells.141142 Antibodies to JAM cause decreasedd transendothelial migration of leukocytes in vitro andd in vivo, strongly suggesting that this molecule might be involvedd in TEM.140143 Ostermann et al have shown that JAM-AA on endothelial cells is a ligand for the /32-integrin LFA-1,, and as such is imohed in tiansendothelial migration off leukocytes.144 The JAM protein family now comprises 3 members:: JAM-A (approximately 32 kDa), present on endo­theliall cejls, epithelial cells, and on all leukocytes, functions as»aa »guktpri|Of TEM and of electrical resistance. JAM-B (or VE-JAM).. which is ^40 kDa in size, is expressed on vascular andd lymphatic endothelium and has been proposed to play a rolee in lymphocyte homing.145146 JAM-B also interacts with thee /3,-integrin receptor VLA-4.147 Most recently, a third familyy member has been identified as JAM-C («» 43 kDa), presentt on endothelial cells, T-lymphocytes, platelets, and

ceJE.1'*7'1*88 JAM-C also functions as a regulator of TEM .es'' and electrical resistance. It interacts with J|c,-11 (aM/32-integnn».present on leukocytes) but

rr VLA-1 findings indicate that yy men^erscouhT^tentially be involved in

ansendötheliall migration but that they are inde-nvolvedd in specific parts of the TEM process.8'

ee integral transmembrane protein CD99 regulates VLA-4-dependentt adhesion of T-lymphocytes to endothelial cells underr physiological shear stress.151 In contrast to VLA-4, j32-integrinss (ie, LFA-1) were not influenced by CD99 stim­ulation.. Recently, CD99 was found to be present on endothe­liall cells as a homophilic binding molecule at cell-to-cell junctions.1522 Antibodies to CD99 were found to block trans­migrationn of monocytes, particularly during diapedesis. Blockingg antibodies to PECAM-1 inhibit monocyte migration beforee diapedesis started, indicative for a sequential engage­mentt of these proteins during transmigration across the junction.1522 Thus, whereas JAM and PECAM-1 are involved inn the first stages of transmigration across endothelium, CD99 seemss to regulate further passage through the endothelial junctions.. A note should be made that, as mentioned, PECAM-11 is required for migration through the basement membranee but is apparently also involved in early steps of diapedesis. .

vann Buul and Hordijk 20 0

Figur ee 3. Schematic overview of the signaling pathways that occur during TEM in endothelial cells. Adherent leukocytes activate endo-theliall cells through binding to adhesion molecules such as ICAM-1 and VCAM-1. The small GTPase Rad induce^ ruffles, possibly leadingg to docking structures and ROS production. ROS inhibit phosphatase activity, which results in increased kinase activity and loss off VE-cadherin-mediated cell-cell junctions. Subsequently, the small GTPase RhoA induces activation of MLÓ and cell contraction. Togetherr with junctional molecules such as PECAM-1, JAM, and CD99, these actions are likely to facilitate translBgration of leukocytes acrosss an endothelial monolayer. *"

Previouss reports have shown that adhesion of neuttopnils too endothelium disrupts the VE-cadherin complex at cell-cell junctions.1"1544 However, it was subsequently shown that preparationn of cell lysates under relatively fhild conditions allowss neutrophil proteases to cause breakdown of the VE-cadherin-cateninn complex.1'5 However, the latter study by Molll et al did not indicate whether proteases are involved in . thee physiological process; it only clarified that such experi- ; mentss could not demonshate regulated proteolysis of VE-cadherinn on neutrophil attachment. Nevertheless, it has been observedd with GFP fusion proteins and live-cell in VE-cadherinn ana localizedd from cell-cell junctions on passage of monocytes or

thee endothelium.'2156157 Recent evi-CD34++ cells through dencee showed thÉ elasiasè, a- protease present in neutrophil granuless and released on activation, is able to degrade the extracellularr part of VE-cadherin, keeping the hypothesis alivee that neutrophils disrupt endothelial cell-cell junctions byy releasing elastase and possibly other proteases to migrate throughh the endothelial cleft.158 However, studies with neu­trophilss from elastase-deficient or MMP-9-deficient mice havee shown that these proteases are not required for rolling over,, adhesion to, or migration across endothelium, although thesee neutrophils were still able to disrupt VE-cadherin function.1599 Together, these studies indicate that proteases mightt be involved in transendothelial migration, but that the rolee for elastase and MMP-9 seems to be limited. Figure 3 summarizess schematically the endothelial signaling that ac­companiess and drives TEM.

Conclusions s Littlee more than one decade since the seminal studies that startedd this field of research, it has become increasingly clear

thatt the 'emigration of leukocytes from the bloodstream IP P

imoivess complex signaling in the migrating leukocyte and in thee endomeliufn. This signaling needs to be carefully con­trolled,, not onI| to ensure proper and efficient extravasation butt also to limit leukocyte transendothelial migration to those sites^Wheree the cells are required. Mislocalized or excessive leukocytee exit damages me endothelium and underlying tissues.. Therefore, leukocyte migration and adhesion repre­sentt important areas for drug targeting. This is even more underscoredd by the realization that tumor cell metastasis ^ymfess leukocyte also that some tumor cells

onn specific ch'emokines, such as SDF-1, for their spreadingg through the body. The events that take place in the endotheliumm and accompany or even allow leukocyte diape-dèsiss have everything to do with control of endothelial integrity.. Given the essential role of the endothelium and its barrierr function for normal physiology, detailed knowledge of thee intracellular signaling and adhesion molecules involved is off key importance. The unique interactions between activated leukocytess and endothelial cells will likely, despite their complexity,, help us to understand the interplay between cell-surfacee molecules, intracellular signaling cascades, and thee control of motility and cell-cell adhesion.

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