The role of beta2-glycoprotein I in liposome-hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.     

The role of β2-glycoprotein I in liposome-hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The ∼55-kDa protein was identified as β₂-glycoprotein I (β₂GPI) by Western blotting. Despite the high affinity of β₂GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, β₂GP1 inhibits, rather than enhances, liposome uptake by liver cells.     

The role of apolipoprotein E in the elimination of liposomes from blood by hepatocytes in the mouse

We evaluated the role of apolipoprotein E (apoE) in the clearance of neutral and negatively charged liposomes by hepatocytes in apoE-deficient mice. Negatively charged liposomes were cleared at identical rates in apoE-deficient and wild-type mice; neutral liposomes were cleared at a 3.6-fold slower rate in apoE-deficient mice. ApoE deficiency did not affect hepatic uptake of negatively charged liposomes but lowered that of neutral liposomes >5-fold. Hepatocyte uptake of neutral liposomes was reduced >20-fold in apoE-deficient mice; that of negatively charged liposomes remained unchanged. We conclude that uptake of neutral liposomes by hepatocytes is nearly exclusively apoE-mediated.     

Oligonucleotide Therapy for Hematological Malignancies

Abstract

In this contribution we describe and discuss (mostly published) experiments providing evidence favoring a decisive role of opsonizing plasma proteins in the removal of liposomes from the vascular compartment. Our conclusion is that cells will only bind and take up liposomes if they are anatomically accessible for the liposomes and if, in addition, they possess (specific) receptors for one or more proteins adsorbing to the liposomal surface. The relative contribution of each cell type fulfilling these criteria to over-all liposome clearance is dictated by the total number of cells in that population, the density of the receptor(s) involved, the affinity of those receptors for their respective ligands and the localization in the vascular system. It is concluded that only a few cell populations meet the criteria. Most are excluded because of inaccessibility while of the accessible ones several lack the proper opsonin receptors for significant liposome uptake. The significance of localization in the vasculature is illustrated by the hepatocytes whose accessibility is limited by the fenestrations in the endothelial lining of the liver sinusoids. The opsonin concept is extrapolated to cells other than macrophages; for example, the existence of hepatocyte-specific opsonins is proposed in order to explain the efficient uptake of small liposomes by this cell population. Because of their virtually complete lack of participation in plasma elimination of liposomes, some readily accessible cell types, such as the circulating blood cells and the vascular endothelial cells, are proposed to lack appropriate receptors. According to the views developed in this contribution the specialty of cells involved in liposome clearance therefore lies in the condition that they possess one or more receptors for plasma-derived proteins that spontaneously adsorb to the liposomal surface. One possible exception to the opsonin-determined concept is the fate of phosphatidylserine-containing liposomes. These may be cleared without or even in spite of involvement of opsonins, by virtue of a PS-specific receptor on macrophages.     

Comparison of two liposome fusion assays monitoring the intermixing of aqueous contents and of membrane components

Divalent cation-induced fusion of large unilamellar vesicles (approx. 0.1 micron diameter) made of phosphatidylserine (PS) or phosphatidylglycerol (PG) has been studied. Intermixing of aqueous contents during fusion was followed by the Tb/dipicolinic acid fluorescence assay, and intermixing of membrane components by resonance energy transfer between fluorescent lipid probes. Both assays gave identical threshold concentrations for Ca2+, which were 2 mM for PS and 15 mM for PG. The dependencies of the initial rate of fusion on the concentration of PG vesicles determined by either assay were identical, the order of this dependence being 1.2 in the concentration range of 5-200 microM lipid. For PS liposomes, this order was found to be 1.5 in the fluorescent lipid assay. No leakage of contents was detected during the fusion of PG vesicles. Mg2+ inhibited the Ca2+-induced fusion of PS vesicles, but did not cause any fusion by itself, consistent with previous results with the Tb/dipicolinic acid assay.     

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE^−/− mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.     

