Time course of expression of mRNA of inducible nitric oxide synthase and generation of nitric oxide by ultraviolet B in keratinocyte cell lines.
BACKGROUND
Nitric oxide (NO), the ubiquitous free radical, has been implicated in the pathogenesis of various inflammatory diseases, including sunburn and ultraviolet (UV) radiation-induced pigmentation, and it also seems to play an important part in host defence against bacterial infection.


OBJECTIVE
The purpose of this study was to determine the time course of production of NO and time course of expression of inducible NO synthase (iNOS) by UVB irradiation and lipopolysaccharide (LPS) stimulation in keratinocyte cell lines. Furthermore, we intended to elucidate the relationship between iNOS and NO in various stimulated conditions.


METHODS
Normal human keratinocytes (NHK), HaCaT cells and PAM212 cells were irradiated with UVB at a dose of 50 mJ cm-2 and 100 mJ cm-2. Separately, the cell lines were stimulated with 20 micro g of LPS. NO was measured by the Griess assay and iNOS mRNA was isolated by reverse transcriptase-polymerase chain reaction at 12, 24, 48 and 72 h after stimulation.


RESULTS
The generation of NO was induced by UVB irradiation and LPS stimulation. NO production was significantly increased at 72 h after irradiation of UVB 100 mJ cm-2 in NHK, and at 48 and 72 h in HaCaT cells. In PAM212 cells, NO production was significantly increased at 12, 24, 48 and 72 h by UVB 100 mJ cm-2 and at 72 h by LPS. Induction of iNOS mRNA peaked at 48 h and then decreased to basal level at 72 h when treated with UVB irradiation. The time course of production of NO was approximately correlated with the timing of induction of iNOS mRNA.


CONCLUSIONS
These results suggest that the expression of iNOS mRNA is upregulated by UVB irradiation, and that NO produced by this inducible enzyme may play a part as a mediator or an immunomodulator in UV-induced skin reactions such as sunburn reaction and photo-induced immune alterations.
D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism / Q000528:radiation effects*; D008070:Lipopolysaccharides / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014466:Ultraviolet Rays; D015854:Up-Regulation / Q000528:radiation effects*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II