Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

Abstract

During inflammation, E-selectin expressed on cytokine-activated endothelial cells mediates leukocyte rolling under flow. E-selectin undergoes endocytosis and may associate with lipid rafts. We asked whether distribution of E-selectin in membrane domains affects its functions. E-selectin was internalized in transfected CHO cells or cytokine-activated human umbilical vein endothelial cells (HUVECs). Confocal microscopy demonstrated colocalization of E-selectin with ␣-adaptin, a clathrin-associated protein.Deleting the cytoplasmic domain of Eselectin or disrupting clathrin-coated pits with hypertonic medium blocked internalization of E-selectin, reduced colocalization of E-selectin with ␣-adaptin, and inhibited E-selectin-mediated neutrophil rolling under flow. Unlike CHO cells, HUVECs expressed a small percentage of E-selectin in lipid rafts. Even fewer neutrophils rolled on E-selectin in HUVECs treated with hypertonic medium and with methyl-␤-cyclodextrin, which disrupts lipid rafts. These data demonstrate that E-selectin clusters in both clathrin-coated pits and lipid rafts of endothelial cells but is internalized in clathrin-coated pits. Distribution in both domains markedly enhances E-selectin's ability to mediate leukocyte rolling under flow. IntroductionRecruitment of leukocytes into secondary lymphoid organs or inflamed tissues requires that the cells first tether to and roll on vascular surfaces. Interactions of selectins with their glycoconjugate ligands mediate tethering and rolling, 1 which precedes integrindependent deceleration, arrest, and transmigration of leukocytes across the endothelial cell layer. 2 L-selectin, expressed on leukocytes, binds to ligands on other leukocytes and on endothelial cells in lymph nodes and in some regions of inflammation. P-and E-selectin, expressed on activated platelets or endothelial cells, bind to ligands on leukocytes. The leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) is the dominant ligand for P-and L-selectin and an important ligand for E-selectin. 3,4 The manner in which selectins or their ligands distribute on cell surfaces has major influence on rolling behavior. 5 For example, L-selectin and PSGL-1 are located on tips of microvilli, 6,7 which enhances the initial tethering of leukocytes. 8 The extended length of P-selectin helps capture flowing neutrophils and slow their rolling velocities, presumably by increasing encounters with PSGL-1. 9 The dimeric structures of P-selectin and PSGL-1 allow them to form dimeric bonds that prolong tether duration and strength. 10 Membrane anchorage of L-selectin and P-selectin through binding of their cytoplasmic domains to cytosolic proteins favors rolling. L-selectin anchors through interactions of its cytoplasmic domain with the cytoskeleton to effectively engage its ligands under flow. 11 P-selectin uses a different mechanism to cluster on the cell surface. Thrombin or histamine mobilizes P-selectin from the membranes of Weibel-Palade bodies to the plasma membranes of endothelial cells. 12,13 Sequences in t...

CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin

“…Similar findings have been reported for the transcriptionally up-regulated leukocyte receptor E-selectin. 47 This concentration of the selectins into patches presumably increases the number of bonds formed within a tether, lengthen the lifetime of the interaction and enhance rolling stability. How the counterintuitive demonstration of a reduction in clathrin-mediated internalization efficiency, (ie, an increase in surface residency) of P-selectin correlates with reduced leukocyte recruitment relates to our own data are unclear.…”

Novel Mechanism for FcϵRI-mediated Signal Transducer and Activator of Transcription 5 (STAT5) Tyrosine Phosphorylation and the Selective Influence of STAT5B over Mast Cell Cytokine Production

“…Field et al established that Fc⑀RI aggregates into lipid rafts upon ligation with antigen-IgE complexes (22). The cholesterol-depleting, chelating agent M␤CD has been utilized for the disruption of lipid rafts (23,24). BMMCs were sensitized to IgE (0.5 g/ml) for 18 h in their normal growth medium.…”

Association of FcγRIIa (CD32a) with Lipid Rafts Regulates Ligand Binding Activity

“…These regions of the plasma membrane, also termed detergent-resistant membranes (DRM) due to their characteristic insolubility following cold detergent extraction, act as docking sites for several intracellular signaling proteins. Association of a number of immunoreceptors with lipid rafts is necessary for receptor signaling and functional responses upon ligand binding (10), including AgR, such as BCR (11), TCR (12), adhesion molecules, like P-selectin glycoprotein ligand 1 (13), E-selectin (14), LFA-1 (15,16), chemokine and cytokine receptors, such as CXCR4 (17), IL-2R (18), as well as key signaling proteins, including Ras (19), Kit (20), and Lyn (21) kinase.…”