Lichtenberger C, Zakeri S, Baier K, Willheim M, Holub M, Reinisch W
Clinic of Internal Medicine IV, Department of Gastroenterology and Hepatology, University of Vienna, Austria.
J Immunol Methods. 1999 Jul 30;227(1-2):75-84. doi: 10.1016/s0022-1759(99)00076-9.
In the past few years, the role of polymorphonuclear neutrophils (PMN) in specific immune responses has gained significance due to their ability to express a variety of immunoregulatory molecules. However, controversial results concerning the potential of neutrophils for cytokine production have been obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutrophil suspensions as outlined by our study. We have established a novel method yielding highly purified neutrophils by combining a discontinuous Percoll gradient with fluorescence activated cell sorting of CD16bright cells. The latter step exploits the exceptionally high expression of Fc gammaRIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viability exceeding 90%. Contamination with NK cells or other lymphocytes, monocytes and eosinophils could be excluded as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD52. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class II molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method should permit studies of PMN at the mRNA level and future investigations concerning the specificity of immunoregulatory molecule synthesis by neutrophils.
在过去几年中，多形核中性粒细胞（PMN）在特异性免疫反应中的作用因其能够表达多种免疫调节分子而变得重要。然而，通过灵敏的分子生物学技术，关于中性粒细胞产生细胞因子潜力的结果存在争议。正如我们的研究所概述的，这个问题可能与传统分离的中性粒细胞悬液中存在污染的白细胞有关。我们建立了一种新方法，通过将不连续的Percoll梯度与CD16bright细胞的荧光激活细胞分选相结合，获得高度纯化的中性粒细胞。后一步利用了PMN上FcγRIIIB的异常高表达。中性粒细胞可以富集到同质状态（>99.9%），活力超过90%。通过使用针对HLA-DR、c-fms和CD52的引物进行逆转录-聚合酶链反应（RT-PCR）评估，可排除NK细胞或其他淋巴细胞、单核细胞和嗜酸性粒细胞的污染。在用粒细胞-巨噬细胞集落刺激因子（GM-CSF）刺激后，这些纯化的中性粒细胞表达MHC II类分子的能力证实了其转录潜力。我们的方法应该能够在mRNA水平上研究PMN，并有助于未来关于中性粒细胞免疫调节分子合成特异性的研究。