Intrahepatic Distribution of Long-Circulating Liposomes Containing Polyethylene Glycol) Distearoyl Phosphatidylethanolamine

Abstract

We investigated the intrahepatic distribution in rats of liposomes of 85 or 130 nm diameter, which were sterically stabilized with a polyethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) so as to increase their circulation time in blood. Various times after intravenous injection of radiolabeled ([³H-]cholesterylether) liposomes, parenchymal and non-parenchymal cells of the liver were isolated and their radioactivity content was determined. Control liposomes of 85 nm without PEG-PE distributed in an approximately 80:20 ratio to hepatocytes (H) and macrophages (M), respectively; the 130-nm control liposomes showed a 50:50 H/M distribution. Incorporation of PEG-PE reduced the rate of total liver uptake about 4-fold for liposomes of either size and shifted the H/M ratio to 60:40 for the smaller vesicles and to 40:60 for the larger ones. For both liposome sizes, PEG-PE apparently causes a shift in intrahepatic distribution in favor of the macrophages. It is concluded that PEG-PE has a stronger inhibitory effect on liposome uptake by hepatocytes than on uptake by macrophages. Attempts to shift liposome uptake more in favor of hepatocytes, by incorporation of lactosylceramide, failed. This compound, although causing an increase in hepatic uptake, particularly for the 130-nm liposomes, shifted the H/M ratio further towards the macrophages. We conclude that the galactose moiety of the glycolipid is sufficiently exposed on the surface of (PEG-PE)-containing liposomes to allow interaction with the galactose-binding lectin at the surface of the liver macrophage and that the extent of exposure is dependent on vesicle size.     

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.     

Formation of Fenestrae in Murine Liver Sinusoids Depends on Plasmalemma Vesicle-Associated Protein and Is Required for Lipoprotein Passage

Liver sinusoidal endothelial cells (LSEC) are characterized by the presence of fenestrations that are not bridged by a diaphragm. The molecular mechanisms that control the formation of the fenestrations are largely unclear. Here we report that mice, which are deficient in plasmalemma vesicle-associated protein (PLVAP), develop a distinct phenotype that is caused by the lack of sinusoidal fenestrations. Fenestrations with a diaphragm were not observed in mouse LSEC at three weeks of age, but were present during embryonic life starting from embryonic day 12.5. PLVAP was expressed in LSEC of wild-type mice, but not in that of Plvap-deficient littermates. Plvap^-/- LSEC showed a pronounced and highly significant reduction in the number of fenestrations, a finding, which was seen both by transmission and scanning electron microscopy. The lack of fenestrations was associated with an impaired passage of macromolecules such as FITC-dextran and quantum dot nanoparticles from the sinusoidal lumen into Disse''s space. Plvap-deficient mice suffered from a pronounced hyperlipoproteinemia as evidenced by milky plasma and the presence of lipid granules that occluded kidney and liver capillaries. By NMR spectroscopy of plasma, the nature of hyperlipoproteinemia was identified as massive accumulation of chylomicron remnants. Plasma levels of low density lipoproteins (LDL) were also significantly increased as were those of cholesterol and triglycerides. In contrast, plasma levels of high density lipoproteins (HDL), albumin and total protein were reduced. At around three weeks of life, Plvap-deficient livers developed extensive multivesicular steatosis, steatohepatitis, and fibrosis. PLVAP is critically required for the formation of fenestrations in LSEC. Lack of fenestrations caused by PLVAP deficiency substantially impairs the passage of chylomicron remnants between liver sinusoids and hepatocytes, and finally leads to liver damage.     

Liposome Internalization by Isolated Rat Hepatocytes

Abstract

We studied the in vitro interaction and the endocytic process of peroxidase-loaded liposomes with isolated rat hepatocytes maintained in suspension culture. We report morphological (both at light and electron microscopy) and biochemical evidence that cationic egg PC (egg phosphatidylcholine)-liposomes are taken up by isolated rat hepatocytes via an endocytic pathway. The incubation of 2.5 mM liposomal lipids for at least 4 hours was not cytotoxic to the cells. The uptake of peroxidase-fluoresceine isothiocyanate conjugate-liposomes was not distributed homogeneously among the hepatocyte population. However the hepatocytes which have apparently internalized greater amount of probe were not damaged since cell shape and integrity of the membrane are retained as evaluated by conventional light microscopy. Therefore the fusion between liposomes and hepatocytes seems to be dependent on the viability of the isolated hepatocytes.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.     

