'Docking structures' or 'transmigratory cups' are terms used to describe fingerlike projections of endothelial apical surface membrane reported to surround the lower portion of adherent leukocytes. The membrane is enriched in the adhesion molecules ICAM–1 and VCAM–1 and overlies cytoplasm enriched in f–actin and actin binding proteins. The formation of these structures makes intellectual sense and many of the data demonstrating these structures are visually compelling. However, these structures are not seen by all investigators studying the same cells under fairly similar conditions and have not been demonstrated (yet) in vivo. This raises the question of whether these structures are ubiquitous or unique to certain transmigration conditions. The purpose of this piece is not to question the published data, which have all been generated by respected laboratories and published in top–tier journals. We hope to start an interactive online discussion to explain some of the discrepancies in the literature.

Sánchez–Madrid and co–workers first used the term 'docking structures' to describe these finger–like projections that engaged polyclonally activated lymphocytes and lymphoblasts adherent to cytokine–activated human umbilical vein endothelial cells (HUVECs)¹. Most of their observations were made with a cell line (4M7) that expressed integrin VLA-4 but not LFA–1. These cells could adhere strongly but could not transmigrate, and thus there was plenty of time to strengthen VLA–4–VCAM–1 interactions and recruit more VCAM–1, actin and ERM proteins to the site of adhesion. However, similar extensions bearing ICAM–1 were seen around adherent LFA–1—expressing lymphoblasts.

Subsequently, Carman et al.² demonstrated similar projections rich in ICAM–1 that seemed to rise up off the endothelial surface and surround at least the lower part and sides of leukocytes engaging cytokine–activated endothelial cells or ICAM–1 transfected CHO cells. Disruption of the cytoskeleton abolished these structures but had no effect on leukocyte adhesion. The authors commented that this might belie a role in transmigration. Interestingly, however, Barreiro et al. had found that the docking structures rapidly vanished as lymphocytes began to migrate through the monolayers¹. The Carman–Springer team went on to show that these projections were associated with transmigrating neutrophils, monocytes and lymphocytes, at least under their experimental conditions that involved apical application of a chemokine or leukocyte activator and interaction with activated endothelium. They referred to these structures as 'transmigratory cups'³.

The experimental conditions used for these studies involved monolayers of tumor necrosis factor–activated HUVECs grown on glass coverslips. Transmigration experiments were performed in the presence or absence of fluid shear, but this did not seem to make a difference^3,4. In all cases the investigators reported that a ring enriched in ICAM–1 (and VCAM–1) fluorescence was seen on the apical surface of the endothelial cell at the point of contact with the adherent or transmigrating leukocyte.

However, not everyone who reports rings of ICAM–1 enrichment around transmigrating leukocytes has seen docking structures or transmigratory cups. For example, Ridley's group, using a similar system (but without application of apical CXCL12) showed distinct ICAM–1 enrichment around transmigrating lymphoblasts, but no docking structures⁵. Luscinskas's group also demonstrated local enrichment of ICAM–1 around transmigrating neutrophils undergoing transmigration⁶ and commented that they did not see such actin–rich microvilli.

At sites of documented ongoing transcellular migration in vivo, cup–like structures were not visualized during migration⁷. Instead, the polymorphonuclear cell appeared to be migrating through a fenestra in the flat endothelial cell. A similar electron micrograph image was shown for a neutrophil migrating through an ICAM–1–enriched zone of endothelial cell cytoplasm in vitro⁸. A true apical cup has been demonstrated⁹ by scanning electron microscopy. This cup was surrounding a differentiated HL60 cell adherent to a tumor necrosis factor–activated HUVEC for 30 min. The cell adhered but did not transmigrate.

What do these docking structures represent, and why are they not universally seen? One possibility is that they represent a response of the endothelial cell to leukocytes that are either highly activated or tightly adherent. The structures were seen under conditions in which the leukocytes were adherent but could not transmigrate, allowing time for recruitment of additional ICAM–1 and/or VCAM–1 molecules^1,9, or under conditions in which the leukocytes were additionally activated by the exogenous application of platelet–activating factor or chemokines on the apical surface of the endothelial cells^2,3. One could easily imagine that under these conditions, enhanced leukocyte integrin activation could result in greater recruitment of counter–receptors from the endothelial surface. The scanning electron micrograph⁹ is reminiscent of ligand–mediated phagocytosis in macrophages, and endothelial cells are known to be phagocytic under certain conditions. In contrast, under conditions in which ICAM–1 enrichment was not accompanied by formation of transmigratory cups, the transmigrating neutrophils⁶ or lymphoblasts⁵ were activated by interactions with the cytokine–activated endothelium without additional apical chemokine provided.

