Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements

Background

It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed.

Methods and Findings

Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge.

Conclusion

CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.     

An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion

Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDL_DMPC) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDL_DMPC+Chol). The low-resolution shape of the lipid phase in both nHDL_DMPC and nHDL_DMPC+Chol were oblate ellipsoids, and fit well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDL_DMPC, making a large hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDL_DMPC+Chol, the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDL_DMPC and nHDL_DMPC+Chol, an open conformation (i.e., not a closed belt) of apoA1 is observed. Collectively, these data show that full length apoA1 retains an open architecture that is dictated by its lipid cargo. The lipid is likely predominantly organized as a bilayer with a micelle domain between the open apoA1 arms. The apoA1 configuration observed suggests a mechanism for accommodating changing lipid cargo by quantized expansion of hairpin structures.     

DNA-containing liposomes as a model for the study of cell membrane permeation by anthracycline derivatives

The uptake of anthracycline derivatives into large unilamellar vesicles (LUV) in response to a driven force provided by DNA encapsulated inside the LUV has been investigated. Four anthracyclines have been used: adriamycin, 4'-O-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR), and carminomycin. No quenching of the drug fluorescence is observed through interaction of the drugs with the lipidic bilayer. Rapid quenching of drug fluorescence occurs when drugs intercalate between the base pairs of DNA. The kinetics of the decay of anthracycline fluorescence in the presence of DNA-containing liposomes can thus be used to follow the diffusion of the drug through the membrane. The initial rates of uptake, as a function of pH, and lipid bilayer permeability coefficients have been calculated for the neutral forms of THP-ADR and DNR. This system suggests that anthracycline may gain access to cells by passive diffusion of the neutral form of the drug under the action of a driven force provided by DNA in the nucleus.     

Comparison of cholesterol-phospholipid interaction in bilayers of liposomes and the red cell membrane

The energetics of interactions of cholesterol with phospholipid in simple liposome bilayers were compared with those in the bilayer of the human erythrocyte membrane, by measuring cholesterol distribution between erythrocytes and liposomes prepared from their whole phospholipid extract. With liposomes of a range of initial cholesterol contents, the equilibrium value for $\text{r}$, the ratio of cholesterol/phospholipid in the liposomes to that in the cells, is in the range 1.1–1.2. The closeness of this value to 1.0 indicates that overall cholesterol-phospholipid interaction in the cell membrane is similar to that in liposomes. However, while the deviation from 1.0 is small, and could arise from average cholesterol-phospholipid interactions in the membrane being only 0.06 to 0.1 kcal · mol^?1 weaker than in liposomes, it could also result from 10 to 20% of the cell membrane phospholipid being unavailable to mix with cholesterol.     

A vaccine formulated with a combination of TLR-2 and TLR-9 adjuvants and the recombinant major outer membrane protein elicits a robust immune response and significant protection against a Chlamydia muridarum challenge

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen in the World and there is a need for a vaccine. To enhance the immunogenicity of a vaccine formulated with the Chlamydia muridarum (Cm) mouse pneumonitis recombinant major outer membrane protein (MOMP), we used combinations of Pam₂CSK₄ + CpG-1826 and Montanide ISA 720 VG + CpG-1826 as adjuvants. Neisseria gonorrhoeae recombinant porin B (Ng-PorB) was used as the antigen control with the same adjuvants. Female BALB/c mice were immunized twice in the nares (i.n.) or in the colon (cl.) and were boosted twice by the intramuscular plus subcutaneous (i.m. + s.c.) routes. Based on the IgG2a/IgG1 ratio in sera, mice immunized with MOMP + Pam₂CSK₄ + CpG-1826 showed a strong Th2 response while animals vaccinated with MOMP + Montanide ISA 720 VG + CpG-1826 had a Th1 response. Both groups of mice also developed robust Cm-specific T cell proliferation and high levels of IFN-γ. Four weeks after the last immunization, the mice were challenged i.n. with 10⁴ inclusion-forming units (IFU) of Cm. Using changes in body weight and number of IFU recovered from the lungs at 10 days post-challenge mice immunized i.n. + i.m./s.c. with MOMP + Pam₂CSK₄ + CpG-1826 were better protected than other groups. In conclusion, MOMP adjuvanted with Pam₂CSK₄ + CpG-1826, elicits strong humoral and cellular immune responses and induces significant protection against Chlamydia.     

Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity

These experiments characterize the effects of hemoglobin and erythrocyte membrane lipids on the dynamic surface activity and adsorption facility of whole lung surfactant (LS) and a calf lung surfactant extract (CLSE) used clinically in surfactant replacement therapy for the neonatal respiratory distress syndrome (RDS). The results show that, at concentrations from 25 to 200 mg/ml, hemoglobin (Hb) increased the minimum dynamic surface tension of LS or CLSE mixtures (0.5 and 1.0 mumol/ml) from less than 1 to 25 dyn/cm on an oscillating bubble apparatus at 37 degrees C. Similarly, erythrocyte membrane lipids (0.5-3 mumol/ml) also prevented LS and CLSE suspensions (0.5-2.0 mumol/ml) from lowering surface tension below 19 dyn/cm under dynamic compression on the bubble. Surface pressure-time adsorption isotherms for LS suspensions (0.084 and 0.168 mumol phospholipid/ml) were also adversely affected by Hb (0.3-2.5 mg/ml), having a slower adsorption rate and magnitude. Significantly, these inhibitory effects of Hb and membrane lipids could be abolished if LS and CLSE concentrations were raised to high levels. In complementary physiological experiments, instillation of Hb, membrane lipids, or albumin into excised rat lungs was shown to cause a decrease in pressure-volume compliance. This decreased compliance was most prominent in lungs made partially surfactant deficient before inhibitor delivery and could be reversed by supplementation with active exogenous surfactant. Taken together, these data show that molecular components in hemorrhagic pulmonary edema can biophysically inactivate endogenous LS and adversely affect lung mechanics. Moreover, exogenous surfactant replacement can reverse this process even in the continued presence of inhibitor molecules and thus has potential utility in therapy for adult as well as neonatal RDS.     

Characterization of mixed monolayers of phosphatidylcholine and a dicationic gemini surfactant SS-1 with a langmuir balance: effects of DNA

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N;N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (pi-A) curves and determined the elastic area compressibility modulus (C) as a function of lateral packing pressure and the mole fraction of the cationic lipid (X(SS-1)), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (X(SS-1) < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P(-)-N(+) dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X(SS-1) and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P(-)-N(+) dipole is further supported by the pronounced increase in monolayer dipole potential psi due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 microM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X(SS-1) < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in DeltaA/molecule, was observed at X(SS-1) > 0.5. At a higher concentration of DNA (1.88 microM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X(SS-1) = 0.5, condensation was observed at all values of X(SS-1).     

Exploring the interaction of the surfactant N-terminal domain of gamma-Zein with soybean phosphatidylcholine liposomes

Zeins are maize storage proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner surface of the protein body membrane, and its N-terminal, proline-rich, repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation and forms cylindrical micelles in aqueous solution. Here we explore the interaction of (VHLPPP)(8) with soybean phosphatidylcholine unilamellar lipid vesicles and examine its effect on the stability and permeability of the liposome membrane. The amphipathic N-terminal domain of gamma-zein interacts with the membrane and assembles to form extended domains over the phospholipid membrane. The interaction between the peptide and the membrane increases the stability and permeability of the liposome membrane. The spontaneous amphipathic aggregation of (VHLPPP)(8) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies.     

Molecular interaction of cationic gemini surfactant with bovine serum albumin: A spectroscopic and molecular docking study

Herein, we report the effect of N,N′-bis(dodecyloxycarbonylmethyl)-N,N,N′,N′-tetramethyl-1,2-ethanediammonium dibromide (dodecyl betainate gemini or DBG) on the structure and function of bovine serum albumin (BSA) by using fluorescence, time resolved fluorescence, circular dichroism and dynamic light scattering techniques. The Stern–Volmer quenching constants K_SV and the corresponding thermodynamic parameters viz ΔH, ΔG and ΔS have been estimated by the fluorescence quenching method. The results indicated that DBG binds spontaneously with BSA through hydrophobic interaction. Time resolved fluorescence data show that the quenching follows the static mechanism pathway. It can be seen from far-UV CD spectra that the α-helical network of BSA is disrupted and its content increases from 71% to 79% at lower concentrations which again decreases to 38% at higher concentration. DLS measurements suggested that hydrodynamic radius (R_h) decreases in the presence of 30 and 40 μM of DBG while it increases when the concentration of DBG was 70 and 100 μM. The molecular docking study indicated that DBG is embedded into subdomain IIA of BSA and binds with the R-914, R-195 and R-217 residues by hydrogen bonding and by hydrophobic interaction.     

