Review: Leucocyte-endothelial cell crosstalk at the blood-brain barrier: A prerequisite for successful immune cell entry to the brain

Leukocyte-endothelial cell crosstalk at the blood-brain barrier: A

prerequisite for successful immune cell entry to the brain

J. Greenwood1*, S.J. Heasman1, J.I. Alvarez2, A. Prat2, R. Lyck3 and B. Engelhardt3

1Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London

EC1V 9EL, UK. 2Neuroimmunology Research Laboratory, Faculty of Medicine, Université de

Montréal, Montréal, Québec, Canada. 3Theodor Kocher Institute, University of Bern, CH-3012

Bern, Switzerland

* Correspondence

Leukocyte-endothelial crosstalk at the BBB, Greenwood et al. 2010 Page 2

Brief summary

Leukocyte migration into the central nervous system (CNS) is a key stage in the development of

multiple sclerosis. Whilst much has been learnt regarding the sequential steps of leukocyte

capture, adhesion and migration across the vasculature the molecular basis of leukocyte

extravasation is only just being unravelled. It is now recognised that bi-directional cross-talk

between the immune cell and endothelium is an essential element in mediating diapedesis during

both normal immune surveillance and under inflammatory conditions. The induction of various

signalling networks, through engagement of cell surface molecules such as integrins on the

leukocyte and immunoglobulin superfamily cell adhesion molecules on the endothelial cell, play a

major role in determining the pattern and route of leukocyte emigration. In this review we discuss

the extent of our knowledge regarding leukocyte migration across the blood-brain barrier and in

particular the endothelial cell signalling pathways contributing to this process.

Introduction

It is widely recognised that in multiple sclerosis (MS) exacerbated leukocyte traffic into the central

nervous system (CNS) represents a key stage in disease development. How leukocytes traffic in

and out of the CNS, therefore, is of considerable interest as it represents a potentially exploitable

therapeutic target [1]. It should be recognised, however, that many other diseases of the CNS

exhibit aspects of inflammation and as such leukocyte-endothelial interactions and migration may

also contribute to the pathogenesis of non-autoimmune diseases. For example, leukocyte traffic

into the CNS may be important in a diverse range of disorders including stroke [2],

neurodegeneration [3], vasculitis [4] and infection [5,6]. Moreover, in the retina, leukocyte adhesion

and infiltration are known to play a major part in diabetic retinopathy and posterior uveitis

respectively [7,8]. The potential importance of leukocyte traffic in many of these diseases remains

to be fully elucidated as investigations into the mechanisms governing leukocyte extravasation

have, in the main part, been conducted solely within the context of MS. Such studies have now

provided the biomedical research community with a valuable set of general principles describing

this phenomenon. Nevertheless, many gaps still exist in our knowledge and the presence of

specialised endothelial cells (ECs), expressing unique complex tight junctions (TJs) and forming


the blood-brain barrier (BBB) in the CNS, adds an additional degree of complexity to the process.

This is especially pertinent given the increasing recognition that the EC plays an active and

important role in the recruitment of leukocytes from the blood. In order to migrate into the CNS,

immune cells must engage in dynamic cross-talk with the ECs lining the vessel wall which results

in the activation of outside-in and inside-out signalling pathways essential for successful

transmigration. For example, through signalling initiated by chemokines sequestered on the

endothelium leukocytes undergo changes that enhance their adhesive properties. Upon leukocyte

integrin binding to EC immunoglobulin superfamily (IgSF) cell adhesion molecules (CAMs) outside-

in signals are then initiated in the endothelium that further support adhesion and the subsequent

process of transendothelial migration. However, many other activation pathways are also likely to

occur resulting in both immediate and longer-term effects on cell function, although our knowledge

of many of these signalling outcomes remains superficial.

Our understanding of leukocyte migration has been further complicated by the re-emergence of the

notion that leukocytes can transmigrate through the body of the EC via pore formation or a

phagocytic-like process (transcellular diapedesis) [9-11] as well as through the EC cell-cell junction

(paracellular diapedesis). Indeed, in the CNS it was suggested as long ago as 1990 by Raine and

colleagues that both these routes were utilised and that they support the differential transvascular

migration of distinct leukocyte subsets [12]. Furthermore, at the tight vascular barrier of the retina

(an outpost of the brain) much of the ultrastructural evidence also pointed towards substantial

transcellular pathway of diapedesis [13,14] (Figure 1). Now that it is generally accepted that both

paracellular and transcellular leukocyte diapedesis occurs it begs the question what regulates the

differential migratory pathways. Again, it is likely that the EC in combination with specific leukocyte

signals will determine the path.

In this review we will report on current knowledge regarding the two-way signalling between

leukocyte and EC with a particular focus on the latter and how such signalling contributes to

leukocyte diapedesis in the CNS.

