Abstract
Background: Mature red blood cells lack protein synthesis and are unable to restore inactivated enzymes, damaged cytoskeleton and membrane proteins. An oxidation breakdown of band 3 is probably part of the mechanism leading to the generation of a senescent cell antigen. This specific signal serves for the clearance of RBCs by inducing the binding of autologous IgG and C3, leading to phagocytosis. In addition, phosphatidilserin molecules appear in the outer membrane and the CD47 expression diminishes. Methods: Erythrocytes of different ages from whole blood were studied by flow cytometry analysing light scatter proprieties, binding of autologous IgG, C3 complement deposits, externalization of phosphatidylserine and CD47 expression. Dot-plot analysis based on forward scatter versus side scatter parameters showed two RBCs populations of different sizes and density. RBCs were further incubated with Alexa 488 IgG, APC-anti-C3, PE-annexin-V and PE-CD47. The comparison of the values obtained for the different variables studied in SeRBC and YRBC populations was carried out by the Student t-test for matched samples or by the Wilcoxon test (after verification of the normality assumption). Results: The percentage of IgG and C3 positive cells was significantly higher in senescent red blood cells population. The fraction of annexin-V positive RBCs was also larger in SeRBCs while the CD47 expression was lower in this population. Conclusions: These results indicate that flow cytometry allow differenciation of erythrocytes populations of different ages, turning this tool into an useful alternative option to study erythrocyte aging process. These findings will contribute to a better understanding of the process and mechanisms involved in erythrocyte senescence process.
Highlights
In healthy individuals, the number of erythrocytes exceeds 4 × 1012/L in blood stream and has a mean lifespan of 115 days with a variation between 70 and 140 days, and a difference of 15% between individuals
Based on data from Kay [19] [20] [21], showing that autologous IgG plays a role in the selective clearance of senescent red blood cells (RBCs), we searched for naturally occurring IgG antibodies (NAbs) directed against RBC proteins
In SeRBCs population, the average values of IgG+ cells percentages were significantly higher than those observed in YRBCs (p < 0.002) when studying erythrocyte populations obtained by both methods
Summary
The number of erythrocytes exceeds 4 × 1012/L in blood stream and has a mean lifespan of 115 days with a variation between 70 and 140 days, and a difference of 15% between individuals. All individuals have physiologic autologous IgG antibodies to band 3 that bind to senescent (Se) RBCs and initiate their removal by macrophage The accumulation of this specific antibody at the end of erythrocyte life represents an accessible evidence to identify the antigen and subsequently the aging mechanism [4] [5]. Conclusions: These results indicate that flow cytometry allow differenciation of erythrocytes populations of different ages, turning this tool into an useful alternative option to study erythrocyte aging process. These findings will contribute to a better understanding of the process and mechanisms involved in erythrocyte senescence process
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