Introduction
To reach the site of infection, leukocytes need to adhere to the vessel wall, migrate across the endothelium and infiltrate into the underlying tissues. Most of the adhesion molecules that mediate leukocyte rolling and arrest on the vascular surface have been identified and studied in detail. In contrast, leukocyte diapedesis through the endothelium is still poorly defined and remains a matter of debate. The most accepted view is that leukocytes find their way through endothelial cell–cell junctions which, acting as sliding doors, would open to allow their passage and then rapidly close to maintain the integrity of the endothelium1, 2, 3, 4.
However, some studies have challenged this model and support the idea of an alternative pathway whereby leukocytes may migrate through single endothelial cells (reviewed in ref. 5). This possibility is supported by a series of morphological studies in vivo. Already in the 1960s, Marchesi and Florey6 described leukocyte penetration through the endothelial cytoplasm, and Williamson and Grisham7 observed leukocytes present in endothelial cytoplasmic vacuolar structures. Feng et al.8 described the transcellular passage of neutrophils through the microvasculature of the skin in response to chemotactic agents8. However, these papers were then contradicted by other morphological observations showing leukocytes migrating through inter-endothelial junctions (reviewed in ref. 5). Thus, the controversy of whether migration in vivo uses the 'para-' or 'trans-' cellular pathway remains unresolved. Now two papers in this issue — Millàn et al.9 on page 113 and Nieminen et al.10 on page 156 — analyse the possible mechanisms of leukocyte transendothelial migrations further, and bring new strength to the idea that the transcellular and paracellular pathways may co-exist.
Once the molecular organization of endothelial junctions became clear11, it became possible to develop new tools with which to modulate leukocyte diapedesis. For example, blocking endothelial junctional proteins such as the JAMs (junctional adhesion molecules) or PECAM (platelet endothelial cell adhesion molecule) significantly impaired leukocyte extravasation, and supported the concept that leukocytes traverse the junctions by binding to these proteins1, 2, 3, 4 (Fig. 1).
Figure 1: Diapedesis of leukocytes through endothelial cell junctions.
Leukocytes can cross the endothelium by interacting with adhesive proteins at junctions. Some have been identified, including the PECAM (platelet endothelial cell adhesion molecule) and JAM (junctional adhesion molecules) family of adhesive proteins, as well as other factors such as CD99. For further details, see refs 1–4.
Full size image (12 KB)Firm adhesion of leukocytes to endothelial cells is mediated by the endothelial adhesion molecules ICAM-1 (intercellular adhesion molecule) and VCAM-1 (vascular cell adhesion molecule), which bind beta2 and beta1 integrins on leukocytes1, 2, 3, 4. These proteins concentrate in an actin-rich, cup-like structure formed by endothelial projections that embrace the leukocytes. This 'docking' structure is enriched not only in adhesive proteins but also in cytoskeletal components12, 13. Carman and Springer14 previously showed that this cup-like structure — the transmigratory cup — is important not only for leukocyte arrest but also for their migration through individual endothelial cells.
In the first of the new studies, Millan et al.9 show that the clustering of ICAM-1 (and to a lesser extent that of VCAM-1) by specific antibodies induces their redistribution into caveolin-1-rich regions and their subsequent internalization. Upon internalization, ICAM-1 is delivered to the basal side of the endothelial cell membrane where it remains in vesicular structures that have the characteristics of caveolae.
The use of clustering antibodies is a strategy that mimicks leukocyte binding to these proteins. The investigators, therefore, went on to study the effect of ICAM-1 engagement by leukocytes. They found that, after adhering to endothelial cells, lymphoblasts extend protrusions that penetrate into the endothelial cell cytoplasm. ICAM-1 and caveolin co-cluster in a 'ring' around the penetrating lymphoblast pseudopod and follow the passage of the lymphoblast by moving towards the basal side of the membrane (Fig. 2a). The overall picture is that leukocyte adhesion induces ICAM-1 clustering and recruitment to caveolae. These, in turn, may fuse into multivesicular structures that form a sort of channel through which leukocytes squeeze and cross the endothelial cell body. These observations fit with a previous publication8 showing that leukocytes migrate through the endothelial cell cytoplasm in vivo through multivesicular structures called vesiculo-vacuolar organelles. Interestingly, junctional proteins such as PECAM or VE-cadherin are absent in areas of ICAM-1 and caveolin clustering, suggesting that the para- and trans-cellular pathways are regulated by different molecular mechanisms. This is further supported by the evidence that silencing caveolin with short-interfering RNA (siRNA) only inhibits the transcellular, not the paracellular, pathway of leukocyte migration.
