MOLECULAR MECHANISMS OF ERYTHROPHAGOCYTOSIS - CHARACTERIZATION OF THESENESCENT ERYTHROCYTES THAT ARE PHAGOCYTIZED BY MACROPHAGES

We have recently developed a flow cytometric assay for the quantitatio n of erythrophagocytosis, using PKH 26-labeled erythrocytes as the tar get cells. Using this assay we have shown that there is extensive phag ocytosis of desialylated erythrocytes. Furthermore, we have demonstrat ed that it is the densest population of erythrocytes obtained on a sel f-forming gradient of Percoll that shows the greatest susceptibility t o phagocytosis. We designate this population of erythrocytes as fracti on X; it is even denser than the fraction 5 found previously. This pop ulation of erythrocytes correspond to zone X previously seen in the do t-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reac tivity with annexin V, which is specific for the detection of phosphat idylserine (PS) exposed oft the outer leaflet of the erythrocyte membr ane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two p opulations of erythrocytes: (A) spheroechinocytes with filipodes and ( B) echinocytes without filipods. After a 2-h period of phagocytosis, t he cells remaining in fraction X show a decrease in population A, comm ensurate with a decrease in reactivity with FITC-labeled annexin V fro m 65.5 to 24%.