Flow cytometric analysis of cytokine production by normal human peripheralblood dendritic cells and monocytes: Comparative analysis of different stimuli, secretion-blocking agents and incubation periods

In this paper, we comparatively analyze the effects of the following differ ent stimuli on the production and intracellular accumulation of the interle ukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) den dritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Sta phylococcus aureus cowan 1 (SAC) in the presence or absence of interferon(I FN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus br efeldin A plus monensin), and incubation periods (6, 12, and 24 h), For thi s purpose, a four-color multiple-staining direct immunofluorescence techniq ue analyzed by flow cytometry was systematically used in all experiments (n = 19), Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produc e significant amounts of all cytokines analyzed under each of the experimen tal conditions assayed. In contrast, CC33(-/+lo) lymphoplasmocytoid DC fail ed to produce detectable levels of any of the above-mentioned cytokines und er the same stimulatory conditions. Upon comparing the different stimuli us ed, LPS was associated with higher percentages of cytokine-producing cells compared with SAG, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS a nd SAG. As regards the secretion-blocking agents, brefeldin A alone was sup erior to the combination of brefeldin A and monensin, This is because it wa s frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential anal ysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed a mong DC, which became even undetectable at 24 h, in the absence of a signif icant increase in cytokine secretion. In summary, our results show that fro m the experimental conditions assayed in this paper, to induce cytokine pro duction by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone. Cytometry (Comm, Clin. Cytometry) 46:33- 40, 2001, (C) 2001 Wiley-Liss, Inc.