Nitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM-CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal-skin-derived dendritic cell line (FSDC) and, consequently, increases the nitrite production

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... m 11.9 ± 3.2 µmol/L (basal level) to 26.9 ± 4.2 µmol/L. Pyrrolidinedithiocarbamate (PDTC) inhibits nitrite production, with a half maximal inhibitory concentration (IC 50 ) of 19.3 µmol/L and the iNOS protein expression in FSDC. In addition, western blot assays revealed that exposure of FSDC to GM-CSF induces the phosphorylation and degradation of the inhibitor of NF-κB (IkB), with subsequent translocation of the p50, p52 and RelB subunits of the transcription nuclear factor kappa B (NF-κB) from the cytosol to the nucleus. Electrophoretic mobility shift assays (EMSA) showed that FSDC exposure to GM-CSF activates the transcription factor NF-κB. Together, these results show that GM-CSF induces iNOS expression in skin dendritic cells by a mechanism involving activation of the NF-κB pathway. Figure 4 Granulocyte-macrophage colony-stimulating factor induced nuclear factor kappa B (NF-κB) activation in fetal-skinderived dendritic cell line (FSDC) cells. FSDC cells (2 × 10 6 cells) were incubated, for the time periods indicated, in culture medium alone (control, lane 2), or in the presence of 200 ng/mL GM-CSF, for 30 min and 1 h (lane 6-7) . Nuclear extracts were subjected to electrophoretic mobility shift assay (EMSA) analysis as described in the experimental procedures. Supershift experiments were done by using specific antip50, antip52 and anti-RelB antibodies (lanes 3-5). To demonstrate specificity of induced bands, binding was carried out in the presence of a molar excess (100×) of non-radioactive NF-κB consensus containing oligonucleotide (lane 8). The gel shown is representative of three gels yielding similar results. The gel was digitally generated using an HP ScanJet 5p and processed in the Corel Photo-Paint program.