Báo cáo khoa học: Endothelial signalling events during leukocyte transmigration pptx

Abbreviations CAM, cellular adhesion molecule; EC, endothelial cells; ERK, extracellular signal regulated kinase; ICAM, intercellular adhesion molecule; JAM, junctional adhesion molecule

Endothelial signalling events during leukocyte

transmigration

Peter L Hordijk

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, the Netherlands

Transendothelial migration (TEM) is an essential

aspect of the trafficking of leukocytes, as well as of

malignant cells Much pathology is associated with

uncontrolled TEM, for instance in chronic

inflamma-tory disorders (asthma, rheumatoid arthritis, psoriasis)

and in metastasis On the other hand, regulated

leuko-cyte trafficking is required for immune surveillance

and stem cell homing following transplantation

proce-dures The multistep model for TEM [1] is well

estab-lished and probably applies to most transmigration

events, albeit that tissue specificity may result in

addi-tional complexity For example, in the brain, the

endo-thelium constitutes the blood–brain barrier, which is

tighter than the endothelium in other tissues, resulting

in additional requirements for both leukocytes and the endothelium to allow efficient transmigration

Seminal work by the groups of Silverstein & Bender [2,3] has triggered an ever-growing list of studies [4] confirming the notion that endothelial cells (EC) not only mediate leukocyte adhesion, but also actively par-ticipate in the transmigration event Most of these studies have focussed on regulation of the paracellular pathway, although, recently, analysis of transcellular migration has also become fashionable This review will focus on events in the EC that have been implica-ted in these different routes of TEM

Keywords

adhesion; ICAM-1; migration;

transendothelial migration; VCAM-1

Correspondence

P L Hordijk, Department of Molecular Cell

Biology, Sanquin Research and Landsteiner

Laboratory, Academic Medical Center,

University of Amsterdam, Plesmanlaan 125,

1066 CX Amsterdam, the Netherlands

Fax: +31 20 5123474

Tel: +31 20 5123263

E-mail: p.hordijk@sanquin.nl

(Received 15 May 2006, accepted 17 July

2006)

doi:10.1111/j.1742-4658.2006.05440.x

The notion that it takes two to tango is certainly true for leukocyte trans-endothelial migration A growing pallet of leukocyte adhesion-induced signaling events in endothelial cells have been identified, mediating both short-term (i.e permeability) as well as long-term (i.e regulation of tran-scription) effects Efficient paracellular migration (i.e through endothelial cell–cell junctions) requires both intracellular calcium and the actin cyto-skeleton, but also involves small GTPases, reactive oxygen species and pro-tein kinases The alternative route of transcellular migration appears to depend on components such as caveolae and intermediate filaments This minireview discusses our current knowledge on the regulation of leukocyte transmigration through endothelial signalling

Abbreviations

CAM, cellular adhesion molecule; EC, endothelial cells; ERK, extracellular signal regulated kinase; ICAM, intercellular adhesion molecule; JAM, junctional adhesion molecule; MAPK, mitogen-activated protein kinase; NOX, NADPH oxidase; PECAM-1, platelet ⁄ endothelial cell adhesion molecule-1; ROS, reactive oxygen species; TEM, transendothelial migration; VCAM-1, vascular cell adhesion molecule-1;

VE-cadherin, vascular endothelial cadherin.

Endothelial docking structures

Rolling and adhesion of leukocytes over activated

endothelium (i.e at sites of inflammation) is

accompan-ied by a complex response from the endothelial cells

Initially, this comprises engagement and subsequent

clustering of endothelial adhesion molecules These

include E-selectin and Ig-like cell adhesion molecules,

such as intercellular adhesion molecule (ICAM)-1,

ICAM-2, vascular cell adhesion molecule-1 (VCAM-1),

platelet⁄ endothelial cell adhesion molecule-1

(PECAM-1) and members of the junctional adhesion

molecule (JAM) subfamily Subsequent to leukocyte

adhesion, the EC show a pronounced morphological

response by forming ‘docking structures’ [5] or

‘trans-migratory cups’ [6] These are actin-rich membrane

extensions that form around the adherent leukocyte In

these structures, not only are integrin ligands such as

ICAM-1 and VCAM-1 concentrated, but also adaptor

and linker molecules, such as ERM (ezrin, radixin,

moesin) proteins, vinculin, talin and a-actinin [5]

