Eosinophils isolated by magnetic cell sorting respond poorly to lipid chemoattractants.

Abstract

Eosinophils play an important role in allergic inflammation. In vitro methods to isolate human eosinophils for the study of chemotactic responses are essential in understanding the mechanisms involved in tissue eosinophilia. We compared LTB4 and PAF-induced chemotactic responses of eosinophils isolated by the standard Percoll (positive selection) versus the magnetic cell separation systems (MACS) (negative selection) technique. 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 the lipid mediators, LTB4 and PAF, in a dose-responsive fashion. Although MACS isolation provided a greater number and higher purity of eosinophils, these eosinophils migrated less to LTB4 and PAF. Neither dextran sedimentation, dextran and Ficoll-Paque, nor dextran Ficoll-Paque and Percoll prior to MACS isolation reversed the decreased chemotactic responses observed with MACS isolated eosinophils. Further, Percoll-isolated eosinophils further purified with CD16 MicroBeads did not respond as well to LTB4 or PAF. The technique used to isolate eosinophils clearly affects the chemotactic responsiveness of this cell to LTB4 and PAF. Since several in vivo studies suggest that LTB4 and PAF are eosinophil chemoattractants, Percoll isolation of these cells might be more appropriate for studies involving eosinophil chemotactic responses to these lipid mediators.