SPOTLIGHT REVIEW

Cardiovascular Research (2015) 107, 310–320 doi:10.1093/cvr/cvv145

The regulation of transendothelial migration: new knowledge and new questions William A. Muller* Department of Pathology, Northwestern University Feinberg School of Medicine, Ward Building 3-140, 303 East Chicago Avenue, Chicago, IL 60611, USA Received 26 November 2014; revised 13 March 2015; accepted 1 April 2015; online publish-ahead-of-print 18 May 2015

Abstract

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Adhesion molecules † Cell junctions † Diapedesis † Lateral border recycling compartment † Transendothelial migration

----------------------------------------------------------------------------------------------------------------------------------------------------------This article is part of the Spotlight Issue on Leucocyte Trafficking.

1. The process of transendothelial migration Leucocyte extravasation involves a series of adhesive and signalling interactions between leucocytes and endothelial cells.1 – 3 Many of these interactions are reviewed in other contributions to this issue. Rolling, activation, adhesion, and locomotion (intraluminal crawling) are all critical for leucocyte extravasation, but they are reversible. Most leucocytes that enter a venule at the site of inflammation do not roll; most leucocytes that roll do not adhere, and most that adhere do not extravasate.4 However, once leucocytes make the commitment to cross the endothelium into the tissue, with notable exceptions,5 they never go back—at least not as the same type of cell. Thus, diapedesis or transendothelial migration (TEM) is a logical process to study if one hopes to control an ongoing inflammatory response.3,6 Electron microscopic studies of leucocytes in the process of TEM show that they squeeze in an amoeboid fashion between tightly apposed endothelial cells.7,8 This process does not compromise the

integrity of the vascular barrier. 9 Considering that it only takes 1–2 min for the leucocyte to completely traverse the endothelial border in vitro 10 or in vivo,5 this suggests that very specialized membrane– membrane interactions are taking place. On the endothelial side, two major movements of membrane and/or membrane proteins are critical for TEM to occur efficiently. Both processes are discussed in more detail below, but are introduced here for clarity. At the site of transmigration, the physical junctions that provide the vascular barrier must be broken down temporarily and there must be an influx of membrane from the lateral border recycling compartment (LBRC). The former is seen as a transient gap in the staining of vascular endothelial cell-specific cadherin (VE-cadherin) or its associated catenins.9,11,12 The LBRC is a reticulum of tubulovesicle-like membrane that is continuous with the plasmalemma at the lateral borders of endothelial cells. It contains all of the membrane proteins known to play a positive role in TEM.13 – 15 During TEM, membrane from this compartment is targeted to the site of engagement with the leucocyte, bringing with it more membrane surface area and

* Corresponding author. Tel: +1 312 503 8144; fax: +1 312 503 8249, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected].

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Leucocyte transendothelial migration (TEM) involves a co-operative series of interactions between surface molecules on the leucocyte and cognate counter-ligands on the endothelial cell. These interactions set up a cascade of signalling events inside the endothelial cell that both allow for the junctions to loosen and for membrane to be recruited from the lateral border recycling compartment (LBRC). The LBRC is thought to provide an increased surface area and unligated receptors to the leucocyte to continue the process. The relative importance of the individual adhesion/signalling molecules that promote transmigration may vary depending on the type of leucocyte, the vascular bed, the inflammatory stimulus, and the stage of the inflammatory response. However, the molecular interactions between leucocyte and endothelial cell activate signalling pathways that disengage the adherens and tight junctions and recruit the LBRC to the site of transmigration. With the exception of disengaging the junctions, similar molecules and mechanisms promote transcellular migration as paracellular migration of leucocytes. This review will discuss the molecular interactions and signalling pathways that regulate transmigration, and the common themes that emerge from studying TEM of different leucocyte subsets under different inflammatory conditions. We will also raise some unanswered questions in need of future research.

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unligated adhesion molecules to engage the leucocyte.13 – 15 Blocking either VE-cadherin gap formation or targeted recycling of the LBRC will significantly inhibit TEM. An increase in vascular permeability and leucocyte extravasation both occur in acute inflammation. However, they are mechanistically distinct. In fact, the increase in permeability can be distinguished from TEM on a genetic basis.16 Furthermore, it is important to point out that, although intercellular gaps that are visible in the light microscope can be produced on endothelial cells cultured on glass coverslips, in vivo the gaps produced between endothelial cells by even the strongest inducers of vascular permeability (e.g. histamine and serotonin) are in the order of hundreds of angstroms.17 This does not mean that these gaps and the associated increase in vascular permeability are not important. However, it means that leucocytes must still crawl through closely adherent endothelial cells; they do not fall into holes between endothelial cells. This review will first introduce the molecules on both the leucocyte and the endothelial cell that interact during TEM. Then, it will review some of the known signalling pathways stimulated by these molecular interactions. Finally, we will discuss some of the macromolecular changes that take place during TEM.

