Maintenance of phosphatidylserine asymmetry in erythroid cells during differentiation, aging, and its significance for macrophage recognition

Phosphatidylserine (PS) is distributed almost entirely in the inner leaflet of the erythrocyte membrane bilayer, and appears to be maintained by a 32 kDa integral membrane protein (PS translocase). The expression of PS on the outer leaflet may serve as a recognition signal for macrophages, since insertion of PS into erythrocytes enhances their adherence to macrophages and clearance from the circulation. Therefore I have hypothesized that erythroid cells display PS on their outer leaflet early in differentiation and upon aging. Analysis of murine erythroleukemia cells (MELC, undifferentiated erythroid progenitor cells) showed high levels of PS on the outer leaflet that decreased during differentiation, correlating with the pattern of macrophage adherence. The activity of the PS translocase during differentiation appears to be unchanged although the equilibrium distribution of PS differs. This difference may be due to qualitative changes in the PS translocase. $\sp{125}$I-Bolton/Hunter-labeled-pyridyldithioethylamine ($\sp{125}$I-B/H-PDA), a radiolabeled probe for the PS translocase, labeled a 32 kDa protein in mature erythrocytes whereas in MELC a 45 kDa protein as well as a 32 kDa protein was identified. The abundance of the 45 kDa protein in relation to the 32 kDa protein declined during differentiation, possibly indicating this protein was a precursor of the 32 kDa protein. Analysis of the 45 kDa protein by N-glycosidase F and endoproteinase cleavage suggested this protein was not a glycosylated form of the 32 kDa protein but appeared to share some structural homology. Aged murine erythrocytes had elevated levels of PS on their outer leaflet, as well as decreased PS translocase activity. $\sp{125}$I-B/H-PDA labeled a 32 kDa protein in both normal and aged erythrocytes. However, the latter cells also contained a 28 kDa protein. Experimental evidence suggests that the appearance of the 28 kDa protein may be due to increased oxidation of aged erythrocytes. Examination of PS distribution showed that the levels of PS on the outer leaflet were elevated early in differentiation, decreased during the mature state, and returned to high levels as the erythrocyte aged. In conclusion,the levels of outer leaflet PS correlated with the differentiation status and macrophage recognition of erythroid cells. ^

Redistribution of plasma membrane phosphatidylserine during erythroid development

Phosphatidylserine (PS) is not only one of the structural components of the plasma membrane, it also plays an important role in blood coagulation, and cell-cell interactions during aging and apoptosis.^ Here we studied some alterations that occur in membrane phosphatidylserine asymmetry during erythroid differentiation-associated apoptosis and erythrocyte aging and characterized some aspects in the regulation of PS asymmetry.^ Erythroleukemia cells, frequently used to study erythroid development, undergo apoptosis when induced to differentiate along the erythroid lineage. In the case of K562 cells induced to differentiate with hemin, this event is characterized by DNA fragmentation that correlates with downregulation of the survival protein BCL-xL and ultimately the result is cell death. We showed here that reorientation of PS from the inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase are also events observed upon hemin treatment. We observed that constitutive expression of BCL-2 did not inhibit the alterations caused by hemin in membrane lipid asymmetry and only slightly prevented hemin-induced DNA fragmentation. On the other hand, BCL-2 effectively inhibited actinomycin D and staurosporine-induced DNA fragmentation and the appearance of PS at the outer leaflet of these cells. z.VAD.fmk, a widely used caspases inhibitor, blocked DNA fragmentation induced by both hemin and actinomycin D but only inhibited PS externalization in cells treated with actinomycin D.^ These results showed that PS externalization occurs during differentiation-related apoptosis. Unlike the pharmacologically-induced event, however, hemin-induced PS redistribution seems to be regulated by a mechanism independent of BCL-2 and caspases.^ Membrane PS is externalized not only during apoptosis but also during red blood cell senescence. To study this event, we artificially induced cellular aging by in vitro storage or vesiculation in the presence of the amphipathic lipid dilauroylphosphatidylcholine. These cells were monitored for age-dependent changes in cell density by Percoll gradient centrifugation and assessed for alterations in membrane lipid asymmetry and their ability to be cleared in vivo. These experiments demonstrated a progressive increase in red cell density upon vesiculation and in vitro aging. The clearance rate of cells obtained after vesiculation, was biphasic in nature, showing a very rapid component together with a second component consistent with the clearance rates of control populations. Quantitation of PS in the outer leaflet of red cells revealed that membrane redistribution of PS occurred upon in vitro storage and vesiculation. Inhibition of the aminophospholipid translocase with the sulfhydryl-oxidant reagent pyridyldithioethylamine resulted in higher PS externalization and enhanced clearance of vesiculated RBC.^ These observations not only suggest that vesiculation may be the mechanism responsible for some of the characteristic changes in cell density and PS asymmetry that occur upon cell aging, but also confirm the role of PS in the recognition and clearance of senescent cells. ^