Co-culture with reconstituted epidermis formed by normal human epidermal keratinocytes (RhE) increases the expression of the skin sensitization markers CD54 and CD86 on the human monocytic leukemia cell line THP-1 without chemicals. Therefore, we investigated the effects of culture media [RPMI1640 for RhE
keratinization induction (KI) medium for THP-1], co-culture, and the responses to the skin sensitizer 2,4-dinitrochlorobenzene (DNCB) on gene expression in mono- and cocultures of RhE and THP-1 cells. Microarray and pathway analyses revealed that in mono-RhE, RPMI medium induced epidermal differentiation-related genes, whereas in monoculture THP-1 cells, KI medium upregulated inflammation-related genes. Surprisingly, the medium composition had a more significant impact than co-culture in both cells. However, crosstalk between RhE and THP-1 cells was observed upon DNCB exposure by comparing the differentially expressed gene sets. DNCB-treated THP-1 cells showed increased expression of NR4A1, NR4A2, NR4A3, SIK1, and HMOX1 in co-culture than in monoculture, and these gene expression patterns were confirmed by real-time RT-PCR. It has been suggested that danger signals from RhE, in response to DNCB, enhance the expression of these genes in THP-1 cells. We clarified the effects of the medium and co-culture and proposed these five genes as potential markers for skin sensitization evaluation.