While selectins and their ligands play an important role in the initial steps of adhesion cascade (reviewed in detail elsewhere18 – 20 and this issue), most of the downstream activation and migration steps are mediated by several molecules on the endothelial cell, almost all of which are members of the immunoglobulin (Ig) superfamily of adhesion molecules.2,3 These molecules and their leucocyte counter-receptors are summarized in Table 1, and a commentary on their function follows here. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) both act downstream of the selectinmediated loose adhesion step and function in facilitating strong adhesion45 by interacting with activated leucocyte integrins. Lymphocyte

Table 1 Major endothelial cell adhesion molecules and leucocyte molecules they interact with in TEM Endothelial CAM

Leucocyte ligand(s)

Distribution on EC

References

ICAM-1 (CD54)

LFA-1 (CD11a/CD18) Mac-1 (CD11b/CD18)

Diffuse

21 – 24

VCAM-1 (CD106) ICAM-2 (CD102)

VLA-4 (CD49d/CD29) LFA-1

Diffuse Junctional

25 – 28

JAM-A (CD321)

LFA-1

Junctional

31 – 33

JAM-B JAM-C

VLA-4 Mac-1

Junctional Junctional

34

PECAM (CD31)

PECAM CD177 on PMN

Junctional

4,36 – 38

PVR (CD155)

DNAM-1

Junctional

39 – 41

CD99 CD99L2

CD99 CD99L2 unknown PMN ligand?

Junctional Junctional

42

.......................................................................................................................................................................................

29,30

35

43,44

Except for CD99 and CD99L2, all are members of the Ig superfamily. CAM, cell adhesion molecule; EC, endothelial cell; ICAM-1 and -2, intercellular adhesion molecule-1 and -2; VCAM-1, vascular cell adhesion molecule-1; JAM-A, -B, and -C, junctional adhesion molecule-A, -B, and -C; PECAM, platelet/endothelial cell adhesion molecule-1; PVR, poliovirus receptor; LFA-1, lymphocyte function-associated antigen-1; Mac-1, macrophage 1 antigen; PMN, neutrophil; DNAX accessory molecule-1 (DNAM-1, CD226).

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2. Molecular interactions between leucocytes and endothelial cells

function-associated antigen-1 (LFA-1) interaction with ICAM-1 is important for adhesion to endothelium, whereas macrophage 1 antigen (Mac 1) interaction with ICAM-1 is more important for locomotion to the junction.10,46 Both ICAM-1 and VCAM-1 are expressed diffusely on the endothelial surface, but become enriched under the leucocyte as it moves across the endothelial cell.47,48 The enrichment of ICAM-1 is dependent on the actin cytoskeleton and remodelling events that include the Src-mediated phosphorylation of cortactin, an actin binding protein.16,49,50 In the local environment of clustered ICAM-1 in TEM, src-mediated phosphorylation of cortactin enhances transmigration, whereas in response to global stimulation by sphingosine-1 phosphate (S1P), a rapid increase in cortactin phosphorylation is associated with a Rac1-dependent association of cortactin with myosin light chain kinase (MLCK) and a tightening of the endothelial barrier.51,52 This is just one of several examples of how localized signalling between leucocyte and endothelial cell results in different outcomes from when the same signalling molecules are activated by global activation by soluble mediators that affect permeability such as thrombin and S1P. Another occurs with Rac1, discussed below. ICAM-2 is a 55 kDa Ig superfamily proteoglycan that has 35% similarity to ICAM-1 expressed on endothelial cells, and, to a lesser extent, leucocytes and platelets.29 ICAM-2 is expressed at especially high levels in high endothelial venules.30 Compared with ICAM-1, it generally seems to play a lesser and partially redundant role in TEM.53 The junctional adhesion molecule (JAM) family consists of three closely related proteins (JAM-A, -B, and -C) that engage in both homophilic interactions with molecules on endothelial cells and heterophilic interactions with leucocyte integrins.54,55 JAM-A, -B, and -C are present in both adherens and tight junctions; JAM-A and -C are also expressed on leucocytes.54,56 The role of JAM-A in TEM is the best studied.31 – 33 However, antibodies against JAM-C and soluble JAM-C extracellular domain both decreased leucocyte emigration in vivo.5,57,58 Recent reports also suggest that JAM-B may interact with JAM-C to support its function in TEM.59,60 Platelet/endothelial cell adhesion molecule-1 (PECAM) is concentrated at the endothelial cell borders and expressed diffusely on leucocytes and platelets.36 It has six extracellular Ig repeats and contains

