Correlating the Kinetics of Cytokine-Induced E-Selectin Adhesion and Expression on Endothelial Cells

AbstractMany human diseases are mediated through the immune system. In chronic inflammatory disorders, the processes ordinarily involved in tissue healing become destructive. Endothelial cells normally recruit leukocytes to inflamed tissue using cytokine-induced adhesion receptors on the surfaces of interacting cells. Leukocyte capture depends on specialized characteristics of these receptors, particularly the binding kinetics. This study is designed to clarify the relationship between cytokine-induced changes in cell properties and binding kinetics. Here, we measure the kinetics of expression and monoclonal antibody binding for E-selectin in interleukin-1α-stimulated microvascular endothelium in vitro and incorporate the data into kinetic models. Quantitative flow cytometry is used to determine molecular density (expression), and micropipette assays are used to find the probability of adhesion (function). Within five hours of interleukin-1α stimulation, E-selectin density increases from 0 to 742 sites/μm2, and antibody-E-selectin adhesion probability increases from a baseline of 6.3% to 64%. A kinetic model is applied to find an apparent association rate constant, kf, of 3.7×10−14cm2/sec for antibody-E-selectin binding. Although the model successfully predicts experimental results, the rate constant is undervalued for a diffusion-limited process, suggesting that functional adhesion may be modified through cytokine-induced changes in microtopology and receptor localization