Expression of nitric oxide from cultured human skin cells and punch biopsies in response to retinoic acid and cytokines

Nitric oxide (NO) is a strong vasodilator produced by a variety of different cell types . Inhibition of nitric oxide synthase (NOS) by N^G-monomethyl-L-arginine (L-NMMA) leads to vasoconstriction which has long been thought to be endothelium-dependent. Constitutive nitrite/nitrate (NO₂^-/NO₃^-) production was inhibited in punch biopsies taken from sun-protected skin of normal adult volunteers following culture with 1 mM L-NMMA. NO₂^-/NO₃^- production by cultured punchs was significantly increased with 25 U/ml of IFNγ and 10 μg/ml LPS (IFNγ/LPS) and was inhibited with 1 mM L-NMMA. Topical application of 0.1% retinoic acid (RA) for 1, 2 and 4 days induced erythema and punchs from treated skin generated a significant increase in NO₂^-/NO₃^- in a time-responsive manner that paralleled the generation of erythema and was inhibited with 1 mM L-NMMA. Erythema induced by topical application of 0.1% RA was partially inhibited with a simultaneous application of L-NMMA and the subsequent NO₂^-/NO₃^- was concurrently significantly reduced. To identify the cell types involved , keratinocytes, fibroblasts and endothelial cells were isolated from normal skin and asayed for NO₂^-/NO₃^- after cytokine stimulation in vitro. Fibroblasts were found to express cNOS but not iNOS while keratinocytes expressed neither. Endothelial cells did not express cNOS but produced significant amounts of NO₂^-/NO₃^- after IFNγ/LPS stimulation. While no detectable NO₂^-/NO₃^- was produced by peripheral human monocytes, macrophage-like cells in dermal sections of RA treated volunteers were found to stain positive for iNOS. Together, these data indicate that NO activity in the skin is a product of both cNOS and iNOS and that a multiple of cell types are responsible. Redness seen following RA application may be due to either or both forms of NOS.