Blood-to-brain leukocyte traffic is a multi-step process


A hallmark feature of relapsing remitting MS, and its animal correlate experimental autoimmune

encephalitis (EAE), is the formation of focal inflammatory lesions within the CNS. These are

comprised of perivascular cuffs consisting of mainly T lymphocytes and monocyte/macrophages,

but also of dendritic cells (DCs) and B cells. As highlighted above, the migration of these cells

represent a key stage in the natural history of the disease but what initiates this event remains

unclear. Under normal conditions antigen-activated lymphocytes are capable of low level

surveillance throughout the CNS and this limited entry is regulated not by the presence of a

vascular barrier but largely by the restricted expression of endothelial CAMs required for leukocyte

capture from the blood. The level of immune surveillance under normal conditions is a fraction of

that observed in other tissues [12,15]. For example, it has been estimated that traffic into the

normal CNS is 100 fold less than that observed for spleen or lung [16,17] and following

intravascular injection of 5x107 labelled antigen-activated T cells in mice an average of only 14.2 (±

3.0 SEM) extravasated cells were found per 100μm coronal brain section after 14 h [18]. However,

once adherent, appropriately activated leukocytes such as antigen specific CD4+ T cells can cross

with the support of the EC. The EC may in turn become activated in response to leukocyte

engagement or to leukocyte-derived cytokines such as tumour necrosis factor (TNF)-α, interferon

(IFN)-γ, interleukin (IL)-17, IL-22 and IL-1β which induce or up-regulate CAM expression and

hence further recruitment of leukocytes leading to an escalation of the inflammatory cascade.

Indeed, in EAE and MS the IgSF molecules intercellular CAM-1 (ICAM-1/CD54), vascular CAM-1

(VCAM-1/CD106) and activated leukocyte CAM (ALCAM/CD166) are all induced or up-regulated

on the endothelium [19-23]. How these events unfold during the initiating phase is not entirely

clear, but leukocyte recruitment is undoubtedly of fundamental importance and continues to be a

predominant feature during the active life of the lesion. It is important to note that adhesion

molecules such as ICAM-1 are also up-regulated and may be involved in the recruitment of

immune cells in non-autoimmune inflammatory conditions of the CNS, such as spinal cord injury

[24,25] and stroke [26], as well as in infectious processes affecting the CNS such as

neurocysticercosis [27], neurosyphilis [28], pneumococcal meningitis [29], streptococal meningitis

[30,31] and cerebral malaria [32,33]. Despite such indications, remarkably little is known regarding

the control of leukocyte traffic into the CNS in these diseases.


In general, leukocyte recruitment by endothelia is regulated through the expression of surface

molecules which are responsible for slowing and securing circulating immune cells. The extent to

which this occurs is governed by the degree of expression and activity of these surface molecules

which, in turn, depends on the activation state of both the leukocyte and the EC. In addition, other

factors such as chemokines and their receptors may also play a pivotal role in orchestrating

leukocyte migration patterns. A substantial literature can be found describing the general molecular

mechanisms controlling leukocyte traffic out of the blood (reviewed in: [34,35]). In the CNS our

current understanding is that the recruitment of leukocytes conforms to similar principles that

govern recruitment to other organs but with some possible differences (reviewed in: [36,37] (Figure

2). Accordingly, the first stage of recruitment involves overcoming the shear forces imparted by

blood flow and entails the temporary capture of circulating leukocytes through cell-cell interactions

mediated by cell surface molecules. In most tissues this initial step is performed by L-selectin,

expressed on the majority of leukocytes, and E‑ and P‑selectin on activated ECs. These selectins

bind to glycosylated ligands, such as P‑selectin glycoprotein ligand 1 (PSGL1), and mediate the

early stage of recruitment characterised by the formation of transient associations (tethering)

resulting in leukocyte rolling along the vessel wall in the direction of flow. In the healthy CNS,

however, T cell interaction with the spinal cord microvasculature has been demonstrated to be

unique due to the lack of rolling and a predominant role of -integrins mediating abrupt T cell

capture and subsequent firm adhesion to the vascular wall [38]. In contrast during EAE the role of

selectins remains unclear. Tethering and rolling of T cells to the inflamed BBB has been reported

to be mediated by PSGL-1 interacting with upregulated endothelial P-selectin [39-42]. On the other

hand the importance of this interaction in immune cell trafficking to the CNS has been largely

dismissed based on the observations that in EAE models functional lack of PSGL-1, or its ligands

E-and P-selectin, showed little impact on the development of the disease [43,44].

