Membrane fusion without cytoplasmic fusion (hemi-fusion) in erythrocytes that are subjected to electrical breakdown

There are many reports of hemi-fusion in phospholipid vesicles but few published studies on hemi-fusion in cells. We report evidence from both fluorescence microscopy and freeze-fracture electron microscopy for hemi-fusion in the electrofusion of human erythrocytes. We have also characterised the conditions that favour hemi-fusion as opposed to complete fusion, and discuss the possibility that hemi-fusion might precede complete electrically-induced cell fusion. A membrane probe (DiIC16) and a cytoplasmic probe (6-carboxyfluorescein) were used to investigate the behaviour of doubly-labelled human erythrocytes which were aligned in chains by dielectrophoresis and then exposed to high voltage breakdown pulses. Some of the cells were fused by the pulses, as shown by diffusion of both membrane and cytoplasmic probes from labelled to unlabelled cells. With other cells, the membrane probe diffused into unlabelled cells after the breakdown pulses, without the cytoplasmic probe diffusing into unlabelled cells or leaking into the medium. Membrane fusion (hemi-fusion) thus occurred without cytoplasmic fusion in these erythrocytes. Such cells were irreversibly, but fragilely, attached to their neighbours by the breakdown pulses. There was an inverse relationship between conditions that permit complete fusion and those that favour hemi-fusion, with respect to breakdown pulse length, breakdown voltage and, in particular, osmolarity and temperature. The incidence of hemi-fusion in 250 mM erythritol was twice that in 150 mM erythritol, and hemi-fusion was 5-fold greater at 25 degrees C than at 20 degrees C. Hemi-fused erythrocytes occasionally fused completely on heating to 50 degrees C, demonstrating that hemi-fusion can proceed to complete cell fusion. Freeze fracture electron micrographs of preparations of hemi-fused cells revealed long-lived, complementary depressions and protrusions on the E- and P-fracture faces, respectively, of tightly apposed cells that may mediate hemi-fusion. The possibility that the fusion of closely adjacent human erythrocytes by electrical breakdown pulses may involve an intermediate, shared bilayer structure, which is stable in certain conditions but which can be ruptured by osmotic swelling of the permeabilised cells, is discussed.     

Temperature and external electric field influence in membrane permeability of Babesia infected erythrocytes

1. Modification of erythrocyte membrane properties infected by Babesia canis was studied using the effect of electric pulses of short duration. 2. This process induces the formation of pores in the membrane and the releasing of hemoglobin and other cytoplasmic proteins into the external medium. 3. The rate of molecular permeation across the electrically perforated membranes depends on several factors: electric-field strength, pulse number, pulse duration, temperature and cellular concentration. 4. Even for low parasitemia, differences in the effect of these parameters were observed between infected and non-infected erythrocytes. 5. Here we describe an influence of electric field intensity and temperatures on the opening pores.     

Effect of cholesterol on the stability of human erythrocyte membranes to electric breakdown

Electrical stability of human erythrocyte membranes with different cholesterol content was studied. Breakdown in the cell membranes was generated by application of electric pulses with field strengths of 1.4-3.2 kV/cm. The share of perforated cells was registered by measuring hemolysis level. The red blood cells from patients with psoriasis and normal erythrocytes after incubation in the presence of liposomes were used as a model of cells with cholesterol-rich membranes. It was discovered that an increase of cholesterol content in the membranes moved the field-dependent curves to a higher field range. The obtained effect is attributed to the increase of the breakdown membrane potential. Application of high-pulse-electric-field technique for investigating the properties of cell membranes is discussed.     

Homokaryons from animal and plant cells generated by electrofusion

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.     

