The selectins are type I membrane glycoproteins composed of an amino terminal C-type lectin domain, a single epidermal growth factor (EGF)-like domain, two to nine short consensus repeat (SCR) domains, a membrane spanning region, and a cytoplasmic tail 41. The family includes three molecules that display different patterns of expression and function.
L-selectin is expressed on almost all circulating leukocytes and is involved in lymphocyte homing 75 and leukocyte recruitment to sites of inflammation 57. Following activation of leukocytes, L-selectin can be shed by proteolytic cleavage near the cell surface. A disintegrin and metallopeptidase (ADAM)-17 and at least one other enzyme are involved in constitutive and activated L-selectin shedding 85. E-selectin expression is limited to inflamed endothelial cells and is induced at the level of transcription, as inhibitors of either transcription or translation inhibit E-selectin expression 10. P-selectin is inducibly expressed on activated endothelium and platelets. P-selectin is stored preformed in the α-granules and Weibel-Palade bodies of platelets and endothelium, respectively. Following activation, P-selectin is rapidly expressed at the cell surface as a result of fusion of these granules with the plasma membrane. Further, P-selectin expression on endothelium is also regulated transcriptionally 31, but the regulation is different in mice and humans 107. In many assays, P-selectin is the dominant selectin in mice, but it is not clear whether this is also true in humans.
Capturing and rolling of neutrophils, which greatly facilitate subsequent arrest and recruitment, are mediated by selectins. Indeed, triple-selectin knockout mice 16 have a severe defect in neutrophil recruitment and other defects. All three selectins can mediate rolling, but the rolling behavior of neutrophils on the selectins is different. In venules of the cremaster muscle, the rolling velocity of leukocytes on L-selectin (130 μm/s) 39 is faster than the velocity on P-selectin (40 μm/s) 39, whereas E-selectin mediates slower rolling (3–7 μm/s) 48109. Ex vivo data suggest that the simultaneous presence of E- and P-selectin has a synergistic effect, with P-selectin increasing the number of rolling cells and E-selectin reducing rolling velocity 87. This mechanism may explain why neutrophil recruitment is enhanced when both selectins are expressed. However, knocking out the Sele gene encoding E-selectin has little effect on neutrophil recruitment 50 and knocking out Selp only delays recruitment by two to four hours 60.
In addition to the direct interaction of neutrophils with the endothelium, neutrophils can also be recruited by “secondary capturing” 110. PSGL-1 on free-flowing neutrophils can bind to P-selectin presented by adherent platelets 18 and L-selectin on free-flowing neutrophils can interact with PSGL-1 presented by adherent leukocytes 22 or leukocyte-derived fragments 88.
P-selectin glycoprotein ligand (PSGL)-1 is a homodimeric mucin-like 220–240-kDa glycoprotein that consists of an extracellular, transmembrane, and cytoplasmatic domain 61. It is expressed on all leukocytes and is mainly located in lipid rafts on the top of microvilli 1. PSGL-1 can bind L- 88, P- 66, and E-selectin 105. The post-translational modifications of PSGL-1 are important for optimal selectin-binding capacity. PSGL-1 requires α2,3-sialylated and α1,3-fucosylated core2 O-glycans to bind P-selectin 61. Core2 N-acetylglucosaminyl transferase-I is required to increase the binding affinity of PSGL-1 to P- and L-selectin 21, whereas the sulfation of tyrosine residues near the N-terminus optimizes the binding of PSGL-1 to P-selectin 61. E-selectin binding to PSGL-1 requires sialylated and fucosylated O-glycans but not tyrosine sulfation 61. The manipulation of the core-type protein glycosylation of PSGL-1 by eliminating the polypeptide N-acetylgalactosamine transferase-1 reduces the binding capacity of PSGL-1 to P- and E-selectin in vitro and in vivo under flow 96. Due to the differences in the amino-acid sequence of the extracellular domain and glycosylation pattern of mouse and human PSGL-1 61106, the binding affinities and, consequently, the signaling characteristics of the two molecules might be different.
