Motility of human polymorphonuclear leukocytes : An image processing approach

Abstract: Cell motility is essential for polymorphonuclear leukocyte (PMN) function in the defense against invading microorganisms and in inflammatory processes. Using digital video microscopy and image processing, individual cells were studied during random locomotion, chemotactic locomotion and phagocytosis. When PMN moved in a specific direction, the lipid membrane was found to flow in the same direction, contradicting the rearward lipid flow model of cell locomotion. This was assessed with video photobleaching of a fluorescent lipid probe, Dii. The motility of PMN could not be blocked by ca2+ depletionand /or inhibition of myosin light chain kinase, suggesting that ea2+ and myosin are not necessary for locomotion. Temporal and spatial characteristics of intracellular free ea2+ and intracellular pH were assessed by loading the cells with esters of Fura-2 and the fluorcscein derivative BCECF, respectively. There was a distinct correlation between the direction of PMN locomotion and the slope of the Ca2+ gradient of the cells. PMN moving randomly on a surface often exhibited a rearward gradient with higher ca2+ concentration in the rear of the cell. Experimental reversal of the gradient, however, did not change the direction of motility. During phagocytosis of yeast particles, Saccharomyces cerevisiae, intracellular free calcium rose within seconds after contact with the prey. Intracellular pH varied between 7.1 and 7.3 and was uniform across the cells. After phagocytosis, phagosomal pH first decreased and then returned to neutraL A role for pH in phagosome-lysosome formation is proposed. PMN loaded with self-quenching concentrations of BCECF exhibited a strict correlation between pseudopod protrusions and increase in fluorescence, indicating water influx and dilution of the probe. This finding may be essential for the understanding of actin cytoskeleton dynamics during cell locomotion.