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

Abstract

SUMMARYThe 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-a), IL-6 and IL-1b. 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 þ ) and monocytes (CD14 þ ). TNF-a, IL-6 and IL-1b production in cell culture supernatants was also measured using ELISAs. In older subjects, flow cytometry detected significant increases in intracellular T cell TNF-a and IL-6 (P < 0·05). IL-1b 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.

Exercise Effects on Methylation ofASCGene

“…TNF-α, IL-6 levels [19] and IL-1, TNF-α levels [3,6] have been reported to increase age-dependently as well. Thus, the development and progression of several age-related diseases are associated with chronic inflammation.…”

Individual susceptibility to periprosthetic osteolysis is associated with altered patterns of innate immune gene expression in response to pro‐inflammatory stimuli

“…We aimed to mitigate against these possibilities in the design and analysis of the study with the following measures: the PBMC stimulation period was limited to 3 h to minimize the potential for adaptive, lymphocyte-mediated cytokine expression and for secondary feedback modulation of the response observed. 25 We included LPS as an inflammatory stimulus that it exerts its biological effects through innate TLR signaling pathways. We used titanium particles as the particulate challenge rather than polyethylene as previous studies have shown that both titanium and polyethylene particle stimulate cytokine …”

Innate immunity in aging: impact on macrophage function

“…The data regarding cytokine and chemokine secretion are ambiguous in several studies using human monocytes from aged or young individuals activated in vitro with mitogens or LPS. Although the majority of the studies indicate an increase in the levels of pro-inflammatory factors such as IL-6 and IL-8 (Ershler et al ., 1993;Clark & Peterson, 1994;O'Mahony et al ., 1998;Roubenoff et al ., 1998;Sadeghi et al ., 1999;Hasegawa et al ., 2000;Mariani et al ., 2002), others find decreased levels (Gon et al ., 1996;Beharka et al ., 2001) or no changes at all (Ahluwalia et al ., 2001). Differences in the genetic heterogeneity and health status of the test population, in addition to the experimental and assay conditions, may all contribute to the discrepancy in these results.…”