Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.     

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type     

Targeting of liposomes by covalent coupling with ecto-NAD+-glycohydrolase ligands

We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD⁺-glycohydrolase, was substituted at N⁶ with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to preformed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD⁺-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand.     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages.

Serum proteins, acting as opsonins, are believed to contribute significantly to liposome-macrophage cell association and thus regulate liposome uptake by cells of the mononuclear phagocytic system (MPS). We studied the effect of serum protein on binding and uptake of phosphatidylglycerol-, phosphatidylserine-, cardiolipin-, and N,N-dioleyl-N,N-dimethylammonium chloride- (DODAC) containing as well as poly(ethylene glycol)- (PEG) containing liposomes by mouse bone marrow macrophages in vitro. Consistent with the postulated surface-shielding properties of PEG, protein-free uptake of liposomes containing 5 mol% PEG and either 20 mol% anionic phosphatidylserine or 20 mol% cationic DODAC was equivalent to uptake of neutral liposomes. In contrast to previous reports indicating that protein adsorption to liposomes increases uptake by macrophages, the presence of bound serum protein did not increase the uptake of these liposomes by cultured macrophages. Rather, we found that pre-incubating liposomes with serum reduced the uptake of liposomes containing phosphatidylserine. Surprisingly, serum treatment of PEG-containing liposomes also significantly reduced liposome uptake by macrophages. It is postulated that, in the case of phosphatidylserine liposomes, the bound serum protein can provide a non-specific surface-shielding property that reduces the charge-mediated interactions between liposomes and bone marrow macrophage cells. In addition, incubation of PEG-bearing liposomes with serum can result in a change in the properties of the PEG, resulting in a surface that is better protected against interactions with cells.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

Enhanced binding of phosphatidylserine-containing lipid vesicle targets to RAW264 macrophages

Summary Phosphatidylserine was found to significantly enchance the binding of phospholipid vesicles to RAW264 macrophages. We have measured the kinetics of non-specific uptake of unilamellar vesicles as a function of phosphatidylserine concentration in these model target membranes. Dimyristoylphosphatidylcholine was the principle component of these phospholipid vesicles. In most experiments, radiolabeled phospholipid and 1 mol % each of both a fluorescent phospholipid and a hapten-containing lipid headgroup were utilized. In the presence of specific anti-hapten antibody phosphatidylserine-containing vesicles are rapidly taken up via phagocytosis. The antibody-independent non-specific uptake of phosphatidylserine-free vesicles was low, as previously reported. However, the presence of 5 mol % phosphatidylserine dramatically enhanced the uptake of phospholipid vesicles by macrophages. This uptake was shown to be principally due to binding to the macrophage surface. Incubation of macrophages in the presence of sodium azide or at 4°C, conditions which are known to inhibit phagocytosis, do not influence the uptake of the lipid vesicles. Fluorescence video-intensification microscopy was used to observe the interaction of carboxyfluorescein-loaded vesicles with macrophages. Fluorescence could not be observed when using phosphatidylserine-free vesicles. However, phosphatidylserine-containing vesicles can be observed bound to the cell periphery. Intracellular fluorescence could not be observed. The binding of phosphatidylserine-containing vesicles was enhanced roughly four-fold over phosphatidylserine because the effect could not be observed with membranes containing 1 mol % or 2.5 mol% phosphatidylserine. In addition, the binding enhancement required the presence of divalent cations in the incubation medium.Abbreviations DMPC dimyristoylphosphatidylcholine - PS phosphatidylserine - DNP-PE dinitrophenyl---minocaproyl-phosphatidylethanolamime - NBDPE N-4-nitrobenzo-2-oxa-1, 3-diazole phosphatidylethanolamine - EDTA ethylenediaminetetraacetic acid     

Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

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.     

Targeting of lactosylceramide-containing liposomes to hepatocytes in vivo

Incorporation of 8 mol% lactosylceramide in small unilamellar vesicles consisting of cholesterol, dimyristoylphosphatidylcholine and phosphatidylserine in a molar ratio of 5:4:1 and containing [³H]inulin as an aqueous-space marker resulted in a 3-fold decreased half-life of the vesicles in blood and a corresponding increase in liver uptake after intracardial injection into rats. The increase in liver uptake was mostly accounted for by an enhanced uptake in the parenchymal cells, while the uptake by the non-parenchymal cells was only slightly increased. The uptake of both the control and the glycolipid-containing vesicles by the non-parenchymal cell fraction could be attributed completely to the Kupffer cells; no radioactivity was found in the endothelial cells. The effect of lactosylceramide on liver uptake and blood disappearance of the liposomes was effectively counteracted by desialylated fetuin, injected shortly before the liposome dose. This observation supports the notion that a galactose-specific receptor is involved in the liver uptake of lactosylceramide liposomes.     

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.     

Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues.     

Effect of cholesterol on vesicle bilayer geometry of choline plasmalogen and comparison with dialkyl-, alkylacyl- and diacyl-glycerophosphocholines

Small unilamellar vesicles containing alkenylacyl-, alkylacyl-, dialkyl- or diacyl-glycerophosphocholine were prepared by sonication. Their size was determined from the average internal volume after chromatography on Sepharose 2B and from 31P-NMR linewidths. Alkenylacyl glycerophosphocholine (choline plasmalogen) was found to form the largest vesicles. By addition of 30 mol% cholesterol, the size of plasmalogen vesicles, but not of those containing the alkyl and acyl analogue lipids, was significantly increased. The presence of 50 mol% sterol led to highly increased vesicle sizes of alkylacyl, dialkyl and diacyl-glycerophosphocholine. Mixtures of plasmalogens with 50 mol% cholesterol did not form unilamellar vesicles upon sonication. Bilayer thickness and surface area per phospholipid molecule were determined by small angle X-ray scattering and measurement of partial specific volumes. There is little difference between alkenylacyl glycerophosphocholine and the corresponding diacyl-analog, whereas bilayers consisting of dioleoyl glycerophosphocholine are significantly thinner. Correspondingly their molecular surface area is by about 8% larger than that of the mixed-chain diradyl glycerophosphocholine, since the partial molar volumes are similar for all vesicles tested.     

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol)

Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces.     

Lymph node macrophages, but not spleen macrophages, express high levels of unmasked sialoadhesin: implication for the adhesive properties of macrophages in vivo

Sialoadhesin is a macrophage-restricted adhesion molecule that recognizes N-acetylneuraminylalpha2-3galactose structure. We prepared a multivalent neoglycoprotein probe carrying this oligosaccharide and characterized the binding activity of sialoadhesin on native rat macrophages. Macrophages from mesenteric and axillar lymph nodes exhibited 36-fold higher activity than those from the spleen. The K(d) values of the probe binding to macrophages of the two organs were indistinguishable (1-2 nM), whereas the B(max) value of lymph node macrophages was markedly higher than that of splenic macrophages. Western blot analysis revealed that the quantity of sialoadhesin present in lymph node macrophages was 25-fold higher than in splenic macrophages. High cell surface expression of sialoadhesin on lymph node macrophages was also shown by flow cytometry. To examine the "masking" of sialoadhesin by endogenous sialoglycoconjugates, we treated macrophages with sialidase before measuring the probe binding. After sialidase treatment, the binding activity of splenic macrophages increased fourfold, whereas that of lymph node macrophages did not increase. In conclusion, we have identified macrophages expressing high levels of unmasked sialoadhesin in lymph nodes. The unmasked forms on these macrophages are available for sialoadhesin-dependent adhesive functions, unlike the masked forms on the majority of splenic macrophages.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.     

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).