The overall objective of this project is to determine the mechanisms by which regions of individual human polymorphonuclear neutrophils (PMN) are triggered by a gradient of mediators of inflammation and to study the factors which modulate this response. PMN respond to a gradient of attractant and transform this information into signals which determine the magnitude and direction of cellular response. Spacial separation of these signals must develop across the surface of the cell and between surface (cortical) and internal (subcortical) regions of the cell. The stimulus induced changes in plasma membrane responses such as transmembrane potential or right angle scatter will be related to subcortical responses such as the plasma membrane component of continuously monitored fluorescent signals that reflect membrane potential or right angle scatter will be related to subcortical responses such as secretion or release of internal calcium. This will be accomplished by determining the plasma membrane component of continuously monitored fluorescent signals that reflect membrane potential, membrane or cytosolic calcium and secretion. The effect of biologic substrates on this response will be assessed by performing these measurements on surfaces coated with fibronectin, collagen, laminin or extracellular matrix. Heterogeneity of PMN interaction with biologic substrates will be studied by relating the rate of spreading and morphological polarization of PMN attached to these substrates to membrane potential, cytosolic calcium, and membrane viscosity measured in single PMN. These parameters will be measured by quantitative video intensification microscopy (QVIM) The distribution of membrane potential, membrane bound calcium, and cytosolic calcium will be measured in single PMN as a function of time by QVIM and computerized image analysis. These parameters will be determined as vectors in a coordinate system centered at the nucleus or the centroid of the cell and changes in their magnitude and direction will be related to subsequent movement and morphological polarization. Changes in membrane viscosity which occur during secretion will be related to changes in PMN activation and functional heterogeneity using novel fluorescent probes of surface membrane viscosity and lipid lateral mobility. Results in vitro will be corroborated by study of PMN from patients with known abnormality in cell membrane viscosity.
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