Cholesterol-dependent tetanolysin damage to liposomes.

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24 degrees C compared to 32 degrees C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.     

Cholesterol-dependent tetanolysin damage to liposomes

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24°C compared to 32°C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.     

Influence of temperature on complement-dependent immune damage to liposomes

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.     

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.     

The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes.

This paper describes experiments showing the importance of the fatty acid chain length on the barrier properties of liposomal bilayers, prepared from saturated lecithins, under conditions of lateral phase separation. 1. Above the gel to liquid crystalline phase transition temperature, liposomes prepared from saturated lecithins with 14 or more carbon atoms per acyl chain exist as stable bilayers, which are practically impermeable to ions. 2. At temperatures well above the transition temperature dilauroyl phosphatidylcholine liposomes exhibited osmotic shrinkage, which was dependent on the ionic size of the solute used to bring about the osmotic gradient, indicating that the permeation through these less stable bilayers takes place mainly via individual diffusion of the permeating ions. 3. An enhanced release of trapped potassium from liposomes was demonstrated in the vicinity of the transition temperature. The extent of the increase, however, depended strongly on the length of the paraffin chain. 4. From measurements of the shrinkage behaviour of liposomes in the vicinity of the transition temperature it is concluded that the increased permeability decreases with increasing diameter of the permeating ion. This finding implies that the increased permeability at the transition temperature cannot be ascribed to "macroscopic" rupture of the liposomal membrane. The maximum permeability in the vicinity of the Tc is discussed in terms of probability and size distribution of statistical pore formation at the boundaries of liquid and solid domains.     

C-reactive protein-mediated suppression of nephrotoxic nephritis: role of macrophages, complement, and Fcgamma receptors

C-reactive protein (CRP) is a member of the pentraxin family of proteins and an acute phase reactant. CRP modulates the response to inflammatory stimuli including LPS and C5a. We recently demonstrated that CRP prevents and reverses proteinuria in accelerated nephrotoxic nephritis (NTN). NTN is a model of active inflammatory immune complex-mediated nephritis induced by injection of antiglomerular basement membrane. CRP treatment prevented the induction of NTN in C57BL/6 (B6) mice, increased survival, and reversed ongoing nephritis. Protection was associated with a decrease in IL-1beta and chemokines in the kidney and peritoneal cells as measured by quantitative RT-PCR. However, IL-10(-/-) mice were not protected by CRP either when given before disease onset or when disease activity was maximal. FcgammaRI(-/-) mice developed NTN, but were only transiently protected by CRP treatment. This transient protection was abrogated by cobra venom factor depletion of complement from FcgammaRI(-/-) mice. However, complement depletion did not prevent CRP-mediated protection in B6 mice, and CRP was protective in C3(-/-) mice. The role of macrophages in the protection provided by CRP was tested by treating B6 mice with liposomes containing clodronate. Clodronate-containing liposomes deplete mice of splenic and hepatic macrophages for 5-7 days. Pretreatment of NTN mice with clodronate but not control liposomes completely prevented CRP-mediated protection. These studies suggest that CRP mediates protection from NTN through the induction of IL-10 and that macrophages are required. In addition, FcgammaRI plays an important role but is not the sole mediator of CRP-mediated protection.     

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes.

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.     

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes.

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.     

Effect of fatty acyl domain of phospholipids on the membrane-channel formation of Staphylococcus aureus alpha-toxin in liposome membrane.

By use of carboxyfluorescein-loaded multilamellar liposomes prepared from synthetic phosphatidylcholine (PC) or sphingomyelin and cholesterol in a molar ratio of 1:1, we studied whether or not fatty acyl domain of the phospholipids affects the membrane-damaging action (or channel formation) of Staphylococcus aureus alpha-toxin on the phospholipid-cholesterol membranes. Our data indicated: (1) that toxin-induced carboxyfluorescein-leakage from the liposomes composed of saturated fatty acyl residue-carrying PC and cholesterol was decreased with increasing chain length of the acyl residues between 12 and 18 carbon atoms, although toxin-binding to the liposomes was not significantly affected by the length of fatty acyl residue; (2) that unsaturated fatty acyl residue in PC or sphingomyelin molecule conferred higher sensitivity to alpha-toxin on the phospholipid-cholesterol liposomes, compared with saturated fatty acyl residues; and (3) that hexamerization of alpha-toxin, estimated by SDS-polyacrylamide gel electrophoresis, occurred more efficiently on the liposomes composed of PC with shorter fatty acyl chain or unsaturated fatty acyl chain. Thus, hydrophobic domain of the phospholipids influences membrane-channel formation of alpha-toxin in the phospholipid-cholesterol membrane, perhaps by modulating packing of phospholipid, cholesterol and the toxin in membrane.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. II. Effect of membrane lipid composition in a liposome system

