## 4 Discussion
The reason was that the expression levels of CD54 and CD86 were already increased in the control cells owing to the 20:7 mixed medium and the secretion from NHEK cells, so that the expression levels in chemical (DNCB)-treated cells appear relatively lower. Therefore, the effects of media and NHEK cells on the expression levels of CD54 and CD86 on THP-1 cells were examined in detail. KI medium, which is a medium for induction keratinization of NHEK cells, enhances the expression of CD54 and CD86 in monocultured THP-1 cells. There was no significant difference in the level of upregulation of CD54 and CD86 between 100% KI medium and 50% (1:1 mixed medium) or 35% (20:7 mixed medium), that is, decreasing the mixing ratio of KI medium up to 35% did not suppress the up-regulation. The detailed components of the KI medium are not disclosed. KI medium has a higher concentration of calcium ions, which is a requirement for induction of keratinization, than general media. [PERSON] et al. provided evidence that increased extracellular Ca\({}^{2+}\) ions can activate the NLRP3 inflammasome, which plays a role in controlling skin sensitization [30] in monocytes [31]. [PERSON] et al. reported that the NLRP3 inflammasome is involved in the upregulation of CD54 expression in THP-1 cells [32]. Corresponding to these published data, our results revealed that high Ca\({}^{2+}\) in the KI medium may enhance CD54 expression in THP-1 cells. In the future, it will be necessary to verify the identification of the factors of the conditioned medium. The concentration of Ca\({}^{2+}\) in the medium, which greatly affects the keratinocyte keratinization, is a factor that influences the state of keratinocyte, and it can be expected that it will lead to changes in the environment surrounding THP-1 cells. KG2 medium, which is a medium that maintains the undifferentiated state of NHEK cells, enhances CD54 expression in THP-1 cells, whereas it suppresses CD86 expression. KG2 medium is a serum-free medium; therefore, the serum concentration in the medium was considered to be involved in the change of these expression levels. ROS cannot be alleviated in serum-free medium, and its involvement in differentiation, metabolic function, viability, and proliferation of THP-1 cells has been reported [33]. To evaluate skin sensitization in a system using the activation of THP-1 cells as an evaluation index, attention should be paid to serum volume. The increase in CD54 expression and the decrease in CD86 expression were suppressed by 50% of KG2 medium (1:1 mixed medium) rather than 100%; therefore, lowering the ratio of KG2 medium or adjusting the serum volume was considered to be an effective way to reduce the impact of the evaluation index. It is known that keratinocytes produce several cytokines mainly involved in immunity and inflammation, such as TNF-\(\alpha\)[34], IL-10 [35], GM-CSF, and IL-1 [36]. These cytokines cause epidermal Langerhans cells to increase a6 integrin [37], migrate to lymph nodes [38], induce immune tolerance [39], and enhance and assist the ability of antigen-presentation [40; 41], respectively. Primary cells are expected to maintain original functions closer to living tissue than cell lines, for example, the ability to produce a variety of cytokines and metabolic enzymes [42]; therefore, NHEK cells were used in this study. The expression levels of CD54 and CD86 were compared between THP-1 monoculture and co-culture using RPMI medium and increased by co-culturing with keratinized NHEK cells. It was suggested that RPMI-stimulated keratinized NHEK cells release secretionsthat enhance the expression of CD54 and CD86. The results showed that the effect of KI medium on THP-1 cells was greater than that of keratinized NHEK cell secretions. The expression levels of CD54 and CD86 were compared between THP-1 cell monoculture and co-culture using RPMI medium and increased by co-culturing with undifferentiated NHEK cells.