Correlating between expression and functional adhesion: A quantitative study of adhesion receptor kinetics on cytokine-stimulated endothelial cells

Many human diseases are mediated through the immune system, including ischemia/reperfusion injury and rheumatoid arthritis. In these chronic Inflammatory disorders, processes ordinarily involved in tissue healing can become destructive. Microvascular endothelial cells (MVEQ normally recruit leukocytes to inflamed tissue through a highly regulated process orchestrated by distinct combinations of intercellular signaling and adhesion receptor binding. A therapeutic strategy with tremendous potential is targeted immune suppression. This includes experimental treatments used to inhibit adhesion receptors using antibodies, soluble ligands or blocking peptides to prevent the activation or progression of damaging immune cells. This investigation is designed to use quantitative measurements to develop a better understanding of the fundamental relationship between cytokine-induced changes in cell properties and the kinetics of adhesion receptor binding.; MVEC are stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha (TNF-alpha) while expression and binding kinetics are characterized for two cytokine induced adhesion receptors: (1) E-selectin, which promotes the initial rolling of leukocytes along cytokine-stimulated endothelium, and (2) intercellular cell adhesion molecule 1 (ICAM-1), which mediates firm adhesion between cells prior to transendothelial migration. Micropipette assays of endothelial receptor adhesion are combined with flow cytometry measurements of receptor and ligand density, and the results are integrated into kinetic models of adhesion to extract effective kinetic rates for binding.; IL-1alpha or TNF-alpha activation of MVEC results in increased adhesion of E-selectin and ICAM-1, respectively. Both E-selectin and ICAM-1 bind to mAb or ligand and have forward rate constants, kf, on the order of 10--17 --- 10--14 cm2/sec, typical of a diffusion limited process but several orders of magnitude lower than predicted. This suggests that other factors may be critical to binding efficiency, such as cytokine induced changes in receptor localization and membrane topology. ICAM-1 binding is probed using purified Mac-1, a natural ligand for ICAM-1. Adhesion rates increase with elevated ligand concentrations and prolonged contact duration. Kinetic models are applied to find an apparent kf of 3.2 x 10--17 cm2/sec and a reverse rate constant, 0.058 sec--1, 10-fold smaller than published values for E-selectin, and consistent with the prevailing theory of high affinity ICAM-1/beta 2-integrin binding.