Mitachi Takafumi, Kouzui Mai, Maruyama Ryo, Yamashita Kunihiko, Ogata Shinichi, Kojima Hajime, Itagaki Hiroshi
Department of Chemical and Energy Engineering, Yokohama National University.
Corporate Research Center, Daicel Corporation.
J Toxicol Sci. 2019;44(3):213-224. doi: 10.2131/jts.44.213.
The human cell line activation test (h-CLAT) is a skin sensitization test that measures the expression of cell surface proteins CD86 and CD54 to evaluate the skin sensitization potential of test chemicals. However, some skin irritants have been reported to induce dramatically high CD54 expression leading to false-positive h-CLAT results. Furthermore, CD54 expression is strongly induced by cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, or danger signals that activate its signaling pathways. In this study, we focused on the relationship between CD54 expression and the Nucleotide binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome, a protein complex that plays a pivotal role in intra-cellular inflammation. We observed the activation of caspase-1 and production of IL-1β after exposure of THP-1 cells to 2,4-dinitrochlorobenzene (DNCB, sensitizer), octanoic acid (OA, non-sensitizer), and salicylic acid (SA, non-sensitizer), implying NLRP3 activation. These observations confirmed the activation of the inflammasome by CD54-only positive chemicals. CD54 expression, induced by OA and SA, was suppressed by potassium chloride, a typical inhibitor of NLRP3 inflammasome activation. These results suggested that the NLRP3 inflammasome may be activated in THP-1 cells resulting in the expression of CD54, and subsequently leading to false-positive results.
人细胞系激活试验（h-CLAT）是一种皮肤致敏试验，通过检测细胞表面蛋白CD86和CD54的表达来评估受试化学物质的皮肤致敏潜力。然而，据报道，一些皮肤刺激物可诱导CD54表达显著升高，导致h-CLAT结果出现假阳性。此外，细胞因子如白细胞介素（IL）-1β和肿瘤坏死因子（TNF）-α，或激活其信号通路的危险信号可强烈诱导CD54表达。在本研究中，我们重点关注了CD54表达与核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎性小体之间的关系，NLRP3炎性小体是一种在细胞内炎症中起关键作用的蛋白质复合物。我们观察到，THP-1细胞暴露于2,4-二硝基氯苯（DNCB，致敏剂）、辛酸（OA，非致敏剂）和水杨酸（SA，非致敏剂）后，半胱天冬酶-1被激活，IL-1β产生，这意味着NLRP3被激活。这些观察结果证实了仅CD54呈阳性的化学物质可激活炎性小体。OA和SA诱导的CD54表达受到NLRP3炎性小体激活的典型抑制剂氯化钾的抑制。这些结果表明，NLRP3炎性小体可能在THP-1细胞中被激活，导致CD54表达，进而导致假阳性结果。
