Leukocyte TEM occurs primarily at EC-junctions (12
), but recent studies suggest that changes in EC morphology, and consequently junctional arrangement, or disruption of β2
-integrin-ICAM-1 interactions, can lead to transcellular migration (12
). Thus here we asked whether differences in EC morphology in straight and converging venular regions affect leukocyte trafficking and TEM in-situ. We showed that leukocytes, in their innate environment, preferentially use tri-cellular junctional regions as “portals” for TEM. Our data suggest that the “portals” are junctions that are surrounded by enriched regions of ICAM-1. These enriched ICAM-1 regions mark the location of a nearby portal, as well as preparing the EC-junctions to accommodate leukocyte passage. Thus, discontinuities in junctional molecules need not pre-exist, but can be initiated by approaching leukocytes via ICAM-1-mediated signaling. Supporting our findings, others have found no evidence for pre-existing discontinuities of the endothelial barrier at tri-cellular junctions in retinal wholemounts, but observed loss of tight junction proteins during leukocyte TEM (34
). While we can not unequivocally demonstrate that all leukocytes transmigrating via these portals were taking a junctional route (vs a paracellular route in very close apposition to the actual junction), our findings that the ICAM-1 tail peptide blocked leukocyte TEM and VE-Cadherin rearrangement at the EC junctions, together with the findings that ICAM-1 mediated signaling directly affects VE-Cadherin phosphorylation and mobility (30
), strongly argues that route taken is indeed junctional.
Rearrangement of junctional molecules such as VE-Cadherin, and formation of transient gaps during leukocyte TEM has been demonstrated in-vitro (21
). We confirmed these findings in-situ, and, further, showed that signaling mediated via the ICAM-1 cytosolic tail is essential for the formation of observed gaps in VE-Cadherin. As the ICAM-1 tail is a potential binding target for Src (37
), the likely signaling mechanism for the disassembly of the homologous VE-Cadherin interactions is the activation of proteins Src- and Pyk2 upon engagement of ICAM-1, leading to phosphorylation of VE-Cadherin (31
). However, the ability of leukocytes to exert force on EC contacts during TEM (38
) could also contribute to the formation of these gaps, and cannot be ruled out.
We add another dimension to the complexity of recruitment by showing that the arrangement of EC-junctions in different venular regions greatly contributes to leukocyte recruitment and TEM. ECs in venular convergences are significantly smaller, more rounded and aligned in a more random fashion than ECs in straight regions (). This arrangement produces more tri-cellular junctions in convergences, apparently making convergences optimally equipped to support TEM. In previous work we also showed that in venular convergences the two inlet vessels are predicted to create a region of low velocity, increasing leukocyte adhesion probability (26
), as was indeed demonstrated in . Increased leukocyte adhesion together with a higher number of tri-cellular junctions in venular convergences, resulted in 2.6-fold higher leukocyte TEM compared to straight regions. Thus, while fluid shear likely contributes to leukocyte accumulation in convergences, it is unlikely to affect leukocyte TEM directly. This is because not only the absolute number (higher as a result of more adhered cells in the region), but also TEM efficiency (%TEM/all adhered cells) was significantly higher in convergences (). Similarly we ruled out the differential expression of adhesion molecules as key contributors for increased TEM, as the expression of ICAM-1 (Fig S4B
) and E-selectin (unpublished) is not different in straight versus converging regions, confirming local EC morphology as a prime candidate.
Previous analysis of leukocyte adhesion in-vivo (10
), and work in monolayers (39
), showed that due to the EC morphology, most adhered leukocytes overlap EC-junctions. We have extended these findings to show that differences in leukocyte adhesion distribution with respect to junctional type in convergences versus straight regions are solely due to differences in EC size, shape and alignment, producing a higher ratio of tri-cellular to bi-cellular junctions in venular convergences.
