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


METHODS
Specific mRNA levels of human UV-irradiated skin were determined by real time quantification (TaqMan RT-PCR). Highly efficient PCR-reaction conditions were obtained by designing very short PCR-templates (72-87 bp). 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-1beta, IL-10, IL-10r, TNFalpha, IL-7) allowed the use of deltadelta C(T)-method to quantify comparable mRNA levels.


RESULTS
Interleukin-1beta (IL-1beta), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNFalpha) mRNA levels were increased in a time- and dose-dependent manner. Interleukin-1beta induction reached a maximum (approx. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythemal doses just perceptible (MEDjp). Maximal TNFalpha 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 MEDjp. 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-1beta, TNFalpha and IL-1beta is involved in UV-induced immune reactions, but no considerable changes were found for IL-10r or IL-7.
Beiersdorf AG, Paul Gerson Unna Skin Research Center, Hamburg, Germany.
N
N
Brink
M
M
Szamel
A R
AR
Young
K P
KP
Wittern
J
J
Bergemann
D000328:Adult; D004307:Dose-Response Relationship, Radiation; D005786:Gene Expression Regulation; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics*; D016753:Interleukin-10 / Q000235:genetics*; D015851:Interleukin-7 / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012867:Skin / Q000378:metabolism / Q000528:radiation effects*; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D014466:Ultraviolet Rays
D007375:Interleukin-1; D015851:Interleukin-7; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10