Development of a new, predictive in vitro test for screening of allergic co ntact sensitizers

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To develop a simple predictive in vitro test for screening of chemical substances that induce allergic contact dermatitis, we examined candidate markers using the HaCaT epidermal keratinocyte cell line, and the ELD-1Langerhans cell line.Expression of cytokine and chemokines mRNA by ELD-1 and HaCaT cells exposed to known allergic contact sensitizers was analyzed quantitatively by real-time reverse transcription-polyinerase chain reaction (RT-PCR).In HaCaT cells, level of mRNA for interlurkin-1α(IL-α), tumor necrosis factor(TNF) and chemokine MIP-3α were increased by allergic contact sensitizers, whereas mRNA levels were altered only slight by ELD-1 cells.When HaCaT and ELD-1 cells were exposed by IL-1α and TNF, which are secreted by keratinocytes, mRNA level and secretion of IL-1α, TNF, and MIP-3α were increased remarkably in HaCaT cells, and IL-6 and IL-8 were detected in cultured media of ELD-1 cells.These findings suggest that keratinocytes and Langerhans cells interact closely during induction of allergic contact dermatitis.To analyze more comprehensively changes in RNA expression in response to allergens, we exposed HaCaT and ELD-1 cells to dinitrochlorobenzene(DNCB), an allergic contact sensitizers, and benzalkonium chloride(BC), a skin irritant, and we then examined changes in mRNA expression change by cDNA microarray method.DNCB increased changes in expression of more genes than did BC in both cell lines. Expression of four genes was increased significantly and expression of two genes was decreased significantly in HaCaT cells.Expression of four genes was decreased significantly in ELD-1 cells.In the present study, we used cutaneous cell lines to investigate expression of several candidate genes as indices of allergic sensitization.We plan to investigate marker molecules in a keratinocyte-Langerhans cell co-culture system.