Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.

Identification and purification of aminophospholipid flippases.

Transbilayer phospholipid asymmetry is a common structural feature of most biological membranes. This organization of lipids is generated and maintained by a number of phospholipid transporters that vary in lipid specificity, energy requirements and direction of transport. These transporters can be divided into three classes: (1) bidirectional, non-energy dependent 'scramblases', and energy-dependent transporters that move lipids (2) toward ('flippases') or (3) away from ('floppases') the cytofacial surface of the membrane. One of the more elusive members of this family is the plasma membrane aminophospholipid flippase, which selectively transports phosphatidylserine from the external to the cytofacial monolayer of the plasma membrane. This review summarizes the characteristics of aminophospholipid flippase activity in intact cells and describes current strategies to identify and isolate this protein. The biochemical characteristics of candidate flippases are critically compared and their potential role in flippase activity is evaluated.

Glutathione loading prevents free radical injury in red blood cells after storage.

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6 degrees C for 0, 42 and 84 days in a conventional additive solution (Adsol) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (approximately 50%), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.

Lipid structure and not membrane structure is the major determinant in the regulation of protein kinase C by phosphatidylserine.

This study addresses the molecular basis for protein kinase C's specific activation by phosphatidylserine. Specifically, we ask whether protein kinase C's phospholipid specificity arises from specific protein/lipid interactions or whether it arises from unique membrane-structuring properties of phosphatidylserine. We measured the interaction of protein kinase C betaII to membranes that differed only in being enantiomers to one another: physical properties such as acyl chain composition, membrane fluidity, surface curvature, microdomains, headgroup packing, and H-bonding with water were identical. Binding and activity measurements reveal that protein kinase C specifically recognizes 1, 2-sn-phosphatidyl-L-serine, independently of membrane structure. High-affinity binding and activation are abolished in the presence of enantiomeric membranes containing 2,3-sn-phosphatidyl-L-serine, 2, 3-sn-diacylglycerol, and 2,3-sn-phosphatidylcholine. Our data also show that the stereoselectivity for 1,2-sn-diacylglycerol is not absolute; 2,3-sn-diacylglycerol modestly increases the membrane affinity of protein kinase C provided that 1, 2-sn-phosphatidyl-L-serine is present. We also find that the stereochemistry of the bulk phospholipid, in this case phosphatidylcholine, has no significant influence on protein kinase C's membrane interaction. These data reveal that specific molecular determinants on protein kinase C stereospecifically recognize structural determinants of phosphatidylserine.

Induction of apoptosis by phosphatidylserine.

Treatment of Chinese hamster ovary (CHO) cells with phosphatidylserine (PS) caused cell death in a dose-dependent manner. Other phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid, had no effect on cell viability. The cells incubated with PS became round and underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. The PS-treatment induced chromatin condensation and extensive DNA fragmentation, with a pattern characteristic of internucleosomal fragmentation on agarose gel electrophoresis. These results indicate that PS-treatment induces apoptosis of CHO cells. This apoptosis-inducing activity was highly specific for PS, and neither of the synthetic PS analogs 1,2-diacyl-sn-glycero-3-phospho-D-serine (D-PS) and 2,3-diacyl-sn-glycero-1-phospho-L-serine induced apoptosis. Analysis using fluorescence-labeled phospholipids showed that both PS and D-PS were taken up equally and then transported to intracellular membranes, suggesting that the PS-specific induction of apoptosis was not the result of its specific internalization. These observations suggest that certain molecules which may recognize the stereo-specific configuration of PS are involved in the apoptotic process triggered by PS.

Effect of a glycerol-containing hypotonic medium on erythrocyte phospholipid asymmetry and aminophospholipid transport during storage.

