Tuschl, H., and R. Kovac. An In Vitro System for the Identification of Contact Allergens. ATLA. 1999. 27: 362.

dinitrochlorobenzene - 25567-67-3; sodium dodecyl sulfate - 00151-21-3

Proper risk management of chemicals requires the identification of their skin sensitising potential. Despite considerable research there is still a lack of in vitro assays for defining chemical induced allergic reactions. Contact allergy is accompanied by a specific spectrum of cytokines, thus cytokine fingerprinting should allow differentiation between the irritating and sensitising effects of chemicals. Cytokines are mediators of multiple biological reactions. They regulate amplification, maintenance and suppression of immune processes. After topical application of substances, only sensitisers induce the expression of the cytokine interleukin-1ß (IL-1ß) mRNA in Langerhans cells. These cells play a critical role in contact hypersensitivity reactions; they take up the hapten, process it and migrate to the regional lymph nodes, where the antigen is presented to naïve T-cells. In the present project, an in vitro model is elaborated, based on human skin explants, that allows the simultaneous determination of intracellular cytokines (primarily IL-1ß) and cell surface markers of Langerhans cells by flow cytometric methods. Skin biopsies derived from corrective plastic surgery are placed on stainless steel grids epidermal side up to allow topical application of test substances while being nourished with medium from the dermal side. Model substances with well-documented sensitising potential are applied to the epidermis. Solvent treated explants act as negative controls. Single cell suspensions are prepared after dispase and trypsin treatment and Langerhans cells enriched by gradient centrifugation and magnetic cell separation. After surface labelling with fluorescence-conjugated monoclonal antibodies, cells are fixed and permeabilised, and intracellular cytokines are detected with monoclonal antibodies by flow cytometry. After the application of the contact allergen, dinitrochlorobenzene (DNCB), and the irritrant, sodium dodecyl sulphate (SDS), a significant difference in the production of IL-1ß was registered: in DNCB-treated explants, 2% of the total cell population were positive for anti- IL-1ß antibody. When Langerhans cells were gated by CD1a and HLA-DR positivity, 12-17% of IL-1ß positive cells were detected after application of DNCB. SDS or solvent treated explants, and cell suspensions labelled with recombinant interleukin-inhibited monoclonal antibody, did not show any IL-1ß positive cells. The preliminary data obtained with the presented model indicate that, after corresponding standardisation and interlaboratory evaluation, it might offer a quick and reliable in vitro test for prescreening chemicals for their potential sensitising effect.