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3'-dipentyloxacarbocyanine (di-O-C5(3)) or 3,3'-dipropylthiadicarbocyanine (di-S-C3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 microgram/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP+) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P less than 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P less than 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na⁺, with a positive cooperativity. Na⁺ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.     

Adenylate cyclase toxin translocates across target cell membrane without forming a pore

The adenylate cyclase toxin‐haemolysin of Bordetella (CyaA) targets CD11b⁺ myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation‐selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch‐clamp assays, we show that a combination of substitutions within the pore‐forming (E570Q) and acylation‐bearing domain (K860R) ablates selectively the cell‐permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC^‐ toxoid on CD11b‐expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8⁺ T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I‐restricted presentation and induce in vivo an OVA‐specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.     

Incorporation of some glycoconjugates into the membrane of liposomes and interaction of ganglioside-containing liposomes with rat hepatocytes in vitro

Inclusion of some glycosides, gangliosides and ceruloplasmin into large (300-400 nm in diameter) unilamellar liposomes was performed. About 100% of the gangliosides, 30-50% of ceruloplasmin and 3-5% of the glycosides were incorporated into the phospholipid vesicles under these conditions. The liposomes containing ceruloplasmin or gangliosides, in contrast to the glycoside-containing vesicles, were precipitated in the presence of agglutinin from Ricinus communis. The interaction of phospholipid vesicles containing gangliosides with rat hepatocytes "in vitro" was studied. It was found that the incorporation of gangliosides into the liposomal membrane increased the liposomal lipid uptake by 50% as can be judged from the uptake of radioactive cholesterol. Possible mechanisms of incorporation of carbohydrate-containing compounds into liposomes are discussed. It is concluded that beside the density of carbohydrates the degree of their exposure on the liposomal membrane is important for specific interactions of the vesicles with lectins.     

Enhanced electron transfer for hemoglobin entrapped in a cationic gemini surfactant films on electrode and the fabrication of nitric oxide biosensor

The direct electrical communication between hemoglobin (Hb) and GCE surface was achieved based on the immobilization of Hb in a cationic gemini surfactant film and characterized by electrochemical techniques. The cyclic voltammograms showed that direct electron transfer between Hb and electrode surface was obviously promoted and then a novel unmediated nitric oxide (NO) biosensor was constructed in view of this protein-based electrode. This modified electrode showed an enzyme-like activity towards the reduction of NO and its amperometric response to NO was well-behaved with a rapid response time and displaying Michaelis-Menten kinetics with a calculated Km(app) value of 84.37 micromol L(-1). The detection limit was estimated to be 2.00 x 10(-8)mol L(-1). This biosensor was behaving as expected that it had a good stability and reproducibility, a higher sensitivity and selectivity and should has a potential application in monitoring NO released from biologic samples.     

Cationic and anionic channels of apical receptive membrane in a taste cell contribute to generation of salt-induced receptor potential

• 1.1. After ionic composition of superficial fluid (ISF) and interstitial fluid (ISF) of the frog Rana catesbeiana) tongue had mostly been changed with a low Na⁺ saline solution, the relations between membrane potentials and receptor potentials in a frog taste cell evoked by various concentrations of NaCl and various types of salts were analyzed to examine permeability of the taste receptive membrane to cations and anions.
• 2.2. The mean reversal potentials for depolarizing potentials of a taste cell in response to 0.05 M, 0.2 M and 0.5 M Nad were -40.0, 6.4 and 28.8 mV, respectively.
• 3.3. When adding an anion channel blocker, SITS, to a NaCl solution the reversal potential for receptor potential with NaCl plus SITS became about twice as large than with NaCl alone.
• 4.4. Reversal potentials for 0.2 M NaCl, LiCl, KCl and NaSCN were 6.4, 25.4, −1.0 and −7.8 mV, respectively, indicating that permeability of the apical taste receptive membrane to cations of Cl⁻ salts is arranged in the order of Li⁺ > Na⁺ > K⁺ and that the permeability to anions of Na⁺ salts is arranged as SCN⁻ > Cl⁻
• 5.5. It is concluded that in the case of NaCl stimulation, Na⁺ and Cl⁻ of NaCl stimulus permeate NaCl-gated cationic and anionic channels at the apical taste receptive membrane in generating receptor potentials.

     

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.