An unlikely possibility, but one that must be excluded, is that these structures are an artifact arising from the three–dimensional reconstruction of confocal images. In all cases, x–y plane images showed an intense ring of ICAM–1 fluorescence. This was maximal at the cell surface, but when intense, such fluorescence can be detected above the plane of the endothelial cell. Each x–y image in a z–stack has some out-of-focus light that is not recognized by our eyes when we see the image. However, it is recorded by the computer. When an orthogonal plane is taken through the z–series, this out–of–focus light is present and, if multiple frames in the stack contain it, will reconstruct a fluorescent image that appears to rise above the endothelial surface. My lab has not seen transmigratory cups in our transmigration experiments^10,11. However, we can make them appear artifactually by turning up the gain so that the image intensity is near saturation (unpublished data). I hasten to point out that I am not disputing the validity of anyone's published data nor claiming that docking structures or transmigratory cups are a microscopic artifact. I am merely stating that this is something for the uninitiated to be wary of.

The most likely explanation for the inconsistency in finding transmigratory cups is probably neither of the above. It is a sad truth that despite attention to details in the Methods sections, some apparently trivial but critical experimental details may be omitted. It is quite possible that if detailed experimental protocols are exchanged in this forum, the secret to getting these structures to form consistently will become obvious. The resulting benefit would be that we would understand more about their nature and, with further experimentation, their purpose.

1. Barreiro, O. et al. Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes. J. Cell Biol. 157, 1233-45 (2002).| PubMed | Article |
2. Carman, C. V., Jun, C. D., Salas, A. & Springer, T. A. Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1. J. Immunol. 171, 6135-44 (2003).| PubMed | Article |
3. Carman, C. V., Jun, C. D., Salas, A. & Springer, T. A. Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1. J. Cell Biol. 167, 6135-44 (2003).| PubMed | Article |
4. Carman, C. V. et al. Transcellular diapedesis is initiated by invasive podosomes. Immunity 26, 784-97 (2007).| PubMed | Article |
5. Millan, J. et al. Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nat. Cell Biol. 8, 113-23 (2006).| PubMed | Article |
6. Shaw, S. K. et al. Coordinated redistribution of leukocyte LFA-1 and endothelial cell ICAM-1 accompany neutrophil transmigration. J. Exp. Med. 200, 1571-80 (2004).| PubMed | Article |
7. Feng, D., Nagy, J.A., Pyne, K., Dvorak, H.F. & Dvorak, A.M. Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP. J. Exp. Med. 187, 903–915 (1998).| PubMed | Article |
8. Yang, L. et al. ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow. Blood 106, 584-92 (2005).| PubMed | Article |
9. van Buul, J. D. et al. RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration. J. Cell Biol. 178, 1279-93 (2007).| PubMed | Article |
10. Mamdouh, Z., Chen, X., Pierini, L. M., Maxfield, F. R. & Muller, W. A. Targeted recycling of PECAM from endothelial cell surface-connected compartments during diapedesis. Nature 421, 748-753 (2003).| PubMed | Article |
11. Mamdouh, Z., Kreitzer, G. E. & Muller, W. A. Leukocyte transmigration requires kinesin-mediated microtubule-dependent membrane trafficking from the lateral border recycling compartment. J. Exp. Med. 205, 951-966 (2008).| PubMed | Article |

Transcellular migration seems to occur more often in certain vascular beds (such as the microvasculature, blood brain barrier and high endothelial venules). Therefore, an extensive search for molecules expressed differently in these susceptible vascular beds versus those more resistant to the migration process is critical for defining the mechanism of transcellular migration.

A requirement for specific molecules has been postulated both for the transcellular and the paracellular routes, with a central function proposed for the adhesion molecule ICAM–1. The blockade of molecules involved in the paracellular route does not substantially increase the rate of transcellular migration, and it would therefore seem that transcellular migration requires molecular machineries distinct from the junctional molecules that participate in heterophilic leukocyte–endothelial interactions during paracellular migration. If this is not the case, the 'choice' of one route rather than the other may be an issue of stimulatory signal threshold, with no qualitative changes in signaling between leukocytes and endothelial cells during the transition from adhesion to transmigration. In this scenario, leukocytes would proceed with transcellular migration only if the signal reached an optimal amount. Another possibility is that chemoattractant gradients may be established across the endothelium. These gradients from the ablumenal to the apical endothelial cellular side might be generated either by intracellular transcytosis, promoting transcellular migration, or by extracellular diffusion through the cell–to–cell junctional space, favoring migration by the paracellular route. These gradients could involve different chemoattractant species and/or concentration ranges.