The conserved cytoplasmic tail of PSGL-1 comprises 63 amino acids and interacts with cytoskeletal proteins 61. Proteins of the ERM (ezrin-moesin-radixin) family link the juxtamembrane region of the cytoplasmatic tail of PSGL-1 with the cytoskeleton in the uropod of migrating cells 497. Further, the interaction between the proteins of the ERM-family with the cytoplasmatic tail of PSGL-1 is important for the formation of protrusive membrane structures 12. In addition to the interaction with ERM proteins, a juxtamembrane region of 18 amino acids forms a constitutive complex with Nef-associated factor 1 (Naf1), which is involved in P-selectin-induced signaling through PSGL-1 100. A recent study has identified a new molecule interacting with the cytoplasmatic tail of PSGL-1 79. The selectin ligand interactor, cytoplasmic-1 (SLIC-1; human ortholog of the mouse sorting nexin 20), binds phosphoinositides and targets PSGL-1 to endosomes, but does not participate in PSGL-1-induced signaling or leukocyte recruitment 79.
The E-selectin ligand, ESL-1, is a 150-kDa glycoprotein, which is localized in the Golgi apparatus and on the cell surface of leukocytes 91. In contrast to PSGL-1 and L-selectin, ESL-1 is not located on the tips of microvilli 91. ESL-1, which can bind E-selectin in vitro and in vivo, is carrier of the HECA452 carbohydrate epitope, and sialic acid and fucose are required for achieving E-selectin binding capacity 923555.
In addition to PSGL-1 and ESL-1, neutrophils express other E-selectin ligands, including CD44 3542, macrophage antigen (Mac)-1 (αMβ2) 17112, and other unknown and poorly characterized ligands 743. Further, L-selectin from human, but not from mouse, neutrophils is able to bind E-selectin 11473. Sialic acid on L-selectin is necessary for the binding to E-selectin 114.
Signaling Events and Consequences In Vitro
Several lines of evidence show that neutrophil binding to P-selectin in vitro leads to the activation of neutrophils. Isolated human neutrophils stimulated with paraformaldehyde-fixed platelets, P-selectin-IgG fusion protein, or antibody against PSGL-1 show enhanced tyrosine phosphorylation 23. Stimulation of murine bone-marrow-derived neutrophils with P-selectin-IgG or cross-linking PSGL-1 with complete antibodies or F(ab')2 fragments leads to an increased production of reactive oxygen intermediates 11 and Mac-1 activation, which in turn, leads to increased binding of Mac-1 to ligands 1. A recent study dissected the proximal signaling pathway following P-selectin engagement. In vivo and in vitro data demonstrated that dimeric, but not monomeric, purified soluble mouse P-selectin and recombinant mouse P-selectin receptor-Ig fusion protein, which included the lectin domain, the epidermal growth factor domain, and the first four and part of the fifth complement-like repeat domains of mP-selectin fused with the heavy chain of mouse immunoglobulin G, induce integrin activation on leukocytes with a subsequent increase of leukocyte adhesion to fibrinogen and ICAM-1 100. These findings suggest that PSGL-1, like growth-factor receptors 6, requires dimerization. It is unknown whether dimeric P-selectin binds to two P-selectin binding sites in the same PSGL-1 dimer, or whether it induces clustering of adjacent PSGL-1 dimers. In response to PSGL-1 engagement, Src family kinases are activated, which in turn, phosphorylate Naf1 following the stimulation of isolated human neutrophils or 293 cells cotransfected with PSGL-1 and Naf1 with mP-selectin-Ig 100. This is necessary to recruit and activate the phosphoinositide-3-OH kinase p85-p110δ heterodimer and, subsequently, induce downstream signaling 100.
