Comparative quantification of IL-1β, IL-10, IL-10r, TNFα and IL-7 mRNA levels in UV-irradiated human skin in vivo

Abstract

Objective and Design: Ultraviolet (UV) exposure induces local immunosuppression and inflammation in human skin. Cytokines are, in part, responsible for these responses. To investigate the effects of UV-induced gene expression at the molecular level we established a sensitive in vivo/ex vivo method for a comparative quantification of cytokines and receptors involved in the local skin immune reactions.¶Material and Methods: Specific mRNA levels of human UV-irradiated skin were determined by real time quantification (TaqMan^TM RT-PCR). Highly efficient PCR-reaction conditions were obtained by designing very short PCR-templates (72-87bp). The most sensitive PCR-conditions were obtained by optimisation of primer and Mn(OAc)₂-concentrations, which led to significant PCR signals (C_T-value) of less than 36 cycles. A strong correlation between PCR efficiency of the internal control (GAPDH) compared to targets (IL-1β, IL-10, IL-10r, TNFα, IL-7) allowed the use of δδC_T-method to quantify comparable mRNA levels.¶Results: Interleukin-1β (IL-1β), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNFα) mRNA levels were increased in a time- and dose-dependent manner. Interleukin-1β induction reached a maximum (approx. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythemal doses just perceptible (MED_jp). Maximal TNFα mRNA expression (approx. 14-fold) was also detected 6 h after UV exposure. Interleukin-10 mRNA induction reached a maximum of approximately 14-fold 24 h after UV-irradiation of 3 MED_jp. Time- and dose-dependent changes in Interleukin-7 and Interleukin-10 receptor mRNA levels did not occur after UV-irradiation.¶Conclusions: Time-distinct gene induction of IL-1β, TNFα and IL-10 is involved in UV-induced immune reactions, but no considerable changes were found for IL-10r or IL-7.