QUANTITATIVE INTRACELLULAR CYTOKINE MEASUREMENT - AGE-RELATED-CHANGESIN PROINFLAMMATORY CYTOKINE PRODUCTION

The proinflammatory cytokines play a central role in mediating cellula r and physiological responses, and levels may reflect immune system ef fectiveness. In this study, the effect of ageing on the inflammatory r esponse was examined using a novel method to detect production of the proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-alph a), IL-6 and IL-1 beta. Peripheral blood mononuclear cells (PBMC) obta ined from healthy donors of different ages were incubated for 0, 24, 4 8 and 72 h with or without phorbol 12-myristate 13-acetate (PMA) stimu lation. At each time point these cells were permeabilized and incubate d with secondary conjugated FITC MoAbs specific for each cytokine. A f low cytometric system was developed to quantify specific intracellular fluorescence in T cells (CD3(+)) and monocytes (CD14(+)). TNF-alpha, IL-6 and IL-1 beta production in eel; culture supernatants was also me asured using ELISAs. In older subjects, flow cytometry detected signif icant increases in intracellular T cell TNF-alpha and IL-6 (P<0.05). I L-1 beta was not detected in any of the T cell samples. Likewise, the monocytes of older subjects demonstrated increased intracellular level s of all three cytokines, bur these increases were not significant (P > 0.05). These changes in intracellular proinflammatory cytokine level s may explain some of the exaggerated inflammatory responses seen in e lderly patients.