Figure 2: Leukocytes can cross the endothelium by penetrating the cell cytoplasm.
(a) Leukocytes may actively penetrate the endothelial cell cytoplasm by elongating pseudopods inside vesicles containing caveolin and ICAM-1. These vesicles can fuse with vesiculo-vacuolar organelles (VVOs), forming a channel that allows leukocyte migration through the endothelial monolayer. (b) When leukocytes adhere to the endothelial surface, an adhesion/transmigration cup is formed. This docking structure contains microvilli that elongate from both endothelial cells and leukocytes. The microvilli contain adhesion molecules (such as ICAM-1 and VCAM-1) and cytoskeletal proteins (such as vimentin and actin). The adhesion/transmigratory cup may mediate leukocyte phagocytosis and movement towards the basal membrane of endothelial cells.
Full size image (26 KB)All the described changes require remodelling of the cytoskeleton. Actin seems to be implicated and it is likely that ERM proteins (ezrin/radizin and moesin) are also important for linking ICAM-1 at, and controlling its movement on, the cell surface.
In the second study, Nieminen et al.10 put forward the possibility that intermediate filaments, and in particular vimentin, might also be important for leukocyte diapedesis. These authors describe a structure very similar to the docking/transmigration cup discussed by Carman and Springer14 (Fig. 2b). They show that after leukocyte adhesion, both endothelial cells and leukocytes form microvilli that elongate and embrace the cells. This process is followed by the penetration of leukocytes through the endothelial cell cytoplasm. They found that the microvilli, both at endothelial and leukocyte sites of contacts, are enriched in vimentin, but not in actin or tubulin. In addition, lymphocyte homing at peripheral lymph nodes and spleen is reduced in vimentin-/- (vim-/-) mice, suggesting that this protein is necessary for lymphocyte trafficking. This may be explained by the observation that in vim-/- endothelial cells, ICAM-1 and VCAM-1 expression and clustering are strongly reduced, and vim-/- lymphocytes are unable to correctly polarize and maintain directional movement.
Taken together, these observations point to a cup-like structure on the endothelium that embraces leukocytes and directs their passage through the cell body (Fig. 2b). This process is reminiscent of cell phagocytosis of foreign organisms but, whereas phagocytosis is usually followed by death of the pathogen inside the cells, leukocytes can traverse the endothelium without apparent functional alterations.
It is probable that the structures described by Niemen et al.10 are identical to those reported by Millan et al.9, but more work on their molecular organization and functional interaction with the cytoskeleton is needed to clarify this point.
Several questions remain. It is not clear what the trigger is that induces the formation of the transmigratory cup. It is likely that engagement of ICAM-1 or VCAM-1 by adhering leukocytes induces their clustering, which in turn mediates re-shaping of the cytoskeleton and formation of the cup; however, which intracellular signals are responsible for these changes is not known. In addition, it remains to be clarified which cytoskeletal proteins are required and how they might contribute. Actin, tubulin and vimentin all seem to be involved in the process. It could be that they each have separate and specific roles, or because they are interconnected the disorganization of one type of cytoskeleton also affects the others.
Another important question is whether both para- and trans-cellular pathways co-exist. Only a small proportion (approximately 10–30%) of leukocytes follow the transcellular pathways; the majority cross junctions9. Millàn et al.9 show that there are differences when they compare endothelial cells of different origin, and that the percentage of leukocytes migrating through the transcellular pathway increases using microvascular endothelium. This suggests that, depending on the region of the vascular tree, leukocytes may predominantly use one pathway or the other. It is likely that where endothelial cell–cell junctions are particularly tight and well organized (such as in the brain microcirculation), leukocytes might preferentially cross the endothelium through the transcellular pathway. By contrast, in post-capillary venules (where junctions are poorly organized), the paracellular pathway would be favoured.
Niemenen et al.10 show that the choice of the migratory pathway may also be cell specific. In their system, only lymphocytes use the transcellular route, whereas neutrophils cross intercellular junctions. It is possible that the conditions used (for example the activation of endothelial cells or leukocytes), the type of stimuli or the exposure to flow, influence and direct the leukocyte's passage through one or another pathway.
Together, these two studies provide new tools and approaches with which to probe this pathway. But numerous other issues remain to be investigated. For example, it will be important to identify experimental methods to selectively inhibit one, but not the other, pathway. One possibility, however, is that the two pathways are interconnected, so that blocking one will also interfere with the other. On another note, which is the biological significance of these two pathways and why do we need both? Could the leukocyte be activated and instructed by endothelial cells in a different way during passage through each pathway? By addressing these issues, the eventual hope is that we might be able to modulate leukocyte trafficking during inflammation and homing.