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recruitment, but only in TEM.37,39,42 Sequential blocking studies demonstrated that PECAM, PVR, and CD99 function in that order to control TEM.39,42 Based on these in vitro studies, the order of events is ICAM-1 and/or VCAM-1 interacting with their leucocyte integrin partners on the apical surface followed by PECAM, PVR/DNAM-1, and CD99 for diapedesis per se. The sequential control of TEM is may be more complex in vivo. Various investigators report differences in the degree to which blocking specific molecules results in reduction of TEM and the relative order in which these molecules function. Some of these seemingly conflicting results can be reconciled by differences in the inflammatory stimulus, tissue, and especially mouse strains used in the various studies.4 In most mouse strains examined, PECAM deficiency or blockade arrests leucocytes on the apical side of the endothelium, consistent with in vitro findings using human cells.79 In the C57BL/6 strain, however, depending on the stimulus, PECAM knockout may have no effect on leucocyte TEM, but instead leads to leucocyte arrest at the basement membrane, suggesting an additional role for PECAM (in particular extracellular domain 6) in the inflammatory process.63 Similar discrepancies have been reported for the roles of ICAM-1, ICAM-2, and CD99.

3. Signalling events in transmigration 3.1 Phosphorylation and de-phosphorylation Interactions between the leucocyte and endothelial cell adhesion molecules mentioned above do far more than simply mediate adhesion. They trigger a cascade of signalling events that initiate and maintain the transmigration process. The best understood of these signalling events are related to ICAM-1 and VCAM-1, and result in breakdown of interactions of VE-cadherin with its links to the actin cytoskeleton. Similar signalling events triggered by molecules such as PECAM, PVR, and CD99 are beginning to be understood. Stimulation of ICAM-1 leads to phosphorylation of VE-cadherin, which is a prerequisite for adherens junction disassembly. 80 In HUVECs, the kinases Src and Pyk2 phosphorylate VE-cadherin on the p120 and b-catenin-binding sites, tyrosine residues 658 and 731, respectively.81 This inhibits the binding of p120 and b-catenin to VEcadherin. A critical role for tyrosine 731 in TEM was recently demonstrated in vivo. Mice with a specific tyrosine-to-phenylalanine mutation at Y731 had defective leucocyte extravasation.82 Since the interaction of these proteins with VE-cadherin is critical for retaining VE-cadherin at the adherens junction, this destabilizes the junctions. Alternatively, if p120 is overexpressed, VE-cadherin levels at the cell border remain high and transmigration is inhibited.83 It is hypothesized that overexpression of p120 interferes with or outcompetes the kinases that would normally phosphorylate VE-cadherin on Y658 and inhibit p120 binding. Phosphorylation of VE-cadherin may be necessary, but is apparently not sufficient for destabilization of adherens junctions. Conditions that led directly to phosphorylation of Y658, 685, and 731 in VE-cadherin were unable to weaken barrier function.84 Additional signals from the leucocyte and/or endothelial cell (physical and/or chemical) may be necessary to weaken the junctions for TEM. Under resting conditions, the vascular endothelial protein tyrosine phosphatase (VE-PTP) associates with VE-cadherin via plakoglobin (g-catenin), maintaining VE-cadherin in a hypophosphorylated state at the junction. Interaction of leucocytes with cytokine-activated endothelial cells triggers rapid dissociation of VE-PTP from VE-cadherin,

