Mechanism of Leukocyte Diapedesis

The mechanism of leukocyte entrance into tissues requires, first, leukocyte adhesion to the endothelium and, subsequently, migration across the blood-vessel wall.

Leukocyte Adhesion

The leukocyte adhesion cascade is a sequence of adhesion and activation events that precede the extravasation of leukocytes. This multistep event is mediated by the engagement of adhesion receptors on leukocytes and their counter-receptors on endothelial cells. First, leukocytes escape from the bloodstream by capture or tethering to the endothelium. Indeed, during inflammation, endothelial cells are activated by signals emanating from inflammatory mediators such as TNFa, nitric oxide, and complement factors. Within minutes of stimulation, P- and E-selectins are expressed at the surface of endothelial cells and interact with their primary ligand PSGL-1 (P-selectin glycoprotein ligand-1), constitu-tively found on all leukocytes. This allows the capture of leukocytes on the surface of endothelial cells and permits neutrophil celerity to be slowed. Following a rolling phase, neutrophils are arrested on the endothelial cell surface. This adhesion step is mostly mediated by interactions involving b2-integrins on the leukocyte surface and immunoglobulins such as ICAM-1 and VCAM-1 on the endothelium. Subsequently, neutrophils migrate on the endothelial surface by amoeboid movement toward the borders of endothelial cells where transmigration occurs.

For more details on the coordinated interplay between the adhesion and signaling proteins involved in the process of firm adhesion of leukocytes on the endothelium, the reader is referred to an excellent recent review [6].

Leukocyte Transmigration

The multiple steps implicated in the transmigration process require the formation and destruction of homophilic and heterotypic interactions between receptors expressed at the surface of leukocytes and endothelial cells. To investigate the role of each adhesion receptor described earlier, attempts were made to block transendothelial migration of leukocytes using specific antibodies.

Role of PECAM-1 and CD99

Pretreating monocytes or neutrophils with antibodies specific for PECAM-1 inhibited their emigration across endothelial cell monolayers in in vitro assays. Reciprocally, blockage of endothelial cell PECAM-1 results in an effective inhibition of leukocyte transmigration. It was also found that PECAM-1 is required for in vivo leukocyte recruitment, because antibodies against PECAM-1 were able to block the accumulation of neutrophils in the peritoneal cavity in a rat model system. This means that homophilic interactions of PECAM-1 on leukocytes with PECAM-1 on endothelial junctions are involved in transmigration. In fact, PECAM-1 is constitutively recycled along the endothelial cell borders. During the transendothelial migration process, recycling is targeted to the part of the junction across which monocytes are squeezing. Indeed, anti-PECAM-1 antibodies block the recruitment of PECAM-1 to the zones of leukocyte migration and thereby the leukocyte transmigration process.

The role of CD99 in leukocyte transendothelial migration was more recently established by a similar method using anti-CD99 antibodies. Thus, whereas anti-CD99 antibodies have no effect on monocyte adhesion, they are able to block their transmigration significantly. Inhibition of either leukocyte or endothelial cell CD99 blocked leukocyte transmigration with equal efficiency, suggesting that homophilic interactions between CD99 expressed on both monocytes and endothelial cells are required for transmigration [7]. This effect is more drastic than the inhibition induced by anti-PECAM-1 antibodies. Simultaneous use of anti-PECAM-1 and anti-CD99 antibodies completely blocks the ability of monocytes to transmigrate. This additive effect suggests that PECAM-1 and CD99 possess distinct roles in the transmigration process. In fact, leukocytes arrested by anti-PECAM-1 antibodies adhere to the apical surface of the endothelium, whereas those arrested by anti-CD99 antibodies are trapped inside the endothelial cell-cell junctions. This indicates that PECAM-1 and CD99 are differentially located along the interendothelial junction and are sequentially involved in the transmigration process. PECAM-1 -mediated homotypic interactions are probably relayed by CD99-mediated ones during leukocyte migration. To date, it is difficult to ascertain whether such a sequential process can be extended to the other leukocyte subfamilies, the surface expression of CD99 of the various types of leukocytes being only partially known.

Role of JAMs

Concerning the role of JAMs on the transendothelial migration of leukocytes, several antibodies directed against JAM A were described to have the capability to inhibit the transmigration of monocytes, T cells, and neutrophils in vitro and in vivo. Thus, in a model of meningitis in mice, it was demonstrated that the addition of anti-JAM A antibodies strongly reduced monocyte and neutrophil infiltration into the cerebrospinal fluid and brain parenchyma. Moreover, anti-JAM B or a soluble fragment of JAM B were also able to block chemokine-induced lymphocyte transmigration.

In fact, JAM A plays a critical role in the diapedesis of leukocytes. After stimulation of endothelial cells with both TNFa and IFNg, JAM-A is redistributed from the junctions to the endothelial surface. Subsequently, it becomes available to interact with the activated form of the ß2-integrin LFA-1 (ß2aL), thus contributing to the adhesion of leukocytes on the activated endothelium. At this stage, hom-ophilic interactions involving JAM A expressed both on leukocytes and on endothelial cells are not crucial to firm adhesion of leukocytes. This is particularly true for monocytes and neutrophils that express very low amounts of JAM A at their surfaces. The interaction of JAM A with LFA-1 contributes first to the chemokine-triggered adhesion and second to the transmigration of memory T cells, neutrophils, and lymphocytes.

The binding of endothelial JAM A to LFA-1 on leukocytes may disrupt junctional homophilic JAM A interactions, thereby unlocking interendothelial junctions. Complementary data are needed to understand the interplay between the homophilic and heterophilic JAM A-mediated interactions occurring during leukocyte transmigration.

