Quantifying rolling adhesion with a cell-free assay: E-selectin and its carbohydrate ligands

Abstract

Rolling of neutrophils over stimulated endothelial cells is a prerequisite to firm attachment and subsequent transendothelial migration during the inflammatory response. The selectin family of adhesion molecules are thought to mediate rolling by binding counter-receptors that present carbohydrates, such as sialyl Lewis(x) (sLe[x]). Recently we described a cell-free system for rolling using sLe(x)-coated microspheres and E-selectin molecules on inert substrates. We showed that sLe(x)-coated microspheres rolled over E-selectin-IgG chimera substrates with dynamics that are similar to those of leukocytes rolling over stimulated endothelium. In this paper we provide a thorough quantitative description of the dynamics of adhesion for this system. We find that particle rolling velocity increases with increasing wall shear stress and decreases with increasing E-selectin or sLe(x) surface densities. Large changes in the average rolling velocity can occur with small changes in sLe(x) or E-selectin density; however, rolling velocity is more sensitive to E-selectin surface coverage than to the number of sLe(x) molecules on the microspheres. Aided by dimensional analysis, we show that decreasing the wall shear stress or increasing either receptor (E-selectin) or ligand (sLe[x]) surface coverage results in an equivalent decrease in particle rolling velocity. In addition, we find that different Lewis carbohydrates are more effective in mediating rolling on E-selectin, with effectiveness following the trend sialyl Lewis(a) > sialyl Lewis(x) >> sulfated Lewis(x) >> Lewis(x). Rolling velocity fluctuated with time for all carbohydrate-selectin pairs tested, and the magnitude of the velocity fluctuations was linearly proportional to the mean rolling velocity for all combinations of E-selectin site density, sLe(x) site density, wall shear stress, and carbohydrate chemistry tested.

L- and P-Selectins Collaborate to Support Leukocyte Rolling in Vivo When High-Affinity P-Selectin-P-Selectin Glycoprotein Ligand-1 Interaction Is Inhibited

“…One strategy for studying the contributions of individual components of that surface is to coat artificial microspheres with purified or recombinant versions of molecules of interest. We and others have used this strategy in the past to investigate the capacity of PSGL-1 and other adhesion molecules to mediate rolling in vitro [27][28][29][30] and in vivo. 20,22,31 Ideally, the properties (size, ligand density, deformabilty, and so forth) of microspheres used for such investigations will closely match those of leukocytes, allowing direct comparisons to be made.…”

Catch Strip Assay for the Relative Assessment of Two-Dimensional Protein Association Kinetics

“…It has been shown that the rolling velocity of microparticles is a sensitive reporter of relative site density, 15 so analyzing average rolling velocities mediated by the P-rIgG/PSGL-1 pair enabled us to investigate local variations in adsorbed protein density.…”

Distinct kinetic and mechanical properties govern selectin-leukocyte interactions

“…This does suggest, however, that E-selectin/PSGL-1 binding in vivo mediates the initial tethering and rolling of leukocytes before other ligands become involved. It is also noteworthy that in vitro experiments involving completely reconstituted cell-free systems consisting of sialyl-Lewis x -coated microspheres rolling on Pselectin (Rodgers et al, 2000) or E-selectin (Brunk and Hammer, 1997) substrates also found that E-selectin-mediated rolling is slower. Taken together, whereas several diverse mechanisms might explain the subtly different rolling characteristics of PMNs on E-and P-selectin, the distinct kinetic and mechanical properties of participating adhesive groups are likely to be crucial determinants of this phenomenon.…”