Previous studies from our laboratory have shown that under blood bank storage conditions red blood cell (RBC) ATP and lipid content were better maintained in a glycerol-containing hypotonic experimental additive solution (EAS 25) than in the conventional storage medium Adsol. The objective of this study was to determine the mechanism of the protective effect of EAS 25, by measuring transmembrane phospholipid asymmetry and the membrane integrity of stored RBCs. Split units of packed RBCs were stored in either EAS 25 or Adsol. RBCs were analyzed after 0, 42, and 84 days and vesicles shed from stored RBCs were analyzed after 84 days of storage. Phospholipid asymmetry was measured by phospholipase A2 digestion (RBCs) and activation of the prothrombinase complex (RBCs, vesicles). RBC membrane exhibited a significantly greater (P < 0.01) amount of phosphatidylethanolamine externalized after storage in Adsol than in EAS 25 (44.3% +/- 11.7 vs. 25.3% +/- 5.7, respectively). Prothrombin converting activities in RBCs were significantly lower than in shed vesicles (P < 0.001) suggesting the presence of phosphatidylserine in the outer monolayer of vesicle, but not in RBC membranes. The rates of inwardly-directed aminophospholipid transport in RBCs decreased by 50% and glutathione levels decreased by approximately 50% in both media. RBC cholesterol and phospholipid content of stored RBCs remained significantly greater (P < 0.01) in EAS 25 than in Adsol. The results indicate that despite comparable reduction in the rate of aminophospholipid transport and reduced GSH concentrations, RBC phospholipid asymmetry was better maintained during storage in EAS 25 than in Adsol. The data suggest that glycerol in the hypotonic EAS helps preserve RBC lipid organization and membrane integrity during storage.

Glutamine- and phosphate-containing hypotonic storage media better maintain erythrocyte membrane physical properties.

We have shown that red blood cell (RBC) adenosine-5'-triphosphate (ATP) is better maintained and that there is less hemolysis and K+ leakage in hypotonic experimental additive solutions (EASs) containing glutamine and glutamine plus phosphate (Pi) than in the conventional additive solution Adsol during blood bank storage. The objective of this study was to determine if the beneficial effect produced in these media correlates with better preservation of RBC membrane properties including lipid content, phospholipid organization, aminophospholipid transport (flippase), and prothrombin converting activity. Aliquots of packed RBCs were stored in EASs containing adenine, glucose, sodium chloride, and mannitol, with 10 mmol/L glutamine (EAS 44) or with 10 mmol/L glutamine and 20 mmol/L Pi(EAS 45), or in Adsol. RBC membranes were studied after 0, 28, 42, and 84 days of storage, and vesicle membranes were studied after 84 days. RBC cholesterol and phospholipid content remained significantly greater (P < .01) in EASs than in Adsol. The degree of membrane vesiculation was more than 50% lower in EASs than in Adsol (P < .01). After 42 days of storage, the accessibility of phosphatidylethanolamine to phospholipases was approximately 1.5 times greater for Adsol and EAS 44 samples than for EAS 45 samples (43.5% v 28%). The rates of phosphatidylserine transport were 43% to 70% lower for stored cells but were not dependent on storage media. The amounts of bands 3 and 4.1 in the microvesicle membranes were not statistically different in any of the preparations. These results suggest that storage of RBCs in glutamine and Pi-medium better maintains ATP, lipid content, and phospholipid asymmetry and results in decreased vesiculation.

Synthesis and characterization of N-acylated, pH-sensitive 'caged' aminophospholipids.

