Blom M, Tool A T, Mul F P, Knol E F, Roos D, Verhoeven A J
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
J Immunol Methods. 1995 Jan 27;178(2):183-93. doi: 10.1016/0022-1759(94)00255-u.
Eosinophils can be isolated from a mixed suspension of granulocytes by different procedures. We compared functional responses of human eosinophils purified according to two different principles: (1) an fMLP-induced difference in specific gravity between eosinophils and neutrophils and (2) selective removal of neutrophils by means of immunomagnetic beads coated with CD16 mAb. The results showed that eosinophils isolated with the CD16 beads method have a higher capacity to synthesize platelet activating factor (PAF) after stimulation with serum-treated zymosan (STZ) than eosinophils purified with the fMLP method. Binding of STZ and subsequent activation of the respiratory burst were also increased in CD16-isolated eosinophils. Furthermore, eosinophils isolated with the CD16 beads showed stronger chemotactic responses towards C5a and PAF. The difference in activity of these eosinophil preparations might be explained by a loss of the more active cells during the isolation with the fMLP method: only 30-60% of the eosinophils were recovered with this method, in contrast to a recovery of more than 95% with the CD16 beads method. Indeed, this 'lost' population of eosinophils, subsequently purified with CD16-coated beads, had a higher respiratory burst activity. The alternative explanation, i.e., an enhancement of eosinophil function by the beads method, appeared not to be valid, because repurification of fMLP-isolated eosinophils in the presence of fresh neutrophils and CD16-coated beads did not change the reactivity of the eosinophils. We conclude that the fMLP method leads to selective purification of eosinophils with a resting (or 'unprimed') phenotype.
嗜酸性粒细胞可通过不同程序从粒细胞混合悬液中分离出来。我们比较了根据两种不同原理纯化的人嗜酸性粒细胞的功能反应：（1）fMLP诱导的嗜酸性粒细胞与中性粒细胞之间比重的差异；（2）通过包被CD16单克隆抗体的免疫磁珠选择性去除中性粒细胞。结果显示，与用fMLP方法纯化的嗜酸性粒细胞相比，用CD16磁珠法分离的嗜酸性粒细胞在用血清处理的酵母聚糖（STZ）刺激后合成血小板活化因子（PAF）的能力更高。在CD16分离的嗜酸性粒细胞中，STZ的结合及随后呼吸爆发的激活也有所增加。此外，用CD16磁珠分离的嗜酸性粒细胞对C5a和PAF表现出更强的趋化反应。这些嗜酸性粒细胞制剂活性的差异可能是由于在fMLP方法分离过程中更活跃的细胞丢失所致：用该方法仅回收了30 - 60%的嗜酸性粒细胞，而用CD16磁珠法回收率超过95%。实际上，随后用包被CD16的磁珠纯化的这群“丢失”的嗜酸性粒细胞具有更高的呼吸爆发活性。另一种解释，即磁珠法增强了嗜酸性粒细胞功能，似乎并不成立，因为在新鲜中性粒细胞和包被CD16的磁珠存在的情况下对fMLP分离的嗜酸性粒细胞进行再纯化并没有改变嗜酸性粒细胞的反应性。我们得出结论，fMLP方法可导致选择性纯化具有静止（或“未致敏”）表型的嗜酸性粒细胞。
