Insertion of fluorescent phosphatidylserine into the plasma membrane of red blood cells. Recognition by autologous macrophages

Abstract

The interaction of macrophages with red blood cells (RBC) displaying phosphatidylserine (PS) in their surface membranes was investigated after the transfer of an exogenously supplied fluorescent lipid analog to the RBC. Nonfluorescent (quenched) lipid vesicles were formed by ultrasonication from 1-acyl-2-[(N-4-nitro-benzo-2-oxa-1,3 diazole)aminocaproyl]phosphatidyl-serine (NBD-PS) or 1-acyl-2[(N-4-nitrobenzo-2-oxa-1,3 diazole)aminocaproyl]phosphatidylcholine (NBD-PC). The interaction of these vesicles with RBC was monitored as a function of vesicle concentration by assessment of the degree to which cell-associated lipid fluorescence was dequenched after vesicle treatment. When vesicle concentrations of less than 100 ng/ml were used, lipid fluorescence was largely dequenched, indicating that lipid transfer was the predominant mechanism of both NBD-PS and NBD-PC uptake; however, when vesicle concentrations were increased to greater than 100 ng/ml, a concentration-dependent increase in the fraction of quenched cell-associated lipid was observed, indicating that another mechanism, possibly vesicle-cell adhesion, also occurred. Using NBD-PS at concentrations at which dilution of all the phospholipid analog in the recipient cell membrane could be unequivocally confirmed, we observed that the uptake of NBD-PS-treated RBC by macrophages was increased 5-fold over that of controls, whereas the uptake of RBC containing an equivalent amount of exogenously supplied NBD-PC was unaltered. Furthermore, preincubation of macrophage monolayers with vesicles containing PS resulted in a approximately 60% inhibition in the uptake of NBD-PS-treated RBC, whereas no inhibition in the uptake of control, opsonized, or NBD-PC-treated RBC was observed. These findings suggest that PS in the outer leaflet of RBC might serve as a signal for triggering their recognition by macrophages.