Engagement of L-selectin can also lead to the activation of neutrophils. Early studies demonstrated that L-selectin engagement by antibodies or ligand mimetics increase intracellular calcium levels, induces tyrosine phosphorylation, superoxide production, and production of Interleukin (IL)-8 and tumor necrosis factor alpha (TNF)-α 5398. Although cross-linking of L-selectin by antibody leads to increased Mac-1 adhesiveness 3, L-selectin-dependent rolling of isolated human neutrophils on peripheral-node addressin and ICAM-1 in a parallel-plate flow chamber at a shear stress of 1.8 dyn/cm2 is not sufficient to induce neutrophil arrest under flow 51. This apparent discrepancy suggests that L-selectin cross-linking may be necessary, but not sufficient for signaling. Most of the studies showing neutrophil activation used intact monoclonal antibodies to L-selectin. These antibodies may also engage and activate Fc receptors and induce neutrophil activation through a combination of L-selectin and Fc-receptor-mediated signaling. Some studies used F(ab')2 fragments of L-selectin antibodies and cross-linked them by secondary F(ab')2 fragments 33 in order to stimulate neutrophils. This approach excluded Fc-receptor engagement, but still induced protein tyrosine phosphorylation 99.
In vitro stimulation of human neutrophils with soluble recombinant human E-selectin, lacking the transmembrane and cytoplasmic domains and the last two consensus repeats, for 15 minutes induces an increased β2-mediated adhesion (76), tyrosine phosphorylation-dependent superoxide release 77, and polarization without affecting whole-cell deformability, as measured by filter assay 76. Although soluble E-selectin did not induce calcium mobilization in isolated human neutrophils by itself in vitro, the elevation of intracellular calcium concentration lasted longer in the presence of E-selectin following chemokine stimulation 7762. This effect is mediated by Src-kinase- and PI(3)K-dependent activation of store operated calcium entry 62. Further, in vitro data with isolated human neutrophils and E-selectin transfected 300.19 cells show that the formation of heterotypic aggregates, p38 MAPK phosphorylation, and surface upregulation of integrins are shear stress dependent 34. E-selectin engagement under shear-stress conditions induces calcium influx in human neutrophils 80. However, these studies do not address the question of which E-selectin ligand is responsible for the observed effects. E-selectin engagement under shear-stress conditions also induces the redistribution and clustering of L-selectin and PSGL-1 to the trailing edge of human neutrophils 78. In vivo, CD44 was found to be required for the redistribution of the adhesion molecules in a p38-dependent manner 35 (see Table 1). The redistribution and clustering of these adhesion molecules may provide an additional platform for capturing circulating leukocytes, which in turn, enhances leukocyte recruitment through cell-cell-interactions 88.
Table 1. Known signaling pathways during leukocyte recruitment
Using a new autoperfused flow chamber system 15, which allows the investigation of neutrophils in whole blood on different substrates, demonstrated that E-selectin engagement activates LFA-1 and induces an intermediate affinity state of LFA-1, which transiently binds to ICAM-1 and reduces the rolling velocity on E-selectin and ICAM-1 without inducing arrest 109 (Figure 1). This E-selectin signaling pathway is PSGL-1 and Syk-dependent 109 (see Table 1).
Figure 1. Two modes of neutrophil arrest: slow rolling versus immediate arrest. A. Neutrophils rolling on E-selectin pick up activating signals through P-selectin glycoprotein ligand (PSGL-1) and spleen tyrosine kinase (Syk), resulting in the partial activation of lymphocyte function antigen-1 (LFA-1) to the extended conformation with closed headpiece, indicated by the pink color. As soon as a neutrophil rolling on E-selectin encounters a surface with E-selectin and intercellular adhesion molecule-1 (ICAM-1) (orange), the rolling velocity immediately decreases because LFA-1 now engages ICAM-1. This is schematically represented in the velocity trace on top. Even a small amount of a CXCR2 ligand, such as immobilized CXCL1 (red), leads to arrest. B. Neutrophils rolling on P-selectin show little evidence of LFA-1 activation. Their rolling velocity changes little when ICAM-1 becomes available. A low dose of CXCL1 coimmobilized with P-selectin and ICAM-1 (P+I) cannot induce arrest, but a high dose can (red).
