Phenotyping Renal Leukocyte Subsets by Four-Color Flow Cytometry: Characterization of Chemokine Receptor Expression-猫眼课题宝

通过四色流式细胞术对肾白细胞亚群进行表型分析：趋化因子受体表达的表征

DOI：

10.1159/000068517

发表时间：

2003

期刊：

Nephron Experimental Nephrology

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

V. Vielhauer;H. Anders;G. Pérez de Lema;B. Luckow;D. Schlöndorff;M. Mack

为了研究肾脏炎症性疾病中细胞介导的损伤机制，确定浸润的肾脏白细胞亚群的表面表型是至关重要的。然而，许多白细胞受体的细胞特异性表达难以在体内表征。在这里，我们报告了一个协议的基础上流式细胞术，允许同时表征表面受体表达的不同子集的浸润性肾白细胞。所描述的技术结合了从全肾制备单细胞悬液的适应性方法和随后的四色流式细胞术。我们最近应用这项技术来确定小鼠趋化因子受体CCR2和CCR5的差异表达的浸润肾白细胞亚群。在这篇文章中，我们总结了我们目前的研究结果的有效性的方法相比，免疫组织学和原位杂交在两个小鼠模型的非免疫性（梗阻性肾病）和免疫介导的（狼疮肾炎）炎症性肾脏疾病。流式细胞术分析显示，与外周血相比，炎症肾脏中CCR5阳性淋巴细胞的积累，而不是CCR2阳性淋巴细胞。特别是肾脏CD8+细胞表达CCR5（梗阻性肾脏79%，狼疮性肾炎90%）。在两种模型中，浸润性肾巨噬细胞对CCR2和CCR5呈阳性。这些数据对应于免疫组织学和原位杂交结果。他们证明，从发炎的肾脏制备的单细胞悬液的流式细胞术分析是一种快速和可靠的技术，以表征和定量浸润肾白细胞亚群上的表面受体表达。

To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune (obstructive nephropathy) and immune-mediated (lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8+ cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets.