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Keywords:

  • adhesion;
  • ICAM-1;
  • migration;
  • transendothelial migration;
  • VCAM-1

Abstract

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

The notion that it takes two to tango is certainly true for leukocyte transendothelial 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 transcription) effects. Efficient paracellular migration (i.e. through endothelial cell–cell junctions) requires both intracellular calcium and the actin cytoskeleton, but also involves small GTPases, reactive oxygen species and protein 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

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 inflammatory disorders (asthma, rheumatoid arthritis, psoriasis) and in metastasis. On the other hand, regulated leukocyte trafficking is required for immune surveillance and stem cell homing following transplantation procedures. The multistep model for TEM [1] is well established and probably applies to most transmigration events, albeit that tissue specificity may result in additional complexity. For example, in the brain, the endothelium 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 participate 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 implicated in these different routes of TEM.

Endothelial docking structures

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

Rolling and adhesion of leukocytes over activated endothelium (i.e. at sites of inflammation) is accompanied 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 ‘transmigratory 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 α-actinin [5]. Formation of these structures requires calcium and, according to some [5], but not to others [7], activation of the Rho/p160ROCK pathway. As a result of the concentration 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

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

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 β2-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-α treatment, or expression of an ICAM-1–green fluorescent protein fusion on immortalized EC, increases the relative contribution of transcellular migration to polymorphonuclear cell diapedesis, suggesting that ICAM-1 plays an active role in determining whether polymorphonuclear cells use the paracellular or the transcellular route. Whether VCAM-1 plays a similar role, for example, for monocytes, is not known. Additional regulatory factors that might promote transcellular migration are the polygonal shape of the EC or the levels of β2-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-vacuolar 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 subclass of membrane lipid rafts that may, by invagination, detach from the membrane and mediate vesicular transport. The protein caveolin, a key marker for caveolae, was found to be enriched at the site of leukocyte–endothelial 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 transmigration process remains to be determined.

Although these studies show that the paracellular and transcellular pathways co-exist, considerable variation 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. reported 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 relative importance of one route over the other. Yet, in most of these studies, the contribution of the transcellular pathway was only 10–30% to the total transmigration events. Intriguingly, down-regulation of caveolin expression in human umbilical vein endothelial cells blocked transcellular migration by T lymphoblasts, 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

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

As mentioned above, leukocyte adhesion and the formation of endothelial docking structures is associated with the clustering of adhesion and signaling molecules. 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 efficient 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 mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) activation and the induction 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 studies showed that tyrosine phosphorylation of the E-selectin 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 disruption ablates the activation of phospholipase C gamma, but not of MAPK, indicating that the activation of different signaling pathways can occur in distinct 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 encephalomyelitis [21,22]. Blocking antibodies to PECAM-1 inhibit 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 immunoreceptor 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 phosphatases, with the SH2 domain-containing inositol 5-phosphatase, SHIP, with adapter, proteins such as Grb2, and with β- and γ-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 inflammation, 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 endothelial tight junctions [28]. In addition, JAM proteins are expressed by leukocytes. Several studies have clearly shown that JAM family members are essential for leukocyte 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 transmit 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 endothelium, but up-regulated upon activation by inflammatory stimuli. ICAM-1 has a short cytoplasmic tail of 29 amino acids that associates to ERM (ezrin, radixin, moesin) proteins, as well as to α-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], triggers 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 tyrosine 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 β1-integrin ligand on the endothelium, VCAM-1, is, in contrast to ICAM-1, absent from resting 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 associate with the cytoplasmic domain of VCAM-1 [5].

VCAM-1 clustering leads to the activation of Rac1, production of reactive oxygen species (ROS), activation of p38 MAPK and changes in the actin cytoskeleton (i.e. stress fiber formation) (Fig. 1). These events have all been associated with the increased endothelial permeability (as measured by tracer molecules or transendothelial 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 integrity 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 membrane, 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 cadherin (VE-cadherin) or of the extracellular matrix (Fig. 2A) [39].

image

Figure 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 distribution 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).

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image

Figure 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 phosphorylation of β-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.

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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 β-catenin [41] (Fig. 2B). However, our present knowledge of this pathway remains limited. The molecular mechanism of VCAM-1-triggered activation of Rac1 is completely unknown, as is the potential role for the relatively abundant NOX4 protein in VCAM-1 signalling. Also, the mechanism of VE-cadherin 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, endothelial 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 β1 and β2 integrins for transmigration. This means that the signaling which is induced by these molecules may also be interconnected. The extent of cross-talk between ICAM-1 and VCAM-1 induced signalling events is thus an important issue for future research.

Effects of shear

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

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 endothelial cell signaling and affects endothelial permeability, proliferation, migration and gene expression [42]. Shear stress is strongly associated with the development of atherosclerosis, which is an arterial disease that occurs predominantly at sites of disturbed laminar blood flow. VCAM-1 and E-selectin, in conjunction 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 phosphatidylinositol-3-Kinase/Akt pathway and is required for the activation of nuclear factor-κB at sites of disturbed flow. Apart from affecting the EC, shear also promotes chemokine-induced lymphocyte TEM, an effect coined ‘chemorheotaxis’[13]. Thus, shear force represents an additional level of regulation of both leukocyte migration as well as endothelial signalling.

Targets of endothelial signaling

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

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-κB and c-fos [17]. The subsequent up-regulation of cell adhesion molecules or metalloproteases may have important effects on the amplification 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 regulation of cell adhesion and migration [44]. In addition, ERK is involved in the activation of c-fos and of nuclear factor-κB. Thus, it appears that there is co-operativity between the ERK and p38 MAPK pathways, as well as the ROS that are produced in the EC, in altering the gene expression profile of activated endothelium.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

Along with the increased knowledge on the control of endothelial integrity, the number of signaling components 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 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 implication of a particular event in TEM.

The complexity of this field is further boosted by studies on the transcellular pathway. A major challenge will be to define whether this pathway is dominant in specific tissues, as was proposed for the brain, or perhaps associated with certain (pathological) conditions. Also, it remains to be determined whether transcellular migration is regulated by endothelial signaling, 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 guarantee a more complex, but no less interesting, future for the research on leukocyte TEM.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References

Dr Jaap van Buul is gratefully acknowledged for critical reading. PLH is a fellow of the Landsteiner Foundation 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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  1. Top of page
  2. Abstract
  3. Endothelial docking structures
  4. Transcellular versus paracellular migration
  5. Signaling by endothelial adhesion molecules
  6. Effects of shear
  7. Targets of endothelial signaling
  8. Concluding remarks
  9. Acknowledgements
  10. References
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