Initial transient engagement between the leukocyte and the EC enables crosstalk to take place

through the activation of various signalling pathways initiated through cell surface receptors. For

instance, chemokine activation of leukocytes causes conformational changes to the integrins

lymphocyte function-associated antigen 1 (LFA-1) and very late antigen (VLA-4) resulting in the

formation of a high affinity binding state as well a lateral relocation into high avidity patches


necessary for firm adhesion [45]. Once the leukocyte has arrested on the surface of the

endothelium it may then polarise and begin to crawl through tightly regulated integrin-CAM (eg

ICAM-1, VCAM-1) mediated adhesive events [46,47] (Table 1) that initiate essential signalling

within the EC and deliver it to optimal sites for transmigration [48,49]. Moreover, it has recently

been reported that in vitro diapedesis is preceded by leukocyte probing of the endothelial cell

membrane by invadosome-like protrusions that palpate the surface of the endothelium [11] (Figure

1), although the signalling regulating this has yet to be defined. At the BBB, sites permissive for

diapedesis may be restricted as T cell crawling distances prior to diapedesis are significantly

shorter on immortalized brain ECs, that have lost junctional tightness, compared to those observed

on primary brain ECs exhibiting greater barrier function [50]. ICAM-1 and VCAM-1 are the major

integrin-ligands for shear resistant T cell arrest at the BBB while ICAM-1 and ICAM-2 are the

predominant endothelial ligands mediating directed T cell crawling against the direction of flow to

the site permissive for diapedesis [47]. β2 and α4 integrin dependent firm adhesion and

intraluminal crawling against the direction of blood flow has been observed in vivo during T cell

recruitment across leptomeningeal vessels of the spinal cord in a rat EAE model [46]. This

supports the notion that T cell shear resistant firm adhesion is mediated in an additive manner by

endothelial ICAM-1 and VCAM-1. Crawling of T cells on the BBB-EC surface has been monitored

at a speed of 3 to 12 m/min [46,47,50]. Recently an immobilised intravascular gradient of the

chemokine MIP-2 has been shown to guide crawling neutrophils to transmigration sites [51] and it

would be interesting to know if BBB endothelial chemokines play a similar role by directing T cell

crawling specifically against the direction of blood flow.

The last stage in the recruitment process is diapedesis, that is the penetration by the leukocyte of

the vascular wall, and once again this appears to be regulated by CAMs and chemokine signalling

processes helping to breach the BBB. As important as it is to establish the principles governing

these events, it is essential to recognise that the factors responsible for controlling the recruitment

of distinct leukocyte subsets across different vascular beds and under differing states of activation

will be divergent and that such differences may lend themselves to targeted therapy.

Leukocyte activation by the BBB


Shear resistant arrest of leukocytes on the vessel wall occurs in response to chemokine-induced

G-protein mediated signalling. During inflammation, ECs upregulate expression of

chemoattractants on their luminal surface which bind to their cognate G-protein coupled receptors

on the leukocyte triggering the activation of integrins and strengthening leukocyte adhesion to the

endothelium [34]. In this regard, the CCR7-CCL19/CCL21 axis appears to play an important role

during neuroinflammation. Under homeostatic conditions CCL19 is expressed at the BBB in human

and mice and is upregulated during the course of MS and EAE where it may mediate the activation

of T cells and antigen presenting cells (APCs) expressing the receptor CCR7. In contrast, the other

CCR7 ligand CCL21 has only been shown to be upregulated at the BBB during the course of EAE

and has not been detected in human brain endothelium under homeostatic conditions or during the

course of MS [52-55]. In addition, the interaction between CXCL12 expressed on the BBB and its

receptor, CXCR4, present on leukocytes mediates activation of infiltrating cells. It may also

influence perivascular infiltration and accumulation as increased levels of CXCL12 are detected in

the abluminal side of blood vessels heavily infiltrated in both MS and EAE [56-58]. Similar results

were reported for CCL2/ CCR2 whereby CCL2 was shown to be released from primary cultures of

BBB-ECs both in mouse [59] and human [60-62], to be expressed within EAE and MS lesions [62-

64], to promote lymphocyte and monocyte migration across human BBB ECs [65] and to impact

significantly on the course of EAE [66,67]. Moreover, human brain EC in vitro express particularly

high levels of CXCL10 and CXCL8 [62] which in vivo may contribute to the predominant Th1-type

inflammatory response observed in MS.

Chemokine activation of leukocyte β2 and α4 integrins through induction of signalling cascades is

clearly a critical step in leukocyte recruitment to the CNS as it results in enhanced binding to

endothelial ICAM-1 and VCAM-1, both of which are upregulated in EAE, as well as influencing

leukocyte motility and directionality. However, the outside-in signalling that may occur through

integrin engagement to EC CAMs and the consequences of such signalling in the CNS require

further elucidation.

Endothelial cell signalling at the BBB


Diapedesis of immune cells through the BBB requires the EC to be actively engaged in the

process, for example by forming a docking structure, reorganizing the cytoskeleton, disengaging

TJ complexes or by forming migration pores. These dynamic processes are under the coordinated

control of a wide range of signalling pathways many of which are only just beginning to be

understood. Interestingly, because of the impermeable nature of CNS-ECs, and because it has

been more widely accepted that transcellular migration occurred in the brain [12], much of the early

work to identify potential outside-in signalling initiated through leukocyte engagement was

conducted on CNS-derived ECs.

ICAM-1 signalling

Early studies to investigate the contribution made by CNS-ECs to leukocyte migration were based

on the premise that diapedesis was unlikely to be achieved through rupturing the barrier, which

would lead to undue damage of the BBB, but rather through a cooperative reversible process

facilitated by the EC. This hypothesis dictated that the ECs of the BBB would need to respond to

the adherent leukocyte through a sequence of signalling events resulting in disengagement of the

TJs or formation of a transendothelial pore. As ICAM-1 had been identified as a key adhesion

molecule in the recruitment of lymphocytes to the CNS [68-72], this receptor became the first to be

investigated for its signalling capacity in CNS-ECs. In a seminal study by Couraud and colleagues,

antibody cross-linking of CNS-EC ICAM-1 (to mimic lymphocyte adhesion through LFA-1) resulted

in ICAM-1 mediated outside-in signal transduction leading to activation of the tyrosine kinase p60src

and phosphorylation of cortactin, a substrate involved in cortical actin dynamics [73]. This work

was followed by further studies where cross-linking ICAM-1 on brain ECs resulted in the

mobilisation of intracellular calcium [74] and activation of the small GTPase Rho [75,76], a key

regulator of the actin cytoskeleton. Crucially, these studies also showed that clamping intracellular

EC calcium with BAPTA or inhibiting Rho GTPase with C3 transferase prevented transendothelial

lymphocyte migration, establishing these downstream ICAM-1-mediated signalling events as

essential to diapedesis. However, it was not until ICAM-1 mutants were employed that definitive

evidence was obtained linking EC ICAM-1 signalling pathways directly to lymphocyte migration. In

these studies deletion of the C-terminal domain in ECs resulted in the loss of intracellular signalling

and inhibition of lymphocyte migration through CNS-ECs in vitro [77,78].


These were not the only signalling effects initiated by ICAM-1 in CNS-ECs. Other studies

demonstrated ICAM-1-mediated tyrosine phosphorylation of the cytoskeletal-associated proteins

focal adhesion kinase, paxillin and p130cas [75] which also occurred downstream of RhoA. These

tyrosine phosphorylated molecules become organised into a multimolecular complex with paxillin

and p130cas and associate with the adapter protein Crk which in turn combines with the GTP

exchange factor C3G. It is interesting to speculate that the formation of such focal adhesions

provides force-transduction platforms allowing for the structural remodelling of the EC during

leukocyte diapedesis. Indeed, ICAM-1 through its activation of the small GTPase RhoA, causes the

formation of F-actin stress fibres in non-contact inhibited CNS-ECs [76] and cortical actin bundling

in confluent cells [79]. ICAM-1 signalling also results in changes to the status of junctional proteins.

Accordingly, ICAM-1 engagement enhances tyrosine phosphorylation in the vicinity of the cell-cell

junction which correlates with tyrosine phosphorylation of the adherens junction (AJ) protein VE-

cadherin [79]. As VE-cadherin is a key regulator of the EC junction, this suggests that one aspect

of ICAM-1 signalling results in junctional disengagement to allow paracellular diapedesis of

leukocytes. ICAM-1-mediated tyrosine phosphorylation of VE-cadherin in CNS-ECs was found to

involve a signalling cascade in which there was sequential mobilisation of calcium and the

activation of Rho, the actin cytoskeleton, AMPK and eNOS [79,80]. Crucially, this pathway

appeared to be involved in supporting paracellular movement as tyrosine mutations in VE-cadherin

resulted in a significant inhibition of lymphocyte diapedesis. Data derived from non-CNS

endothelium also reveals that ICAM-1-mediated signaling results in VE-cadherin phosphorylation

and is necessary for successful neutrophil transmigration [81,82]. Given the key role of VE-

cadherin in regulating the structure and function of the endothelial junctions, it is likely that other

junctional proteins are affected following ICAM-1 signalling and other CAMs may be involved in this

interplay. Certainly in EAE dephosphorylation of the TJ protein occludin occurs early in the disease

process [83] and in MS lesions there is loss of junctional proteins [23,84]. These effects, however,

may be the consequence of a highly inflammatory environment where vasoactive products lead to

the induction of signalling pathways resulting in structural and functional alterations to the

junctional components (reviewed in: [85]) that differ from those mediated by leukocytes. Therefore,

it is speculated that TJ disengagement in response to leukocyte mediated EC signalling results in a


tightly controlled and reversible opening that causes little, if any, increased permeability whereas

junctional opening by vasoactive mediators results in a more prolonged disruption. Whether the

former is more important during homeostatic leukocyte surveillance and the latter to leukocyte

extravasation during inflammation is not fully understood.

