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

为了建立一种简便的体外筛选过敏接触性皮炎的化学物质的方法，我们使用HaCaT表皮角质形成细胞系和ELD-1Langerhans细胞系检测候选标记物。用实时荧光定量逆转录-多聚酶链式反应分析已知变态反应性接触性致敏剂对ELD-1和HaCaT细胞细胞因子和趋化因子基因表达的影响。在HaCaT细胞中，变态反应性接触性致敏剂可增加白细胞介素1α(IL-α)、肿瘤坏死因子和趋化因子α的表达。当角质形成细胞分泌的IL-1α和肿瘤坏死因子作用于HaCaT和ELD-1细胞时，HaCaT细胞的IL-1α、肿瘤坏死因子和MIP-3α的表达和分泌显著增加，并且在ELD-1细胞的培养上清液中检测到IL-6和IL-8。这些结果表明，在变态反应性接触性皮炎的诱导过程中，角质形成细胞和朗格汉斯细胞之间存在密切的相互作用DNCB是一种皮肤刺激物，然后我们用基因芯片方法检测了mRNA表达的变化。在两种细胞系中，DNCB比BC增加了更多基因表达的变化。在HaCaT细胞中，4个基因的表达显著增加，2个基因的表达显著降低；在ELD-1细胞中，4个基因的表达显著降低。在本研究中，我们使用皮肤细胞系来研究几个候选基因的表达作为过敏反应的指标。

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.