Abstract
Leukocytes are recruited from the bloodstream to sites of inflammation by the selectin family of adhesion receptors. In vivo and in vitro studies reveal distinctive rolling velocities of polymorphonuclear leukocytes over E-, P- and L-selectin substrates. The kinetic and mechanical properties of the selectin-ligand bonds responsible for these differences at the single-molecule level are not well understood. Using single-molecule force spectroscopy, we probe in situ the rupture force, unstressed off-rate and reactive compliance of single selectin receptors to single ligands on whole human polymorphonuclear leukocytes (PMNs) under conditions that preserve the proper orientation and post-translational modifications of the selectin ligands. Single L-selectin bonds to PMNs were more labile than either E- or P-selectin in the presence of an applied force. This outcome, along with a higher unstressed off-rate and a higher reactive compliance, explain the faster L-selectin-mediated rolling. By quantifying binding frequency in the presence of a specific blocking monoclonal antibody or following enzyme treatment, we determined that P-selectin glycoprotein ligand-1 is a high-affinity ligand for E-selectin on PMNs under force. The rupture force spectra and corresponding unstressed off-rate and reactive compliance of selectin-ligand bonds provide mechanistic insights that might help to explain the variable rolling of leukocytes over different selectin substrates.
Highlights
Central to all cell-cell communications are the adhesive interactions between biological receptors and their respective ligands
We propose explanations based on the tensile strength still capable of tethering on E- but not P-selectin substrates (Goetz et al, 1997), supporting the notion that P-selectin glycoprotein ligand 1 (PSGL-1) has binding sites for E-selectin other than the crucial 19 amino acid of single selectin-ligand bonds, the intrinsic kinetics of bond dissociation and the reactive compliance, which is a measure of the susceptibility for bond rupture under applied sequence recognized by KPL-1
polymorphonuclear leukocytes (PMNs) cell suspensions at 5×106 cells ml–1 were incubated with 120 μg ml–1 of O-sialoglycoprotein endopeptidase (OSGE; Accurate Chemical & Scientific, Westbury, NY) to cleave proteins that are Oglycosylated on serine and threonine residues (Sutherland et al, 1992)
Summary
Central to all cell-cell communications are the adhesive interactions between biological receptors and their respective ligands. The kinetics regulating receptor-ligand binding impart unique properties that allow specialized cells to interact with one other amidst the challenge of physiological and competitive stresses This phenomenon is perhaps best represented by the remarkable ability of the selectins (E-, Pand L-selectin) to mediate the initial adhesion events during leukocyte recruitment to sites of inflammation. The distinctiveness of selectin binding is attributed to their fast association/dissociation rates, as well as their ability to form high-strength tethers under rapid loading These properties allow polymorphonuclear leukocytes (PMNs) first to tether and roll on activated vascular endothelium under hydrodynamic shear, before firm adhesion and extravasation into the tissue space (Konstantopoulos et al, 1998; McEver, 2002; Springer, 1995). L-selectin, by contrast, is constitutively expressed on most leukocyte subpopulations and is responsible for amplifying the inflammatory response through leukocyte-leukocyte interactions or so-called secondary tethers (Alon et al, 1996; Konstantopoulos et al, 1998)
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