Isolation technique alters eosinophil migration response to IL-8

Disparate reports exist on the eosinophil chemotactic capacity of interleukin-8 (IL-8). We hypothesized that the difference is due to the methods used to purify eosinophils. We therefore compared the eosinophilotactic capacity of IL-8 on human cells isolated by Percoll (positive selection) vs. magnetic cell separation system (MACS) (negative selection). Discontinuous Percoll gradients were preceded by dextran and Ficoll-Paque steps, and followed by gelatin wash and red blood cell (RBC) lysis. MACS isolation included: Percoll 1.090 g/ml layering and RBC lysis; incubation with CD16 antibody conjugated to magnetic beads (to bind neutrophils); and isolation of eluate from column positioned in magnet. Percoll isolated eosinophils migrated to IL-8 in a dose-responsive fashion. Although MACS isolation provided a greater number and higher purity of eosinophils, these eosinophils did not migrate to IL-8. Neither dextran sedimentation, Ficoll-Paque and Percoll prior to, nor Percoll discontinuous gradients subsequent to, MACS isolation reversed the negative chemotactic response. Moreover, Percoll-isolated eosinophils further purified with CD16 MicroBeads no longer chemotactically responded to IL-8. This inhibition was not due to change in eosinophil purity, a loss of eosinophil adhesion molecules or activation markers, the presence of a soluble neutrophil or eosinophil inhibitor or the effect of the magnet. Thus, the technique used to isolate eosinophils clearly affects the chemotactic responsiveness of this cell to IL-8. Since several in vivo studies suggest that IL-8 is an eosinophil chemoattractant, Percoll isolation of these cells might be more appropriate for studies involving eosinophil chemotactic responses to IL-8.