Inserm, U 858, BP 84225, F-31432 Toulouse Cedex, France.
Neuropeptides. 2010 Apr;44(2):155-61. doi: 10.1016/j.npep.2009.12.005. Epub 2009 Dec 31.
The kallikrein-kinin system has been investigated in many experimental models. Dysregulations of the KKS are likely to be involved in pathologies such as inflammation, cancer and cardiovascular diseases. Previous works on the human KKS mostly rely on gene polymorphism and mRNA expression. In order to assess the KKS in human at the protein level, we have developed an approach based on flow cytometric analysis of leukocytes. Whole blood samples were collected and erythrocytes were lysed. Permeabilised leukocytes were incubated with anti-B2R (IgG2b), anti-IgG2b-PE, anti-CD3-PerCP (lymphocytes) and anti-CD14-APC (monocytes) antibodies. FACScalibur analyzed fluorescence intensities. Results were expressed as per cent of B2R-positive cells in each leukocyte subset and as B2R fluorescence intensity per positive cell. Detection of the B2R protein by this methodology was validated by (i) correlation with Western blotting using two different B2R antibodies, (ii) BK-induced Erk activation, (iii) B2R mRNA expression. The methodology was then applied to evaluate variations of B2R expression in a population including young healthy, elderly healthy, and elderly treated hypertensive men and women. In the young healthy subjects, B2R distribution was: monocytes>polymorphonuclear neutrophils (PMN)>lymphocytes and no difference with gender was observed. Moreover, no difference was observed on PMN B2R expression. B2R expression remained unchanged in the elderly healthy or hypertensive men. By contrast, monocytes and lymphocytes B2R expressions were decreased in the elderly healthy women. Finally, FACS analysis of B2R expression on leukocytes subsets provides single cell quantification of B2R expression allowing comparison of cellular sub-populations. This approach provides a new efficient tool to investigate B2R profiling of immune system in pathological states.
激肽释放酶-激肽系统已在许多实验模型中得到研究。KKS 的失调可能与炎症、癌症和心血管疾病等病理学有关。以前关于人类 KKS 的研究主要依赖于基因多态性和 mRNA 表达。为了在蛋白质水平上评估人类的 KKS，我们开发了一种基于白细胞流式细胞分析的方法。采集全血样本并裂解红细胞。用抗 B2R（IgG2b）、抗 IgG2b-PE、抗 CD3-PerCP（淋巴细胞）和抗 CD14-APC（单核细胞）抗体孵育透化的白细胞。用 FACScalibur 分析荧光强度。结果以每个白细胞亚群中 B2R 阳性细胞的百分比和每个阳性细胞的 B2R 荧光强度表示。通过以下方法验证了该方法检测 B2R 蛋白的有效性：（i）使用两种不同的 B2R 抗体与 Western blot 相关联，（ii）BK 诱导的 Erk 激活，（iii）B2R mRNA 表达。然后，该方法用于评估包括年轻健康、老年健康和老年治疗性高血压男女在内的人群中 B2R 表达的变化。在年轻健康受试者中，B2R 分布为：单核细胞>多形核白细胞（PMN）>淋巴细胞，且性别之间无差异。此外，PMN 上的 B2R 表达无差异。在老年健康或高血压男性中，B2R 表达保持不变。相比之下，老年健康女性的单核细胞和淋巴细胞 B2R 表达减少。最后，白细胞亚群上 B2R 表达的 FACS 分析提供了 B2R 表达的单细胞定量，允许比较细胞亚群。这种方法提供了一种新的有效工具，可用于研究病理状态下免疫系统的 B2R 分析。
