Role of cytokine and nitric oxide in the inflammatory response produced by sulfur mustard (HD)†

Abstract

We have determined by immunocytochemistry the levels of interleukin-1 beta; (IL-1β) in cultured human epidermal keratinocytes (NHEK) following exposure to HD. Human skin keratinocytes release significant numbers of IL-1 cytokine as determined by the Quantikine^TM Interleukin-1β kit, an enzyme-linked immunosorbent assay (ELISA) procedure. Exposure of NHEK [∼10⁶–10⁷ cells, to HD (2 mM) and preincubation for 3 h at 37 °C] results in significant changes in IL-1 activation. In neonatal NHEK exposed to HD, IL-1β is decreased. Conversely, in adult breast NHEK exposed to HD, IL-1β is increased. Nitric oxide (˙NO) has been implicated as the effector molecule that mediates IL-1β. To confirm the involvement of ˙NO in the expression of the IL-1β, electron paramagnetic resonance (EPR) spectroscopy was employed. EPR detectable iron–nitrosyl complex in NHEK exposed to HD (18 h post exposure to 1 mM HD) were measured, and the generation of ˙NO and this induced complex was blocked by N ^ω-nitro-L-arginine (L-NOARG), a competitive inhibitor of nitric oxide synthase (NOS). Our results show the release of nitric oxide during IL-1 cytokine expression when keratinocytes are exposed to HD. Based upon this work, it appears possible that IL-1 could be used as a specific marker for epidermal cytoxicity in mechanistic studies of the toxicity of HD and in defining interventive and therapeutic regimens against HD vesication.