For-mation of these structures requires calcium and,

accord-ing to some [5], but not to others [7], activation of the

Rho⁄ p160ROCK pathway As a result of the

concen-tration of adhesion and signaling molecules, docking

structures represent the main signaling ‘platforms’ from

which intracellular signaling into the EC is initiated

There are indications that docking structures, and the

proteins therein, remain associated with the leukocytes

throughout the transmigration process [5,6] This might

well be important for the sustained signaling that is

required for efficient crossing of the endothelial barrier

Transcellular versus paracellular

migration

Leukocyte TEM has classically been considered to

occur at cell–cell junctions It is now clear that, next to

this paracellular pathway, transcellular migration (i.e

through the endothelial cell body) can also be

observed [8] In vivo analysis showed that neutrophils

can cross the endothelial monolayer in a transcellular

manner [9] Recently, a series of studies reported that

transcellular migration can also be observed in vitro

[6,10–13] In particular, ICAM-1 has been associated

with transcellular migration [6,10,11,13] It is obvious

that ICAM-1, being the main endothelial ligand for

b2-integrins, is crucial for TEM in general and for

polymorphonuclear cell transmigration in particular

[14] Yet, Yang et al [10] showed that prolonged

tumor necrosis factor-a treatment, or expression of an

ICAM-1–green fluorescent protein fusion on

immortal-ized EC, increases the relative contribution of

transcel-lular migration to polymorphonuclear cell diapedesis, suggesting that ICAM-1 plays an active role in deter-mining whether polymorphonuclear cells use the para-cellular or the transpara-cellular route Whether VCAM-1 plays a similar role, for example, for monocytes, is not known Additional regulatory factors that might pro-mote transcellular migration are the polygonal shape

of the EC or the levels of b2-integrin occupancy, shear and the presence of chemokines on the EC [10,13] The endothelial structures that mediate transcellular migration were initially suggested to be vesiculo-vacuo-lar organelles, which are abundant in EC and could align to form a channel for macromolecules [15] and perhaps even for migrating leukocytes More recently, transcellular migration was linked to caveolae, a sub-class of membrane lipid rafts that may, by invagination, detach from the membrane and mediate vesicular trans-port The protein caveolin, a key marker for caveolae, was found to be enriched at the site of leukocyte–endo-thelial cell contact [6] Using a caveolin knockdown approach, Millan et al [11] showed that caveolin was required for transcellular, but not for paracellular, TEM Another study, by Nieminen et al [12], has implicated the intermediate filament protein, vimentin,

in the process of lymphocyte transcellular migration However, its regulation and precise role in the transmi-gration process remains to be determined

Although these studies show that the paracellular and transcellular pathways co-exist, considerable vari-ation in the relative contribution of the transcellular pathway to leukocyte TEM has been noted This may depend on the type of leukocyte, as Yang et al repor-ted efficient transcellular migration (up to 50% of the total transmigration events) for neutrophils, whereas

T lymphocytes exclusively used the paracellular route [10] Contrasting findings have also been described (i.e that lymphocytes would preferentially use the transcellular route) [12] Similarly, the source of the endothelium (microvascular versus macrovascular [11]), and the state of activation of the endothelium

or the leukocytes [10,12,13], may also affect the relat-ive importance of one route over the other Yet, in most of these studies, the contribution of the transcel-lular pathway was only 10–30% to the total trans-migration events Intriguingly, down-regulation of caveolin expression in human umbilical vein endothel-ial cells blocked transcellular migration by T lympho-blasts, but did not reduce the overall TEM, suggesting that cells can switch from the transcellular to the paracellular route without a significant reduction in TEM efficiency [11] The factors that determine the choice of leukocytes for one or the other pathway remain to be established

