In vitro determination of the force required for detachment of human polymorphonuclear granulocytes (PMN) from cultured endothelium and immobilized albumin

Abstract

Detachment of adherent human PMN from cultured endothelial cells (human umbilical vein, HUVEC) and from immobilized albumin (bovine serum albumin, BSA) was investigated as a function of shear stress. Non-activated PMN did not adhere to either of the substrates (<2%). 20 min after activation with PMA (phorbol myristate acetate, 10 nM), 48 ± 5% (mean ± SEM, n=15) of the total number of PMN added had adhered to albumin, and 18 ± 4% (n=9) to HUVEC. The dishes with pre-adhered PMN were subjected to shear stress in the range occurring in venules in vivo, using a rotating plate-and-cone (cone angle 2.5°) apparatus made of transparent plastic to allow observation of the cells during the assay. On HUVEC, the majority of adhered PMN (66 ± 3%, n=9) was removed by a shear stress of 0.12 Pa (1.2 dyn/cm²), while the same shear stress removed only 15 ± 5% (n=9) of the PMN adhered to BSA. Further increase of shear stress to 2.3 Pa (23 dyn/cm²) removed 39 ± 4% (n=14) of the PMN adhered to BSA. Using spherical and semispherical approximations for the shape of the adhered PMN, the adhesive forces between PMA-activated PMN and HUVEC were calculated to be on the order of 0.05 nN per PMN, while most PMN on BSA adhered with a force of more than 4 nN per PMN. The force needed to detach PMN from HUVEC is more than one order of magnitude lower than value reported for PMN adhesion to venular endothelium in vivo. The present results suggest that the endothelium may playa regulatory role in PMN adhesion and limit the attachment force to allow emigration.