Cell-Free Rolling Mediated by L-Selectin and Sialyl Lewisx Reveals the Shear Threshold Effect

Abstract

The selectin family of adhesion molecules mediates attachment and rolling of neutrophils to stimulated endothelial cells. This step of the inflammatory response is a prerequisite to firm attachment and extravasation. We have reported that microspheres coated with sialyl Lewis(x) (sLe(x)) interact specifically and roll over E-selectin and P-selectin substrates (Brunk et al., 1996; Rodgers et al 2000). This paper extends the use of the cell-free system to the study of the interactions between L-selectin and sLe(x) under flow. We find that sLe(x) microspheres specifically interact with and roll on L-selectin substrates. Rolling velocity increases with wall shear stress and decreases with increasing L-selectin density. Rolling velocities are fast, between 25 and 225 microm/s, typical of L-selectin interactions. The variability of rolling velocity, quantified by the variance in rolling velocity, scales linearly with rolling velocity. Rolling flux varies with both wall shear stress and L-selectin site density. At a density of L-selectin of 800 sites/microm(2), the rolling flux of sLe(x) coated microspheres goes through a clear maximum with respect to shear stress at 0.7 dyne/cm(2). This behavior, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, is known as the shear threshold effect. We found that the magnitude of the effect is greatest at an L-selectin density of 800 sites/microm(2) and gradually diminishes with increasing L-selectin site density. Our study is the first to reveal the shear threshold effect with a cell free system and the first to show the dependence of the shear threshold effect on L-selectin site density in a reconstituted system. Our ability to recreate the shear threshold effect in a cell-free system strongly suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and largely eliminates cellular features such as deformability or topography as its cause.

Dynamic states of cells adhering in shear flow: From slipping to rolling

“…In addition to specific properties of the molecular bonds, transport processes might also play an important role in creating the shear threshold [10,11]. Both rolling adhesion [12] and the shear threshold [13] have also been demonstrated in cell-free flow chamber experiments with ligand-coated micron-sized beads. Although cell-free rolling is more erratic than leukocyte rolling, this indicates that cellular features (like cell deformability) are not essential to rolling adhesion.…”

L-selectin-mediated leukocyte tethering in shear flow is controlled by multiple contacts and cytoskeletal anchorage facilitating fast rebinding events

“…Its properties are optimized for initial capture and rolling under physiological shear (2, 3), as confirmed by recent experimental data and computer simulations (4, 5). In contrast to tethering through other receptor systems like P-selectin, E-selectin, or integrins, appreciable tethering through L-selectin and subsequent rolling occurs only above a threshold in shear (6), even in cell-free systems (7,8). Down-regulation by low shear is unique for L-selectin tethers and might be necessary because L-selectin ligands are constitutively expressed on circulating leukocytes, platelets, and subsets of blood vessels (9).…”

Science and Engineering Programs at The University of Pennsylvania

“…Our major achievement was the development of a colloidal mimetic, bearing selectins or selectins hgands, which can recreate the adhesion of leukocytes. These "cell-free"systems, in which we can recreate rolling adhesion with any of the molecules of the selectin family, demonstrate that rolling is a function of the physical chemistry of selectin adhesion molecules, and not due to cellular features such as roughness, deformability and signaling [1][2][3][4]. A particular accomplishment with the cell free system are that we can recreate the shear threshold effect -a phenomenon seen in cells and previously attributable to cell features such as microvillitherefore, the shear threshold effect is really due to the physical chemistry of adhesion molecules [4,5].…”

“…These "cell-free"systems, in which we can recreate rolling adhesion with any of the molecules of the selectin family, demonstrate that rolling is a function of the physical chemistry of selectin adhesion molecules, and not due to cellular features such as roughness, deformability and signaling [1][2][3][4]. A particular accomplishment with the cell free system are that we can recreate the shear threshold effect -a phenomenon seen in cells and previously attributable to cell features such as microvillitherefore, the shear threshold effect is really due to the physical chemistry of adhesion molecules [4,5]. We further extended the construction of the "cell-free" systems to biodegradable microspheres that could be used for drug delivery or carrying imaging agents, most recently derivatizing poly-lactic glycolic acid microspheres to have the adhesive properties of leukocytes, but filling them with inflammatory agents [6].…”