Signaling by endothelial adhesion

molecules

As mentioned above, leukocyte adhesion and the

for-mation of endothelial docking structures is associated

with the clustering of adhesion and signaling

mole-cules It is very likely that this clustering is required

for efficient signal transduction into the EC which, at

least for some types of leukocyte, is important for

effi-cient TEM Many cell surface (adhesion) proteins have

been implicated in leukocyte TEM, in particular Ig

family members However, for only some of these has

the induction of intracellular signaling been causally

related to leukocyte transmigration

E-selectin

Although in the classical multistep model for TEM,

selectins are usually depicted as mediating low-affinity

interactions to allow rolling, there is ample evidence for

the signaling capacity of E-selectin (CD62 E), both

towards the actin cytoskeleton [16] as well as to p42

mi-togen-activated protein kinase (MAPK)⁄ extracellular

signal regulated kinase (ERK) activation and the

induc-tion of c-fos [17] Clustering of E-selectin, which is an

adhesion receptor for neutrophils and memory T cells,

results in its association with the actin cytoskeleton In

addition, clustered E-selectin associates, through its

intracellular domain, with Ras, Raf and MAPK⁄ ERK

kinase (MEK) These proteins trigger the downstream

signaling towards MAPK and c-fos [17,18] Later

stud-ies showed that tyrosine phosphorylation of the

E-se-lectin intracellular tail is instrumental in these events

through the recruitment and activation of the SHP-2

phosphatase, which signals, via Shc and Grb2 adapter

proteins, to the Ras-MAPK pathway [19] In addition,

E-selectin resides in caveolin-containing lipid rafts and

associates with phospholipase C gamma [20] Raft

dis-ruption ablates the activation of phospholipase C

gamma, but not of MAPK, indicating that the

activa-tion of different signaling pathways can occur in

dis-tinct membrane subdomains [20]

PECAM-1

The Ig-like CAM, PECAM-1, mediates homotypic

interactions between leukocytes and EC and between

EC themselves at intercellular junctions PECAM-1 has

been implicated in cell survival, angiogenesis, lung

development and experimental autoimmune

encephalo-myelitis [21,22] Blocking antibodies to PECAM-1

inhi-bit neutrophil and lymphocyte TEM in vitro [23,24]

(also see review by Petri & Bixel, this issue of FEBS) In

contrast to most other Ig-like CAMs, PECAM-1 has an extended intracellular tail that encodes two immuno-receptor tyrosine-based inhibition motifs and which is subject to tyrosine phosphorylation by src-like kinases, primarily in response to cell stimulation or PECAM-1 cross-linking [21] These immunoreceptor tyrosine-based inhibition motifs mediate, following phosphorylation, association with the SHP-1 and SHP-2 tyrosine phos-phatases, with the SH2 domain-containing inositol 5-phosphatase, SHIP, with adapter, proteins such as Grb2, and with b- and c-catenin PECAM-1 stimulates integrin adhesion by activating Rap1 [25] and has been associated with cell survival Similarly to E-selectin, PECAM-1 can activate MAPK via its association with SHP2 Remarkably, although the signaling capacities of PECAM-1 have been extensively studied, its relevance

as a signaling molecule in TEM is not clear This may also relate to the fact that PECAM-1-deficient mice showed only limited problems in models of inflamma-tion, although later studies reported that this result may depend on the mice strain used [26] Recently, however, the modulation of cell–cell adhesion by PECAM-1 has been proposed, based on studies in transfected epithelial cells [27] Whether these data can be readily translated

to EC remains to be seen

JAMs The family of JAM molecules concentrate in endothel-ial tight junctions [28] In addition, JAM proteins are expressed by leukocytes Several studies have clearly shown that JAM family members are essential for leu-kocyte TEM [8,29] (also see the review by Petri & Bixel, this issue of FEBS) In addition, JAM proteins have been implicated in cell signaling towards cell polarity and the formation of cell–cell contact JAMs can associate, through C-terminal PDZ-binding motifs, with a series of proteins, including ZO1, AF6, Par 3 and MUPP1 [28] Despite their role in the regulation

of cell–cell adhesion and the fact that the JAMs clearly have relevant signaling capacities, it is, as for PECAM-1, not yet known whether they in fact trans-mit signals into the EC that promote TEM

ICAM-1 ICAM-1 is one of the main integrin ligands involved

in leukocyte TEM, in particular of lymphocytes and neutrophils ICAM-1 is expressed on resting endo-thelium, but up-regulated upon activation by inflam-matory stimuli ICAM-1 has a short cytoplasmic tail

of 29 amino acids that associates to ERM (ezrin, ra-dixin, moesin) proteins, as well as to a-actinin [30,31]

ICAM-1 acts as an adhesion molecule and a signal

transducer in EC ICAM-1 activates the p60src kinase,

which leads to phosphorylation of cortactin [32],

trig-gers release of intracellular calcium and activates the

Rho GTPase, which explains the effects of ICAM-1 on

the actin cytoskeleton and on contractility in EC [16]