In the first paper of this series, it was shown that a toxin from the sea anemone Stoichactis helianthus increased the permeability of black lipid membranes due to transmembrane channel formation. In the present study, we have used liposomes to examine the reactivity of the toxin with different phospholipids. Membrane damage was assessed by measuring the release of ⁸⁶Rb⁺ and ¹⁴C-labeled membrane lipid. For the different lipids, the rank order of marker release was: sphingomyelin > C18: 2 phosphatidylcholine > C18: 1 phosphatidylcholine > C18: 0 phosphatidylcholine > C16: 0 phosphatidylcholine = C14: 0 phosphatidylcholine. In C14: 0 and C16: 0 phosphatidylcholine liposomes there was no ¹⁴C-labeled lipid release and only 13 to 16% ⁸⁶Rb⁺ release which corresponds to the ⁸⁶Rb⁺ content in the outermost aqueous shell of multilamellar liposomes. This indicates that membrane damage was limited to the outermost bilayer. In liposomes prepared with the other lipids, the extent of release of both markers increased proportionately with the length and the degree of unsaturation of the lipids' acyl side chains. Sphingomyelin liposomes were the most susceptible with 47% of the ¹⁴C-labeled lipid marker and 90% of the ⁸⁶Rb⁺ marker being released. The large extent of ¹⁴C-labeled lipid release is attributed to a detergent-like activity of the toxin which presumably is due to the amphipathic nature of the protein. Thus, the toxin can inflict membrane damage in two ways: (1) channel formation, and (2) detergent action. The importance of one mechanism or the other apparently varies depending on membrane structure and lipid composition.     

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 > C18 : 1 phosphatidylcholine > 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°C and 4°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°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.     

Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artificial membrane systems

Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.     

Antibody-dependent phagocytosis of haptenated liposomes by human neutrophils is dependent on the physical state of the liposomal membrane

In the present study we have examined the response of human neutrophils to specific antibody-dependent stimulation by spin-label haptenated fluid phase and solid phase liposomes. Both fluid and solid liposomal antigens are shown to stimulate the neutrophil respiratory burst to approximately equivalent degrees as assessed by measurement of oxygen consumption or oxidation of [1-14C]glucose to 14CO2. In contrast, release of superoxide and hydrogen peroxide from the neutrophils is stimulated to a significantly greater degree by fluid-phase liposomes than by the equivalent solid-phase liposomes. This apparent discrepancy is shown to be due to an inability of the neutrophils to phagocytose fluid-phase liposomes under conditions in which solid-phase liposomes are readily phagocytosed. A fluorescence assay, which does not depend upon binding measurements, has been developed in order to quantitate liposomal phagocytosis.     

Cholesterol superlattice modulates CA4P release from liposomes and CA4P cytotoxicity on mammary cancer cells

Liposomal drugs are a useful alternative to conventional drugs and hold great promise for targeted delivery in the treatment of many diseases. Most of the liposomal drugs on the market or under clinical trials include cholesterol as a membrane stabilizing agent. Here, we used liposomal CA4P, an antivascular drug, to demonstrate that cholesterol content can actually modulate the release and cytotoxicity of liposomal drugs in a delicate and predictable manner. We found that both the rate of the CA4P release from the interior aqueous compartment of the liposomes to the bulk aqueous phase and the extent of the drug's cytotoxicity undergo a biphasic variation, as large as 50%, with liposomal cholesterol content at the theoretically predicted C(r), e.g., 22.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol for maximal superlattice formation. It appears that at C(r), CA4P can be released from the liposomes more readily than at non-C(r), probably due to the increased domain boundaries between superlattice and nonsuperlattice regions, which consequently results in increased cytotoxicity. The idea that the increased domain boundaries at C(r) would facilitate the escape of molecules from membranes was further supported by the data of dehydroergosterol transfer from liposomes to MβCD. These results together show that the functional importance of sterol superlattice formation in liposomes can be propagated to distal targeted cells and reveal a new, to our knowledge, mechanism for how sterol content and membrane lateral organization can control the release of entrapped or embedded molecules in membranes.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

