Direct inhibition of phospholipid scrambling activity in erythrocytes by potassium ions.

The exposure of phosphatidylserine (PS) at the cell surface plays a critical role in blood coagulation and serves as a macrophage recognition moiety for the engulfment of apoptotic cells. Previous observations have shown that a high extracellular [K(+)] and selective K(+) channel blockers inhibit PS exposure in platelets and erythrocytes. Here we show that the rate of PS exposure in erythrocytes decreases by approximately 50% when the intracellular [K(+)] increases from 0 to physiological concentrations. Using resealed erythrocyte membranes, we further show that lipid scrambling is inducible by raising the intracellular [Ca(2+)] and that K(+) ions have a direct inhibitory effect on this process. Lipid scrambling in resealed ghosts occurs in the absence of cell shrinkage and microvesicle formation, processes that are generally attributed to Ca(2+)-induced lipid scrambling in intact erythrocytes. Thus, opening of Ca(2+)-sensitive K(+) channels causes loss of intracellular K(+) that results in reduced intrinsic inhibitory effect of these ions on scramblase activity.

Tumoricidal activity of human monocytes activated in vitro by free and liposome-encapsulated human lymphokines.

Human peripheral blood mononuclear cells from normal donors obtained by separation on a Percoll gradient were incubated with free or liposome-entrapped lymphokines produced from concanavalin A-stimulated lymphocytes and then were tested for cytotoxic activity against tumor cells. The treated monocytes lysed tumorigenic melanoma and glioblastoma target cells, but had no effect on three types of nontumorigenic target cells. The activation of monocytes to become tumoricidal was caused by macrophage-activating factor (MAF) and not by contamination with endotoxins, concanavalin A, or interferon. The endocytosis of liposomes containing MAF, but not of those containing control supernatants, led to the activation of cytotoxic properties in the monocytes. Activation by liposome-encapsulated MAF was very efficient and required less than 1/800th of the amount of free MAF necessary to achieve the same levels of cytotoxicity. Thus, the encapsulation of mitogen-induced MAF in liposomes could provide an effective approach for the activation of blood monocytes in situ.

Increased adherence of sickled and phosphatidylserine-enriched human erythrocytes to cultured human peripheral blood monocytes.

The precise mechanism by which sickle erythrocytes (RBC) are removed from the circulation is controversial, although it is possible that enhanced recognition of these cells by circulating mononuclear phagocytes could contribute to this process. We investigated this possibility by interacting sickle cells with cultured human peripheral blood monocytes. Our results show that both irreversibly sickled cells (ISC) and deoxygenated reversibly sickled cells (RSC) had a higher avidity for adherence to monocytes than did oxygenated sickle and normal RBC. ISC were the most adherent cell type. Adherence of RSC to monocytes was found to be reversible; reoxygenation of deoxygenated RSC resulted in a significant decrease in RSC--monocyte adherence. Concomitant with alterations in sickle RBC adherence were alterations in the organization and bilayer distribution of membrane phospholipids in these cells. Specifically, enhanced adherence was associated with increased exposure of RBC membrane outer leaflet phosphatidylserine (PS) and phosphatidylethanolamine, whereas lack of adherence was associated with normal patterns of membrane phospholipid distribution. To investigate the possibility of whether the exposure of PS in the outer membrane leaflet of these cells might be responsible for their recognition by monocytes, the membranes of normal RBC were enriched with the fluorescent PS analogue 1-acyl-2[(N-4-nitro-benzo-2-oxa-1,3-diazole)aminocaproyl]-phosphatidy lse rine (NBD-PS) via transfer of the exogenous lipid from a population of donor phospholipid vesicles (liposomes). RBC enriched with NBD-PS exhibited enhanced adherence to monocytes, whereas adherence of RBC enriched with similar amounts of NBD-phosphatidylcholine (NBD-PC) was not increased. Furthermore, preincubation of monocytes with PS liposomes resulted in a approximately 60% inhibition of ISC adherence to monocytes, whereas no inhibition occurred when monocytes were preincubated with PC liposomes. These findings strongly suggest that erythrocyte surface PS may be a ligand recognized by receptors on human peripheral blood monocytes and that abnormal exposure of PS in the outer leaflet of the RBC membrane, as found in sickle RBC, might serve to trigger their recognition by circulating monocytes. Our results further suggest that abnormalities in the organization of erythrocyte membrane phospholipids may have significant pathophysiologic implications, possibly including shortened cell survival.

Effects of liposome structure and lipid composition on the activation of the tumoricidal properties of macrophages by liposomes containing muramyl dipeptide.

