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 specific immune responses has gained significance due to their ability to express a variety of immunoregulatory molecules. However, controversial results concerning the potential of neutrophils for cytokine production have been obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutrophil 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 CD16bright cells. The latter step exploits the exceptionally high expression of Fc gammaRIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viability exceeding 90%. Contamination with NK cells or other lymphocytes, monocytes and eosinophils could be excluded as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD52. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class II molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method should permit studies of PMN at the mRNA level and future investigations concerning the specificity of immunoregulatory molecule synthesis by neutrophils.
Lichtenberger
C
C
Clinic of Internal Medicine IV, Department of Gastroenterology and Hepatology, University of Vienna, Austria.
Zakeri
S
S
Baier
K
K
Willheim
M
M
Holub
M
M
Reinisch
W
W
D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D006684:HLA-DR Antigens / Q000096:biosynthesis; D006801:Humans; D009504:Neutrophils / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D017452:Receptors, IgG / Q000032:analysis; D012680:Sensitivity and Specificity
D006684:HLA-DR Antigens; D012333:RNA, Messenger; D017452:Receptors, IgG