L-SELECTIN SHEDDING DOES NOT REGULATE HUMAN NEUTROPHIL ATTACHMENT, ROLLING, OR TRANSMIGRATION ACROSS HUMAN VASCULAR ENDOTHELIUM IN-VITRO

Current models of the multistep adhesion cascade for leukocyte-endothe lial interactions predict loss of L-selectin from the leukocyte surfac e before transendothelial migration. We have tested this hypothesis us ing in vitro adhesion and transendothelial migration assays and a zinc -dependent metalloproteinase inhibitor, Ro yl)-4-methylvaleryl]-N-1,3- dimethyl-L-valinamide), which prevents chemoattractant-induced (e.g., IL-8, FMLP, C5a, platelet-activating factor) L-selectin endoproteolyti c cleavage from isolated human neutrophils. Inhibitor and vehicle-trea ted neutrophils exhibited identical behavior during both adhesive inte ractions with 4- and 24-h TNF-alpha-activated HUVEC monolayers under f low, (including rate of initial attachment, rolling velocities, stable adhesion, and transmigration) and in static adhesion assays, Flow cyt ometric analysis of transmigrated neutrophils with mAb to L-selectin r evealed that vehicle treated neutrophils had minimal detectable surfac e L-selectin, whereas inhibitor-treated neutrophils retained comparabl e levels of L-selectin on their surface as neutrophils maintained at 3 7 degrees C, In addition, mAb to L-selectin that induce rapid shape ch ange and homotypic adhesion (LAM1-116) did not enhance the rate or ext ent of neutrophil transmigration under flow or static conditions, Neut rophils preincubated with LAM1-116 displayed similar behavior to neutr ophils preincubated with the control anti-L-selectin mAb, LAM1-101. In summary, these results demonstrate that there is no requirement for L -selectin to be shed from the surface of neutrophils before, or during , their migration across endothelial monolayers, and that prevention o f surface L-selectin proteolytic cleavage does not enhance or inhibit neutrophil-endothelial cell adhesive interactions.