Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8- T cells, IL-2 and interferon-gamma (IFN-gamma) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8+ T cells IL-2 was optimally induced after 10 h and IFN-gamma after 6 h. The levels of IL-2 and IFN-gamma in CD8+ and CD8- T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2+ (range 9.7-41.3) and CD3/IFN-gamma+ cells (10.1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3+/CD8+ peripheral blood T cells expressing IFN-gamma and an increased percentage of CD3+/CD8- T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.