Various vesicle structures and lipid compositions have been studied to identify the optimal type of liposome for delivery of the macrophage-activating agent muramyl dipeptide (MDP) to macrophages. Evaluation of the ability of liposomes to be phagocytosed by macrophages established that optimal initial rates of engulfment were obtained when multilamellar vesicles (MLV) composed of distearoylphosphatidylcholine (18:0 PC): phosphatidylserine (PS) (7:3 mol ratio) were used. MLV composed exclusively of 18:0 PC were phagocytosed at rates greater than or equal to those of MLV composed of egg phosphatidylcholine (PC):PS, whereas MLV composed only of egg PC were very poorly phagocytosed. Although phagocytosis was enhanced by incorporation of PS into MLV, the inclusion of PS brought about significant enhancement in liposome permeability in the presence of serum. The inclusion of PS, however, was a requirement for the delivery of MLV to the lungs following i.v. injection into mice whether used in conjunction with 18:0 PC or egg PC. Activation of macrophages to become tumoricidal against syngeneic tumor cells wtih liposome-encapsulated MDP was superior in both degree and duration when MLV composed of 18:0 PC:PS (7:3 mol ratio) were used. MLV were found to be superior to large unilamellar vesicles containing equal amounts of lipid and entrapped MDP. On the other hand, higher levels of macrophage activation were obtained when an equivalent amount of a lipophilic MDP derivative, muramyltripeptide:phosphatidylethanolamine, was incorporated into the liposome bilayer irrespective of whether the adjuvant was incorporated in liposomes composed of 18:0 PC:PS or egg PC:PS.

Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes.

We determined whether the presence of phosphatidylserine (PS) in the outer membrane leaflet of human tumor cells correlated with their recognition by activated human monocytes. Three tumorigenic cell lines, A375 melanoma and A431 and Colo-16 carcinomas, and a normal human epidermal keratinocyte line (NHEK) were incubated with monocytes activated to the tumoricidal state by gamma-interferon and lipopolysaccharide. Activated human monocytes bound to and lysed all tumorigenic targets, while the nontumorigenic NHEK were neither bound nor killed. Semiquantitative analysis of PS in the outer leaflet of the cells revealed that the tumorigenic cells expressed 3-7-fold more PS than did the nontumorigenic NHEK. To determine whether enhanced PS expression on the tumor cells was responsible for their recognition by activated monocytes, NHEK were supplemented with exogenously supplied analogues of PS and phosphatidylcholine. PS-labeled NHEK but not phosphatidylcholine-labeled nor control NHEK bound to activated human monocytes. These results suggest a role for PS in monocyte recognition of tumor cells.

Activation of tumoricidal properties in human blood monocytes by liposomes containing lipophilic muramyl tripeptide.

Peripheral blood monocytes were isolated from normal human donors by separation on a continuous Percoll gradient and adherence to yield preparations of blood monocytes with a high degree of purity (greater than 99%). The monocytes were incubated in vitro with medium alone or with multilamellar liposomes that contained either a lipophilic derivative of muramyl dipeptide, muramyl tripeptide (MTP-PE), or medium. The cytotoxic properties of the monocytes were assessed by an in vitro radioisotope release assay against various allogeneic targets. Monocytes that have phagocytosed liposomes containing MTP-PE were rendered tumoricidal. These monocytes lysed cells of three different tumorigenic lines but not cells of two nontumorigenic lines. The ability of MTP-PE-activated human blood monocytes to recognize and selectively lyse neoplastic cells was also demonstrated under cocultivation conditions. We conclude that human blood monocytes can be rendered tumoricidal after interaction with liposomes containing MTP-PE.

Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer.

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Transbilayer movement of phosphatidylserine in erythrocytes. Inhibitors of aminophospholipid transport block the association of photolabeled lipid to its transporter.

The ability to cross-link [125I]iodo-azido-phosphatidylserine (125I-N3-PS) to the putative 32-kDa aminophospholipid transporter of human red blood cells (RBC) has been examined by SDS-PAGE. In the absence of transport inhibitors, 125I-N3-PS preferentially labeled the 32-kDa polypeptide, whereas treatment of the cells with pyridyldithioethylamine (PDA), a potent inhibitor of the aminophospholipid translocase, abrogated the association of the probe to this protein. ATP-depletion, low temperature, and diamide or 5,5'-dithiobis(2-nitrobenzoic acid), inhibitors that oxidize an endofacial sulfhydryl distinct from the PDA-sensitive site (Connor, J. and Schroit, A.J. (1990) Biochemistry 29, 37-43), also blocked association of the PS analogue to the protein. Once 125I-N3-PS became associated with the transporter, however, only PDA was able to partially displace it. These data suggest that sulfhydryl reactive reagents inhibit PS transport by blocking the association of PS with its transporter, a process that is also ATP- and temperature-dependent.

Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems.

Large unilamellar vesicles (LUV) that contained a fluorescent analog of phosphatidylserine (NBD-PS) were used in model systems to determine the feasibility of employing phosphatidylserine decarboxylase (PS-decarboxylase) to generate asymmetric vesicles and to determine the transbilayer distribution of PS. PS-decarboxylase prepared by sonication of Escherichia coli JA 200 pLC 8-47 was found to be stable in detergent-free buffers and catalyzed the conversion of NBD-PS to NBD-phosphatidylethanolamine (NBD-PE). PS-decarboxylase was capable of decarboxylating virtually all of the NBD-PS present in the outer leaflet of LUV containing a symmetric or asymmetric (outside only) distribution of NBD-PS, but not NBD-PS present in the inner leaflet of the vesicles. The ability of PS-decarboxylase to decarboxylate only NBD-PS located in the outer leaflet of the vesicles was independently verified by resonance energy transfer (between NBD-PS and (lissamine) rhodamine B-labeled phosphatidylethanolamine) and by derivatization with trinitrobenzenesulfonic acid (TNBS). These techniques revealed that the exchangeable pool (the fraction of NBD-PS on the outer leaflet) and the respective fraction of Tnp-(NBD-PS) formed were equivalent to the extent of PS-decarboxylase-mediated decarboxylation of NBD-PS to NBD-PE. These results show that PS-decarboxylase can be used to generate asymmetric vesicles (i.e., PS inside, PE outside) and determine the intrabilayer distribution of PS in model membranes.

Maintenance of lipid asymmetry in red blood cells and ghosts: effect of divalent cations and serum albumin on the transbilayer distribution of phosphatidylserine.

The maintenance of lipid asymmetry in the plasma membrane of human red blood cells (RBC) was investigated by assessing the equilibrium distribution of exogenously inserted NBD-labeled phosphatidylserine (PS) and endogenous PS in RBC and hypotonically lysed ghosts. PS distribution was determined by the ability to 'back-exchange' NBD-lipids into acceptor membranes and bovine serum albumin, and by prothrombinase complex assay for endogenous PS. To maintain the normal asymmetric distribution of PS in RBC, ghosts required Mg2+ in the lysis buffer. The inclusion of Ca2+, even in the presence of Mg2+ resulted in complete randomization of endogenous and exogenously inserted PS. These results indicate that NBD-labeled PS analogs faithfully monitor the distribution of endogenous PS during ghost preparation. In contrast, treatment of RBC with bovine serum albumin had no effect on the distribution of endogenous PS, although it resulted in a time-dependent movement of NBD-labeled PS from the inner to the outer leaflet (flop). This phenomenon was dependent on continuous incubation in the presence of albumin and could not be duplicated when pure acceptor membranes were used.

beta(2)-glycoprotein I-dependent alterations in membrane properties.

beta(2)-Glycoprotein I (beta(2)GP1), a 50 kDa serum glycoprotein, binds anionic phospholipids and plays a role in phosphatidylserine (PS)-dependent coagulation and apoptotic processes. To characterize the molecular consequences that occur to target membranes upon binding of beta(2)GP1, the interaction between beta(2)GP1 and PS-containing vesicles was investigated by fluorescent spectroscopy. Membranes containing pyrene-labeled lipid showed that binding of beta(2)GP1 induced a decrease in excimer/monomor ratios (E/M) of the target membrane. Although these membrane alterations occurred in isotonic buffer, the effects were greater in low ionic strength buffer and were coincident to membrane precipitation. In contrast, increases in membrane polarization were only seen in low ionic strength buffer. Analysis of beta(2)GP1 binding kinetics by resonance energy transfer between fluorescein-labeled beta(2)GP1 and rhodamine-containing PS vesicles revealed a two-component process: (1) a primary and rapid binding via the C-terminus that occurred <2 s in both isotonic and low ionic strength buffers, and (2) a sequential binding of the N-terminus that was approximately 100-fold slower in low ionic strength solution. Taken together, these data suggest that beta(2)GP1 alters the fluidity and membrane polarization of its target membrane, which in low ionic strength buffer is of sufficient magnitude to induce precipitation.

Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity.

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.

Colocalization of Rh polypeptides and the aminophospholipid transporter in dilauroylphosphatidylcholine-induced erythrocyte vesicles.