Most data on transcellular migration in vivo have been provided by ultrastructural analyses of fixed samples by series of micrographs obtained with electron microscopy. One of the key factors that still impedes understanding of how transcellular migration works in vivo is that the achievable resolution with advanced techniques of fluorescence optical microscopy is insufficient to allow evaluation of the molecular dynamics of this route. It may be that definition of the mechanism of leukocyte–endothelium transcellular migration will require the identification of an appropriate vascular bed that would allow investigation of this process by fluorescence intravital microscopy. In this context, several studies have attempted to address this point with in vitro cellular models, but how different these models are from the pathophysiological situations involved in this process in vivo remains unknown.

The seminal studies of Gowans and colleagues in the early 1960's described two important aspects of leukocyte migration across the vascular wall. First, they observed that circulating lymphocytes extravasate into the lymph node across the wall of a specific segment of the vascular tree, namely the high endothelial (postcapillary) venules (HEVs), and second, they observed that lymphocyte diapedesis across the HEVs takes place via a transcellular route¹. Subsequent research in the field has confirmed that in most tissues leukocyte extravasation specifically takes place at the level of postcapillary venules and follows a multistep interaction of the leukocyte with the luminal surface of the endothelium involving a well defined sequence of rolling, activation and firm adhesion, crawling, and subsequent diapedesis. Focusing on the molecular mechanisms involved in each step, a distinct set of molecules, which were found to mediate leukocyte diapedesis across the endothelium, are preferentially located in endothelial cell junctions: namely, platelet endothelial cell adhesion molecule (PECAM–1, also known as CD31)², endothelial cell selective molecule (ESAM–1)³, junctional adhesion molecules A and C (JAM–A, JAM–C)⁴ and CD99 (refs. 5,6). As these molecules are usually also expressed by the leukocyte itself, the general view presently held by researchers in the field is that the leukocyte migrates through the endothelial cell junction from the luminal to the basolateral side by engaging homophilic interactions between the leukocyte and two endothelial cell partners in a zipper-like fashion. The early observations on transcellular leukocyte diapedesis by Gowans and others (summarized in ref. 7) have not been seriously considered subsequently, with one exception. Those researchers investigating leukocyte diapedesis across the endothelial blood-brain barrier (BBB) in vivo during central nervous system inflammation (summarized in ref. 7) have repeatedly questioned the in vivo relevance of paracellular leukocyte diapedesis. Performing ultrathin serial section electron microscopy, we and others have invariably found leukocyte extravasation to occur through the brain endothelial cells, leaving the BBB tight junctions—which are usually found in close proximity to the extravasating leukocyte—morphologically intact (summarized in ref. 7). Because BBB endothelial cells are phenotypically unique and connected by an elaborate network of complex tight junctions, transcellular leukocyte diapedesis has been regarded as a rare exception not applicable to other vascular beds. Even compelling evidence that neutrophils pass through the endothelial cells of the skin via vesiculo–vacuolar organelles was questioned and even considered artifactual owing to the strong chemotactic stimulus (fMLP) used in this study⁸. Finally, as these in vivo studies solely relied on ultrastructural evidence and lacked evidence for molecular pathways involved in transendothelial leukocyte migration, this concept has never reached the level of being fully accepted.

Molecular evidence for paracellular leukocyte diapedesis has mostly been derived from in vitro studies. Despite our knowledge that in vivo leukocyte diapedesis—with the exception of that in the lung and liver—takes place at the level of postcapillary venules but not in other segments of the vascular tree, human umbilical vein endothelial cells (HUVECs) have been the workhorse for studying leukocyte diapedesis across human endothelium—driven by their availability rather than any other reasons. Using these embryonic and large–vein endothelial cells ignores the fact that endothelial cell phenotypes and functional characteristics greatly vary during development and along the vascular tree^9,10. Thus, one may boldly question whether HUVECs represent an appropriate model for studying leukocyte diapedesis when its cellular and molecular mechanisms are to be investigated. In fact, recent studies employing human microvascular endothelium were able to demonstrate transendothelial diapedesis of leukocytes in vitro (summarized by ref. 11), providing in vitro evidence for this pathway of leukocyte diapedesis.