A series of acid-labile 'caged' phosphatidylserine (PS) and phosphatidylethanolamine (PE) molecules have been synthesized by N-acylation of the aminophospholipid with maleic, citraconic, dimethylmaleic, phthalic, or 3,4,5,6-tetrahydrophthalic anhydride. N-citraconyl-dioleoylphosphatidylethanolamine (C-DOPE) and N-citraconyl-dioleoyl-phosphatidylserine (C-DOPS) exhibited the highest degree of sensitivity to acidic pH; incubation at pH 5.5 and 6.5, respectively, resulted in 50% cleavage to the native aminophospholipid within 60 min. Significant cleavage of the phthalyl- and maleyl-PE derivatives did not occur at physiologically relevant pH values (pH 5.5-8), while tetrahydrophthalyl-PE and dimethylmaleyl-PE could not be isolated, reflecting their inherent instability. At pH 5.5, the half time for cleavage of C-DOPE and C-DOPS was 110 min and 85 min, respectively. The utility of these 'caged' lipids was demonstrated in human erythrocytes. When mixed with cells, C-DOPS, or the short chain analog, N-citraconyl-dilauroylphosphatidylserine (C-DLPS), transferred from liposome membranes into erythrocytes and remained in the cell outer monolayer. Low pH treatment released the citraconyl group and the free PS was transported to the inner monolayer by the aminophospholipid transporter. These novel 'caged' phospholipids may be useful for the study of transmembrane aminophospholipid transport, protein-lipid interactions and membrane fusion.

Protein kinase C as a measure of transbilayer phosphatidylserine asymmetry.

The endogenous phosphatidylserine of normal erythrocytes is confined to the cytoplasmic leaflet of the membrane. However, under pathologic conditions transmembrane asymmetry can be altered and cytofacial phosphatidylserine may appear on the cell surface. A sensitive alternative method for the measurement of the exposed phosphatidylserine content of erythrocyte membrane was developed using the activation of exogenous protein kinase C. Erythrocytes containing exogenous phosphatidylcholine incorporated into the outer membrane monolayer do not stimulate protein kinase C activity more than untreated cells. In contrast, red cells that have exogenous phosphatidylserine incorporated into their membrane outer monolayer, by prior inhibition of the aminophospholipid transporter, stimulate protein kinase C significantly more than red cells in which exogenous phosphatidylserine is allowed to translocate to the inner membrane monolayer. Kinase activation is comparable for normal cells and cells not exposed to lipid in which the aminophospholipid transporter is inhibited with sulfhydryl reagents (diamide or N-ethylmaleimide). However, Ca(2+)-loading results in an increase in activation of protein kinase C over control cells, consistent with previous reports that Ca2+ induces the exposure of erythrocyte and platelet phosphatidylserine. By reference to protein kinase C activation by phosphatidylserine in model systems, the quantity of phosphatidylserine on the cell surface may be estimated. Thus, protein kinase C activation affords a sensitive and specific measure of phosphatidylserine in the outer monolayer of biological membranes.

Regulation of a candidate aminophospholipid-transporting ATPase by lipid.

The effect of lipid environment on the activation of a vanadate-sensitive Mg(2+)-ATPase purified from human erythrocytes was studied in detergent-lipid-protein mixed micelles. ATPase activity was stimulated maximally by phosphatidylserine. Other anionic diacylglycerophospholipids (phosphatidic acid, cardiolipin, phosphatidylglycerol, and phosphatidylinositol) supported 25-100% of the phosphatidylserine-stimulated activity. Another aminophospholipid, egg PE, supported 38% of the phosphatidylserine-stimulated activity. The phosphoinositides (phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate) also stimulated the ATPase; however, activity decreased with increasing lipid phosphorylation. Monoacyl negatively charged lipids (lysophosphatidylserine, fatty acids) and zwitterionic lipids (phosphatidylcholine and sphingomyelin) did not activate the enzyme. ATPase activation was dependent on phospholipid fatty acyl chain composition: ATPase activity increased with increasing PS acyl chain length, and the optimal fatty acid composition was one saturated and one unsaturated fatty acid. However, the long, unsaturated acyl chain requirement could be satisfied by nonactivating lipids. The characteristics of this ATPase are similar to those of the Mg(2+)-ATP-dependent aminophospholipid flippase, suggesting that it may be associated with the transporter.

Hyperglycemia induces a loss of phospholipid asymmetry in human erythrocytes.

