Quantitative intracellular cytokine measurement: age-related changes in proinflammatory cytokine production

Abstract

SUMMARY: The proinflammatory cytokines play a central role in mediating cellular and physiological responses, and levels may reflect immune system effectiveness. In this study, the effect of ageing on the inflammatory response was examined using a novel method to detect production of the proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-α), IL-6 and IL-1β. Peripheral blood mononuclear cells (PBMC) obtained from healthy donors of different ages were incubated for 0, 24, 48 and 72 h with or without phorbol 12-myristate 13-acetate (PMA) stimulation. At each time point these cells were permeabilized and incubated with secondary conjugated FITC MoAbs specific for each cytokine. A flow cytometric system was developed to quantify specific intracellular fluorescence in T cells (CD3+) and monocytes (CD14+). TNF-α, IL-6 and IL-1β production in cell culture supernatants was also measured using ELISAs. In older subjects, flow cytometry detected significant increases in intracellular T cell TNF-α and IL-6 (P < 0.05). IL-1β was not detected in any of the T cell samples. Likewise, the monocytes of older subjects demonstrated increased intracellular levels of all three cytokines, but these increases were not significant (P > 0.05). These changes in intracellular proinflammatory cytokine levels may explain some of the exaggerated inflammatory responses seen in elderly patients.