The data described above, along with other contemporaneous studies on non-CNS vascular ECs,

established irrefutably that ICAM-1 acts as a major signalling adhesion receptor [86,87]. Over the

last few years, however, it has become clear that there is greater complexity to the signalling

pathways than can be accounted for by simple linear cascades. For example, blocking RhoA

activity with C3 does not block lymphocyte adhesion but does prevent ICAM-1 clustering

suggesting that initial ICAM-1 engagement results in Rho activation and subsequent ICAM-1

aggregation [88]. Clustered ICAM-1 in turn then activates other signalling pathways, including

further sustained RhoA activation and subsequent changes in actin dynamics. In support of such a

dual ICAM-1 signalling paradigm engagement of un-clustered ICAM-1 on CNS-ECs with primary

antibody alone is sufficient to activate a number of very early and rapid responses including

calcium oscillations as well as Rho and eNOS activation [80], the latter being lost upon ICAM-1

clustering. The signalling initiated upon leukocyte engagement and ICAM-1 clustering is likely to

depend on the interrelationship between the multiple components of the newly formed signalling

complex, which incorporates not only ICAM-1 and VCAM-1 but other potential CAMs and signalling

partners. Clearly the clustering of EC adhesion molecules at the point of leukocyte contact is

necessary for firm adhesion and arrest but the presence of other recruited proteins indicates

additional function. Thus, the clustering of ICAM-1 into a multimolecular signalling domain

coincides with the formation of the recently described EC docking structure whereby EC actin-rich

membrane protrusions embrace the lower portion of the adherent leukocyte [9,89,90]. What is now

testing the research community are which signals initiated through this multimolecular complex

mediate the development of the docking structure and which are responsible for facilitating

leukocyte diapedesis or whether they are part of a signalling and functional continuum. Whilst our

understanding of the downstream signalling initiated via ICAM-1 and VCAM-1 residing within this

complex is reasonably advanced, our knowledge of how they may feedback and contribute to the

assembly of the docking structure is poorly defined. Nevertheless, work conducted on non-CNS


EC has revealed that ICAM-1 clustering activates RhoG which in turn is essential for cup formation

[90], possibly through induction of membrane ruffling either directly or via activation of Rac1. It

should be pointed out, however, that whilst the presence of these structures has been shown to

exist in vitro in some studies, other studies using non-CNS endothelium and non-activated T cell

populations have failed to demonstrate the formation of such docking structures suggesting that

the activation stage of the immune cell population may be critical [91]. Also, the evidence for the

occurrence of docking structures in vivo, in particular in the CNS, is less compelling (Figure 1).

ICAM-1 has a very short intracellular domain (28 amino acids in human) which poses the question

of how it propagates its intracellular signal. Following crosslinking, ICAM-1 concentrates as a

detergent-insoluble cell fraction suggesting that there is either an increase in association with the

cytoskeleton [92] or with lipid-raft domains [93]. Other studies have investigated potential binding

partners and have reported the association of ICAM-1 with various cytoskeleton-associated

proteins in both CNS and non-CNS ECs. These include α-actinin 1 and 4 [94,95], β-tubulin [96],

ezrin [97], moesin [89,98] and filamins A and B [99]. Knockdowns of α-actinin 4, which links actin to

the membrane [95], and filamin B, which is an actin crosslinking molecule [99] have been shown to

inhibit transendothelial migration. It has also been demonstrated that the membrane-associated

signaling protein caveolin-1 is required for transcellular lymphocyte migration [100], that filamin-A

and caveolin-1 bind ICAM-1 [99] and that filamin-A regulates internalization of caveolae [101].

Together this provides the first insight into the molecular regulation of transcellular migration.

The ezrin/radixin/moesin (ERM) proteins ezrin and moesin may also be key signalling partners as

they serve as linkers between the plasma membrane and the actin cytoskeleton. These molecules

are involved in the organisation of cortical actin and the formation of lamellipodia, possibly through

downstream activation of Rho. As described above, ICAM-1 clustering activates the tyrosine

kinase Src resulting in phosphorylation of one of its substrates, cortactin and both have also been

identified in ICAM-1 immunoprecipitates from crosslinked EC extracts [102]. In addition, the

observation that β-tubulin associates with ICAM-1, as does glyceraldehyde-3-phosphate

dehydrogenase [96] which is involved in bundling of microtubules, indicates that EC microtubules

may also be involved in the observed cell remodelling. Destabilization of microtubules by

nocodazole treatment, inhibition of the microtubule dependent motor protein kinesin or knock-down


of the F-actin/microtubule adapter protein IQGAP1 decreased lymphocyte or monocyte diapedesis

across HUVECs [103,104]. These findings further support a possible involvement of endothelial

microtubule dynamics in the extravasation process of T cells or monocytes. Indeed, we have found

in an in vitro migration assay that treatment of brain EC with colchicine (2μm for 1h) is a powerful

inhibitor of T lymphocyte diapedesis (JG unpublished observations).

