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

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.