Induction of low density and up-regulation of CD11b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation

Abstract

Neutrophils and eosinophils circulating in an activated state are of low density. However, purification procedures such as dextran sedimentation and centrifugation may influence the density and function of cells. In the present study we have evaluated the effect of dextran sedimentation and subsequent centrifugation on the density and CD11b expression of neutrophils and eosinophils. Direct density separation of whole blood resulted in 17.7 ± 9.0% low density neutrophils (< 1.090 g/ml) and 8.7 ± 3.5% low density eosinophils (< 1.093 g/ml). Dextran sedimentation at room temperature prior to density separation yielded 57.8 ± 14.7% low density neutrophils and 43.2 ± 8.0% of low density eosinophils. Additional centrifugation after dextran sedimentation resulted in an increase of these numbers to 91.7 ± 3.1 and 69.8 ± 11.7% respectively. The density shifts were found with hypertonic as well as isotonic Percoll. Furthermore, it was shown that dextran sedimentation resulted in an increased CD11b expression on neutrophils as well as eosinophils. During subsequent washing by centrifugation, a further increase in CD11b expression was observed together with lactoferrin release. The effects of dextran sedimentation on density and CD11b expression were independent of extracellular calcium. These results indicate that dextran sedimentation induces the release of specific granule compartments with subsequent expression of CD11b, resulting in changes in granulocyte density.