Further development of the NCTC 2544 IL-18 assay to identify in vitro contact allergens

Several European Union legislations request the use of in vitro methods for toxicological evaluations, including sensitization, in order to increase consumer safety but also to reduce the use of animals. The EU project SENS-IT-IV addresses the need of developing predictive in vitro tests to assess contact and respiratory hypersensitivity reactions. In this context, we have recently reported the possibility to use IL-18 production in the human keratinocyte cell line NCTC 2544 to discriminate contact sensitizer from irritants and low molecular weight respiratory allergens. The aims of the present study were to further develop this assay in order to optimize experimental conditions; to develop a 96-well plate format to establish a high throughput assay; to test the performance of other available keratinocyte cell lines, and to understand the signal transduction pathway involved in p-phenylenediamine (PPD)-induced IL-18 production. If cells reach confluence at the moment of treatment, the ability to identify contact allergens is lost; therefore a careful check for the optimal cell density using PPD as reference contact allergen is critical. In our hands, a cell density of 1-2.5×105cells/ml gave optimal stimulation. In order to develop a high throughput test, cells seeded in 96-well plate were exposed to contact allergens (2,4-dinitrochlorobenzene, p-phenylenediamine, isoeugenol, cinnamaldehyde, tetramethylthiuram disulfite, resorcinol, cinnamic alcohol and eugenol), irritants (phenol, sodium laurel sulphate, lactic acid and salicylic acid) and respiratory allergens (hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride). A selective increase in total (intracellular plus released) IL-18 was observed 24h later in cells treated with contact allergens, whereas no changes were observed following treatment with respiratory allergens and irritants, confirming previous results obtained in a 24-well format assay. A selective induction of IL-18 was also obtained testing with PPD other keratinocyte cell lines, namely HPKII and HaCaT, with the HPKII showing the highest stimulation index. Regarding the signal transduction pathway, we could demonstrate using selective inhibitors a role for oxidative stress, NF-κB and p38 MAPK activation in PPD-induced IL-18 production. In conclusion, results obtained suggest that the production of IL-18 represents a promising endpoint for the screening of potential contact allergens. The assay can be performed in a 96-well plate format, different keratinocyte cell lines can be used, and a role for oxidative stress in contact allergen-induced IL-18 was demonstrated.