Adhesion Molecules

Leukocyte adhesion molecules are defined as transmembrane receptor-ligand pairs that transmit mechanical force. Adhesion molecules are required for leukocyte capture or initial tethering, rolling, firm adhesion, and transendothelial migration (Figure 1). All known adhesion molecules relevant for leukocyte adhesion are type I single-spanning transmembrane molecules (N terminus outside the cell), some are homo- or heterodimers. Although it is formally possible that type II or multispanning or GPI-linked molecules could be involved in leukocyte adhesion, there is no evidence that these molecules actually mediate leukocyte rolling or adhesion under in vivo conditions. Notably, a chemokine-chemokine receptor interaction, a variant of fibroblast growth factor receptor, a GPI-anchored receptor, and a secreted molecule have all been proposed to serve as leukocyte adhesion molecules, but none of these proposed interactions have been substantiated in vivo.

Selectins

The selectins are a class of three C-type (calcium-binding) lectins that are expressed on leukocytes (L-selectin), inflamed endothelial cells (E-selectin), and activated platelets and endothelial cells (P-selectin) [4]. They share a common genomic organization and protein structure with an N-terminal lectin domain followed by an epidermal growth factor (EGF) domain, a number (two to nine) of consensus repeats, a transmembrane domain, and a cytoplasmic domain that is homologous among selectins from different species, but unique to each type of selectin [4]. Selectins bind glycosylated selectin ligands (see later discussion) with high to intermediate affinities (nanomolar range) and high on-rates and off-rates. This unique combination of properties enables selectins to mediate capture between cells in the flowing blood and the endothelium, and rolling through formation and breakage of reversible bonds. All three selectins are important in inflammation. In addition, L-selectin mediates rolling of (naive) lymphocytes in high endothelial

Figure 1 Neutrophils interacting with the endothelium lined with a 500-nm glycocalyx (blue) in a postcapillary venule. In capillaries, neutrophils are deformed into a near-cylindrical shape (A) and deform the endothelial glycocalyx. Almost all leukocyte rolling is initiated at the beginning of postcapillary venules, where leukocytes are in close physical contact with the endothelial surface (B). Noninteracting deformed leukocytes eventually recover their spherical shape (C), but rolling leukocytes, especially neutrophils, acquire a characteristic teardrop shape that reflects the effect of adhesive forces balanced by shear forces on the cytoskeleton (D). Rolling or adherent cells can nucleate L-selectin-PSGL-1-dependent secondary capture or tethering events (E). A close-up of a rolling neutrophil (bottom right) shows the endothelial glycocalyx (grey 500 nm) with endothelial adhesion molecules (E-selectin, 30 nm, and P-selectin, 40 nm). The black hairlike lines represent the length of a P-selectin-PSGL-1 pair (100 nm) completely buried in the glycocalyx. Rolling leukocytes probably continuously deform the glycocalyx as they roll while selectin (and integrin) bonds are formed at the leading edge (right) and broken at the trailing edge (left). Arrows indicate direction of blood flow. (see color insert)

Figure 1 Neutrophils interacting with the endothelium lined with a 500-nm glycocalyx (blue) in a postcapillary venule. In capillaries, neutrophils are deformed into a near-cylindrical shape (A) and deform the endothelial glycocalyx. Almost all leukocyte rolling is initiated at the beginning of postcapillary venules, where leukocytes are in close physical contact with the endothelial surface (B). Noninteracting deformed leukocytes eventually recover their spherical shape (C), but rolling leukocytes, especially neutrophils, acquire a characteristic teardrop shape that reflects the effect of adhesive forces balanced by shear forces on the cytoskeleton (D). Rolling or adherent cells can nucleate L-selectin-PSGL-1-dependent secondary capture or tethering events (E). A close-up of a rolling neutrophil (bottom right) shows the endothelial glycocalyx (grey 500 nm) with endothelial adhesion molecules (E-selectin, 30 nm, and P-selectin, 40 nm). The black hairlike lines represent the length of a P-selectin-PSGL-1 pair (100 nm) completely buried in the glycocalyx. Rolling leukocytes probably continuously deform the glycocalyx as they roll while selectin (and integrin) bonds are formed at the leading edge (right) and broken at the trailing edge (left). Arrows indicate direction of blood flow. (see color insert)

venules of secondary lymphatic organs, P-selectin mediates platelet interactions with leukocytes and endothelial cells, and E-selectin mediates homing of certain lymphocytes to the skin. L-selectin is constitutively expressed on all neutrophils, monocytes, eosinophils, and most lymphocytes. P-selectin is stored in Weibel-Palade bodies of endothelial cells and platelet alpha granules and released upon stimulation by secretagogues. E-selectin is transcriptionally regulated through nuclear factor kappaB (NFKB)-dependent pathways. It is suspected that all three selectins and other adhesion molecules also serve signaling functions. Leukocyte activation upon ligation of L-selectin has been demonstrated, as has endothelial cell remodeling secondary to E-selectin engagement. There is little evidence for signaling through P-selectin, but engagement of endothelial or platelet P-selectin appears to result in activation of the bound cells via selectin ligands.

