Distribution and expression of B2-kinin receptor on human leukocyte subsets in young adults and elderly using flow cytometry

Abstract 

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.

Keywords: Bradykinin B2 receptor, Human, Gender, Aging, Leukocytes, Flow cytometry