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several known and putative phosphorylation sites including two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs).61 During TEM, PECAM on the leucocyte and endothelial cell binds homophilically.37,62 Disruption of this interaction using genetic ablation or function blocking reagents (PECAM-Fc chimeras or antibodies that bind domain 1 of PECAM) arrests leucocytes on the apical surface of the endothelial above the junction in vitro 37,63 and in vivo.62,64,65 When visualized live, these arrested leucocytes are not stationary, but instead migrate up and down along the junction as if poised to undergo TEM but lacking a critical signal.10 Poliovirus receptor (PVR), the key receptor for poliovirus entry into cells,66 is also involved in TEM.39 – 41 PVR localizes to sites of cell– cell and cell –matrix adhesion and binds to DNAX Accessory Molecule-1 (DNAM-1, CD226) on leucocytes. DNAM-1 is a transmembrane Ig superfamily member expressed on NK cells, T-cells, some B-cells, platelets, monocytes, and possibly activated human umbilical vein endothelial cells (HUVECs).67 – 71 A role for PVR/DNAM-1 in TEM has been shown for monocytes41 and effector memory T cells. 40 Sullivan et al. 39 demonstrated that PVR regulates a step in TEM between those regulated by PECAM and CD99 (see below). CD99 is highly O-glycosylated and does not belong to a characterized superfamily72; it exhibits sequence similarity to only one other protein, CD99 antigen-like protein 2 (CD99L2).73 CD99L2 has 32% amino acid similarity to CD99 and is similarly highly O-glycosylated.73,74 Both have short (,40 amino acids) but divergent cytoplasmic domains. Like PECAM, CD99 and CD99L2 are expressed at endothelial cell junctions and diffusely on leucocytes. CD99 facilitates TEM through homophilic interactions between the two cell types.42 Similarly, CD99L2 has been reported to function through homophilic interactions,43 but it may have another unidentified ligand on neutrophils.44 Disruption of CD99 and CD99L2 interactions using function blocking antibodies or genetic knockout (in the case of CD99L2) impairs leucocyte extravasation in vitro and in vivo. While PECAM blockade arrests leucocytes on the apical surface, blocking CD99 function in vitro traps the migrating leucocytes part way through the junctions.42,75 In mice, blocking either CD99 or CD99L2 by polyclonal antibody arrested leucocytes in vivo at a similar step in extravasation, suggesting that the two proteins may function to facilitate the same step.76 In addition to these endothelial surface molecules, antibodies against a multitude of others have been reported to block TEM.3 One major question in the field is, why are so many molecules seemingly involved in this process and why don’t these molecules compensate for the blockade or genetic deletion of each other? Certain molecules have been shown to function sequentially during TEM (see below). However, it is hard to believe that there are over a dozen different sequential steps in TEM. Thus, understanding how these molecules function in relation to each other and why there is not more redundancy in the system is a question for future research. Several of these molecules have been observed to function sequentially during TEM.3,77,78 ICAM-1 and VCAM-1 function upstream of PECAM and CD99. This finding is well supported in the literature both in vivo and in vitro.15 It also fits with the subcellular localization of these proteins with ICAM-1 and VCAM-1 both localized to the apical surface of endothelial cells where they can function in the activation and adherence of captured leucocytes, whereas PECAM and CD99 are localized to the cell border where they facilitate the subsequent migration though the junction. Furthermore, antibody blockade studies have demonstrated that PECAM, PVR, and CD99 play no role in the firm adhesion step of

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3.2 Calcium The VCAM-1- and ICAM-1-mediated changes in VE-cadherin phosphorylation have been shown to occur through the activation several other signalling pathways. Cross-linking VCAM-195 and also ICAM-1 in some reports,96 but not others,95 stimulates an increase in cytosolicfree calcium ions, which has long been known to be a requirement for diapedesis.97 The increase in cytosolic-free calcium ion has been shown to activate MLCK, leading to actin – myosin fibre contraction which is believed to help disrupt the adherens junction complex.98 VCAM-1 clustering recruits ezrin and moesin.99

3.3 Rho family small GTPases The role of Rac1 in transmigration is somewhat paradoxical. It is well established that the integrity of endothelial junctions in microvascular endothelial cells is maintained by activated Rac1.100 This is particularly true when cAMP-mediated signalling predominates to activate both protein kinase A and the exchange protein directly activated by cAMP (Epac).101 However, when leucocytes are involved, either localized signal transduction or other signalling events may subvert Rac1 activity to destabilize junctions. Cross-linking VCAM-1 activates Rac1, which stimulates an increase in reactive oxygen species (ROS) in endothelial cells through NADPH oxidase;102 – 104 when this pathway is blocked, leucocyte TEM is arrested.105 ROS activate a number of protein tyrosine kinases (and inactivate phosphatases) that phosphorylate VE-cadherin and other substrates leading to loosening of adherens junctions. These differences could be attributed to the differences between Rac1 function in macrovascular endothelium used for the TEM studies vs. microvascular endothelium used for the permeability studies. However, a recent report using microvascular endothelial cells also reported that interaction of lymphocyte VLA-4 with endothelial cell VCAM-1 led to activation of Rac1, which activated NADPH to produce

ROS that activated Pyk2 to promote dissociation of VE-PTP from VEcadherin.106 The net result is ‘loosening’ of junctional structures. In a similar manner, clustering of ICAM-1 activates RhoA through Rho GEF 12; RhoA activates Rho kinase (ROCK) (reviewed in Cernuda-Morollon and Ridley107). This signalling is particularly enhanced by mechanical forces exerted on ICAM-1 by leucocytes engaging it.108 ROCK phosphorylates and inactivates the myosin phosphatase targeting subunit (MYPT1) of the trimeric myosin phosphatase, the major phosphatase inactivating MLCK. The end result is potentiation of actin–myosin contraction. Activation of RhoA also induces the phosphorylation myosin light chain through the activation MLCK.109,110 Inhibition of either Rho or MLCK has no effect on leucocyte adhesion, but substantially blocks TEM.105,110 Similarly, ICAM-1 stimulation leads to phosphorylation of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide.111 Signalling through the eNOS pathway has been shown to be required for leucocyte TEM both in vitro 111 and in vivo.87 Although this growing body of evidence suggests that these pathways are important to TEM, much remains to be determined about how they relate to each other and how they fit in to the overall process.

