MEDIATORS OF THE SELECTIVE CLEARANCE OF SENESCENT ERYTHROCYTES

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

Ciudad del Cabo - Sudáfrica.

Congreso; XXIX International Congress of the International Society of Blood Transfusion; 2006

International Society of Blood Transfusion

Background: After a life span of 120 days, Senescent Red Blood Cells (SeRBCs) expose removal markers that account for their selective recognition by macrophages and clearance from the circulation. The determination of the erythrocyte lifespan is a process affected by many cellular parameters. Aims: In the present study we characterized the RBCs membrane proteins, mainly band 3, and quantified membrane-bound IgG in SeRBCs and Young RBCs (YRBCS). We also investigated through a functional assay the interaction between SeRBCs and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of RBCs desialiniysed. Methods: ACD anticoagulated blood samples (n=31) were centrifuged to concentrate RBCs to a hematocrit of 80%. Density separation was effected by short--duration, high speed centrifugation of these concentrated RBCs. The top and bottom 10% fractions were removed and designated as YRBCs and SeRBCs, respectively. The membranes were prepared from washed cells by lysis with PBS haemolysis buffer (pH 8.0). Protein concentration in each suspension was determined by the Lowry method. Electrophoresis was carried out on 8% acrylamide gels. Gels were stained with Coomassie brilliant blue R-250 and scanned using the GelPro Analyser program. The amount of membrane-bound IgG was measured on intact SeRBCs and YRBCs using an enzyme-linked antiglobulin test. Purified RBCs (8x109 cells/mL) were incubated with goat anti-human IgG alkaline phosphatase conjugate in a microtiter plate. Aliquots were then transferred to new wells coated with human IgG. After washing to remove cell-bound antibody, phosphatase substrate was added and the remaining amount of antibody was measured by reading the absorbance. The interaction between monocytes and SeRBCs and YRBCs was evaluated by the erythrophagocytosis assay. Blood monocytes were obtained through their glass-adhering property and incubated with 0.5% different suspensions of SeRBCs and YRBCs. The unbound erythrocytes were washed out and the cells on the glass were fixed with methanol, stained by the May Grünwald Giemsa method and observed under the light microscope. Two hundred cells taken from different glass spots were analyzed to determine the percentage of monocytes with phagocytozed and adherent RBCs (AM). This functional assay was also performed with RBCs treated with neuraminidase and trypsin. Results: The results showed no changes in the protein content between SeRBCs and YRBCs and no differences when examining membrane proteins by SDS-PAGE. The IgG content of intact RBCs using an enzyme linked anti-immunoglobulin test showed that the number of IgG molecules bound to SeRBCs was significantly higher than that observed for YRBCs. We demonstrated an increased rate of erythrophagocytosis by monocytes of SeRBCs compared to YRBCs. The significative increase in the percentage of AM with SeRBC and SRBCs showed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher % AM obtained with neuraminidase treated RBCs than that obtained with YRBCs. The trypsin activity no modified the percentage of AM. Conclusions: The results obtained show that there are at least two erythrophagocytosis pathway for the removal of SeRBCs, one that is immunoglobulin-dependent and another involving the loss of sialic acid. e-mail: aracca@fbioyf.unr.edu.ar