Effects of interferon on natural killer (NK) cells assessed by fluorescent probes and flow cytometry

The effects of interferon-alpha and -gamma (IFN) on natural killer (NK) cells was investigated by labelling cells with fluorescent membrane and intracellular probes and analysing these by flow cytometry. Peripheral blood mononuclear cells (PBMCs), sorted NK cells and non-NK cells were labelled with one of the fluorescent probes, 3,3'-dihexyloxacarbocyanine (DiOC6(3], N-phenyl-1-naphthylamine (NPN) or Quin 2, subsequent to incubation with IFN-alpha or IFN-gamma and the change in their fluorescence was monitored by flow cytometry. NK activity after treatment with IFN-alpha or -gamma was also monitored in parallel using a standard 51Cr-release assay. IFN-alpha treatment of PBMCs caused an apparent depolarisation and subsequent hyperpolarization of the cell membranes. Such changes reflect movement of ions across the cell membrane and also the binding of IFN-alpha to the cell surface receptor. Molecular conformational changes in the cell membrane due to IFN-alpha and IFN-gamma were monitored by labelling cell populations with NPN. Changes in NPN-labelled cell fluorescence intensity, indicating changes in membrane conformation, were greatest in NK cell populations activated by IFN-gamma. IFN-alpha had a more profound effect on non-NK cell populations. The concentration of free intracellular Ca2+ ions is also affected by IFN-alpha and IFN-gamma activation, as monitored by the fluorescent probe, Quin 2. There is an apparent decrease in intracellular Ca2+ ion concentration in the NK cell population when treated with IFNs, with the greatest effect being shown by IFN-gamma. These data indicate that the effects of IFNs on NK cells can be monitored at a cellular level using fluorescent probes and flow cytometry. As analysed by these probes, IFN-alpha and IFN-gamma appear to affect NK cells via different mechanisms.