4. The lateral border recycling compartment When anti-PECAM antibody is added to endothelial cells at 378C, it is seen by immunoelectron microscopy not only along the endothelial border but also in a sub-junctional structure of interconnected 50 nm vesicles and tubules13 (Figure 1). PECAM from this compartment exchanges with the surface membrane at the cell border with a halftime of 10 min. Therefore, this compartment was called the lateral border recycling compartment or LBRC.13 The LBRC is contiguous with the plasma membrane, suggesting that this compartment is a complex invagination of the junctional membrane that extends several hundred nanometers into the cell. Interestingly, the compartment is labelled well at 378C, but incubating endothelial cells with the same antibodies at 48C only labelled the junction.13,39 Electron micrographs clearly show that the compartment still exists under this condition, indicating that PECAM in the LBRC is protected from large molecules at 48C. This is not because the vesicles have pinched off, as protons and small molecules can still enter (Mamdouh et al. 13 and unpublished results). Under resting conditions, roughly 30% of the total PECAM is in the LBRC.13 The LBRC is distinct from other endothelial vesicular organelles such as the vesiculo-vacuolar organelle (VVO) and caveolae. VVOs are much larger (.150 nm) in size, more heterogeneous in shape, and typically communicate between the apical and basal surfaces.112 VVOs open and close in response to VEGF, whereas the LBRC is completely and continuously accessible to the exterior of the cell.113 VVOs have not been observed in vitro. Likewise, caveolae are rare in endothelial cells in vitro and are typically observed as single vesicles or single invaginations on the apical and basal membranes, seldom present at the junction. By IF, caveolin-1 (a marker of both caveolae and VVOs114,115) does not colocalize with PECAM at the junction or during TEM.13,15 In addition, biochemical analysis of PECAM and caveolin-1 shows that the two localize to different membrane microdomains.13 This was examined using sucrose gradients to purify cholesterol-rich membrane microdomains from endothelial cells solubilized with cold non-ionic detergent.13 In

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allowing it to be phosphorylated on tyrosine, increasing junctional permeability and facilitating TEM.85 The phosphatase SHP2 also plays a major role in signalling during TEM, as might be expected given the presence of ITIM domains on the cytoplasmic tails of so many of the molecules involved. SHP2 is recruited to the cytoplasmic tail of ICAM-1 upon stimulation.86 Minshall and co-workers reported that SHP2 dephosphorylates src tyrosine 530. In doing so, it removes an inhibitory phosphate that blocks the activity of src. The expression of a dominant-negative form of SHP2 prevented the src activation upon ICAM-1 cross-linking, indicating that this dephosphorylation event is required for ICAM-1-mediated signalling.87,88 SHP2 also plays a role in VE-cadherin and adherens junction remodelling. SHP2 associates with b-catenin in VE-cadherin/ p120/b-catenin/plakoglobin complexes. Ablation of SHP2 expression in endothelial cells causes increased phosphorylation of all of the components in the complex and, consequently, increased permeability in response to thrombin.89 It is possible that similar events are taking place on a local scale during TEM. SHP2 in endothelial cells also associates with the phosphorylated ITIM motifs of both PECAM90 – 92 and PVR.39,93 Although the downstream functions of this association in LBRC function have yet to be determined, it is interesting to note that src kinase phosphorylation of PECAM facilitates its efficient entry into the LBRC,92,94 whereas PVR is phosphorylated by src upon ligand stimulation with DNAM-1 or cross-linking.39

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Figure 1 PECAM, CD99, and JAM-A, but not VE-cadherin, are in the LBRC. Endothelial cells were incubated with HRP-conjugated mAb specific for PECAM, CD99, JAM-A, or VE-cadherin for 1 h at 378C, then fixed, and reacted with diaminoabenzidine-H2O2 as described.13 – 15 As a control for nonspecific labelling, free HRP was added at the concentration present on the antibodies. En face sections were cut for EM analysis. In addition to being present along the cell border (dark electron-dense staining), PECAM, CD99, and JAM-A are all present in interconnected vesicular structures of the LBRC. VE-cadherin is present at the cell border (arrowhead), but not in the interconnected vesicular structures adjacent to it (arrows). Arrowheads indicate cell borders; arrows indicate the LBRC. Scale bar: 200 nm. Reproduced from Mamdouh et al. 15 with permission.