The evidence provided above points to ICAM-1 associating with partner proteins via its intracellular

C-terminal domain and subsequent propagation of the intracellular signal. However, other

signalling modalities may also occur through molecular interactions between different receptors

within the complex. One such postulated co-receptor is CD47. This pentaspanin protein has

previously been shown to associate with the plasma membrane supramolecular signalling complex

which contains the integrin αvβ3 [105] and whilst αvβ3 and CD47 have their own extracellular

ligands and generate unique signals outside the complex, upon association within the complex

their signalling properties change. This type of cis interaction involving other accessory receptors

may also exist for ICAM-1. CD47 is highly expressed on CNS ECs and is required for monocyte

migration through endothelial monolayers [106]. Moreover, lymphocyte transendothelial migration,

but not adhesion, can be inhibited under flow conditions by blocking the interaction of CD47 with its

ligand SIRPγ [107]. This implicates EC CD47 in both monocyte and lymphocyte diapedesis and as

CD47 crosslinking results in actin reorganisation [106] implicates it in outside-in signalling. The

detailed nature of the signalling pathway and whether it is mediated through an association with

ICAM-1, or possibly an integrin, has yet to be established.

The tetraspanins, a group of membrane-spanning molecules, are also thought to form an integral

part of the leukocyte docking structure [108]. One of these proteins, CD9, is responsible for ICAM-

1-mediated co-recruitment of VCAM-1 [109]. It is proposed that CD9 forms a web linking ICAM-1 to

VCAM-1 (and most possibly other receptors) via its extracellular domain and helps to establish a

plasma membrane signalling domain. The so-called tetraspanin web microdomains are formed by

transient external homophilic and heterophilic interactions and intracellular associations with

signalling proteins that provide a scaffold for induction of specific outside-in signalling. The

importance of tetraspanins in leukocyte migration to the CNS has been shown by CD81 antibody

blockade which inhibits monocyte migration and reduces EAE disease severity [110]. The exact


nature of the role of tetraspanins in ICAM-1-mediated signalling and leukocyte migration warrants

further investigation.

Other downstream functional endpoints besides the docking structure and the cell-cell junction

have also been proposed for ICAM-1 signalling. Regulation of EC gene expression is also a likely

outcome of ICAM-1 signalling [111]. ICAM-1 activates Rho GTPases which in turn are known to

modulate gene expression through activation of transcription factors such as serum response

factor (SRF) and NFκB. This may be achieved through various pathways including the induction of

MAL translocation to the nucleus and its association with SRF to activate a subset of SRE-

containing genes [112], activation of c-Jun N-terminal kinase (JNK) [75] which can then stimulate

AP-1 inducing reactive oxygen species (ROS) that further activate the transcription factors AP-1

and NFκB. If, as intial evidence indicates, ICAM-1 and other CAMs can regulate gene expression

and hence cell phenotype this raises the possibility that these pathways are involved in the overall

regulation of the inflammatory cascade and contribute to the so-called “beginning programs the

end” phenomena [113].

VCAM-1, PECAM-1 and ALCAM signalling

Other CAMs, such as VCAM-1, platelet endothelial CAM (PECAM-1) and ALCAM also contribute

to the signalling networks that facilitate leukocyte recruitment across the BBB and the relative

contribution of these CAMs likely determines the extent of recruitment for different leukocyte sub-

populations. Whilst ICAM-1 signalling has been especially associated with T lymphocyte

diapedesis across the BBB, a major player in the regulation of monocyte migration through the

endothelium is VCAM-1 [114]. As with ICAM-1 the intracellular domain of VCAM-1 lacks any

recognized signaling motifs, apart from a putative PDZ-domain-binding region, but is clearly able to

initiate intracellular signalling pathways. Outside the CNS, VCAM-1 antibody mediated cross-

linking or addition of VCAM-1-antibody coated beads to ECs results in the activation of Pyk2

tyrosine kinase, PKC and Rac-1, ROS generation and matrix metalloproteinase activity which are

necessary for successful leukocyte transendothelial migration [115-119]. Moreover as observed

with ICAM-1, VCAM-1 also associates with the ERM proteins ezrin and moesin [89] signifying that

VCAM-1 is closely linked to the actin cytoskeleton, and that actin remodelling is important in


monocyte migration across brain EC as it can be inhibited with a Rho-kinase (ROCK) inhibitor

[120]. The downstream target of these signalling pathways appear predominantly to be the cell-cell

junction which is consistent with the observation that VCAM-1 engagement contributes to leukocyte

transendothelial migration by stimulating gap formation between cells in the endothelial monolayer

[115]. This gap formation is mediated by VCAM-1-induced Rho and Rac-1 activation and is

dependent on Rac-1-mediated ROS generation. Many of the signalling events attributed to ICAM-1

engagement also appear to be similar to those described following VCAM-1 clustering. Moreover,

the recent report that ICAM-1 clustering results in eventual recruitment of VCAM-1 to the signalling

molecule (as indeed is observed at the docking structure) makes it difficult to disentangle the

signalling generated by these two adhesion molecules.

