The effect of chronological age on the inflammatory response of human fibroblasts

Abstract

The immune system undergoes profound age-related changes, including a gradual increase in the production and circulation of proinflammatory cytokines. Despite the known capacity of fibroblasts to produce cytokines, little is known so far about the inflammatory response of fibroblasts to cellular stress such as viral and/or bacterial infection in the context of aging. Therefore the aim of this study was to analyze the levels of IL6 and IL8 secretion in supernatants of human skin fibroblasts from young and elderly persons. Cytokine and chemokine secretion was analyzed before and after in vitro infection of the cells with Cytomegalovirus (CMV) and/or stimulation with Lipopolysaccharide (LPS). The exposure of fibroblasts to these agents caused inflammatory changes, reflected by the secretion of both the cytokine IL6 and the chemokine IL8 by fibroblasts from young as well as elderly persons. The cytokine/chemokine production induced by either agent alone could be further increased by co-stimulation of the cells with both stimuli. The level of protein secretion was dependent on the chronological age of the fibroblasts. Stimulated human skin fibroblasts from elderly donors produced higher amounts of IL6 as well as IL8 than fibroblasts from young donors. These differences were more pronounced for IL6 than for IL8. The inflammatory response of fibroblasts to stimulation differed among donors and did not correspond to the responsiveness of whole blood derived from the same person. In summary lifelong CMV-infection may act as an in vivo trigger for inflammatory changes by increasing the inflammatory response to bacterial products such as LPS. It may thus contribute to age-related inflammatory processes, referred to as 'inflamm-aging'.

Fig. 1

(A) Secretion of the inflammatory proteins IL6 and IL8 by human skin fibroblasts from young and elderly donors following infection with CMV and/or stimulation with LPS. IL6 (left) and IL8 (right) concentration in the supernatant of human skin fibroblasts after infection with CMV (MOI = 1) and/or stimulation with LPS (10 ng/mL) was determined by ELISA after 48 h. The data represent mean ± S.E.M of n = 5 (young)/n = 8 (old) donors. Statistical analyses were carried out using the Mann–Whitney-U-Test and p values < 0.05 were considered as statistically significant. * significant differences compared to mock-infected cells. Δ significant differences between LPS stimulated and co-stimulated (CMV/LPS) fibroblasts. (B) Human skin fibroblasts from elderly donors secrete higher amounts of the inflammatory proteins IL6 and IL8 following infection with CMV and/or stimulation with LPS. IL6 (left panels) and IL8 (right panels) concentration in the supernatant of human skin fibroblasts after infection with CMV (MOI = 1) (upper part) or stimulation with 10 ng/mL LPS (middle part) or a combination of both (lower part) was determined by ELISA every 24 h for six days. The data represent mean ± S.E.M of n = 5 (young)/n = 8 (old) donors. Significant differences (Mann–Whitney-U-Test) between young and old human skin fibroblasts are marked by * (p < 0.05).

Fig. 2

p16 protein expression of cultivated human skin fibroblasts of young and elderly persons as well as early and late passage human foreskin fibroblasts. The protein expression of p16 was assessed in cultivated human skin fibroblasts of young and elderly donors by Western Blot analysis. As controls early and late passage (replicative senescent) human foreskin fibroblasts (HFF) were used. (A) The image shows one representative Western blot analysis of p16 expression of early and late HFF and human skin fibroblasts of young and elderly donors. (B) The graph shows the relative expression of p16 compared to late passage (senescent) HFF. Shown are mean values ± S.E.M; n = 3 (early HFF)/n = 5 (young)/n = 8 (elderly). All values were normalized to expression of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Fig. 3

LPS induced IL6 and IL8 secretion of human skin fibroblasts from elderly donors participating in the Leiden 85-plus study. IL6 (left panel) and IL8 (right panel) concentration in the supernatant of human skin fibroblasts from elderly donors was determined by ELISA after 24 h of stimulation with LPS (10 ng/mL). White bars: ‘low proinflammatory’ responders, black bars: ‘high proinflammatory’ responders as defined on the basis of the production of proinflammatory cytokines (TNFα, IL1B and IL6) by blood cells following stimulation with LPS for 24 h (van den Biggelaar et al., 2004).