Correlating the kinetics of cytokine-induced E-selectin adhesion and expression on endothelial cells

Many human diseases are mediated through the immune system. In chronic infl ammatory disorders, the processes ordinarily involved in tissue healing bec ome destructive. Endothelial cells normally recruit leukocytes to inflamed tissue using cytokine-induced adhesion receptors on the surfaces of interac ting cells. Leukocyte capture depends on specialized characteristics of the se receptors, particularly the binding kinetics. This study is designed to clarify the relationship between cytokine-induced changes in cell propertie s and binding kinetics. Here, we measure the kinetics of expression and mon oclonal antibody binding for E-selectin in interleukin-1 alpha -stimulated microvascular endothelium in vitro and incorporate the data into kinetic mo dels. Quantitative flow cytometry is used to determine molecular density (e xpression), and micropipette assays are used to find the probability of adh esion (function). Within five hours of interleukin-1 alpha stimulation, E-s electin density increases from 0 to 742 sites/mum(2), and antibody-E-select in adhesion probability increases from a baseline of 6.3% to 64%. A kinetic model is applied to find an apparent association rate constant, k(f), of 3 .7 x 10(-14) cm(2)/sec for antibody-E-selectin binding. Although the model successfully predicts experimental results, the rate constant is undervalue d for a diffusion-limited process, suggesting that functional adhesion may be modified through cytokine-induced changes in microtopology and receptor localization.