Leukocyte crawling is presumably a tool to get adhered leukocytes to the nearest EC junction, where TEM occurs. Confirming previous observations (10
), we showed that nearly 90% of leukocytes undergo intraluminal crawling (). This suggests that the initial location of leukocyte adhesion is simply a way station where leukocytes pause before crawling to a “portal” to undergo TEM. Leukocyte crawling, in control venules and monolayers (28
) or in MIP-2 stimulated venules (12
) is random (independent of blood flow). However, in our study, leukocyte crawling in the presence of fMLP was parallel or perpendicular to blood flow, but very rarely against it, suggesting some degree of directionality. The finding that blockade of the luminal portion of the ICAM-1 molecule, but not blockade of the cytoplasmic tail, resulted in loss of the observed directionality (), argues that ICAM-1 might play a role in directing leukocyte crawling.
We showed that 50 minutes after fMLP application ICAM-1 expression was significantly increased (Fig S4A
) compared to control conditions, indicating endothelial activation. The fMLP evoked response in this time frame is surprising as fMLP is considered to be a leukocyte activator, and there is very little evidence for its ability to affect ECs. Whether the increase in ICAM-1 expression was directly induced by fMLP treatment or whether it was a consequence of fMLP-induced leukocyte activation and adhesion is not clear and will require new studies. Importantly, upon exposure of the tissue to fMLP, otherwise homogenously expressed ICAM-1 on individual ECs became enriched near EC-junctions (primarily tri-cellular, ). The presence of ICAM-1enriched regions near tri-cellular junctions could explain the shorter crawling distances that leukocytes exhibit in venular convergences, as well as their lower crawling velocities ().
In our preparation fMLP-induced TEM occurred mainly at or very close to EC-junctions. Our results differ from those previously reported (40
), possibly due to the route of fMLP administration or the different tissue that was investigated. Intriguingly, leukocytes that originally adhered at bi- or tri-cellular junctions were often observed to crawl away and either detach from the endothelium or transmigrate elsewhere. This argues that initial adhesion doesn't determine whether the leukocyte will undergo TEM, or affect the route it will use, although this requires future studies. It also suggests that not all EC-junctions are the same; some EC-junctions can act as “portals” for leukocyte TEM and some apparently cannot. Our study implicates ICAM-1 in making some EC-junctions act as portals for TEM.
Many molecules make up EC-junctions, and most have been implicated in leukocyte TEM (14
). Importantly, the nature of endothelial cell-cell contacts varies with the need to regulate vessel barrier function. For example, arterioles have a significantly higher number of tight junctions compared to venules (42
), and are significantly less permeable (43
). Likewise, there are differences in expression and subcellular localization of junctional adhesion molecules (JAM) (22
). In the current work we suggest that the junctions that could act as portals for TEM are those junctions that are surrounded by high ICAM-1 expression, but an alternative/additional explanation could be that active portals have a different assembly or different array of junctional proteins compared to elsewhere. The local microanatomy of these junctions might also be different.
As mentioned above, not all junctions are used by leukocytes for TEM. In fact, only a small portion of the junctions act as active portals, and often the same junctional region (bi- or tri-cellular) is used by multiple leukocytes. Supporting the idea that portals are EC-junctions that are enriched in ICAM-1, only ~43% of all tri- and 23% of all bi-cellular junctions became enriched in ICAM-1, correlating with the percentages of leukocytes undergoing TEM in these regions. Moreover, we showed that gaps in VE-Cadherin staining were primarily formed at tri-cellular junctions (), and were mediated via ICAM-1 signaling, again suggesting that the observed leukocyte behavior is directly connected to ICAM-1 enriched regions.
Some regions within the venular wall express lower levels of key extracellular matrix proteins comprising the basal lamina than other regions in the same vessel (44
). These regions are also associated with gaps between the pericytes and are preferentially used by migrating leukocytes (44
). We speculate that localization of these regions with EC-junctions could also make these locations optimal for leukocyte TEM.
In summary, we show that leukocytes in their innate environment preferentially target tri-cellular EC junctions to traverse the vessel wall. We show that EC morphology plays an important role in determining these portals, making venular converging regions optimally equipped to support leukocyte TEM. Moreover, in exploring the nature of the active portals, we suggest that enrichment of ICAM-1 surrounding some EC-junctions makes these locations preferred for TEM, by signaling to leukocytes the location of the portal, and further by mediating rearrangement of junctional molecules at these locations.