A new simple whole blood flow cytometry-based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation

摘要

The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor (TNF) at the cell surface through the use of a specific inhibitor of the TNF-converting enzyme. This inhibitor does not affect the release of cytokines other than TNF and makes it possible to assess, in the same measurement, the phenotype of TNF+-secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNF+ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3h with PMA plus ionomycin, cellular responses were associated with surface TNF expression on the majority of CD3+ T cells and secretion of Th1-associated cytokines: interferon , interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4h of LPS-stimulation, the majority of CD14+ monocytes showed surface TNF expression; in parallel, high amounts of soluble IL1, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viral-specific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNF+ monocytes, TNF+/CD8+ T cells and TNF+/CD8- T lymphocytes in association with an increased secretion of IFN, IL6 and TNF.