Download figure to PowerPoint
In contrast to the autoperfused flow chamber system, where rolling is observed at 6.0 dyn/cm2 and more, flow chamber experiments with isolated human neutrophils on E-selectin (site density of up to 885 sites/μm2) do not show neutrophil-substrate interactions at shear stresses above 3.6 dyn/cm252. Neutrophils in whole blood interact with E-selectin in vivo under higher wall shear stress conditions 48109, but additional molecules may contribute. Further, parallel-plate flow chambers with isolated human neutrophils show that neutrophils, in the absence of chemoattractants, adhere to L cells coexpressing E-selectin and ICAM-1 84. This may be caused by the isolation procedure known to activate neutrophils and induce increased expression of Mac-1 and decreased surface expression of L-selectin 302447. The differences seen in studies using whole blood on recombinant proteins, compared with studies using isolated neutrophils on transfected L cells, may also be due to the expression of other adhesion molecules and/or cytokines by L cells. In addition, species differences between human and mouse neutrophils may explain some of the differences.
Signaling Events and Consequences During Neutrophil Rolling
In the absence of additional stimuli, such as other selectins, cytokines, and chemokines, P-selectin binding can prime neutrophils, but not fully activate integrins and induce arrest. These data are supported by in vivo experiments in uninflamed dermal microvessels 103 and flow chamber experiments that showed that the rolling velocity of neutrophils in whole blood is reduced on P-selectin and ICAM-1, compared to P-selectin alone 10915. However, in the presence of other proinflammatory stimuli, P-selectin acts synergistically and contributes to full integrin activation 100. Elimination or blocking of P-selectin by gene targeting or antibody reduces neutrophil recruitment into the peritoneal cavity following thioglycollate injection. However, whether this reduction of neutrophil recruitment is only caused by a reduction of capturing or by decreasing the signaling input remains to be elucidated.
The activation of neutrophils by L-selectin engagement is also important in vivo. Blocking L-selectin shedding by a hydroxamic acid-based protease inhibitor increases L-selectin expression on the surface of neutrophils and augments signal input through L-selectin 33. These changes were associated with a reduced rolling velocity 32, increased the “smoothness” of rolling, enhanced arrest, and transmigration 33 (see Table 1). Interstingly, this influence of L-selectin shedding on rolling velocity is cell specific, since increased L-selectin surface expression on T-lymphocytes does not influence the rolling velocity 27. However, subphysiological L-selectin levels on T-lymphocytes increased the rolling velocity in vitro and in vivo as well as reduced homing to lymph nodes 27. These data suggest that there is a threshold density of L-selectin on T-lymphocytes that is required for optimal homing to peripheral lymph nodes. Elimination of ADAM17, which is involved in activated L-selectin shedding, by gene targeting increases the presence of L-selectin on the surface of neutrophils and enhances neutrophil rolling, arrest, and recruitment in a peritonitis model (K. Ley, E. Raines, J. Tang, A. Zarbock, unpublished observation). These data suggest that L-selectin has an important signaling role in neutrophil activation and recruitment. This may partially explain the substantial neutrophil recruitment defect seen in L-selectin-deficient mice 95, which is more severe than would be expected from the small contribution of L-selectin to neutrophil rolling.
Slow rolling in vivo can be induced by the injection of TNF-α and requires E-selectin and the engagement of β2 integrins 4840, as blocking both integrins involved in slow rolling, Mac-1 and LFA-1, increases leukocyte rolling velocity 20. In contrast to the requirement of PSGL-1 for E-selectin-mediated slow rolling in the autoperfused flow chamber system, Hidalgo and colleagues showed that the rolling velocity on P- and E-selectin under inflammatory conditions in vivo is also dependent on CD44 35.