Role of ROS and HMGB1 in Contact Allergen–Induced IL-18 Production in Human Keratinocytes

Keratinocytes have a key role in all phases of allergic contact dermatitis. We have recently identified the possibility to use IL-18 production for the in vitro identification of contact allergens. The purpose of this study was to characterize the molecular mechanisms underlying allergen-induced IL-18 production, in order to identify the cellular source of reactive oxygen species (ROS) and the danger signals involved. The NCTC2544 cell line was exposed to three contact allergens, namely p-phenylenediamine (PPD), 2,4-dinitrochlorobenzene (DNCB), and citral, in the presence or absence of diphenylene iodonium (DPI), allopurinol, and rotenone to identify the source of ROS, and to anti-Toll-like receptor 4 antibody and glycirrizic acid to characterize the danger-associated molecular pattern molecules. In the case of PPD, the induction of IL-18 can be modulated by rotenone, allopurinol, and DPI. In the case of DNCB, rotenone completely prevents the induction of IL-18, whereas for citral, DPI completely prevents the induction of IL-18. We demonstrated the ability of all allergens tested to induce the release of high-mobility group protein B1 (HMGB1). Its sequester by glycirrizic acid significantly modulates PPD-induced IL-18 production and completely prevents DNCB- and citral-induced IL-18. We found that different intracellular sources of ROS are triggered by contact allergens, and an important role for HMGB1 in chemical allergen-induced IL-18 production was demonstrated.