BACKGROUND Human neutrophils are key players of innate immunity, and influence inflammatory and immune reactions through the production of numerous cytokines and chemokines. Despite major advances in our understanding of this important functional response of neutrophils, the short lifespan of these cells and their resistance to transfection have always been an obstacle to the detailed dissection of signaling pathways and effector responses that is often possible in other cell types. RESULTS Here, we report that granulocytic differentiation of human PLB-985 cells with DMSO yields cells that are neutrophil-like with respect to surface markers, acquisition of responsiveness to physiological neutrophil stimuli (fMLP, LPS), cytokine expression and production profile, and transcription factor activation profile (NF-kappaB, C/EBP, AP-1, STAT). We also show that granulocytic PLB-985 cells can be reliably tranfected by nucleofection in a rapid and efficient manner. Indeed, we overexpressed several proteins and luciferase constructs into these cells. In particular, overexpression of a dominant negative IkappaB-alpha confirmed the central role of NF-kappaB in the production of cytokines by granulocytes. Moreover, the use of PLB-985 granulocytes in which the NADPH oxidase is inactive due to the targeted disruption of a key component (gp91phox) revealed that NF-kappaB activation and kappaB-dependent responses are independent of endogenous reactive oxygen intermediates in these cells. Antioxidant studies performed in primary human neutrophils support this conclusion. CONCLUSION Our results unveil a new facet of the NF-kappaB system of human granulocytes, and pave the way for deciphering signal transduction pathways and promoter activation in these cells.

中文翻译：

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可转染的人类中性粒细胞模型中的细胞因子生成、启动子激活和不依赖于氧化剂的 NF-kappaB 激活。

背景人类中性粒细胞是先天免疫的关键参与者，并通过产生多种细胞因子和趋化因子影响炎症和免疫反应。尽管我们对中性粒细胞这一重要功能反应的理解取得了重大进展，但这些细胞的短寿命及其对转染的抵抗力一直是详细剖析信号通路和效应器反应的障碍，而这在其他细胞类型中通常是可能的。结果 在这里，我们报告了人类 PLB-985 细胞与 DMSO 的粒细胞分化产生的细胞在表面标志物、对生理中性粒细胞刺激（fMLP、LPS）的反应性获得、细胞因子表达和产生特征以及转录方面类似于中性粒细胞因子激活谱（NF-kappaB、C/EBP、AP-1、STAT）。我们还表明，粒细胞 PLB-985 细胞可以通过核转染以快速有效的方式可靠地转染。事实上，我们在这些细胞中过度表达了几种蛋白质和荧光素酶构建体。特别是，显性负 IkappaB-alpha 的过度表达证实了 NF-kappaB 在粒细胞产生细胞因子中的核心作用。此外，使用 PLB-985 粒细胞，其中 NADPH 氧化酶由于关键组件 (gp91phox) 的靶向中断而处于非活动状态，表明 NF-kappaB 激活和 kappaB 依赖性反应独立于这些细胞中的内源性活性氧中间体。在原代人类中性粒细胞中进行的抗氧化研究支持这一结论。结论我们的结果揭示了人粒细胞 NF-kappaB 系统的一个新方面，