Cytoskeleton-free vesicles released from human red blood cells (RBC) transport exogenously supplied aminophospholipid analogues from the vesicle's outer to inner leaflet at rates comparable to those of normal RBC (Beleznay et al. (1993) Biochemistry 32, 3146-3152). Because polypeptides associated with the Rh blood group system have been implicated in the transbilayer movement of phosphatidylserine (PS), we investigated the relationship and co-localization of the aminophospholipid translocase and Rh in dilauroylphosphatidylcholine-induced RBC vesicles. The transbilayer movement of fluorescent (NBD-PS) and photoactivatable (125I-N3-PS) PS in RBC vesicles was ATP-and temperature-dependent. Inhibition of PS transport by sulfhydryl reagents could be accomplished by direct vesicle treatment or by treating RBC before vesiculation. In the case of diamide- and pyridyldithioethylamine-mediated inhibition, NBD-PS transport could be restored by reduction with dithiothreitol, indicating that the movement of the PS transporter into the emerging vesicle was independent of the oxidative status of membrane sulfhydryls. The presence of Rh polypeptides in the vesicles was verified by direct immunoprecipitation of isotopically-labeled Rh and semi-quantified by antibody adsorption assays. Similar to the movement of the PS transporter, localization of Rh polypeptides in the vesicle membrane was independent of the red cell's oxidative status. These results show that the PS translocase and Rh-related proteins colocalize in RBC vesicles suggesting that these proteins may be members of a multicomponent complex that plays a role in lipid movement and the generation of membrane lipid asymmetry.

Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase.

Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes.

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.

Phosphatidylserine externalization during differentiation-triggered apoptosis of erythroleukemic cells.

K562 erythroleukemia cells undergo apoptosis when induced to differentiate along the erythroid lineage with hemin. This event, characterized by DNA fragmentation, correlated with downregulation of the survival protein, BCL-xL, and decrease in mitochondrial transmembrane potential (deltapsi[m]) that ultimately resulted in cell death. Reorientation of phosphatidylserine (PS) from the cells inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase was observed upon hemin-treatment. Constitutive expression of BCL-2 did not inhibit hemin-induced alterations in lipid asymmetry or decrease in deltapsi[m], and only moderately prevented DNA fragmentation. BCL-2, on the other hand, effectively inhibited actinomycin D-induced DNA fragmentation, the appearance of PS at the cells outer leaflet and the decrease in deltapsi[m]. The caspase inhibitor, z.VAD.fmk, blocked DNA fragmentation by both hemin and actinomycin D, but inhibited PS externalization only in the actinomycin D-treated cells. These results suggest that, unlike pharmacologically-induced apoptosis, PS externalization triggered by differentiation-induced apoptosis occurs by a mechanism that is associated with a decrease in deltapsi[m], but independent of BCL-2 and caspases.

Membrane phospholipid asymmetry: host response to the externalization of phosphatidylserine.

Membrane phospholipid asymmetry is an important regulator of cellular function and homeostasis. The activities of lipid transporters are contributing factors to the regulation of membrane lipid composition over the lifespan of the cell. Alterations in the activities of these proteins result in the movement of phosphatidylserine to the cell's outer leaflet. This promotes several physiologic responses including initiation of the coagulation cascade and cell recognition by the reticuloendothelial system.

Quantitative in vitro phagocytic rate measurements.

A model for the calculation of phagocytic rate constants based on the assumption that the process of phagocytosis can be treated in a manner analogous to chemical reactions of the first order is suggested. By employing such an approach, accurate rate constants of ingestion of in vitro cultivated macrophages can be obtained following minimal time intervals from the onset of phagocytosis.

A rapid and sensitive technique for the detection of Fc receptors on macrophages.

A rapid sensitive technique for the production of macrophage EA rosettes employing centrifugal sedimentation of opsonized erythrocytes is described. Using this method, a sensitive rosette formation-inhibition assay for the determination of anti-macrophage antibody titers has been developed.

Estimation of plasma beta-2-glycoprotein levels by competitive ELISA.

Beta-2-glycoprotein I (beta2GPI), a 50-kDA serum glycoprotein that binds negatively charged phospholipids plays a role in coagulation, thrombosis, and the clearance of phosphatidylserine expressing cells. Because of its recently recognized role in several autoimmune responses, we have developed a method that quantifies plasma beta2GPI levels by using a competitive ELISA assay. When combined with data from a standard ELISA, this method determines the concentration of free beta2GPI and the fraction of antibody-bound beta2GPI thereby facilitating quantification of total antigen in individuals with autoimmune antibodies. Standard competitive inhibition ELISA was compared with this method, which uses known amounts of standard beta2GPI added to the plasma as an internal standard. Identical results were obtained with both methods for plasma samples from normal individuals that did not contain blocking antibodies. Analysis of plasma from antiphospholipid syndrome patients (patients with autoantibodies to beta2GPI) by the internal standard method, however, resulted in significantly lower apparent beta2GPI levels indicating that a substantial fraction of the plasma beta2GPI was bound by antibody.