The influence of retinal on complement-dependent immune damage to liposomes

Retinal was incorporated into liposomes containing dipalmitoyllecithin, cholesterol, dicetyl phosphate and galactocerebroside; the latter substance served as antigen. They were compared to control liposomes, lacking retinal, with regard to glucose release due to complement-dependent immune damage in the presence of anticerebroside serum. The liposomes were indistinguishable from each other in the amount of total glucose trapped, light scattering characteristics and phosphate content. The rate and extent of glucose release in 30 min was inhibited by the incorporation of retinal. In addition, inhibition was directyl related to retinal concentration and was also observed in the presence of a wide range of concentrations of antigen and complement. Damage to liposomes in the presence of either guinea pig or human complement was inhibited by retinal; this was in contrast to the erythrocyte system in which the hemolytic activity of guinea pig complement was inhibited while that of human complement was enhanced by retinal. Addition of retinal to performed liposomes did not influence complement-dependent damage. Inhibition occurred only when retinal was present during the initial formation of the model membranes. Inhibition persisted even after washing the liposomes free of any unincorporated retinal. The data indicate that liposomes may be an excellent model for studying the influences of retinal on complement mechanism in membranes.     

Permeability and integrity properties of lecithin-sphingomyelin liposomes.

The properties of multibilayered liposomes formed from mixtures of sphingomyelin and phosphatidylcholine in varying mole ratio (all containing one mole dicetylphosphate per 10 moles of phospholipids) have been studied. The principal findings are: (1) Over the range 0 to 1 mole fraction sphingomyelin the liposomes exhibit multibilayer structure as visualized by electron microscopy using negative staining. (2) The two phospholipids differ in their interaction with dicetylphosphate in a bilayer structure. In mixtures of the two the effect of sphingomyelin is dominant. (3) The ability of sphingomyelin to form osmotically active liposomes depends on its fatty acid's composition. (4) Liposomes of all mole fractions of sphingomyelin are osmotically active if the C24: 1 fatty acid content of sphingomyelin exceeds 10% of the total acyl residues. The degree of osmotic activity, however, depends upon the molar ratio between the two phospholipids. The highest initial rate of water permeability was found for lecithin liposomes. The maximal change of volume by osmotic gradients was obtained for liposomes composed of 1:1 lecithin to sphingomyelin (mole ratio). (5) Permeability to glucose increased with increasing lecithin mole fraction. (6) Liposomes composed of 1:1 lecithin to sphingomyelin have the largest aqueous volume per mole of phospholipid as measured by glucose trapping. (7) The osmotic fragility of liposomes made of sphingomyelin is higher than for those made of lecithin but the highest osmotic fragility was obtained for liposomes containing lecithin and sphingomyelin in 1:1 molar ratio. (8) When the temperature is abruptly lowered to about 2 degrees C, lipsomes formed from phosphatidylcholine release about 20% of trapped glucose during a transient increase in permeability. Liposomes containing 0.5 mole fraction sphingomyelin release about 30% of the trapped glucose under these conditions. Liposomes composed of sphingomyelin alone do not exhibit this phenomenon.     

Haptenic activity of galactosyl ceramide and its topographical distribution on liposomal membranes. I. Effect of cholesterol incorporation

The relation between the immune-reaction of phosphatidylcholine liposomes containing spin-labeled galactosyl ceramide with or without cholesterol and the topographical distribution of the glycolipid in membranes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and antibody binding occurred, irrespectively of the presence of cholesterol, though the motion of the fatty acyl chain of spin-labeled galactosyl ceramide was restricted by cholesterol. In dipalmitoyl phosphatidylcholine liposomes, unlike in egg yolk phosphatidylcholine liposomes, the immune-reaction depended on the cholesterol content. The electron spin resonance (ESR) spectra of spin-labeled galactosyl ceramide in dipalmitoyl phosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled galactosyl ceramide in the liposomes. Without cholesterol, most of the spin-labeled galactosyl ceramide was clustered on the dipalmitoyl phosphatidylcholine membrane, but with increase of cholesterol, random distribution of hapten on the membrane increased. The cholesterol-dependent change in the topographical distribution of hapten on the membranes was parallel with that of immune reactivity. 'Aggregates' composed solely of galactosyl ceramide did not show any binding activity with antibody. The findings suggest that the recognition of galactosyl ceramide by antibody depended on the topographical distribution of hapten molecules. Phosphatidylcholine and/or cholesterol may play roles as 'spacers' for the proper distribution of 'active' haptens on the membranes. The optimum density of haptens properly distributed on liposomal membranes is discussed.