In postcapillary venules, leukocytes, endothelial cells, shear forces, and adhesion molecules interact to allow leukocyte recruitment to sites of inflammation and lymphocyte recruitment to secondary lymphatic organs. The physiological processes discussed next have been observed in many model tissues and probably apply to skeletal muscle, cardiac muscle, skin, and lymph nodes, but not to the liver, spleen, or lung. In these latter organs the physiology of leukocyte recruitment is not well understood.
Leukocyte adhesion is initiated by leukocyte-endothe-lial, leukocyte-platelet, and leukocyte-leukocyte contact. Direct capture of leukocytes by endothelial cells is rarely observed, probably because the endothelial cells are covered by a thick glycocalyx layer (500 nm) that dwarfs the adhesion molecules, the longest of which (P-selectin) extends a mere 40 nm from the endothelial plasma membrane. In post-capillary venules, most leukocyte-endothelial interactions are initiated at the point where leukocytes enter the venules from capillaries. Since capillary diameters are smaller than leukocyte diameters in most organs, leukocytes must deform to pass, and in the process squeeze down the endothelial surface layer, which brings them in close contact with the endothelial adhesion molecules. Once engaged, these adhesion molecules support stable rolling, during which the endothelial surface layer is probably continuously flattened. The layer appears to recover after the leukocyte has passed. Most primary leukocyte capture or tethering is mediated by P-selectin on the endothelial cell and PSGL-1 on the leukocyte.
Capture in other parts of postcapillary venules is rare. The few interactions initiated along the venule and not at its beginning involve leukocytes attaching to already adherent or rolling leukocytes in a process called secondary capture or tethering. This process requires L-selectin on the flowing leukocytes and PSGL-1 (and possibly other molecules) on the adherent leukocytes. Sometimes, adherent or rolling leukocytes may leave behind fragments in the form of tethers or cytoplasts, which can nucleate more leukocyte rolling through an L-selectin-dependent process similar to secondary tethering. Endothelial E-selectin does not support much tethering, but its limited tethering function appears to require PSGL-1. Integrins and immunoglobulins have no known role in capture, but LFA-1 stabilizes adhesion events initiated by selectins in plate-and-cone model systems.
Under mild inflammatory conditions, P-selectin is rapidly expressed on the surface of endothelial cells and supports stable neutrophil rolling at a characteristic velocity of 20 to 50 mm/sec. During rolling, molecular bonds form and break continuously, and the cell is rolled forward by the torque resulting from the blood flow until the next available bond is pulled taut. In ideal rolling, cells remain in continuous contact with the postcapillary endothelium without ever breaking free, but occasional hops and skips with reattachment are also observed. The velocity of neutrophil, but not lymphocyte, rolling is regulated by proteolytic cleavage of L-selectin from the leukocyte surface.
Lymphocyte rolling in high endothelial venules of the inguinal lymph node, Peyer's patches, and probably other lymphatic organs is achieved by transient L-selectin binding to sulfated and glycosylated endothelial ligands. Although early studies identified GlyCAM-1 and CD34 as potential L-selectin ligands, later studies in gene-targeted mice did not confirm this. Potential L-selectin ligands relevant for rolling include podocalyxin and an incompletely defined molecule called sgp200. In mesenteric lymph nodes, interaction of a4b7 integrin on lymphocytes with MAdCAM-1 on endothelial cells also contributes to rolling.
When inflammatory conditions persist, for example, in the presence of TNFa, E-selectin is also expressed on endothelial cells. E-selectin supports rolling at a markedly reduced velocity of about 5 mm/sec by binding through an unknown leukocyte ligand distinct from PSGL-1. This ligand requires sialylation by sialyl transferase 3-Gal-IV for full functionality. E-selectin engagement may also serve to activate rolling neutrophils.
During severe inflammation induced by TNFa or other cytokines, the leukocyte integrins participate in the rolling process, presumably by engaging in transient bonds with ligands on endothelial cells . Specifically, Mac-1 has been shown to engage ICAM-1 during rolling, LFA-1 engages an unknown ligand other than ICAM-1 or ICAM-2 [6a]. There is a strong synergy between E-selectin and ß2 integrins in leukocyte rolling  so that in the absence of both almost no rolling occurs, and leukocyte recruitment is severely compromised.
Rolling leukocytes must arrest to become firmly adherent. Interestingly, this arrest mechanism is fundamentally different for neutrophils and lymphocytes . Lymphocyte arrest was studied in postcapillary venules of Peyer's patches and lymph nodes. It requires activation by the chemokine CCL21 (SLC) through the G-protein-coupled receptor CCR7. In flow chamber systems, CXCL12 (SDF-1) interacting with its receptor CXCR4 has also been shown to mediate arrest of lymphocytes and monocytes. In large blood vessels such as the carotid artery, monocyte arrest is mediated by CXCL1 (Gro-a) binding to its receptor CXCR2 and by CCL5 (RANTES) binding to CCR1,3,5 or a combination thereof.
For neutrophils, the arrest situation is much less clear. Although the textbook paradigm proposes activation through chemokines, and there are plenty of chemokines immobilized on inflamed endothelial cells and at least two relevant chemokine receptors, CXCR1 and 2 (only CXCR2 in mice) expressed on resting neutrophils. Recently, CXCR2 was identified as a ventrophil arrest chemokine receptor in postcapillary venules . Neutrophils roll for a long time (1 to 3 minutes) and distance (several hundred micrometers), apparently scanning the endothelial surface for activating signals, before they arrest. This is in contrast to lymphocyte, which arrest within less than a second of activation . The difference may be found in other activating signals that neu-trophils, but not lymphocytes, may receive while rolling. Most prominently, such signals have been shown to emerge from engagement by E-selectin, P-selectin, and L-selectin.
Transendothelial migration proceeds through interen-dothelial clefts and through the body of endothelial cells (transcellular). The molecular requirements for each pathway are likely different. ß2 integrins such as LFA-1 and Mac-1 have long been known to be required for transendothelial migration. Recent evidence suggests that they bind junctional adhesion molecules. Certainly, transendothelial migration requires an active participation by endothelial cells, because it can be blocked by chelating intracellular free calcium or by blocking other signaling pathways. Transendothelial migration appears to involve retraction of vascular endothelial (VE)-cadherin. Another candidate molecule possibly involved in transendothelial migration is PECAM-1. Antibody blockade blocks transmigration, possibly by endothelial signaling, and PECAM-
1-deficient mice have a significant defect in transendothelial migration.
Transmigrated neutrophils (and possibly other leukocytes, although this has not been investigated) acquire a different phenotype after transmigration. They express a4ßb a2ß1, a6ß1, and other ß1 integrins on their surfaces that enable them to engage extravascular matrix components. For migration, a2ß1 appears to be most important.
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