Quantitative intracellular cytokine measurement: age-related changes in proinflammatory cytokine production.
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.
O'Mahony
L
L
Department of Surgery, St James's Hospital, Dublin, Ireland.
Holland
J
J
Jackson
J
J
Feighery
C
C
Hennessy
T P
TP
Mealy
K
K
D000328:Adult; D000368:Aged; D000375:Aging / Q000276:immunology*; D000704:Analysis of Variance; D017252:CD3 Complex; D016207:Cytokines / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology*; D018950:Lipopolysaccharide Receptors; D008875:Middle Aged; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D017252:CD3 Complex; D016207:Cytokines; D007375:Interleukin-1; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D014409:Tumor Necrosis Factor-alpha; D013755:Tetradecanoylphorbol Acetate