The role of PECAM-1 (CD31), which is expressed on ECs and leukocytes, in regulating migration at

the BBB is less well understood. Nonetheless, it has been shown to be directly involved in

diapedesis via homophilic interactions between migrating leukocytes, particularly monocytes and

neutrophils, at the endothelial junctions [121]. In the CNS, PECAM-1 knockout leads to an increase

in the number of activated leukocytes crossing the BBB due to a defect in junctional integrity of the

ECs [122]. As PECAM-1 is widely recognised as a signalling adhesion receptor which can

transduce both inhibitory and stimulatory signals [123,124], it is probable that along with ICAM-1

and VCAM-1 its signalling combines to regulate the trafficking of different leukocyte populations. In

support of signalling cross-talk between IgSF molecules it has been reported that in EC derived

from the BBB PECAM-1 acts as an antagonist of ICAM-1-induced tyrosine phosphorylation of

cortactin and actin cytoskeleton remodelling [125]. These findings suggest a novel role for PECAM-

1 at the BBB whereby its engagement results in signalling that “resets” the ECs into a non-

activated state after ICAM-1-induced activation.

ALCAM is also a member of the IgSF consisting of five extracellular Ig domains and a short (32-aa)

cytoplasmic tail. ALCAM is expressed by immune cells, particularly monocytes and DCs as well as

by various non-hematopoetic cells, including CNS-ECs, where it is found in membrane

microdomains (lipid rafts) [22]. The role played by endothelial ALCAM at the level of the BBB and

under homeostatic conditions remains to be established, but under neuroinflammatory conditions

like EAE and MS it is thought to facilitate extravasation of immune cells. Thus, blockade of ALCAM


in an in vitro BBB model results in the inhibition of transendothelial migration of CD4+ T cells,

CD19+ B cells and monocytes but not of CD8+ T cells. This is mediated via heterotypic interactions

with CD6 [22] and, in all probability, through homotypic ALCAM-ALCAM interactions [126,127]. In

terms of intracellular signalling only limited data are available, although the docking structure

(transmigratory cup) is rich in ALCAM molecules [22], suggesting that the signalling mechanisms

described for ICAM-1 and VCAM-1 are most likely mirrored by ALCAM. Analysis of the intracellular

pathways activated upon ALCAM ligation and changes induced to the cytoskeleton are mostly

limited to transfected cell lines and tumours. These findings demonstrate that ALCAM engagement

results in the activation of various intracellular signalling pathways. Accordingly, in non-endothelial

cell lines transfected with ALCAM homotypic interaction leads to ALCAM redistribution to the cell-

cell contacts, ALCAM-ALCAM interaction strengthening and reorganization of the cell architecture

via actin cytoskeleton-dependent clustering that is stabilized by protein kinase C in a Rac-

dependent, RhoA-independent manner [128-130] (reviewed in [131]). The effect of inflammatory

conditions on this elaborate network in CNS-ECs remains to be established, but assessment of

ALCAM and junctional complex protein expression in primary cultures of human CNS-ECs and in

blood vessels associated with MS lesions indicates that ALCAM upregulation is associated with a

disturbed junctional and cytoskeletal architecture [23]. In this regard, ALCAM organization at the

cellular membrane also depends on components of the junctional complex as it requires interaction

with -catenin, -catenin, the catenin p120 and VE-cadherin, all known components of the

adherens junctions stabilizing the BBB [132,133]. This close interaction may also play a role in

leukocyte transmigration as the phosphorylation state of VE-cadherin, which is regulated by p120,

is involved in the formation of the “short” lived gaps purportedly required for paracellular

transmigration [82]. Thus, to establish the intracellular pathways in ALCAM signalling during

homotypic or heterotypic interactions at the levels of the BBB will provide a better understanding of

the role played by this CAM during homeostasis and neuroinflammation. Of interest will also be to

distinguish the intracellular pathways activated upon binding with CD6 versus those activated

during homophilic interactions, as well as the differential activation depending on the immune cell

type bound to the BBB endothelium. In addition, ALCAM-CD6 interactions also influenced

immunological responses, particularly at the level of the immunological synapse between


monocyte-T cell as well as DC-T cell. These have been proposed to result in stable adhesion

during the early activation phase and later in the proliferative stage of the immune response

[134,135] where ALCAM-CD6 interaction could contribute to the T helper type 1 subset

commitment and the enhancement of IL-2 sensitivity. These findings shed light into possible

mechanisms of immune activation at the level of the BBB, as monocyte and T cell interactions with

the endothelium via ALCAM may modulate the immune cell phenotype and the transmigratory

mechanisms taking place in this particular niche.