These effects are mediated by the C-terminus of

ICAM-1 and are required for efficient TEM of

lymphocytes [31,33,34] Moreover, ICAM-1 has been

shown to activate p60src via the activation of xanthine

oxidase, in a SHP2-dependent manner, leading to

tyro-sine phosphorylation of ezrin and p38 MAPK [35]

Finally, cell-permeable versions of the cytoplasmic tail

of ICAM-1 were found to block leukocyte TEM

[10,34,36] In conclusion, ICAM-1 activates a series of

signaling events through its intracellular C-terminal tail

that are likely to increase endothelial permeability,

resulting in enhanced leukocyte TEM

VCAM-1

The main b1-integrin ligand on the endothelium,

VCAM-1, is, in contrast to ICAM-1, absent from

rest-ing cells but greatly up-regulated by inflammatory

stimuli Similarly to ICAM-1, VCAM-1 not only acts

as an adhesion receptor, but also as a signal transducer

upon binding of leukocytes The cytoplasmic domain

of VCAM-1 is only 19 amino acids long and comprises

a type I PDZ-binding motif However, whether specific

interactions are mediated by this motif is unknown; to

date, only ezrin and moesin have been shown to

asso-ciate with the cytoplasmic domain of VCAM-1 [5]

VCAM-1 clustering leads to the activation of Rac1,

production of reactive oxygen species (ROS),

activa-tion of p38 MAPK and changes in the actin

cytoskele-ton (i.e stress fiber formation) (Fig 1) These events

have all been associated with the increased endothelial

permeability (as measured by tracer molecules or

trans-endothelial resistance) that is induced by VCAM-1

cross-linking VCAM-1-mediated leukocyte TEM is

also dependent on some of these signaling events,

including Rac1 and Rho activation [37] Of particular

interest is the role of VCAM-1-induced production of

ROS ROS are known to impair cell–cell adhesion in

EC and are important regulators of endothelial

integ-rity through their indirect stimulation of tyrosine

kinase activity In addition, vascular ROS play an

important role in the development of cardiovascular

disease [38] Conversely, scavenging ROS preserves

endothelial barrier function, prevents endothelial cell

migration and angiogenesis, and is atheroprotective

The source of endothelial ROS has been suggested to

be the NADPH oxidase 2 (NOX2) which is, like its

relative NOX4, also expressed in EC [39,40] NOX2 is supposedly localized in the endothelial plasma mem-brane, resulting in an extracellular release of ROS These have been proposed to activate metalloproteases that would promote endothelial permeability by proteolytic degradation of vascular endothelial

cadher-in (VE-cadherin) or of the extracellular matrix (Fig 2A) [39]

Our laboratory has shown that ROS production in response to VCAM-1-mediated activation of Rac1 can

be observed intracellularly, and we have proposed a role for the redox sensitive proline-rich tyrosine kinase

2 in the control of endothelial integrity through the phosphorylation of b-catenin [41] (Fig 2B) However, our present knowledge of this pathway remains limited The molecular mechanism of VCAM-1-triggered acti-vation of Rac1 is completely unknown, as is the poten-tial role for the relatively abundant NOX4 protein in VCAM-1 signalling Also, the mechanism of VE-cadh-erin inactivation through ROS (i.e either by proteolytic breakdown, or by reducing its homophilic adhesion through reduction of its link to the actin cytoskeleton),

be it from the inside or the outside of the cells, requires further analysis In addition to ROS signaling, endo-thelial integrity is also subject to regulation by the Rap1 GTPase, microtubule dynamics and by proteins that control VE-cadherin internalization To what extent these events are also part of the process of leukocyte TEM is presently unclear

It is important to underscore that engaged, clustered ICAM-1 and VCAM-1 may be in very close proximity

on the endothelial cell surface, in particular following adhesion of leukocytes that use b1 and b2 integrins for transmigration This means that the signaling which is induced by these molecules may also be intercon-nected The extent of cross-talk between ICAM-1 and

Fig 1 Clustered vascular cell adhesion molecule-1 (VCAM-1) aligns with actin stress fibers Transient expression of the VCAM-1–green fluorescent protein (GFP) fusion (green) shows its diffuse distribu-tion over the endothelial cell surface (left panel) Cross-linking by a VCAM-1 antibody induces clustering of the protein and alignment

of the VCAM-1–GFP clusters with actin stress fibers (right panel; F-actin in blue).