Selectin Ligands

Many selectin ligands have been proposed, but only P-selectin glycoprotein ligand-1 (PSGL-1) has been fully characterized and confirmed to be relevant in vitro and in vivo. PSGL-1 is a homodimer with functional groups near the N terminus, including three (in mouse, two) sulfated tyrosine residues and a critical threonine decorated with an O-linked carbohydrate chain terminating in the tetrasaccha-ride sialyl Lewisx (sLex). sLex biosynthesis requires fucosyl transferase VII and a sialyl transferase (ST), possibly ST3GalIV, and is most likely directly involved in selectin binding. The critical O-linked side chain is located on a core2 structure so that core2 A-acetylglucosaminyl transferase is required for biosynthesis of fully functional PSGL-1. PSGL-1 functions much better as a dimer than as a monomer, possibly because P-selectin is also present as a dimer on the cell surface. Cocrystals between PSGL-1 and P-selectin show that both the tyrosine sulfate and the carbohydrate interact with the lectin domain simultaneously.

Although PSGL-1 was initially discovered as a P-selectin ligand, it was later shown to also bind L-selectin and E-selectin. PSGL-1 knockout mice have a significant defect in P-selectin mediated rolling, a complete absence of transient leukocyte-leukocyte interactions known as secondary tethering, and a moderate defect in E-selectin-dependent rolling. This suggests that PSGL-1 may be the only relevant L-selectin ligand on leukocytes, although other L-selectin lig-ands have been postulated. PSGL-1 is clearly the major P-selectin ligand, accounting for 80 to 90 percent of P-selectin mediated rolling. PSGL-1 appears to mediate leukocyte capture on E-selectin, but is not the E-selectin lig-and responsible for the characteristic slow rolling.

Other proposed selectin ligands include mouse GlyCAM-1, which mediates L-selectin-dependent leukocyte rolling when coated on a solid substrate, but is normally secreted from high endothelial venules in secondary lymphatic organs, and in milk. No ortholog of GlyCAM-1 has been found in humans so far. The GlyCAM-1 knockout mouse showed no evidence of defective lymphocyte homing. E-selectin glycoprotein ligand-1 (ESL-1) was shown to bind E-selectin with high affinity, but most ESL-1 is present in the Golgi system and not accessible from the cell surface. CD24 is a GPI-linked glycoprotein that can function as a P-selectin ligand and can mediate rolling of cancer cells on P-selectin, but a function in leukocyte adhesion or rolling was not demonstrated. Other molecules on neutrophils including the integrin Mac-1 express sLex, but the function of these molecules as selectin ligands in leukocyte recruitment remains unclear. A number of other putative L-selectin ligands have been characterized in high endothelial venules, but it is unclear which of these molecules are physiologically relevant.

Integrins

Integrins are heterodimeric transmembrane molecules expressed by almost all cells except erythrocytes. Most if not all integrins exist in two conformations, active (ligand-binding) and inactive. In some integrins, dramatic changes in molecular shape have been demonstrated upon activation [5], but some of these changes may be secondary to isolation conditions. Although these and other results are suggestive of a conformational change, the exact nature of integrin activation remains an area of ongoing investigation. In addition to conformational activation, integrins cluster into groups upon cell activation, leading to a change in avidity of interaction with clustered ligands that may be present on interacting cells.

Integrins are classified by their (smaller) b chain. For leukocyte adhesion, Pj, b2, b3, and b7 integrins are relevant. bi integrins are expressed on the surface of most cells including endothelial and smooth muscle cells. On memory lymphocytes, several b1 integrins are expressed, with an increased expression after cell activation. a4b1 integrin (VLA-4) is also expressed on monocytes and appears to be important in monocyte rolling, adhesion, and recruitment. a4b7 is involved in lymphocyte homing to gut-associated tissues b2 integrins are almost leukocyte-specific, the only exception being glial cells. All leukocytes express LFA-1 (aLb2). Monocytes, neutrophils, and some effector lymphocytes also express Mac-1 (aMb2). Dendritic cells express axb2, and some lymphocytes express adb2. Whereas LFA-1 serves important functions in leukocyte adhesion (see later discussion) and in forming the immunological synapse, Mac-1 is mostly engaged in phagocytic functions of macrophages and other phagocytes. The functions of axb2 and adb2 in leukocyte adhesion are unknown.

Immunoglobulins

Most cellular integrin ligands are immunoglobulins. Intercellular adhesion molecule-1 (ICAM-1) is a five-domain immunoglobulin-like molecule expressed on endothelial cells, most lymphocytes, and most other cells after exposure to proinflammatory cytokines such as inter-leukin-1 (IL-1) or tumor necrosis factor-a (TNFa). ICAM-1 binds LFA-1 and Mac-1 through its first and third immunoglobulin domain, respectively. ICAM-2 contains two immunoglobulin domains and is expressed on resting endothelial cells and platelets. It also binds LFA-1 and Mac-1. Vascular cell adhesion molecule-1 (VCAM-1) is the main endothelial ligand for VLA-4 and is also expressed on inflamed smooth and cardiac muscle cells.

Another class of immunoglobulins is engaged in homophilic interactions. Platelet-endothelial adhesion molecule-1 (PECAM-1) is a six-domain immunoglobulin expressed by endothelial cells, where it localizes to inter-endothelial borders, and on platelets and most leukocytes. Antibodies to PECAM-1 inhibit monocyte and leukocyte transendothelial migration, but PECAM-1 knockout mice show a clear defect in transendothelial migration only on some backgrounds. The junctional adhesion molecule (JAM) family encompasses three members, JAM1, JAM2, and JAM3, that also localize to interendothelial borders and regulate endothelial monolayer permeability. In addition, JAM1 interacts with LFA-1, JAM2 (also called JAM-3) with Mac-1, and JAM3 (also called JAM-2) with VLA-4. The JAMs are likely involved in regulating transendothelial migration.

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