Comparative quantification of IL-1 beta, IL-10, IL-10r, TNF alpha and IL-7mRNA levels in UV-irradiated human skin in vivo

Objective and Design: Ultraviolet (UV) exposure induces local immunosuppres sion and inflammation in human skin. Cytokines are, in part, responsible fo r these responses. To investigate the effects of UV-induced gene expression at the molecular level we established a sensitive in vivo/ex vivo method f or 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 efficie nt PCR-reaction conditions were obtained by designing very short PCR-templa tes (72-87 bp). The most sensitive PCR-conditions were obtained by optimisa tion 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 PC R efficiency of the internal control (GAPDH) compared to targets (IL-I beta , IL-10, IL-10r, TNF alpha, IL-7) allowed the use of Delta Delta C-T-method to quantify comparable mRNA levels. Results: Interleukin-1 beta (IL-1 beta), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNF alpha) mRNA levels were increased in a time- an d dose-dependent manner. Interleukin-1 beta induction reached a maximum (ap prox. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythe mal doses just perceptible (MEDjp). Maximal TNF alpha mRNA expression (appr ox. 14-fold) was also detected 6 h after UV exposure. Interleukin-10 mRNA i nduction reached a maximum of approximately 14-fold 24 h after UV-irradiati on of 3 MEDjp. Time- and dose-dependent changes in Interleukin-7 and Interl eukin-10 receptor mRNA levels did not occur after W-irradiation. Conclusions: Time-distinct gene induction of IL-1 beta, TNF alpha and IL-10 is involved in UV-induced immune reactions, but no considerable changes we re found for IL-10r or IL-7.