Although these recent studies have revived discussions on transendothelial diapedesis, most researchers consider paracellular diapedesis as the more plausible and simply straightforward mechanism and are therefore still hesitant in accepting the biological relevance of this pathway. I wish to challenge this view with our in vivo observations on leukocyte diapedesis across the BBB during neuroinflammation, which clearly show endothelial cell junctions to be overlapping and thus often very long (up to 100 µm), and occasionally even tortuous¹². In contrast, the distance between the luminal and abluminal endothelial cell membrane at the BBB and other postcapillary vascular beds is typically below 1 µm. Thus, from the view of the leukocyte the transcellular extravasation pathway is much shorter and must appear straightforward. In addition, from the perspective of a continuous postcapillary endothelium, allowing leukocytes to diapedese through cellular junctions, which are fundamental for maintaining functional and structural integrity of the endothelial monolayer at the vascular wall, seems a fairly adventurous endeavor. Supporting the concept that intact endothelial junctions may even be relevant during leukocyte diapedesis is the observation that VE–cadherin, which establishes endothelial adherens junctions, provides a barrier for leukocyte diapedesis in vivo¹³.

Thus, we need a fresh and open view on the cellular and molecular mechanisms of transendothelial leukocyte migration to be able to define the biological relevance of paracellular versus transcellular diapedesis of leukocytes. It will be challenging but exciting to find out the following:

One, whether the currently defined junctional molecules involved in leukocyte diapedesis serve as signaling cues or act rather to maintain endothelial junctional integrity during transcellular leukocyte dipedesis by triggering membrane fusions and actin cytoskeleton rearrangements. Two, whether the very last step of the multistep leukocyte–endothelial interaction cascade displays molecular diversity depending on the leukocyte and the vascular bed involved or if it is instead governed by a master mechanism.

1. Marchesi, V.T. & Gowans, J.L. The migration of lymphocytes through the endothelium of venules in lymph nodes: an electron microscope study. Proc. R. Soc. Lond. B 159, 283–290 (1964).| PubMed |
2. Muller, W.A., Weigl, S.A., Deng, X. & Phillips, D.M. PECAM-1 is required for transendothelial migration of leukocytes. J. Exp. Med. 178, 449–460 (1993).| PubMed | Article |
3. Wegmann, F. et al. ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability. J. Exp. Med. 203, 1671–1677 (2006).| PubMed | Article |
4. Nourshargh, S., Krombach, F. & Dejana, E. The role of JAM-A and PECAM-1 in modulating leukocyte infiltration in inflamed and ischemic tissues. J. Leukoc. Biol. 80, 714–718 (2006).| PubMed | Article |
5. Schenkel, A.R., Mamdouh, Z., Chen, X., Liebman, R.M. & Muller, W.A. CD99 plays a major role in the migration of monocytes through endothelial junctions. Nat. Immunol. 3, 143–150 (2002).| PubMed | Article |
6. Bixel, G. et al. Mouse CD99 participates in T-cell recruitment into inflamed skin. Blood 104, 3205–3213 (2004).| PubMed | Article |
7. Engelhardt, B. & Wolburg, H. Mini-review: Transendothelial migration of leukocytes: through the front door or around the side of the house? Eur. J. Immunol. 34, 2955–2963 (2004).| PubMed | Article |
8. Feng, D., Nagy, J.A., Pyne, K., Dvorak, H.F. & Dvorak, A.M. Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP. J. Exp. Med. 187, 903–915 (1998).| PubMed | Article |
9. Aird, W.C. Phenotypic heterogeneity of the endothelium: II. Representative vascular beds. Circ. Res. 100, 174–190 (2007).| PubMed | Article |
10. Aird, W.C. Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ. Res. 100, 158–173 (2007).| PubMed | Article |
11. Carman, C.V. & Springer, T.A. Trans-cellular migration: cell-cell contacts get intimate. Curr. Opin. Cell Biol. published online, doi:10.1016/j.ceb.2008.05.007 (30 June 2008).| PubMed | Article |
12. Wolburg, H., Wolburg-Buchholz, K. & Engelhardt, B. Diapedesis of mononuclear cells across cerebral venules during experimental autoimmune encephalomyelitis leaves tight junctions intact. Acta Neuropathol. 109, 181–190 (2005).| PubMed | Article |
13. Gotsch, U. et al. VE-cadherin antibody accelerates neutrophil recruitment in vivo. J. Cell Sci. 110, 583–588 (1997).| PubMed | Article |

The recruitment of leukocytes to sites of injury, infection or immune-mediated reaction is essential for homeostasis. Evidence from both in vivo and in vitro models now indicates that leukocytes can traverse the endothelium lining blood vessels either at cell-cell junctions ('paracellular' migration) or by migrating through the endothelial cell ('transcellular' migration), or by both routes. The extent to which the various leukocyte types use either or both routes remains unanswered. This will require the development of in vivo and in vitro models that provide robust levels of both routes of transendothelial migration (TEM). Here we provide a brief recap of recent studies of mechanisms regulating paracellular TEM and highlight some of the important unanswered questions.

