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

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 after short term culture with polyclonal activators in the presence of protein secretion inhibitors. Three-colour immunofluorescence analysis was used to examine cytoplasmic cytokines, IL-4 and IFN-, in CD4 or CD8 T cells, obtained from healthy individuals and patients with allergic asthma. The kinetics of the expression of proinflammatory cytokines, TNF- and IL-6, in LPS-activated monocytes was analysed following the back gating approach to include monocytes only according to theri 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 intracullular 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 of brefeldin A, increased the percentage of cytokine positive cels. Coexpression analysis of IL-4 and IFN- production showed the CD4 subset to produce more IFN- and IL-4, while CD8 subset produced predominantly IFN- . Allergic individuals had a lower percentage of IFN- producing cells than healthy controls and therefore demonstrated higher IL-4/IFN- ratio. The analysis of TNF- 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.

CD4; CD8; monocytes; intracellular cytokine; IL-4; IFN-; TNF-; IL-6; flow cytometry; allergy; human

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