Roberts R L, Gallin J I
Blood. 1985 Feb;65(2):433-40.
Previous studies on human eosinophils often have used cells from patients with hypereosinophilia syndrome or parasitosis owing to the difficulty in isolating pure populations of eosinophils from normal individuals. In the present study, human eosinophils were isolated with a purity of 97%, with 70% recovery from normal individuals with blood eosinophil counts of less than 3%. Human eosinophils are denser than neutrophils, but the range of densities of the two cell types overlap, making purification of eosinophils by density-gradient centrifugation difficult. However, if neutrophils were exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate eosinophils, the neutrophils' density decreased, shifting them away from the density of eosinophils. Whole normal blood anticoagulated with EDTA was incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe and then layered over a discontinuous Percoll gradient (65% and 75% in diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes at 22 degrees C. The cell layer between the 65% and 75% Percoll was collected and washed, and hypotonic lysis was used to remove erythrocytes. This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of 4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total eosinophil count. The isolated eosinophils were in a quiescent state but responded to Escherichia coli endotoxin-activated serum with shape change and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium (96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did not respond chemotactically, alter membrane potential, or reduce nitroblue tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu-[3H]Phe showed that normal eosinophils lacked expression of receptors for f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed to f-Met-Leu-Phe during purification also failed to respond to f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils and neutrophils. Thus, exposure of whole blood to f-Met-Leu-Phe, followed by separation on Percoll is a simple method for rapid isolation of normal human eosinophils.
由于难以从正常个体中分离出纯嗜酸性粒细胞群体，以往关于人类嗜酸性粒细胞的研究通常使用来自高嗜酸性粒细胞增多综合征或寄生虫病患者的细胞。在本研究中，人类嗜酸性粒细胞的分离纯度为97%，从血液嗜酸性粒细胞计数低于3%的正常个体中回收率为70%。人类嗜酸性粒细胞比中性粒细胞密度大，但两种细胞类型的密度范围有重叠，使得通过密度梯度离心法纯化嗜酸性粒细胞变得困难。然而，如果将中性粒细胞暴露于不会刺激嗜酸性粒细胞的趋化肽（f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸），中性粒细胞的密度会降低，使其密度与嗜酸性粒细胞的密度分开。用EDTA抗凝的全血在37℃下与10⁻⁶mol/L f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸孵育15分钟，然后铺在不连续的Percoll梯度（在稀释的磷酸盐缓冲盐水中为65%和75%）上，在22℃下以400g离心25分钟。收集65%和75%Percoll之间的细胞层并洗涤，用低渗裂解去除红细胞。该细胞层含有97.3±0.7%的嗜酸性粒细胞（N = 8），每毫升全血的嗜酸性粒细胞产量为4.9×10⁴个，占嗜酸性粒细胞总数的70%。分离出的嗜酸性粒细胞处于静止状态，但在用佛波酯肉豆蔻酸酯刺激时，对大肠杆菌内毒素激活的血清有形态变化和趋化性、膜去极化反应，并能还原硝基蓝四氮唑（96.0±1.0%）。在吞噬试验中，89.3±1.3%的嗜酸性粒细胞吞噬白色念珠菌，而中性粒细胞为96.0%±1.0%。相反，当用f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸处理时，嗜酸性粒细胞没有趋化反应、膜电位改变或硝基蓝四氮唑还原反应，并且用f - 甲硫氨酰 - [³H]亮氨酰 - 苯丙氨酸进行的研究表明，正常嗜酸性粒细胞缺乏f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸受体的表达。在对照研究中，纯化过程中未暴露于f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸的正常嗜酸性粒细胞对f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸也无反应，这表明正常嗜酸性粒细胞与中性粒细胞存在内在差异。因此，全血暴露于f - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸后再通过Percoll分离是一种快速分离正常人嗜酸性粒细胞的简单方法。
