Howard T H, Wang D, Berkow R L
Department of Pediatrics, School of Medicine, University of Alabama, Birmingham.
J Leukoc Biol. 1990 Jan;47(1):13-24. doi: 10.1002/jlb.47.1.13.
To study the effect of endotoxin (LPS) on the basal and chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP)-induced alterations in neutrophil cytoskeleton, we purified (greater than 98%) LPS-free neutrophils (LPS- less than 10 pg/ml LPS), compared their cytoskeletal organization to that of circulating neutrophils, and examined the effect of LPS exposure on the basal and fMLP-induced change in the cytoskeleton as reflected by F-actin content and distribution. Shape, F-actin content and distribution were monitored by FACS analysis and fluorescence microscopy of NBDphallicidin-stained cells. The F-actin content of basal and fMLP-activated, purified LPS- cells is similar to that of circulating neutrophils (defined as cells drawn in LPS- buffers at 37 degrees C and analyzed after less than 10 seconds of ex vivo manipulation). LPS- cells are round with a diffuse F-actin distribution. Exposure of LPS- cells to LPS causes cell polarization and F-actin redistribution without net gain in F-actin content. Peptide activation of the LPS- cell causes actin polymerization, which is preceded by a brief lag time. Exposure of LPS- cells to LPS (LPS+) enhances fMLP-induced actin polymerization by: 1) increasing the maximal extent of polymerization; 2) shortening the lag time preceding polymerization and increasing the rate of polymerization; and 3) lowering fMLP dose required for half maximal F-actin response. The enhancement depends on LPS dose, duration of exposure, and temperature. To examine the mechanism whereby LPS enhances fMLP-induced actin polymerization, we determined the predominant end for filament growth in LPS- and LPS+ cells, the number of actin nuclei generated in LPS- and LPS+ by fMLP activation, and the number and affinity of fMLP receptors on LPS- and LPS+ cells by 3[H]fMLP binding. Actin polymerization in both LPS- and LPS+ occurs predominantly by monomer addition to the barbed ends of nuclei, and the number of actin nuclei in basal and fMLP-activated LPS- and LPS+ cells is similar. LPS+ cells express three times more fMLP receptors than LPS- cells. The results show that LPS- cells are similar in cytoskeletal organization to circulating neutrophils, LPS causes shape change without change in F-actin content, and LPS enhances fMLP-induced actin polymerization response in neutrophils. The results suggest that LPS enhancement of actin polymerization response is associated with an increase in the number of fMLP receptors expressed on the cell surface.
为研究内毒素（LPS）对基础状态及趋化肽甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）诱导的中性粒细胞细胞骨架改变的影响，我们纯化了（纯度大于98%）不含LPS的中性粒细胞（LPS含量小于10 pg/ml LPS），将其细胞骨架结构与循环中的中性粒细胞进行比较，并检测LPS暴露对基础状态及fMLP诱导的细胞骨架变化的影响，这种变化通过F-肌动蛋白含量及分布来反映。通过FACS分析及对NBD鬼笔环肽染色细胞的荧光显微镜观察来监测细胞形态、F-肌动蛋白含量及分布。基础状态及fMLP激活的纯化LPS-细胞的F-肌动蛋白含量与循环中的中性粒细胞相似（循环中的中性粒细胞定义为在37℃下于不含LPS的缓冲液中采集，并在离体操作少于10秒后进行分析的细胞）。LPS-细胞呈圆形，F-肌动蛋白分布弥散。将LPS-细胞暴露于LPS会导致细胞极化及F-肌动蛋白重新分布，但F-肌动蛋白含量无净增加。LPS-细胞的肽激活会导致肌动蛋白聚合，这之前有一个短暂的延迟期。将LPS-细胞暴露于LPS（LPS+）可通过以下方式增强fMLP诱导的肌动蛋白聚合：1）增加聚合的最大程度；2）缩短聚合前的延迟期并增加聚合速率；3）降低达到最大F-肌动蛋白反应一半所需的fMLP剂量。这种增强取决于LPS剂量、暴露持续时间及温度。为研究LPS增强fMLP诱导的肌动蛋白聚合的机制，我们确定了LPS-和LPS+细胞中肌动蛋白丝生长的主要末端、fMLP激活在LPS-和LPS+细胞中产生的肌动蛋白核数量，以及通过3[H]fMLP结合确定LPS-和LPS+细胞上fMLP受体的数量及亲和力。LPS-和LPS+细胞中的肌动蛋白聚合主要通过单体添加到核的带刺末端发生，基础状态及fMLP激活的LPS-和LPS+细胞中的肌动蛋白核数量相似。LPS+细胞表达的fMLP受体比LPS-细胞多两倍。结果表明，LPS-细胞的细胞骨架结构与循环中的中性粒细胞相似，LPS导致细胞形态改变但F-肌动蛋白含量不变，且LPS增强中性粒细胞中fMLP诱导的肌动蛋白聚合反应。结果提示，LPS对肌动蛋白聚合反应的增强与细胞表面表达的fMLP受体数量增加有关。
