Detection of intracellular cytokines in human lymphocytes and monocytes at the single cell level by flow cytometry

Background and purpose: Intracellular assessment of cytokines at the single cell level by flow cytometry has recently become available. Several critical parameters such as the cell type used, mode of activation, intensity and timing of cytokine generation should be adjusted for each cytokine tested We evaluated T cell and monocyte cytokine production alter short term culture with polyclonal activators in the presence of protein secretion inhibitors. Material and methods: Three-colour immunofluorescence analysis was used to examine cytoplasmic cytokines, IL-4 and IFN-gamma in CD4 or CD8 T cells, obtained from healthy individuals and patients with allergic asthma. The kinetics of the expression of proinflammatory cytokines, TNF-alpha and IL-6, in LPS-activated monocytes was analysed following the backgating approach to int elude monocytes only according to their size, granularity and expression of CD14 and CD45. Results and conclusions: Proper activation with PMA and ionomycin, determined by expression of CD69, is a prerogative for the detection of intracellular cytokines in T cells. Transient internalisation of CD4 molecule caused by PMA, could be partially circumvented by using the highly sensitive fluorochromes such as phycoerythrin. The addition of secretion inhibitor monensin or brefeldin A, increased the percentage of cytokine positive cells. Coexpression analysis of IL-4 and IFN-gamma production showed the CD4 subset to produce more IFN-gamma than IL-4, while CD8 subset produced predominantly IFN-gamma Allergic individuals had a lower percentage of IFN-gamma producing cells than healthy controls and therefore demonstrated higher IL-4/IFN-gamma ratio. The analysis of TNF-alpha and IL-6 in LPS-stimulated monocytes enabled us to demonstrate the kinetics of proinflammatory cytokine production at the single cell level by flow cytometry.