DeLeo F R, Jutila M A, Quinn M T
Department of Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA.
J Immunol Methods. 1996 Oct 30;198(1):35-49. doi: 10.1016/0022-1759(96)00144-5.
The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.
人类中性粒细胞产生超氧化物（O2-）的NADPH氧化酶由膜结合蛋白和胞质蛋白组成，这些蛋白在活化细胞的质膜中组装。迄今为止，我们对NADPH氧化酶组装的大部分了解都是通过无细胞检测获得的，并且一些模拟NADPH氧化酶蛋白区域的肽已被证明使用该检测可阻断氧化酶组装。然而，无细胞检测无法完整呈现NADPH氧化酶在体内的组装和调节情况，因此有必要开发将生物分子（如肽）引入完整中性粒细胞的方法，以便在其中研究它们的作用。电穿孔就是这样一种方法。尽管该方法此前已用于人类中性粒细胞，但尚未得到充分表征。我们在此报告电穿孔中性粒细胞检测系统的详细表征，包括膜电穿孔的最佳条件及功能容量的最大保留、分析实验肽作用的最佳条件、内化肽浓度的定量以及分子扩散进入这些细胞的分子大小限制。我们的结果表明，使用比先前报道的电场强度低得多的电场即可实现中性粒细胞的最佳通透化（98 - 100%），从而保留更高水平的O2（-）生成活性。我们还发现，高达2.3 kDa的生物分子很容易扩散到通透化细胞中。对先前在无细胞检测中显示可抑制NADPH氧化酶活性的黄素细胞色素b肽的分析表明，这些肽也能阻断电穿孔人类中性粒细胞中的O2-产生；尽管有效浓度高于无细胞系统。因此，电穿孔中性粒细胞提供了一个模型系统，用于评估肽和其他药物制剂在完整细胞中的作用，这些细胞在体内与中性粒细胞非常相似。