LBRC move in concert during TEM; there is no separation of membrane enriched in PECAM or CD99. During TEM, the LBRC moves along microtubules to engage the transmigrating leucocyte in a process called ‘targeted recycling’. This is a critical step in diapedesis. Disrupting PECAM– PECAM homophilic interactions ablates both TEM and targeted recycling.13 The enrichment of the LBRC at the leucocyte is similarly blocked when the endothelial cells are treated with reagents demecholcine or taxol, which depolymerize microtubules or cause microtubule bundling, respectively.14 Neither treatment affected the size or the constitutive recycling of the compartment, suggesting instead that microtubules are essential for LBRC movement during TEM. This is supported by the finding that LBRC targeted recycling is also impaired upon microinjection of function blocking antibodies against the motor domain of kinesin, a molecular motor that facilitates intracellular traffic along microtubules.14 The presence within the LBRC of so many molecules that are involved in the endothelial cell’s role in TEM suggests that it could act as a reservoir of these molecules in TEM as well as a reservoir of junctional membrane. If one calculates the membrane surface area necessary to surround the transmigrating leucocyte, the plasma membrane at the cell borders immediately adjacent to the leucocyte can only contribute about two-third.121 The additional one-third could come from the LBRC; this is precisely the fraction of junctional membrane that resides in the LBRC.13 In this way, transmigration could proceed without wholesale retraction of the endothelial cells. Furthermore, the available data suggest that the molecules in the LBRC, not those on the surface of the junction, are the ones that are required for TEM. When endothelial cells were treated with blocking antibodies against PECAM, PVR, or CD99 at 48C, which leaves the protected LBRC pool of PECAM untouched, TEM remained unaffected, while the same antibodies given at 378C (conditions under which they enter the LBRC) blocked TEM.37,39 Still there is much to be learned about the LBRC: which signals recruit it to the site of TEM? Are these signals the same for PECAM, PVR/ DNAM-1, and CD99 interactions? What happens to the membrane

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this analysis, caveolin-1 was predominantly recovered in the buoyant fractions that correspond to lipid rafts, whereas PECAM was recovered from the dense fractions which contained the solubilized proteins that are excluded from membrane microdomains. (This is in distinction to leucocytes, where activation of PECAM led to its phosphorylation and partition into cholesterol-rich microdomains116 and in platelets where a small fraction of palmitoylated PECAM can be recovered in such domains.117) The LBRC also contains JAM-A15 and CD9915 (Figure 1), PVR,39 and nepmucin,118 which regulates lymphocyte TEM in high endothelial venules. The LBRC is also observed in vivo as EM examination blood vessels from mice injected intravenously with anti-mouse PECAM-HRP shows an identical sub-junctional staining pattern (unpublished results). The same immunoelectron microscopy experiments show that VEcadherin is not in the LBRC.15,39 We have recently developed a way to isolate the LBRC from endothelial cells.119 A proteomic approach was taken to identify proteins unique to or enriched in the LBRC. These studies confirmed that caveolin-1 is not a component of the LBRC and revealed that in addition to VE-cadherin, its associated catenins were also excluded from the LBRC. We were unable to identify any unique proteins in the LBRC; however, both isoleucine glutamine motif GTPase-activating protein 1 (IQGAP1) and vimentin were specifically enriched in the LBRC fraction.119 We recently used chimeric molecules of CD25, PECAM, and VEcadherin to study the signals for inclusion of membrane proteins into the LBRC.120 Inclusion of proteins from the lateral border of the endothelial cell plasma membrane into the LBRC seems to be the default pathway. VE-cadherin and its associated catenins are actively excluded by its homophilic interaction motif [peptide arginine valine aspartate alanine glutamate (RVDAE)]. Removal of this domain from VE-cadherin or competition with a soluble RVDAE peptide allowed its entry into the LBRC. This explains why no unique proteins were found in the isolated LBRC and enriched proteins were associated with the cytoplasmic side.119 This study also demonstrated that the constituents of the

Regulation of leucocyte diapedesis

after targeted recycling; how is membrane retrieved for another round of targeted recycling?

5. Special cases (?): transcellular migration and TEM across tight junctions Most TEM takes place at the level of postcapillary venules where adherens junctions predominate. Paracellular transmigration (between endothelial cells) is the predominant pathway taken by leucocytes.5 Transcellular TEM (migration through the endothelial cell body) is a distinct process and may occur up to 10% of the time outside of the CNS.122,123 Transcellular TEM is thought to occur more often at the blood –brain barrier (BBB), where intercellular tight junctions are complex124,125 and leucocytes might take the path of least resistance.126 Transcellular TEM also occurs in situations where leucocytes are highly activated.15,127,128 Transcellular TEM still requires targeted trafficking of the LBRC (Figure 2).15,126 It can be inhibited by agents that disrupt microtubules or block PECAM or CD99 function, even after ICAM clusters around the transmigrating leucocyte.15 Aside from the fact that junctions remain intact, the mechanisms of paracellular and transcellular TEM are quite similar (reviewed in Muller3).