Phospholipid asymmetry in biological membranes is maintained by an aminophospholipid-specific Mg(2+)-ATPase that transports PS and PE from the outer to the inner monolayer. Recent evidence indicates that a loss of phospholipid asymmetry occurs in erythrocytes from diabetic individuals, resulting in the appearance of PS in the membrane outer leaflet. We show that hyperglycemic treatment of normal erythrocytes duplicates this effect. Erythrocytes incubated for 18-24 h in the presence of glucose were assayed for PS transport and transmembrane phospholipid asymmetry. Phospholipid asymmetry in erythrocytes treated with high concentrations of glucose (> 5 mM) showed a time-dependent (t1/2 approximately 12 h) and concentration-dependent (half-maximal concentration approximately 7.5 mM) increase in the accessibility of PS and PE, and a decrease in the accessibility of SM and PC, to exogenous phospholipases. After an 18 h incubation with 20 mM glucose, 40% of the endogenous PS and PE was found in the outer monolayer concomitant with a decrease in the outer monolayer content of SM (from 80% to 50%) and PC (from 75% to 65%). These values are consistent with an almost complete transbilayer scrambling of erythrocyte phospholipids. The loss of PS asymmetry was verified using an assay based on the activation of the prothrombinase complex. The observed loss of asymmetry is not due to inhibition of PS transport or glucose-induced Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Human erythrocyte shape regulation: interaction of metabolic and redox status.

The echinocyte-to-discocyte shape recovery of metabolically depleted erythrocytes is compromised by sulfhydryl reducing agents (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214-221). In the presence of dithiothreitol (DTT) and sugars, crenated cells recover normal discoid shape transiently, but then develop the invaginations and intracellular inclusions of stomatocytes. The stomatogenic effects of DTT were investigated in erythrocytes recovering from crenation induced by several independent mechanisms. Cells crenated by direct manipulation of the membrane bilayer (lysophosphatidylcholine incorporation) recovered discoid shape similarly in the presence and absence of the reducing agent. In contrast, resealed ghosts and cells crenated by Mg2+ depletion or Ca2+ loading did not maintain stable discoid morphology in the presence of DTT, proceeding further to form stomatocytes. Thus cell crenation by expedients that involve cellular metabolic processes develop a redox-related morphological instability that is not found in amphipath-crenated cells.

Dithiothreitol stimulates the activity of the plasma membrane aminophospholipid translocator.

Metabolic depletion induces human erythrocytes to crenate, a shape change that is reversed when ATP is regenerated by nutrient supplementation. In the presence of the sulfhydryl reducing agent dithiothreitol (DTT), this shape reversal is exaggerated, proceeding beyond normal discoid morphology to stomatocytic forms. DTT-induced stomatocytosis does not correlate consistently with alterations in cell ATP, spectrin phosphorylation, or phosphoinositide metabolism (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214-221). The effect of DTT on outer-to-inner-monolayer transport of aminophospholipids was examined by monitoring shape changes induced by dilauroylphosphatidylserine (DLPS). Stomatocytosis induced by transport of this exogenous lipid to the membrane inner monolayer is accelerated and exaggerated by DTT. The effect of DTT on DLPS translocation is reversible and temperature dependent, consistent with the intervention of reducing agents in the activity of the aminophospholipid translocator. These findings bear on the relationship between cell redox status and shape regulation.

Phosphatidylserine transport in Rhnull erythrocytes.

Phosphatidylserine transport in normal and Rhnull red blood cells was determined by measuring characteristic morphologic changes induced by synthetic phospholipids. Treating normal A+ cells with commercial anti-A antisera, anti-Rho(D) antisera, or with saturating concentrations of purified Rho(D) antibodies had no effect on phosphatidylserine transport. Normal B- cells treated with purified anti-B antibodies transported phosphatidylserine at rates equal to those of cells not treated with antibody. Rhnull cells, deficient in the protein bearing the Rho(D) antigen, incorporated dimyristoylphosphatidylcholine and dimyristoylphosphatidylserine at rates and to extents similar to normal cells. Furthermore, incorporated phosphatidylserine, but not phosphatidylcholine, was rapidly transported across the membrane bilayer. Energy depletion or treatment with sulfhydryl reagents inhibited phosphatidylserine transport equally in normal and Rhnull cells. These results indicate that, although Rhnull cells have numerous membrane defects, they are capable of adenosine triphosphate-dependent transport of exogenously added dimyristoylphosphatidylserine. Normal phosphatidylserine transport in the presence of anti-Rho(D) antibodies or in cells deficient in the Rho(D) polypeptide indicates that this protein is not the aminophospholipid transporter.

