Comparative responses of human polymorphonuclear leukocytes obtained by counterflow centrifugal elutriation and Ficoll-Hypaque density centrifugation. II. Membrane potential changes, membrane receptor analysis, membrane fluidity, and analysis of the effects of the preparative techniques

Abstract

Standard preparative techniques for human polymorphonuclear leukocytes involves the sequential exposure of the PMN to dextran, Ficoll-Hypaque (FH) gradient centrifugation, and hypotonic stress. Counterflow centrifugal elutriation (CCE) allows isolation of PMN from whole blood without exposure to these potentially toxic substances. We have previously reported that PMN isolated by CCE release more superoxide on stimulation than do PMN isolated by standard techniques (FH PMN). In this report, we extend these observations and show that CCE and FH PMN have similar binding kinetics for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and similar membrane fluidity. CCE PMN, however, were noted to have a more marked membrane depolarization on stimulation with FMLP than FH PMN. We show that exposure of purified CCE PMN to dextran increases their superoxide release on stimulation, as well as the available FMLP binding sites. Exposure of FH PMN to elutriation buffer increased their superoxide release on stimulation. It was also noted that both CCE and FH PMN re-exposed to FH increased available binding sites for FMLP. The possible reasons for these findings are discussed.