Inflammatory reactivity of reconstructed human epidermis to metal haptens by integration of TLR4-positive cells

Allergic contact dermatitis is critically determined by the ability of haptens to mount an innate immune signal additionally to a directed T cell response. Reconstructed human epidermis (RhE) is widely employed as replacement assay to avoid animal experiments for safety evaluation of proinflammatory effects of new ingredients in industrial products. Unfortunately, RhE lacks responsiveness for metal haptens, the most relevant human contact allergens, raising concerns about their suitability as adequate assay system for allergen testing. Here we investigated whether the defect of RhE might rely on a lack of functional TLR4 expression, which critically governs proinflammatory sensitivity to nickel and cobalt. By comparing RhE to reconstructed human full skin (RhS) we demonstrate that addition of dermal fibroblasts is sufficient to confer proinflammatory responsiveness for those metals. Unlike cultured epidermal keratinocytes, normal human fibroblasts expressed high levels of TLR4 mRNA and triggered production of IL‐8 upon stimulation with nickel, cobalt or the TLR4 agonist LPS. Consistently, dermal isolates from RhS expressed considerable amounts of TLR4 mRNA whereas RhE or epidermis isolated from RhS or normal or inflammatory activated donor skin failed to express substantial TLR4 mRNA. Co‐culture with human DCs similarly was able to license metal responsiveness of RhE, suggesting that integration of naturally TLR4 positive cells can compensate the defect of RhE. Our data suggest the use of RhS or RhE/DC co‐culture models could circumvent current shortcomings of RhE concerning allergen risk assessment and may allow to combine benefits of complex and monoculture‐based test systems in a single assay.