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

Abstract

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-alpha), IL-6 and IL-1beta. 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-alpha, IL-6 and IL-1beta production in cell culture supernatants was also measured using ELISAs. In older subjects, flow cytometry detected significant increases in intracellular T cell TNF-alpha and IL-6 (P < 0.05). IL-1beta 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.

Fig. 1

Dot plots and histograms representing intracellular cytokine levels in T cells and monocytes. The distinct T cell and monocyte populations can be gated in the dot plot using cell size (FSC) versus cell granularity (SSC). This method of cell identification was confirmed using cell surface CD3 (80–85% positive) or CD14 antibodies (70–80% positive). Using the histograms, intracellular fluorescence can be measured. The ordinate relates to the relevant cell number, while fluorescence intensity can be seen on the abscissa, representing the amount of intracellular cytokine. A shift to the right demonstrates an increase in the amount of cytokine within a cell. Peaks can be observed for non-permeabilized cells, T cells and monocytes. Specific intracellular fluorescence levels can be quantified from these histograms for each of the cell types.

Fig. 2

Differences in intracellular cytokine production between phorbol 12-myristate 13-acetate (PMA)-stimulated and spontaneous cultures are shown for T cells and monocytes (n = 19). The abscissa represents time in culture up to 72 h, while the ordinate represents median fluorescence intensity (MFI), which are the arbritary units being used to quantify intracellular cytokine levels (± s.e.m.). *P < 0.05 PMA-stimulated versus spontaneous values.

Fig. 3

Time course experiments were performed to examine the appearance of extracellular tumour necrosis factor-alpha (TNF-α) production (pg/ml) and its relationship to intracellular (mean fluorescence intensity (MFI)) cytokine staining. Rapid increases in intracellular monocyte TNF-α staining immediately preceded the appearance of extracellular cytokine.

Fig. 4

Intracellular tumour necrosis factor-alpha (TNF-α) levels are shown illustrating the differences between the two age groups. Time in culture (abscissa) is plotted against mean fluorescence intensity (MFI; ordinate). G1 and G2 represent the 20–40 and > 62 age groups, respectively. *P < 0.05.

Fig. 5

ELISAs were carried out on the 72 h supernatants from the phorbol 12-myristate 13-acetate (PMA)-stimulated cultures. The two age groups are shown on the abscissa, with cytokine production (pg/ml) plotted on the ordinate.