315 Owing to the tightness and intricacy of junctions at the BBB, enhanced transcellular TEM is believed to occur at the BBB.129 However, the more we study adhesion, locomotion, and TEM across brain endothelial cells in vitro or the process or leucocyte extravasation across the BBB in vivo, the more similarities are seen with leucocyte extravasation outside of the CNS in terms of the molecules and signalling pathways that regulate these steps.129 In an attempt to study the mechanisms of transcellular TEM more directly, we recently examined TEM in an in vitro model of the BBB, assuming that the tighter junctions would result in more transcellular migration. Human endothelial cell cultured on hydrated collagen gels in a combination of astrocyte conditioned medium and agents that raised cAMP levels developed junctions that were qualitatively (expressed BBB claudins 3 and 5) and quantitatively (five times higher transendothelial electrical resistance, five times lower solute permeability) different from conventional cultures in ways that mimicked the BBB. Despite this, paracellular TEM was still by far the predominant pathway: ≥98% of the transmigration events were paracellular.9 Furthermore, targeted recycling of the LBRC was still seen, and TEM was still dependent on PECAM and CD99. Most interesting, the tight junctions showed the same plasticity as adherens junctions. Claudin 5 moved out of the junction at the point of transmigration during the transmigration event, just like VE-cadherin, but was rapidly reestablished in the endothelial junction as soon as TEM was completed.9 Downloaded from by guest on November 15, 2015

Figure 2 Targeted trafficking of membrane from the LBRC to surround leucocytes undergoing transcellular migration. PECAM in the LBRC was labelled with Fab fragments of a non-blocking mAb as a surrogate marker for LBRC trafficking to the endothelial surface, as described.13 – 15 Staining of recycled PECAM as a surrogate for recycling LBRC (left panel, red in overlay) surrounding monocytes (upper panel) and a neutrophil (PMN) (lower panel) demonstrates movement of membrane from the LBRC to the endothelial surface. Recycled PECAM is seen around the leucocytes migrating transcellularly (thin arrows) far from the endothelial border (arrowheads). Recycling LBRC enrichment is as great around the monocyte migrating transcellularly as it is around the monocyte migrating paracellularly (thick arrow). Actin staining (middle panel, green in merge) shows relative positions of the cells. Orthogonal (X– Z) projection demonstrates leucocytes in the act of transcellular migration. Arrows indicate recycled PECAM around leucocytes. Bar ¼ 10 mm. Reproduced from Mamdouh et al. 15 with permission.

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Figure 3 Schematic model of some molecular events that regulate TEM. A highly oversimplified diagram of a leucocyte (top) engaging endothelial cells at the start of TEM. Events relevant to weakening of adherens junctions are shown in the left endothelial cell; those relevant to the LBRC are shown in the right endothelial cell. In reality, these events are taking place simultaneously in both cells. Similarly, the clustering of ICAM-1 and VCAM-1 is separated, but these molecules intermix. Clustering of ICAM-1 by leucocyte integrins LFA-1 and Mac-1 and of VCAM-1 by leucocyte integrin VLA-4 leads to the signalling cascades described in the text. These pathways intersect and both lead to increase in actin – myosin tension and phosphorylation of VEcadherin, which facilitates its clearance for the adherens junction. Homophilic interactions between PECAM (hook-shaped surface molecules) on the leucocyte and PECAM on the endothelial cell lead to activation of kinesin and recruitment of the LBRC membrane along microtubules to the site of TEM. Clustering of ICAM-1 activates src, which is required for TEM and brings SHP2 to the site of TEM, perhaps to dephosphorylate PECAM and PVR and reset the system. Other abbreviations: [Ca+2]i , increase in cytosolic-free calcium ion; CaM, calmodulin; MLCK, myosin light chain kinase; MPase, myosin phosphatase; ROCK, Rho kinase; ROS, reactive oxygen species; circled P, phosphorylated state.

Endothelial permeability was not compromised during TEM, so tight junctions may be far more plastic than previously thought. In endothelial cells, adherens junction proteins and tight junctions are intermixed and not spatially distinct, as in epithelial cells. The proteins that comprise these junctions are attached to actin filaments.130 – 132 Thus, similar mechanisms might allow for tight junctions and adherens junctions to undergo lateral movement in the plane of the membrane. Nonetheless, junctions in the BBB in vivo are still five to ten times tighter than in this model, so there may still be substantial transcellular TEM across BBB vasculature in vivo. Furthermore, there is good evidence to support the idea that leucocytes (or at least lymphocytes)

do take the path of least resistance.133 Martinelli et al. showed that the tighter the junctions and the lower the density of local F-actin, the greater the propensity of lymphocytes to migrate transcellularly. The differences in the findings of these two studies could be related to many technical differences including the leucocyte type (monocytes and neutrophils vs. lymphocytes), differences in culture systems, and differences in substratum. An important question for future research is what determines the route of TEM in vivo and whether the stimuli and mechanisms are the same for all classes of leucocytes. Advances in intravital imaging may be able to answer these questions soon.

