Diapedesis the final chapter in a sequence of separate consecutive adhesion events

Elegant intravital microscopy studies revealed that diapedesis of leukocytes is preceded by a coordinated sequence of adhesion events that enables these cells to withstand the hemodynamic forces, known as shear forces, generated by the flow of blood and to make firm contact with the vascular endothelial cells.

At present, three separate consecutive events have been clearly distinguished (Figure 1). Leukocytes first attach to the vessel wall in a rolling interaction (1) and then are arrested, sticking firmly to the surface membrane of a single endothelial cell (2), before diapedesis (3). Specific cell surface glycoproteins on endothelial cells and leukocytes are directly implicated in these events.

1. Leukocyte rolling

The rolling of leukocytes along the endothelial lining of small venules is observed exclusively at inflammatory sites or sites of vascular injury. Leukocyte rolling is a process involving attachment to, and detachment from, the endothelium. Consequently the leukocytes slow down in the main vascular flow. Studies in rodents and in vitro experiments using laminar flow chambers showed that leukocyte rolling is initiated by the expression of specific molecules, designated as selectins, on the surface of leukocytes, i.e. L-selectin (CD62L), or activated endothelial cells, i.e. P-selectin (CD62P) and E-selectin (CD62E). The amino-terminal lectin domain of these adhesion molecules recognizes sialyl Lewis54 (CD15s) or related sulfated, fucosylated or sialylated carbohydrate structures on the opposing cell and is thus critically involved in cell-cell adhesion. Selectin-mediated rolling is an obligatory event before leukocytes can move on to the stage of firm adhesion.

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■ Endothelium

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Figure 1 Consecutive events in the binding and transendothelial migration of leukocytes. (1) Rolling of leukocytes along the endothelium of vessel wall adjacent to an extravascular site of inflammation. (2) Arrest of rolling and firm attachment of leukocytes to the endothelial cells. (3) Lateral migration of leukocytes over the surface of the endothelial cells and subsequent diapedesis. Shear forces generated by the flow of blood, chemokines and adhesion molecules belonging to the family of selectins, immunoglobulins and integrins act in sequence, with some overlap.

2. Arrest of roiling and firm adhesion

The arrest of rolling leukocytes at a particular site of the vascular endothelium involves cell surface adhesion molecules that are different from those required for rolling. At least five heterodimeric cell surface proteins, designated as integrins, have been implicated in the firm adhesion of leukocytes to endothelial cells. These proteins are: LFA-1 (lymphocyte-function associated antigen 1; CD11 a/CD 18); CR3 (complement receptor 3; CDllb/CD18) and pl50,95 (CDtlc/CD18), which belong to the ß2 subclass of the integrin superfamily of adhesion molecules; the ß,-inte-grin VLA-4 (very late antigen-4; CD49d/CD29); and the ß7-integrin LPAM-1 (lymphocyte Peyer's patch adhesion molecule 1; CD49d/ß7). These integrins bind their respective counterstructure on the endothelial surface, which include proteins that structurally belong to the immunoglobulin supergene family, i.e. ICAM-1 (intercellular adhesion molecule 1; CD54), ICAM-2 (CD102) and VCAM-1 (vascular cell adhesion molecule 1; CD106). Most of the adhesion promoting molecules are expressed on the surface of endothelial cells at inflammatory sites and are not or are minimally expressed on resting (nonstimulated) endothelial cells.

The integrin-mediated firm leukocyte-endothelium interaction is triggered or amplified by the action of chemotactic cytokines or chemokines, such as IL-8 (interleukin 8) or MCP-1 (monocyte chemotactic protein I), that are produced by the endothelial cells in response to activation or binding of leukocytes. The present belief is that selectin-mediated rolling of leukocytes will enhance exposure to chemokines that are bound to, and presented by, proteoglvcan-like molecules on the surface of the endothelial cell. This will lead to activation of integrin adhesiveness and firm leukocyte adhesion to endothelium.

Firm adhesion between leukocytes and the endothelium coincides with an increase of the intercellular contact area. These leukocytes change shape, become flattened and move in a lateral direction over the endothelial cell surface. Very little is known about the exact mechanisms and molecules involved in this process, but there are some indications that after initial contact between leukocytes and endothelial cells ICAM-1 diffuses in the plane of the membrane of endothelial cells to the intercellular contact. Whether chemokines provide an additional stimulus is not yet clear.

3. Diapedesis: the actual transendothelial migration

Most endothelia form a continuous and nonten estrated monolayer of endothelial cells. The peripheral membranes of adjacent endothelial cells overlap each other and at these sites, specialized junctions, e.g. tight and gap or communicating junctions, preserve the integrity of the monolayer. Adherent leukocytes cross the vessel wall by squeezing themselves between tightly apposed endothelial cells in an ameboid fashion. Transendothelial migration of leukocytes through the cytoplasmic compartment of the endothelial cells is not a normal pathway. During diapedesis the barrier function of the endothelium is excellently preserved because of the close interaction between the migrating leukocyte and the peripheral membranes of the endothelial cells. The maintained integrity of the endothelium during diap-edesis of leukocytes in vitro can be very well demonstrated by measuring the electrical resistance over the monolayer, which, in fact, does not change during diapedesis. Electron microscopy and time-lapse microcinematography have indicated that the retraction of endothelial cells is restricted to the endothelial cells which are directly involved in the interaction, and that most of the surrounding endothelial cells maintain their integrity and spreading morphology. Although reorganization of the cytoskeletal filaments must be involved, the exact mechanism of endothelial cell retraction, as well as the intracellular signal(s) which coordinate the opening of the inter-endothelial junctions, are still obscure. It is speculated that the interaction of leukocytes with the endothelial cell surface adhesion molecules is an important initiating signal.

Because leukocytes must adhere to the endothelium before subsequent diapedesis, it is difficult to unravel the contribution of a particular adhesion molecule to firm adhesion of leukocytes to the endothelium versus its role in the actual diapedesis. In experimental settings agents that inhibit firm adhesion will also reduce diapedesis. Clearly, fJ2-inte-grin molecules are involved in diapedesis. This is concluded from the profound lack of neutrophil and monocyte diapedesis observed in patients with the leukocyte adhesion deficiency (LAD) syndrome, who are genetically deficient in the functional expression of leukocyte p2-integrin molecules. Beside 32-integrins, other adhesion molecules contribute to the diapedesis. Recent experiments suggest a role for PECAM-1 (platelet-endothelial cell adhesion molecule-1; CD31). This molecule is expressed on leukocytes and it is found in high densities on endothelium at sites of intercellular junctions. An interesting feature is that PECAM-1 can interact with itself. It is suggested that such homophilic binding between PECAM-1 on leukocytes and PECAM-1 on endothelial cells may promote diapedesis, whereby the leukocyte migrates in the direction of the highest density of endothelial PECAM-1, a process called haptotaxis.

Following diapedesis the migrating leukocytes attach to and subsequently traverse the subendothel-ial matrix, or basement membrane, consisting of fibronectin, laminin, elastin and various types of collagen, and accumulate in the extravascular tissues. The endothelial cells rapidly reconstruct an intact monolayer of tightly connected cells. The time required for leukocytes to attach to and traverse the endothelium varies from several minutes to several hours, depending on the type of invading cell, the type of endothelium including their basal matrix and the presence of (inflammatory) stimuli.

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