In this project we focus on the physical and chemical mechanisms that determine the kinetics of neutrophil-endothelial adhesion. Our goal is to understand the role that specific physical characteristics of the adhesive interface have on adhesion. In particular, the deformability of the cell, the microtopography of the cell membrane, the distribution and mobility of receptors, and changes in adhesion molecule affinity will be assessed in relation to the formation of adhesive contacts between neutrophils and endothelium. Micromechanical manipulation of single cells into contact with artificial substrates with well-defined adhesion molecule presentation provides unparalleled ability to control both the chemistry and the mechanical forces in relation to adhesive interactions. This approach, combined with newly implemented fluorescence imaging methods enables us to determine the specific role that cellular mechanics, surface chemistry, and membrane topology play in the formation of adhesive contacts. Once an understanding of the importance of these different mechanisms is reached, this knowledge will be used as a basis for understanding the mechanisms by which different signaling mechanisms work to effect changes in adhesive behavior. Specifically we will examine the effect of selectin ligation, anion transport inhibition, and exposure of cells to chemokines of the CXC family on cellular deformability, surface topography and compliance, and the distribution and mobility of adhesive ligands in relation to the kinetics of neutrophil adhesion. Finally, we will replace artificial substrates presenting endothelial cell adhesion molecules with cultured endothelial cells to identify additional modulating effects of the endothelium. Lateral mobility and distribution of adhesion receptors, cytoskeletal microrheology, and cellular deformability in the vicinity of neutrophil contact will be measured to assess the influence of physical properties of the endothelium on leukocyte adhesion and migration. These studies will result in a clear understanding of the mechanisms of neutrophil adhesion to endothelium and its regulation, and should serve as a basis for developing novel and improved strategies for clinical therapy.
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