Synthesis of phospholipid-conjugated bile salts and interaction of bile salt-coated liposomes with cultured hepatocytes

To examine the possibility of targeting liposomes to hepatocytes via bile salts, the bile salt lithocholyltaurine was covalently linked to a phospholipid. The isomeric compounds disodium 3alpha-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy)-5beta-cholan-24-oyl-2'-aminoethansulfonate and disodium 3beta-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy-5beta-cholan-24-oyl-2'-aminoethansulfonate (DSPE-3beta-LCT) were synthesized and incorporated into liposomal membranes. Confocal laser scanning microscopy studies showed that bile salt-bearing liposomes (BSLs) attach to the surface of rat hepatocytes in culture. Studies with radioactively labeled liposomes revealed that the bile salt linked via the 3beta-conformation resulted in a higher attachment efficiency than that with the 3alpha-derivative. In the presence of BSLs corresponding to 2 mM liposomal phosphatidylcholine, uptake of 50 microM cholyltaurine (CT) into hepatocytes was reduced by approximately 40% by the 3beta-derivative and by approximately 17% by the 3alpha-derivative. When added simultaneously with the liposomes, CT up to 75 microM inhibited the binding of DSPE-3beta-LCT-bearing liposomes. By contrast, increasing concentrations reversed this inhibition and resulted in an increased bile salt-mediated binding. The same was true when CT was added 10 min before the liposomes were added. The attachment of BSLs to the surface of hepatocytes opens up promising possibilities for hepatocyte-specific drug delivery. More generally, not only substrates for cellular endocytosing receptors but also substrates for cellular carrier proteins should be suitable ligands for the cell-specific targeting of nanoscale particles such as liposomes.     

The effect of a water-soluble tris-galactoside terminated cholesterol derivative on the in vivo fate of small unilamellar vesicles in rats

When the water-soluble cholesterol derivative, N-[tris [(beta-D-galactopyranosyloxy)methyl]methyl]-N alpha-[4-(5-cholesten-3 beta-yloxy)succinyl]glycinamide (tris-gal-chol) (Kempen et al. (1984) J. Medicin. Chem. 27, 1306-1312) is added as an aqueous micellar solution to a dispersion of small unilamellar phospholipid vesicles it rapidly associates with the vesicles, without causing significant leakage of liposome contents. Incorporation of 10 mol% tris-gal-chol in the liposomal membrane caused a substantial increase in the rate and extent of rat liver uptake and a shift in intrahepatic distribution of an intravenously administered dose of liposomes. For neutral liposomes composed of equimolar amounts of cholesterol and sphingomyelin incorporation of tris-gal-chol led to a 7-fold increase in total liver uptake, which was mainly accounted for by an increase in uptake by the Kupffer cells (12-fold) and by only a small increase in uptake by the hepatocytes (1.4-fold). The increased liver uptake is blocked by preinjection of N-acetyl-D-galactosamine and not affected by preinjection of N-acetyl-D-glucosamine. This indicates that the increased interaction of liposomes as a result of tris-gal-chol incorporation is mediated by galactose-specific recognition sites on both Kupffer cells and hepatocytes. Targeting of liposomes to the asialoglycoprotein receptor of the hepatocytes is thus frustrated by the highly active galactose-specific receptor on Kupffer cells. Comparable results on lactosylceramide incorporation into liposomes were recently reported by us (Spanjer et al. (1984) Biochim. Biophys. Acta 774, 49-55).     

A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6- NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non- endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.     

Association of Methotrexate Encapsulated in Negatively Charged Liposomes with Cells at Nanomolar Lipid Concentrations

Abstract

An in vitro liposome-cell association system has been developed that will allow the study of uptake and metabolism of liposomes by cultured cells at nanomolar lipid concentrations. The fate of cell associated liposomes is followed through the liposome encapsulated marker, methotrexate. Detection is based on the inhibition of dihydrofolate reductase by methotrexate, after its release from cells through boiling. Methotrexate in phospha-tidylglycerol (PG) liposomes is taken up by cells and then subsequently lost from the cells. Uptake is partially blocked by monensin. Loss from the cells is blocked by metabolic inhibitors, monensin, ammonium chloride, and chloroquine. Methotrexate in distearoylphosphatidylglycerol (DSPG) liposomes is taken up by cells slowly, and there is minimal lost of methotrexate after uptake. Pulse studies show that metabolism of PG liposomes after endocytosis is occurring at a much higher rate than that of DSPG liposomes, and substantial retention of encapsulated methotrexate occurs for both liposome compositions.     

Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6

Gas6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains g-carboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that Gas6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of Gas6 with PS, we examined the effect of Gas6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that Gas6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of Gas6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that Gas6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of Gas6. These findings suggest that Gas6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, Gas6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells.     

Uptake of liposomes containing phosphatidylserine by liver cells in vivo and by sinusoidal liver cells in primary culture: in vivo-in vitro differences

The interaction with liver cells of liposomes containing different mol fractions of phosphatidylserine was investigated in vivo and in vitro. Increasing the amount of liposomal phosphatidylserine from 10 to 30 mol% leads to a faster blood disappearance of the liposomes. Within the liver, which is mainly responsible for this elimination, these liposomes are only taken up by the hepatocytes and Kupffer cells. By contrast, sinusoidal endothelial cells, in vitro, do bind and internalize liposomes containing >/=30% phosphatidylserine at least as actively as Kupffer cells. The uptake by endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules, suggesting the involvement of scavenger receptors. The lack of liposome uptake by endothelial cells under in vivo conditions can be attributed to plasma effects since addition of various sera caused severe reduction of in vitro uptake of liposomes. In vivo the phosphatidylserine head groups may be masked by plasma proteins adsorbed to the liposomal surface, thus preventing recognition by receptors, which are intrinsically able to recognize phosphatidylserine.     

Interaction between phosphatidylserine and the phosphatidylserine receptor inhibits immune responses in vivo

Phosphatidylserine (PS) on apoptotic cells promotes their uptake and induces anti-inflammatory responses in phagocytes, including TGF-beta release. Little is known regarding the effects of PS on adaptive immune responses. We therefore investigated the effects of PS-containing liposomes on immune responses in mice in vivo. PS liposomes specifically inhibited responses to Ags as determined by decreased draining lymph node tissue mass, with reduced numbers of total leukocytes and Ag-specific CD4(+) T cells. There was also a decrease in formation and size of germinal centers in spleen and lymph nodes, accompanied by decreased levels of Ag-specific IgG in blood. Many of these effects were mimicked by an agonistic Ab-specific for the PS receptor. TGF-beta appears to play a critical role in this inhibition, as the inhibitory effects of PS were reversed by in vivo administration of anti-TGF-beta Ab. PS-containing liposomes did not appear to directly inhibit dendritic cell maturation in vitro in response to a variety of stimuli, nor did it prevent their migration to regional lymph nodes in vivo, suggesting that the inhibitory effects may have resulted from complicated interactions between tissue cells and dendritic cells, subsequently inhibiting their ability to productively activate T lymphocytes.     

Effects of apolipoproteins A-IV and A-I on the uptake of phospholipid liposomes by hepatocytes

We examined the effects of apolipoproteins A-IV and A-I on the catabolism of whole particles by hepatoma G2 cells and cultured primary hepatocytes. For this type of experiment, high density lipoprotein is unsuitable, because all of its lipid and protein components independently dissociate and exchange and hence poorly trace whole particle catabolism. We therefore used phosphatidylcholine liposomes with radioactive tracers entrapped within their aqueous cores. Apolipoproteins A-IV, A-I, or E added to liposomes became liposome-associated and produced no detectable release of encapsulated label. As a positive control, apolipoprotein E doubled the uptake of labeled liposomes by hepatoma cells, compared to apolipoprotein-free controls, and this increase could be blocked by the addition of excess unlabeled low density lipoprotein. Degradation of labeled liposomes by hepatoma cells was increased 6-fold by the addition of apolipoprotein E. In contrast, neither apolipoprotein A-IV nor A-I increased cellular uptake or degradation of the particles. Similar results were obtained with primary hepatocytes. In studies using apolipoprotein combinations, apolipoproteins A-IV and A-I were each able to displace apolipoprotein E from liposomes and thereby reduce cellular uptake. Our data indicate that apolipoproteins A-IV and A-I do not facilitate uptake or degradation of whole particles by liver-derived cells in vitro. However, these apolipoproteins may modulate receptor-mediated uptake of particles by reducing the amount of particle-bound apolipoprotein E.