FLOW CYTOFLUOROMETRIC ANALYSIS OF YOUNG AND SENESCENT HUMAN ERYTHROCYTES PROBED WITH LECTINS - EVIDENCE THAT SIALIC ACIDS CONTROL THEIR LIFE-SPAN

Comparing the properties of 'young' and senescent ('aged') O+ erythroc ytes isolated by applying ultracentrifugation in a self-forming Percol l gradient, we demonstrate that the sialic acids of membrane glycoconj ugates control the life span of erythrocytes and that the desialylatio n of glycans is responsible for the clearance of the aged erythrocytes . This capture is mediated by a beta-galactolectin present in the memb rane of macrophages. The evidence supporting these conclusions is as f ollows: (1) Analysis by flow cytofluorimetry of the binding of fluores cein isothiocyanate labelled lectins specific for sialic acids shows t hat the aged erythrocytes bind less WGA, LPA, SNA and MAA than young e rythrocytes. The binding of DSA and LCA is not modified. On the contra ry, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA(120) as well as Erythrina cristagalli and Erythrina corallode ndron lectins specific for terminal beta-galactose residues lead to un expected and unexplained results with a decrease in the number of lect in binding sites associated with increasing desialylation. (3) The gly coconjugates from the old erythrocytes incorporate more sialic acid th an the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive IV-acetylneuraminic acid, using as donors CMP-9-fluoresce inyl-NeuAc and CMP-[C-14]-NeuAc, respectively. (4) Microscopy shows th at the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophag e membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparat ive study of the binding of WGA, LPA, SNA and MAA to the aged erythroc ytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to le ad to their capture by the macrophages.