Mechanical shedding of L‐selectin on neutrophils occurs during rolling in vitro

L‐selectin is constitutively present on the surface of leukocytes and is important in initial capture and rolling of leukocytes on the endothelium, facilitating their migration into inflamed sites or lymph nodes. It is known that leukocyte activation by chemoattractants, hypotonicity or LPS results in rapid shedding of L‐selectin. Here we present a novel mechanism for surface cleavage of L‐selectin on neutrophils during rolling on a sialyl Lewis x (sLe x )‐coated surface that does not require extracellular activation. It was verified using flow cytometry and rolling of neutrophils labeled with Qdot ® ‐L‐selectin Abs that the mechanical shedding of L‐selectin occurs during rolling and depends on the shear applied. We found that shed L‐selectin exists both on the sLe x ‐surface and in the solution using immuno‐fluorescence microscopy and ELISA, respectively. In addition, we found that mechanical shedding is mediated by p38 MAPk activation. To quantify the effect of mechanical L‐selectin shedding on rolling dynamics of neutrophils in vitro , we tracked individual rolling cells for several millimeters within a flow chamber. We found that cell rolling velocity increased with time, suggesting a gradual loss of L‐selectin. Inhibition of L‐selectin shedding using a metalloprotease inhibitor significantly increased the number of rolling cells with no increase of rolling velocity during rolling. Thus, we conclude that mechanical force facilitates the cleavage of L‐selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand‐presenting surface in flow. This mechanical L‐selectin shedding is shear‐ and metalloprotease‐dependent and is controlled by the p38 MAPk pathway. This work was funded by NIH ( HL018208 ).