VCAM-1 induced signalling events is thus an

import-ant issue for future research

Effects of shear

EC in various parts of the vasculature are exposed to

different levels of fluid shear stress There is no doubt

that this shear force triggers and modulates endothel-ial cell signaling and affects endothelendothel-ial permeability, proliferation, migration and gene expression [42] Shear stress is strongly associated with the develop-ment of atherosclerosis, which is an arterial disease that occurs predominantly at sites of disturbed lami-nar blood flow VCAM-1 and E-selectin, in conjunc-tion with the actin cytoskeleton, have been shown to activate ERK2 in a shear-dependent manner [18] Recently, the vascular endothelial growth factor receptor, VE-cadherin and PECAM-1 were identified

as components of a shear detecting complex in EC [43] This complex mediates shear induced and ligand-independent activation of src and of the phosphatidyl-inositol-3-Kinase⁄ Akt pathway and is required for the activation of nuclear factor-jB at sites of disturbed flow Apart from affecting the EC, shear also pro-motes chemokine-induced lymphocyte TEM, an effect coined ‘chemorheotaxis’ [13] Thus, shear force repre-sents an additional level of regulation of both leuko-cyte migration as well as endothelial signalling

Targets of endothelial signaling There appears to be at least two classes of endothelial target downstream of the signaling that is initiated by leukocyte binding There are rapid effects on the actin cytoskeleton and the VE–cadherin–catenin complex and these appear to co-operate in mediating efficient transendothelial migration On the other hand, there

is evidence for activation of transcription factors, such

as nuclear factor-jB and c-fos [17] The subsequent up-regulation of cell adhesion molecules or metallo-proteases may have important effects on the amplifi-cation and⁄ or duration of the inflammatory response Activation of ERK may well play a role in both pathways, as ERK has been implicated in the regula-tion of cell adhesion and migraregula-tion [44] In addiregula-tion, ERK is involved in the activation of c-fos and of nuclear factor-jB Thus, it appears that there is co-operativity between the ERK and p38 MAPK path-ways, as well as the ROS that are produced in the

EC, in altering the gene expression profile of activated endothelium

Concluding remarks Along with the increased knowledge on the control of endothelial integrity, the number of signaling compo-nents that are implicated in the efficient transmigration

of leukocytes is also growing The key players appear

to be small GTPases and the actin cytoskeleton, ROS, MAPKs, cell-matrix adhesion molecules, transcription

A

B

Fig 2 Models for the action of vascular cell adhesion molecule-1

(VCAM-1)-derived reactive oxygen species (ROS) in controlling

transendothelial migration (TEM) According to one model (Fig 2A),

VCAM-1 activates NADPH oxidase 2 (NOX2), which resides in the

plasma membrane, in a Rac1-dependent manner Extracellularly

produced ROS activate metalloproteases, which degrade junctional

and ⁄ or matrix proteins A second model (Fig 2B) proposes

ROS-mediated activation of the proline-rich tyrosine kinase 2 and

phos-phorylation of b-catenin as instrumental in the transient loss of

vascular endothelial cadherin-mediated cell-to-cell contact, which

follows VCAM-1 engagment Intercellular adhesion

molecule-1-mediated RhoA activation is required in both models, for providing

enhanced endothelial contractility See the text for details.

factors, and also enzymes such as calpain or activated

metalloproteases A recurrent theme is that the type of

leukocyte, source of the EC, inflammatory stimulus

and the absence or presence of shear, will affect the

responses measured and thus the consequent

implica-tion of a particular event in TEM

The complexity of this field is further boosted by

studies on the transcellular pathway A major

chal-lenge will be to define whether this pathway is

domin-ant in specific tissues, as was proposed for the brain,

or perhaps associated with certain (pathological)

con-ditions Also, it remains to be determined whether

transcellular migration is regulated by endothelial

sign-aling, or is primarily a result of the protrusive activity

of leukocytes Thus, although we are not quite dancing

in the dark anymore, many issues remain that

guaran-tee a more complex, but no less interesting, future for

the research on leukocyte TEM

Acknowledgements

Dr Jaap van Buul is gratefully acknowledged for

crit-ical reading PLH is a fellow of the Landsteiner

Foun-dation for Blood Transfusion Research I apologize to

all whose work could not be included because of space

constraints VCAM-1-GFP was a kind gift from

Dr Sanchez-Madrid

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