Kinetics of intracytoplasmic Th1 and Th2 cytokine production assessed by flow cytometry following in vitro activation of peripheral blood mononuclearcells

Background: Standard cytokine detection methods are unable to determine whi ch cells are the producing cells. We report on the extent and under which c onditions the multilabeling capability of flow cytometry (FCM) can bring ne w advances into the field. Methods: Five different cytokines, interleukin-2 (IL-2), -4, -5, -10 and in terferon-gamma (IFN-gamma), were assessed simultaneously under five ex vivo stimulation conditions in peripheral blood mononuclear cells from five hea lthy volunteers in a 5-day kinetic study. A second group of 35 volunteers w as assessed for IFN-gamma and IL-2 production. Results: This study showed that (a) intracytoplasmic cytokines were almost undetectable within unstimulated cells, (b) intracytoplasmic cytokines were detected only in CD69(+)T lymphocytes, and (c) intracytoplasmic IL-2 and I FN-gamma were dramatically upregulated after stimulation with phorbol myris tate acetate (PMA)-ionomycin in a biphasic response or with PMA-phytohemagg lutinin (one major peak only at 18 h) but to a lesser extent with other sti muli such as monoclonal antibodies. Th2 cytokines were detected at a later time point and at lower levels. PMA/ ionomycin stimulation after 4 h and 18 h of culture in 35 other volunteers individualized several subgroups accor ding to the frequency of IFN-gamma- or IL-2-producing cells-IFN-gamma delay ed producers (n = 10/35), IFN-gamma low producers (n = 8/35), and IL-2 dela yed producers (n = 16/35)-as opposed to IFN-gamma or IL-2 normal producers. Conclusions: FCM appears to be a good tool to examine cell cytokine status in pathology (allergy, autoimmune disease, etc.) provided that optimal stim ulation conditions and multiple time-point cultures are used. It also seems to be a relevant method to define new Th subsets further. (C) 1999 Wiley-L iss, Inc.