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6. Coordination of molecular events during TEM

7. The next steps A related event that has received relatively little attention is how the endothelial junction reseals after transmigration events. Carman and colleagues134 have provided some exciting and important inroads into this matter. Transmigration induces a loss of isometric tension in the endothelial monolayer. In response, the endothelial cell produces lamellipodia on its ventral surface that move across the substratum to contact the neighbouring endothelial cell and seal the gap. This process required several Rac1 effectors as well as localized production of hydrogen peroxide.134 Once the leucocyte has passed across the endothelial barrier, it still must cross the subendothelial basal lamina. This process generally requires far more time.5 Nourshargh and colleagues have demonstrated that neutrophil (PMN) 135 and monocytes136 move between the

8. Conclusion The transmigration of leucocytes across the endothelium involves unique molecular signalling events and coordinated membrane movements. Yet, in reality, TEM is continuous with the preceding and prerequisite steps of tight adhesion and locomotion as well as the subsequent step of migration across the basement membrane. Indeed, since the diameter of a leucocyte is .10 mm and the thickness of the endothelial cell at the junction is ≤0.5 mm, for a large portion of its transmigatory journey, the front of the leucocyte is already under the endothelium and the rear of the leucocyte is still attached to the apical surface of the endothelial cell. Thus, a careful coordination of signalling events is necessary. During TEM, the leucocyte leaves fragments of its plasma membrane attached to the endothelial cell and the basement membrane, in an a3b1 integrin-dependent process, although the significance of this is not clear.139 Several of the TEM signalling pathways are initiated by the clustering of the selectins, ICAM-1, and VCAM-1 in the early steps of leucocyte capture and adhesion and, as discussed above, may prime the TEM machinery for its function. Diapedisis is mediated by a number of molecules including PECAM, PVR, JAMs, and CD99, which have distinct molecular interactions and signalling pathways, but all reside in the LBRC. TEM, whether across the endothelial junction or through the cell, involves the recruitment of the LBRC and its associated molecules, which are required for efficient TEM. Taken together, these signalling pathways and the LBRC provide the migrating leucocytes with an appropriate conduit through which they can reach their target tissue and carry out their essential functions. Conflict of interest: none declared.

Funding This work was supported by the National Heart, Lung, and Blood Institute at the National Institutes of Health through grants R01 HL046849 and R37 HL064774.

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Several of the molecules and signalling pathways that are activated or recruited during upstream events like ICAM-1/VCAM-1 clustering also have roles in TEM events like LBRC recruitment and junction remodelling (Figure 3). It is possible that the prelude to TEM (e.g. leucocyte adhesion and locomotion on the endothelial surface) primes these signalling pathways for rapid function to facilitate TEM. The recruitment of SHP2 activates Src (by removing the inhibitory phosphorylation of src Y530), which can then phosphorylate cortactin and reinforce ICAM-1 clustering. Src may also then phosphorylate ITIM motifs of PECAM and PVR, brought to the site of TEM by the targeted recycling of the LBRC. These would then bind SHP2; dephosphorylation of PECAM might be important in collapsing the LBRC during targeted recycling.92,94 If the migrating leucocyte is near a junction, VE-cadherin and its associated adherens junction proteins would be phosphorylated, leading to a local disassembly of the junctional barrier. The leucocyte, now presented with both the plethora of unligated adhesion molecules delivered from the LBRC and a loosening of the junction, could extend pseudopods through the opening and migrate across the endothelium. After the leucocyte has passed, reforming the adherens junction could be aided through the activity of SHP2 brought to the TEM site by PECAM and PVR. After dephosphorylation of VE-cadherin and its associated catenins, stable adherens junctions could re-establish their connection across the junction and with the actin cytoskeleton, and the whole mechanism would be reset for another round of TEM. In addition to serving as a reservoir for unligated molecules that participate in TEM, the presence of counter-receptors for numerous leucocyte adhesion molecules may maintain the tight apposition of the membranes of these cells during TEM to maintain the integrity of the endothelial junctions.9 During transcellular migration, the same processes would function except that the LBRC itself serves as the conduit allowing passage across the endothelium in place of a gap at the junction. In both cases, the LBRC, serving as a repository of several critical molecules, functions in TEM by delivering these molecules en mass to the site of TEM. It is tempting to speculate that the LBRC serves as a platform for the recruitment of key cytosolic signalling molecules as well. In doing so, the endothelial cell would then only have to recruit one compartment instead of recruiting each molecule individually, which would be mechanistically more complicated.

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