Measurement of cytokine secretion, intracellular protein expression, and mRNA in resting and stimulated peripheral blood mononuclear cells

Quantitation of cytokine production is a valuable adjunct to standard immun ologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of a ssay should be performed, and which stimulation protocol should he used, As these types of assays enter the clinical arena, there is need for standard ization. There is also a need to maximize the amount of information which m ay be derived from a single sample. We compared secreted interleukin 4 (IL- 4), IL-2, IL-6, tumor necrosis factor alpha (TNF-alpha), and gamma interfer on proteins as measured by enzyme-linked immunosorbent assay with intracell ular cytokine production (IL-2 and gamma interferon) as detected by flow cy tometry and quantitative competitive PCR for IL-2, IL-4, TNF-alpha, and gam ma interferon mRNA and cDNA Results from unstimulated cells and cells stimu lated with phorbol myristate acetate, phytohemagglutinin, and phorbol myris tate acetate plus phytohemagglutin were compared. All three methodologies d etected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-poten t stimulus.