The overall objective of this project is to determine the mechanisms by which regions of individual human polymorphonuclear neutrophils (PMN) respond to a gradient of mediators of inflammation and to study how these regional responses bring about polar shape change and directed movement. Motile PMN exhibit rhythmic extension of pseudopods, assume an elongated shape and seem to glide over the surface powered by barely perceptible cytoplasmic undulations. The rhythmic nature of this process suggests that the biochemical reactions which synchronize cell shape change might also be rhythmic and that their spatial and temporal frequency may ultimately direct net motion. We developed a computerized video image analysis system which records the ion concentration at each point within single moving PMN labelled with a fluorescent Ca++, Na+, or pH-sensitive probe as well as the cell position every 4 seconds. We represent the distribution of fluorescence intensity, which measures intracellular cation distribution, as a vector calculated in a coordinate system which moves with the PMN. The orientation of the intracellular Ca++ vector predicts subsequent PMN velocity. The magnitude of this vector measures asymmetric distribution of [Ca]/i, oscillates at a rate independent of transient elevations of total [Ca]/i and is synchronized with high frequency cytoplasmic waves. Similarly, we have detected waves of [Na]/i than move from front to back during PMN motion. We will determine the mechanism of pseudopod extension and development of morphologic polarity in single human PMN on various biologic substrates. We will induce pseudopod extension by local application of chemotactic peptide (fmlp) and determine if the magnitude of directly measured intracellular gradients of Ca++/Na+/pH/F-actin or the frequency/amplitude of oscillations in these ions/proteins predicts the rate/direction of pseudopod extension. We will corroborate these findings in a model of shape change and f-actin oscillation in suspended PMN. We will determine if uniform concentrations of fmlp increase PMN chemokinetic activity by increasing gradients of cytsolic Ca++, Na+, or Ph or by increasing the frequency and/or amplitude of oscillations/waves of these ions. We will quantify the spatial distribution of cytosolic [Ca++], [Na+], pH in single PMN moving in a gradient of chemo-attractant and determine if the gradient of stimulus changes persistence of motion (probability of going in a straight line) or velocity by inducing changes in internal cation distribution or the frequency/amplitude of intracellular cation/mechanical waves.
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