Effects of density and of dehydration of sickle cells on their adhesion to cultured endothelial cells

Abstract

Abnormal adhesion of sickle cells to vascular endothelium may be a factor in the initiation of painful vaso-occlusive crisis. The sickle cell population contains an unusually large number of less dense reticulocytes that are known to be more adhesive than mature red cells, but there is contradictory evidence regarding the adhesiveness of dense sickle cells. We used a flow-based assay of adhesion to cultured human umbilical vein endothelial cells to test the properties of density fractions of sickle cells, prepared either by density gradient or by centrifugation of packed cells. We also examined the effects of incubating sickle cells with or without cyclical deoxygenation on their adhesion. After fractionation on a Percoll-isopaque gradient, the less dense 10% (reticulocyte-rich) cells and the most dense 10% cells adhered in greater number than the remainder (by about twofold). However, after centrifugation of packed cells, the less dense 10% were again more adhesive than the "middle" cells, but the most dense were not. Exposing sickle cells to constituents of the gradient had no consistent effect on adhesion, while centrifugal packing induced a degree of hemolysis, and tended to reduce adhesiveness of the dense fraction previously obtained from a gradient. Incubation in air at 37 degrees C for 15 hr reduced the number of reticulocytes and the adhesiveness of less dense sickle cells compared to those held at 4 degrees C. On the other hand, incubation at 37 degrees C for 15 hr with cyclical deoxygenation caused formation of dense cells and increased adhesiveness compared to incubation without cyclical deoxygenation. We conclude that young, less dense sickle cells are unusually adhesive, but that this adhesiveness is reduced during maturation. However, repeated sickling in vivo causes formation of an abnormally dense subpopulation of cells which either redevelop an increased tendency to adhere to endothelial cells or preserve their initial adhesiveness. Both adhesive cell populations may be implicated in promoting vascular obstruction.