Moreover, according to the subpopulation of leukocytes, the transmigration step involving the JAM family implicates different couples of adhesion receptors. For example, JAM B expressed at the surface of high endothelial venules interacts with JAM C of T cells and NK and dendritic cells. This molecular pair seems to play a critical role in the trafficking of T, NK, and dendritic cells into and out of high endothe-lial venules.

Role of VE-Cadherin

In contrast to antibodies specific for PECAM-1, JAMs, or CD99, antibodies directed against the extracellular domain of VE-cadherin increase the recruitment of leukocytes to the infection sites. In fact, the anti-VE-cadherin antibodies have the ability to disrupt the VE-cadherin homophilic interactions. This results in the formation of gaps between endothelial cells through which leukocytes can easily squeeze. In physiological conditions, alterations of VE-cadherin-mediated adhesion also occur at endothelial junctions during leukocyte transmigration. Indeed, immunofluorescence experiments reveal that VE-cadherin and a-, ß-, and g- catenins disappear from cell-to-cell contacts following adhesion of neutrophils to endothelial monolayers. This effect appears only where neutrophils firmly adhere while VE-cadherin-based complexes remain intact in areas devoid of adherent neutrophils. By contrast, PECAM-1 distribution remains unaffected.

The mechanism leading to the formation of gaps at cell-cell junctions during leukocyte transmigration is still controversial. To cast some light on this mechanism, move ments of VE-cadherin occurring during leukocyte transmigration were observed using real-time microscopy. Thus, transmigration of fluorescently labeled leukocytes was followed in real time by two-color fluorescence microscopy using HUVECs expressing endogenous VE-cadherin and a VE-cadherin protein fused with its C-terminal part to Green Fluorescent Protein (VE Cad-GFP) [8]. This study showed that transmigration occurs both through preexisting gaps and through de novo gap formation. Gap widening accommodates to the size of the transmigrating leukocyte, allowing a narrow contact between the endothelial cell and the leukocyte. The widening of the gaps seems to be accompanied by a clustering of the VE Cad-GFP molecules on the edge of the forming clefts. After transmigration, the displaced molecules diffuse back to reconstitute the junctions. This leads to a rapid resealing of the junctions within 5 minutes after leukocyte transmigration. These results were confirmed by a real-time study following the differential movements of VE-cadherin in living endothelial cells during transmigration of neutrophils with anti-VE-cadherin antibodies that do not interfere with transendothelial migration. The lateral movement of VE-cadherin was hypothesized to be a consequence of its decoupling from the cytoskeleton, probably initiated by an intracellular signal due to leukocyte adhesion to the endothelium. In both of the studies, only the movement of the cytoplasmic tail of VE-cadherin was followed in real time. It is not known whether the extracellular domain of VE-cadherin remains intact during the lateral movement.

Real-time fluorescent imaging shows that neutrophils only move laterally underneath the vascular endothelium instead of deeply into the vascular tissue. Thus, the question as to how neutrophils go across the endothelium before penetrating into the surrounding tissues remains unanswered. The curtain effect that moves VE-cadherin is necessary but not sufficient to completely support leukocyte transmigration.

The event initiating the lateral movement of VE-cadherin may correspond to the cleavage of this adhesive receptor by proteases expressed at the surface of transmigrating neu-trophils. This hypothesis is based on the fact that elastase, at the surface of transmigrating neutrophils, mainly localizes to the migration front [9]. Thus, after the adhesion to the endothelial cell surface, elastase is expressed on the apical face of neutrophils, avoiding contact between proteases and the endothelial cell surface. When neutrophils reach endothelial cell-cell junctions, elastase moves from the apical face to the basal face where it comes in close proximity with junctional components. An in vivo study suggested that elastase may contribute to lung neutrophil accumulation and microvascular injury during intestinal ischemia-reperfusion (IR). Moreover, following IR, elastase and VE-cadherin fragments were found in the bronchoalveolar fluid.

Despite results obtained in vitro and in vivo, the participation of proteases in the transmigration of leukocytes is still controversial. Indeed, a study published in 2002 suggested that elastase and MMP9 are not essential for neu-

trophil transmigration. This assumption is based on the use of neutrophils depleted in both elastase and MMP9 that show no defect in their transmigration capacity. These results contradict our own study as we assessed that VE-cadherin is cleaved following adhesion of neutrophils to endothelial cell monolayers [10]. Using specific inhibitors of neutrophil proteases, we were able to identify elastase and cathepsin G bound to the neutrophil surface as the major proteases involved in the cleavage of VE-cadherin. Furthermore, inhibition of both membrane-bound proteases inhibited neutrophil transmigration in in vitro assays. In vivo, the surface-bound elastase and cathepsin G may cleave VE-cadherin on very restricted areas close to the sites of neutrophil adhesion. On the endothelium, the redundancy between these two proteases can explain why neutrophils still transmigrate across endothelial monolayers in the presence of specific inhibitors of elastase and also why neutrophils from elastase-deficient mice show no defect in transendothelial migration. Indeed, cathepsin G can replace elastase, thus allowing the neutrophil transmigration to occur despite elas-tase inhibition or deficiency.

The involvement of a paracrine mechanism in neutrophil-mediated alteration of the endothelial barrier function was suggested by Gautam and coworkers. Upon neutrophil adhesion to the endothelium, leukocyte b2-integrin signaling triggered the release of the neutrophil CAP37, a serine protease without catalytic activity that induced a cytoskeletal rearrangement and the formation of gaps in the endothelium in vitro. Alteration of the endothelium may result from the interaction between CAP37 on the surface of neutrophils and proteoglycans present at the endothelial cell surface. This interaction could stimulate endothelial cell contraction by a yet unidentified mechanism.

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