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay.

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of anti-Tac and anti-transferrin receptor-conjugated liposomes for specific drug delivery to adult T-cell leukemia.

Adult T-cell leukemia (ATL) is a rapidly progressive and usually fatal malignancy of mature T cells characterized by the expression of large numbers of interleukin-2 (IL-2) receptors on the cell surface. Anti-Tac, a monoclonal antibody directed against the IL-2 receptor, was conjugated to liposomes and compared with anti-transferrin receptor (anti-TFR) conjugates for specific binding, internalization, and intracellular drug delivery to ATL cells. Two independent assays were used: a fluorimetric assay with liposome encapsulated 1-hydroxypyrene-3,6,8-trisulfonic acid, a pH-sensitive fluorescent dye, and a growth inhibition assay using methotrexate-gamma-aspartate, a liposome-dependent cytotoxic drug. MT-1 and HUT-102 cell lines derived from patients with ATL were compared with Molt-4, a leukemia cell line that does not express IL-2 receptors in an uninduced state. Fluorimetric studies showed specific binding and internalization of anti-Tac-conjugated liposomes by HUT-102 and MT-1 but not by the Tac-negative cell line Molt-4, demonstrating the lack of nonspecific or Fc receptor-mediated uptake. Anti-TFR-conjugated liposomes were effectively bound and internalized by all three cell lines and consistently showed the highest degree of cellular liposome uptake. Drug-containing liposomes conjugated to anti-Tac were more than tenfold more effective in causing growth inhibition of ATL cells than the nonspecific control conjugates. Anti-Tac conjugates caused minimal growth inhibition of Molt-4 cells over the concentration range effective against the ATL cells. Anti-TFR-coupled liposomes gave better growth inhibition of HUT-102 and MT-1 cells (40- to 60-fold) than anti-Tac conjugates. Both anti-Tac-directed and anti-TFR-directed liposomes are effective for intracellular drug delivery to ATL cells and may represent a useful method of treatment in this disease.

Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids.

The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.

Incorporation and translocation of aminophospholipids in human erythrocytes.

Cell morphology changes are used to examine the interaction of exogenous phosphatidylserine and phosphatidylethanolamine with human erythrocytes. Short-chain saturated lipids transfer from liposomes to cells, inducing shape changes that are indicative of their incorporation into, and in some cases translocation across, the cell membrane bilayer. Dioleoylphosphatidylserine and low concentrations of dilauroyl- and dimyristoylphosphatidylserine induce stomatocytosis. At higher concentrations, dilauroylphosphatidylserine and dimyristoylphosphatidylserine induce a biphasic shape change: the cells crenate initially but rapidly revert to a discocytic and eventually stomatocytic shape. The extent of these shape changes is dose dependent and increases with increasing hydrophilicity of the phospholipid. Cells treated with dilauroylphosphatidylethanolamine and bovine brain lysophosphatidylserine exhibit a similar biphasic shape change but revert to discocytes rather than stomatocytes. These shape changes are not a result of vesicle--cell fusion nor can they be accounted for by cholesterol depletion. The reversion from crenated to stomatocytic forms is dependent on intracellular ATP and Mg2+ concentrations and the state of protein sulfhydryl groups. The present results are consistent with the existence of a Mg2+- and ATP-dependent protein in erythrocytes that selectively translocates aminophospholipids to the membrane inner monolayer engendering aminophospholipid asymmetry.