A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain reaction-based mRNA studies

in the past few years, the role of polymorphonuclear neutrophils (PMN) in s pecific immune responses has gained significance due to their ability to ex press a variety of immunoregulatory molecules. However, controversial resul ts concerning the potential of neutrophils for cytokine production have bee n obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutroph il suspensions as outlined by our study. We have established a novel method yielding highly purified neutrophils by combining a discontinuous Percoll gradient with fluorescence activated cell sorting of CD16(bright) cells. Th e latter step exploits the exceptionally high expression of Fc gamma RIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viabi lity exceeding 90%. Contamination with NK cells or other lymphocytes, monoc ytes and eosinophils could be excluded as evaluated by reverse transcriptio n-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD5 2. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class LI molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method shoul d permit studies of PMN at the mRNA level and future investigations concern ing the specificity of immunoregulatory molecule synthesis by neutrophils. (C) 1999 Elsevier Science B.V. All rights reserved.