Epidermal Dysfunction Leads to an Age-Associated Increase in Levels of Serum Inflammatory Cytokines

Abstract

Even though elderly populations lack visible or other clinical signs of inflammation, their serum cytokine and C-reactive protein levels typically are elevated. However, the origin of age-associated systemic inflammation is unknown. Our previous studies showed that abnormalities in epidermal function provoke cutaneous inflammation, and because intrinsically aged skin displays compromised permeability barrier homeostasis and reduced stratum corneum hydration, we hypothesized here that epidermal dysfunction could contribute to the elevations in serum cytokines in the elderly. Our results show first that acute disruption of the epidermal permeability barrier in young mice leads not only to a rapid increase in cutaneous cytokine mRNA expression but also an increase in serum cytokine levels. Second, cytokine levels in both the skin and serum increase in otherwise normal, aged mice (>12 months). Third, expression of tumor necrosis factor-α and amyloid A mRNA levels increased in the epidermis, but not in the liver, in parallel with a significant elevation in serum levels of cytokines. Fourth, disruption of the permeability barrier induced similar elevations in epidermal and serum cytokine levels in normal and athymic mice, suggesting that T cells play a negligible role in the elevations in cutaneous and serum inflammatory cytokines induced by epidermal dysfunction. Fifth, correction of epidermal function significantly reduced cytokine levels not only in the skin but also in the serum of aged mice. Together, these results indicate that the sustained abnormalities in epidermal function in chronologically aged skin contribute to the elevated serum levels of inflammatory cytokines, potentially predisposing the elderly to the subsequent development or exacerbation of chronic inflammatory disorders.

Figure 1. Acute Barrier Disruption Induces Not only Cutaneous, but also Systemic Inflammation in Normal Mice

Both flanks of 8-week old C57BL/6J mice were repeatedly tape-stripped until in transepidermal water loss rates increased 4-fold. One group of non-tape stripped mice served as normal controls. Three hours after tape-stripping, blood was collected for cytokine analysis (see Methods). Skin samples were collected for determination of cytokine mRNA levels in the dermis and epidermis, separated by brief heating (Feingold et al., 1991). Fig 1a: Serum cytokine levels, measured by ELISA assay; 1b: Changes in mRNA levels, measured by Q-PCR in the dermis and epidermis 3 hrs after acute barrier disruption. Data were normalized to non-tape stripped normal controls, setting normal controls as 100% (dotted line in Fig 1b). A Mann Whitney two-tailed test was used to determine the significances between tape-stripped vs. non-tape stripped mice.

Figure 2. Cutaneous and Serum Cytokines Increase in Aged Mice

Both flanks of 12-month old C57BL/6J mice were used in this study. The dermis and epidermis were separated by heat (Feingold et al., 1991). Fig 2a: Expression levels of cytokine mRNA in the skin; Fig 2b: Serum cytokine levels; Fig 2c: Serum amyloid A levels in 7-week old vs. 12-month old mice. Fig 2d: mRNA levels for amyloid A and TNFα in the liver (red dots) and the epidermis (black dots) of 12-month old mice. Data were normalized to normal young controls, setting normal young controls as 100% (dotted lines). A Mann Whitney two-tailed test was used to determine the significances between aged and young mice. P values were vs. the non-tape stripped normal controls.

Figure 3. Acute Barrier Disruption Induces Increases in Cutaneous and Systemic Cytokines in Athymic Mice

Both flanks of 8-week old athymic mice were repeatedly tape-stripped until transepidermal water loss rates ≥4-fold increases. Groups of mice and experimental methods were the same as that in C57BL/6J mice. Fig 3a: Changes in mRNA levels in the dermis and epidermis; Fig 3b: Serum cytokine levels 3 hrs after barrier disruption. Fig 3c: Changes in serum cytokine levels in athymic vs. C57BL/6J mice following acute barrier disruption by sequential tape stripping. Data were normalized to non-tape stripped normal controls, setting normal controls as 100% (dotted line). A Mann Whitney two-tailed test was used to determine the significances between tape-stripped vs. non-tape stripped mice. P values were vs. the non-tape stripped normal controls.

Figure 4. Improvements in Epidermal Function Alleviate Not only Cutaneous, but also Systemic Inflammation

12-month old C57BL/6J mice were treated topically with either petrolatum or glycerol twice-daily for 10 days, followed by collection of blood and skin for analyses of serum cytokines and cutaneous cytokine mRNA. Fig 4a and c: Expression levels of cutaneous pro-inflammatory cytokine mRNA levels in aged mice following treatment with petrolatum or glycerol; Fig 4b and d: Changes in serum cytokine levels in aged mice treated with petrolatum and glycerol, respectively. The data are expressed as % of untreated young mice, setting the level of young mice as 100% (dotted line). One way ANOVA was used to determine the statistical significances. The significances are indicated in the figures. N=8 for all groups. Significances indicated in column bars represent the differences between treated vs. untreated aged mice.