Concluding remarks

Much has been learnt in recent years of the mechanisms controlling immune cell migration into the

CNS. Whilst recent research into the molecular basis of leukocyte migration through the BBB has

provided us with some novel insight into the cell signalling pathways involved it has yet to deliver a

suitable therapeutic target. The therapeutic benefit of targeting leukocyte traffic into the CNS has

already been demonstrated through the relative success of antibody blockade of the leukocyte

integrin VLA-4 [136]. Nevertheless, it is anticipated that by gaining a greater understanding of the

molecular mechanisms pertaining to migration through the BBB more specific targets may

ultimately be identified [1]. Whether such targets emerge from the ICAM-1 [137] or other IgSF

molecule signalling pathways (including VCAM-1, PECAM-1, ALCAM, Junctional Adhesion

Molecules), or from those initiated by other signal transduction receptors is yet to be established.

The hope is, however, that as we gain a greater understanding of the molecular basis of leukocyte

migration through the BBB we will identify targets that will ultimately pay therapeutic dividends.

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Figure 1. (A). Polymorphonuclear cells (thick arrows) migrating through the vascular blood-retinal

barrier in retinal inflammatory disease away from the TJ (thin red arrows) and over the endothelial

cell nucleus (*). (From [14]). Leukocyte (pink) extending invadosome-like protrusions (*) in (B) rat

brain endothelial cell (blue) in vitro and (C) in guinea pig EAE. Examples of inflammatory cells

adhering to CNS vessels in (D) EAE and (E) experimental uveitis of the retina. (F). Micrograph of

leukocyte migrating along the lumen of a microvessel in experimental uveitis. Images C and D

courtesy of Dr P. Munro, UCL Institute of Ophthalmology.


Figure 2. (A). Cartoon demonstrating the major phases of leukocyte capture and migration through

the blood-brain barrier. (B). Detailed summary of some of the identified signaling events associated

with transcellular and paracellular leukocyte migration through the blood-brain barrier.

Abbreviations used: P‑selectin glycoprotein ligand 1 (PSGL-1); intercellular adhesion molecule

(ICAM); vascular cell adhesion molecule (VCAM); platelet endothelial cell adhesion molecule

(PECAM); activated leukocyte adhesion molecule (ALCAM); Ras-related C3 botulinum toxin

substrate 1 (Rac-1); Ras homolog gene family member (Rho); 5' adenosine monophosphate-

activated protein kinase (AMPK); endothelial nitric oxide synthase (eNOS); nitric oxide (NO);

reactive oxygen species (ROS); sarcoma tyrosine kinase (Src); focal adhesion kinas (FAK).

Table 1: Functions of different IgCAMs in T cell interaction with the BBB

Endothelial IgCAM

Protein expression upregulated in response to pro-inflammatory stimuli

Leukocyte ligand Proposed role in leukocyte extravasation across the BBB, key references

VCAM-1 (CD106) yes alpha 4-integrins VLA-4 (α4β1, CD49d/CD29)

(20,34,36-38,46-48,68,69,114)

ICAM-1 (CD54) yes

beta 2-integrins LFA-1 (αLβ2, CD11a/CD18) and Mac-1 (αMβ2, CD11b/CD18)

(19-21,34-37,46-49,68-72)

ICAM-2 (CD102) no beta 2-integrin LFA-1 (αLβ2, CD11a/CD18)

(21,35,37,47,49,70,71)

ALCAM (CD166) yes CD6, ALCAM (CD166) (22,23,128-133)

PECAM-1 (CD31) no PECAM-1 (CD31) (34,36,37,122-125)

*

*

*

A B

C

FED

ROLLING/CAPTURE

CHEMOKINEACTIVATION DIAPEDESIS

Paracellular Transcellular

Bloodvessellumen

PerivascularSpace

PSGL-1

P-Selectin

?VLA-4

VCAM-1

G-proteincoupled

receptors

Chemokines

ARREST/FIRM ADHESION

LFA-1

ICAM-1

VLA-4

VCAM-1

PericyteParenchymalBasementMembrane

1-MACEP

Tight Junction

AdherensJunction

ICAM-1

VCAM-1

ICAM-1clustering

VE Cadherin

Tetraspanins

p130Cas

Paxillin

RhoG

Calcium

Src

CorticalActin

RhoA/B

AMPK

eNOS

NO

Focal Adhesions

Rac1

actinrearrangement

?

Endothelial Cells

A

B

Signalling Platform

ralull ecar aP

noit ar gimsnar T

VE Cadherin

Tight Junction

AdherensJunction

ROS

CD47

ALCAM ALCAM

FAK

Actin

DockingStructure

Actin-associated

proteins

Animal

iF

niloe

vaC

Leukocyte

Leukocyte

2. ParacellularTransmigration

1. Leukocyte interactionwith signalling platform

2. TranscellularTransmigration

EndothelialBasementMembrane

EndothelialCells

Documents

Review: Leucocyte-endothelial cell crosstalk at the blood-brain barrier: A prerequisite for successful immune cell entry to the brain