A whole blood method for measurement of intracellular TNF-alpha, IFN-gamma and IL-2 expression in stimulated CD3+ lymphocytes: differences between young and elderly subjects.

Cytokines, the central regulators of leucocyte growth and differentiation, are produced by a wide variety of cell types, target various cell subsets and exhibit numerous biological activities. Cytokine dysregulation is believed to play a role in the remodelling of the immune system in old age, however, previous reports of cytokine levels in elderly subjects have been conflicting, possibly due to methodologies employed. We used the relatively new technique of intracellular cytokine detection by flow cytometry to measure cytokine production in CD3+ lymphocytes from young and elderly subjects, but applied it to whole blood, thereby eliminating the need for laborious cell separation techniques and maintaining cells in their normal physiological environment. We found the assay to be very reproducible with acceptable intra- (2.9%) and inter- (6.3%) assay CVs. The percentages of CD3+ cells producing TNF-alpha and IFN-gamma were significantly higher in elderly compared to young people (p=0.0049; p=0.0026, respectively) after stimulation with PMA and ionomycin. Absolute counts of CD3+IFN-gamma+ and CD3+TNF-alpha+ cells were also significantly higher in the elderly group (p=0.039; p=0.051) respectively. There was no significant difference between the age groups for the percentage or numbers of IL-2-producing CD3+ cells on stimulation. CD3+ cells expressing TNF-alpha were highly associated with CD3+ cells expressing IFN-gamma in both elderly and young people. In contrast, IL-2 secreting CD3+ cells were associated with TNF-alpha and IFN-gamma producing CD3+ cells in young but not elderly subjects providing further evidence for the remodelling of the cytokine network associated with old age.