The endothelial-cell VE-cadherin complex (VE-cadherin and -, -, - and p120 catenins¹) localizes to cell junctions and is an important gatekeeper in leukocyte transmigration^2–4. During TEM, the VE-cadherin complex disassociates, forming a gap, and leukocytes migrate through this gap. The gap reseals once TEM is complete. Many laboratories have found that tyrosine phosphorylation of the VE-cadherin cytoplasmic tail regulates leukocyte paracellular TEM^2–4 and this may involve regulation by p120 catenin⁴. On the basis of those reports, new models^2–4 propose that inducible adhesion molecules such as ICAM-1 serve many functions in TEM⁵. After binding the leukocyte integrin LFA-1 (Mac-1), ICAM-1 associates with the cytoskeleton through Src-mediated phosphorylation of endothelial cortactin at cell junctions. This stabilizes leukocyte adhesion in conditions of shear flow and, notably, is predicted to facilitate cross-phosphorylation of tyrosine residues at positions 658 and 731 in VE-cadherin through activated Src and Pyk-2 kinases (and perhaps others). Phosphorylation of these residues prevents and/or competes for the binding of p120 catenin and -catenin to VE-cadherin⁶, which stabilize VE-cadherin at cell junctions⁷. Thus, phosphorylation leads to disassociation of the VE-cadherin complex and 'loosening' of cell-cell junctions.

Key unanswered questions: What initiates the phosphorylation of VE-cadherin and how is it terminated (kinases or phosphatases)? Is this a uniform process or does it differ for different leukocyte types? Is the 'VE-PTP' protein, which binds VE-cadherin⁸, involved in gap formation by VE-cadherin and, by extension, in leukocyte TEM? How does the gap in VE-cadherin⁹ open and reseal in such a short time frame (3–5 min)? Is the gap in VE-cadherin mediated by endocytosis or internalization of VE-cadherin? A published report suggests internalization is not involved⁴, but new models to monitor VE-cadherin activity in TEM are needed to extend those findings. Previous studies noted that VE-cadherin and and -catenin are cleaved by leukocyte proteases¹⁰. This raises the issue of whether the gap in VE-cadherin is mediated by enzymatic cleavage. A published report has linked the metalloprotease ADAM10 to the mediation of lymphocyte TEM and VE-cadherin proteolysis¹¹. Further studies along these lines are necessary to firmly establish the context for a contribution by proteases to TEM.

1. Lampugnani, M.G. et al. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, -catenin, and -catenin with vascular endothelial cadherin (VE-cadherin). J. Cell Biol. 129, 203–218 (1995).| PubMed | Article |
2. Allingham, M.J., van Buul, J.D. & Burridge, K. ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration. J. Immunol. 179, 4053–4064 (2007).| PubMed | Article |
3. Turowski, P. et al. Phosphorylation of vascular endothelial cadherin controls lymphocyte emigration. J. Cell Sci. 121, 29–37 (2008).| PubMed | Article |
4. Alcaide, P. et al. p120-catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation. Blood published online, doi:10.1182/blood-2008-03-147181 (18 July 2008). | PubMed | Article |
5. Alcaide, P., Auerbach, S. & Luscinskas, F.W. Neutrophil recruitment under shear flow: it's all about endothelial cell rings and gaps. Microcirculation published online, doi:10.1080/10739680802273892 (19 August 2008).| PubMed | Article |
6. Potter, M.D., Barbero, S. & Cheresh, D.A. Tyrosine phosphorylation of VE-cadherin prevents binding of p120- and -catenin and maintains the cellular mesenchymal state. J. Biol. Chem. 280, 31906–31912 (2005).| PubMed | Article |
7. Xiao, K. et al. Cellular levels of p120 catenin function as a set point for cadherin expression levels in microvascular endothelial cells. J. Cell Biol. 163, 535–545 (2003).| PubMed | Article |
8. Nawroth, R. et al. VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts. EMBO J. 21, 4885–4895 (2002).| PubMed | Article |
9. Shaw, S.K., Bamba, P.S., Perkins, B.N. & Luscinskas, F.W. Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. J. Immunol. 167, 2323–2330 (2001).| PubMed | Article |
10. Vestweber, D. VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arterioscler. Thromb. Vasc. Biol. 28, 223–232 (2008).| PubMed | Article |
11. Schulz, B. et al. ADAM10 regulates endothelial permeability and T-cell transmigration by proteolysis of vascular endothelial cadherin. Circ. Res. 102, 1192–1201 (2008).| PubMed | Article |