Keyel Peter A, Heid Michelle E, Watkins Simon C, Salter Russell D
Department of Immunology, University of Pittsburgh School of Medicine, PA, USA.
J Vis Exp. 2012 Oct 1(68):e4227. doi: 10.3791/4227.
Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type(1). In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines(2). Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms. Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism(3,4). This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation(4). In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines(5,6,7). A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes(8). To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.
细菌毒素与细胞膜中的胆固醇结合，形成孔道，导致细胞内容物泄漏以及外界物质流入。细胞要么从这种损伤中恢复，这需要活跃的膜修复过程，要么根据毒素暴露量和细胞类型死亡(1)。此外，这些毒素通过激活免疫细胞（包括巨噬细胞）在受感染宿主中引发强烈的炎症反应，巨噬细胞会产生一系列促炎细胞因子(2)。许多革兰氏阳性菌产生胆固醇结合毒素，这些毒素已被证明通过 largely uncharacterized机制促进其毒力。暴露于这些毒素的细胞的质膜形态变化包括它们被隔离到富含胆固醇的表面突起中，这些突起可脱落到细胞外空间，这表明存在一种内在的细胞防御机制(3,4)。这个过程在所有细胞中都在没有代谢活性的情况下发生，并且在化学固定后可以通过电子显微镜观察到(4)。在诸如巨噬细胞等介导毒素暴露炎症反应的免疫细胞中，诱导的膜泡被认为含有IL-1家族的细胞因子，可能既负责毒素的脱落又负责这些促炎细胞因子的传播(5,6,7)。有人提出IL-1β释放与一种特定类型的细胞死亡（称为焦亡）之间存在联系，因为两者都是半胱天冬酶-1依赖性过程(8)。为了理清这种巨噬细胞反应的复杂性，包括毒素结合、膜泡脱落、细胞因子释放以及潜在的细胞死亡，我们开发了标记技术和荧光显微镜方法，能够实时观察毒素与细胞的相互作用，包括功能障碍和死亡的测量（图1）。由于其他技术存在局限性，因此需要使用活细胞成像。生化方法无法解析单个细胞中发生的效应，而流式细胞术无法提供单个细胞的高分辨率实时可视化。这里描述的方法可应用于由涉及细胞复杂表型变化的其他刺激诱导的反应的动力学分析。
