Erythrocyte senescence

RUCCI A; ENSINCK A; RACCA L; GARCÍA BORRÁS S; BIONDI C; COTORRUELO C; RACCA A

Cell aging

Nova Publishers

Año: 2011; p. 241 - 256

Mature red blood cells (RBCs) lack protein synthesis and are unable to restore inactivated enzymes or 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 (SCA). It serves as a specific signal for the clearance of these cells by inducing the binding of autologous IgG and phagocytosis. Whole blood samples from volunteer donors were processed. Senescent (Se) RBCs and Young (Y) RBCs were obtained by self-formed Percoll gradients. The separation of both populations was demonstrated by statistically significant changes in hematological parameters and creatine concentration. The antioxidant response in RBCs of different ages was studied. Activities of glucose-6-phosphate dehydrogenase (G6PD), soluble NADH-cytochrome b5 reductase (b5Rs) and membrane-bound b5R (b5Rm) were determined spectrophotometrically. The G6PD and b5Rm activities in SeRBCs were significantly lower than that observed in YRBCs. The decline in those activities would indicate a decrease in the antioxidant response associated to RBC aging. Membrane proteins modifications in RBCs of different ages were assessed. Membrane proteins were analyzed by SDS-PAGE, band 3 by immunoblotting, and protein oxidation by measuring the carbonyl groups. Densitometric analysis showed no differences between mean percentage values obtained from the major bands of SeRBCs and YRBCs membrane proteins. On the contrary an increase in band 3 and its degradation products were found by immunoblotting in SeRBCs. A higher protein oxidation level was also encountered in this population. These results provide experimental evidence about protein modifications occurring during the RBC lifespan. Then, considering that the accumulation of autologous IgG on RBCs membrane provides a direct mechanism for the removal of SeRBCs, the IgG content of intact RBCs was measured using an enzyme linked anti-immunoglobulin test. In addition, the presence of bound IgG was observed by confocal microscopy. It was shown that the amount of IgG bound to SeRBCs was significantly higher than that observed for YRBCs. The interaction between different RBCs populations (SeRBCs, YRBCs and desialiniysed RBCs) and peripheral blood monocytes was further investigated through a functional assay. The increase observed in the percentage of erythrophagocytosis with SeRBCs confirmed the involvement of autologous IgG in the selective removal of erythrocytes. Also, the percentage of monocytes with phagocytosed desialiniysed RBCs was higher than that obtained with YRBCs. This finding suggests that a decrease in sialic acid content of RBCs may be involved in the physiological erythrophagocytosis. In addition, cells of different ages in whole blood were characterized using light scatter, binding of autologous IgG and externalization of phosphatidylserine measurements. Dot-plot analysis based on the forward scatter versus side scatter parameters showed two RBC populations of different sizes and density. RBCs were further incubated with FITC-conjugated mouse anti-human IgG o PE-annexin-V. Binding of IgG to RBCs was analyzed by mean fluorescence intensity. The percentage of IgG positive cells was significantly higher in SeRBCs. The fraction of annexin-V positive RBCs was also larger in SeRBCs. These results indicate that flow cytometry permits differentiating erythrocyte populations of different ages. This methodology could be an alternative tool to study erythrocyte aging. Taken together, these findings will contribute to a better understanding of the process and mechanisms involved in the erythrocyte senescence.