Abstract

Assessment of nanomaterial (NM) induced inflammatory responses has largely relied on rodent testing via measurement of leukocyte accumulation in target organs. Despite observations that NMs activate neutrophil driven inflammatory responses in vivo, a limited number of studies have investigated neutrophil responses to NMs in vitro. We compared responses between the human neutrophil-like HL-60 cell line and human primary neutrophils following exposure to silver (Ag), zinc oxide (ZnO), copper oxide (CuO) and titanium dioxide (TiO₂) NMs. NM cytotoxicity and neutrophil activation were assessed by measuring cellular metabolic activity, cytokine production, respiratory burst, and release of neutrophil extracellular traps. We observed a similar pattern of response between HL-60 cells and primary neutrophils, however we report that some neutrophil functions are compromised in the cell line. Ag NMs were consistently observed to stimulate neutrophil activation, with CuO NMs inducing similar though weaker responses. TiO₂ NMs did not induce a neutrophil response in either cell type. Interestingly, ZnO NMs readily induced activation of HL-60 cells but did not appear to activate primary cells. Our findings are relevant to the development of a tiered testing strategy for NM hazard assessment which promotes the use of non-rodent models. Whilst we acknowledge that HL-60 cells may not be a perfect substitute for primary cells and require further investigation regarding their ability to predict neutrophil activation, we recommend their use for initial screening of NM-induced inflammation. Primary human neutrophils can then be used for more focused assessments of neutrophil activation before progressing to in vivo models where necessary.

摘要

纳米材料 (NM) 诱导的炎症反应的评估在很大程度上依赖于啮齿动物测试，通过测量靶器官中的白细胞积累。尽管观察到 NMs在体内激活中性粒细胞驱动的炎症反应，但数量有限的研究已经在体外研究了中性粒细胞对 NMs 的反应。我们比较了人类中性粒细胞样 HL-60 细胞系和人类原代中性粒细胞在暴露于银 (Ag)、氧化锌 (ZnO)、氧化铜 (CuO) 和二氧化钛 (TiO _{2 ) 后的反应}) 海里。通过测量细胞代谢活性、细胞因子产生、呼吸爆发和中性粒细胞胞外陷阱的释放来评估 NM 细胞毒性和中性粒细胞活化。我们观察到 HL-60 细胞和原代中性粒细胞之间的类似反应模式，但是我们报告某些中性粒细胞功能在细胞系中受到损害。一直观察到 Ag NM 刺激中性粒细胞活化，CuO NM 诱导相似但较弱的反应。_二氧化钛NM 不会在任何一种细胞类型中诱导中性粒细胞反应。有趣的是，ZnO NMs 很容易诱导 HL-60 细胞的激活，但似乎没有激活原代细胞。我们的研究结果与开发用于 NM 危害评估的分层测试策略有关，该策略促进了非啮齿动物模型的使用。虽然我们承认 HL-60 细胞可能不是原代细胞的完美替代品，并且需要进一步研究其预测中性粒细胞活化的能力，但我们建议将其用于 NM 诱导的炎症的初步筛查。然后，原代人类中性粒细胞可用于更集中地评估中性粒细胞活化，然后在必要时进行体内模型。