During the inflammatory response, granulocytes roll on, adhere to, and transverse the endothelial layer that lines blood vessels to exit into tissue and release inflammatory mediators. Among other mechanisms, granulocytes are known to roll via selectins and bind firmly via activated integrins. While it was once thought these two steps were distinct, there is now evidence of overlapping roles of selectins and integrins in the cascade from rolling to firm adhesion. Using several biophysical tools, we aim to elucidate the mechanics and the kinetics of the transition from rolling to firm adhesion. We hypothesize that selectins play two roles in mediating the transition to firm adhesion: a direct activation of integrins through signal transduction, and the dynamic facilitation of firm adhesion by controlling the dynamics of rolling which aids in integrin binding. In the first aim, using beads that we can reconstitute with both selectin ligands and intergins, and which do not support signaling, we will demonstrate quantitatively the selectins' role of dynamic facilitation of firm adhesion by adhering these beads to molecularly pure surfaces and activated endothelial cell layers in flow chambers. A variety of different chemistries will be employed to elucidate the precise coupling between selectin identity and density, integrin binding, and firm adhesion. In the second aim, we will study the ability of human neutrophils to roll and bind firmly on surfaces containing integrins and selectins. Neutrophils will roll on selectin ligands of different densities for distinct periods of time before reaching surfaces coated with selectin and the intregrin ligand ICAM-1. The probabily of neutrophil arrest, the dynamics of rolling, and the internal calcium activation of these rolling neutrophils will be measured. Comparison of the rolling dynamics for neutrophils to bead systems will clearly identify the role of internal signal generation in the transition from rolling to firm adhesion. We will verify the role of intracellular signaling enzymes in supporting firm adhesion transition using pharmacological inhibitors of kinases.
In aim 3, we will use computational models of the transition from rolling to firm adhesion, using a new version of adhesive dynamics that includes interfacial deformation, signal transduction networks, and the switching of integrin adhesion molecules from passive to active conformations. The model will allow us to verify mechanisms of the transition from rolling to firm adhesion, and make predictions about the likelihood of granulocyte firm arrest.
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