Modulation of cytokine and nitric oxide production by keratinocytes, epithelial cells, and mononuclear phagocytes in a co-culture model of inflammatory acne.

INTRODUCTION: Ultraviolet B (UVB, 290 nm to 320 nm) has been reported to modulate the cytokine-mediated inflammatory process in various inflammatory skin conditions, including production of TNF-α, IL-1α, IL-6, IL-8, and IL-10. We constructed an in vitro model system involving co-culture of different cell types to study the effect of UVB on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF)-α as markers of inflammation. OBJECTIVE: This study was conducted to quantitatively assess the products secreted by human epithelial keratinocytes in the presence and absence of macrophages/monocytes. METHODS: Cells were exposed to UVB radiation (50 mJ to 200 mJ per cm2) or treated with bacterial lipopolysaccharide (LPS) as stimulator of inflammatory response. Nitric oxide (NO) was measured by modified Griess assay and TNF-α was measured by quantitative ELISA. For the co-culture system, SC monocytes were seeded in a 24-well Transwell tissue culture plate whereas irradiated keratinocytes were seeded in the individual baskets subsequently placed on top of the monocyte cultures, and samples of culture supernatants were collected at 1 to 6 days. RESULTS: When primary human epidermal keratinocytes (NHEK) were irradiated with UVB, a dose-dependent stimulation of TNF-α production was observed (33% to 200% increase). TNF-α production was not changed significantly in SC monocytes/NHEK co-culture. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, P<0.001) was seen. CONCLUSION: This improved model of cutaneous inflammation could use multiple cells to study their interactions and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.