In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: A Study in a Dorsal Window Chamber in Mice

Electropermeabilization/electroporation (EP) is a physical method that by application of electric pulses to cells increases cell membrane permeability and enables the introduction of molecules into the cells. One of the uses of EP in vivo is plasmid DNA electrotransfer to the skin for DNA vaccination. EP of tissues induces reduction of blood flow and, in combination with plasmid DNA, induction of an immune response. One of the EP protocols for plasmid DNA electrotransfer to the skin is a combination of high-voltage (HV) and low-voltage (LV) pulses. However, the effects of this pulse combination on skin-vessel blood flow are not known. Therefore, using intravital microscopy in a dorsal window chamber in mice and fluorescently labeled dextrans, the effects of one HV and eight LV pulses on skin vasculature were investigated. In addition, a detailed histological analysis was performed. Image analysis of fluorescence intensity changes demonstrated that EP induces a transient constriction and increased permeability of blood vessels as well as a “vascular lock.” Histological analysis revealed rounding up of endothelial cells and stacking up of erythrocytes at 1?h after EP. In addition, extravasation of erythrocytes and leukocyte infiltration accompanied by edema were determined up to 24?h after EP. In conclusion, our results show that blood flow modifying effects of EP in skin contribute to the infiltration of immune cells in the exposed area. When combined with plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins.     

Extracorporeal immunostimulating effect of terrilytin and lysozyme]

The role of splenocytes and erythrocytes in showing an extracorporal action by terrilytin and lysozyme was studied. The extracorporal effect of terrilytin was to a greater extent mediated by the spleen cells adhering to the plastic while the extracorporal effect of lysozyme was mainly mediated by the heavy ("old") erythrocytes. The heat treatment at a temperature of 42 degrees C for 15 minutes did not abolish the terrilytin extracorporal effect mediated by the erythrocytes but completely abolish the similar effect induced by lysozyme which bound to the erythrocyte membrane. After exposure of the erythrocytes to terrilytin, the strength of the lysozyme binding increased and there was a respective increase in the immunostimulating activity of the erythrocytes.     

Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degrees C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipds.     

Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells.

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degree C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipids.     

Response of erythrocytes to heat in the presence of D2O, glycerol, and anisotonic saline

Mammalian erythrocytes that lack cytoplasmic organelles and a nucleus are a useful model for studying the effect of heat on the cell membrane and cytoskeleton. The effect of heat on the membrane bilayer and cytoskeleton of erythrocytes is remarkably similar to that observed in nucleated cells. Some concentrations of D2O and glycerol can effectively protect erythrocytes from heat-induced damage to the membrane and cytoskeleton. These results are similar to observations in nucleated cells. Heating erythrocytes in some concentrations of anisotonic NaCl solutions reduced damage, an observation that does not apply to enhanced killing of nucleated cells. This difference implies that some components of the cytoplasm or nucleus, or both, may contribute to the enhancement of cytotoxicity of nucleated cells when they are heated in the anisotonic NaCl solution. Incremental heating, dividing a heat treatment into two fractions, and preheating of erythrocytes all modify the effect of heat on erythrocytes slightly, but the results suggest little, if any, development of thermotolerance. The response of chicken erythrocytes is similar to that of mammalian erythrocytes, although higher temperatures are required to produce a heat effect in chicken erythrocytes. These observations suggest that the characteristic differences in heat sensitivity in nucleated and enucleated cells involve components other than the cell membrane.     

Dipole interactions in electrofusion. Contributions of membrane potential and effective dipole interaction pressures.

The contributions of pulse-induced dipole-dipole interaction to the total pressure acting normal to the membranes of closely positioned pronase treated human erythrocytes during electrofusion was calculated. The total pressure was modeled as the sum of pressures arising from membrane potential and dipole-dipole attraction opposed by interbilayer repulsion. The dipole-dipole interaction was derived from the experimentally obtained cell polarizability. The threshold electric field amplitude necessary for fusion of pronase-treated human erythrocytes was experimentally obtained at various combinations of pulse duration, frequency, and the conductivity of the external medium. The theoretical values of the critical electric field amplitude compared favorably to the experimentally obtained threshold field amplitudes. Fusion by dc pulses may be primarily attributed to attainment of sufficiently high membrane potentials. However, with decreasing external conductivity and increasing sinusoidal pulse frequency (100 kHz-2.5 MHz), the induced dipole-dipole interactions provide the principal driving force for membrane failure leading to fusion.     

Bioactivity of electric field-pulsed human recombinant interleukin-2 and its encapsulation into erythrocyte carriers

The molecular integrity of human recombinant interleukin-2 (rIL-2), as measured by size exclusion chromatography, was not altered when exposed to high electrical field intensities. In addition, the biological activity was unaffected, as evidenced by the ability of the rIL-2 to stimulate the proliferation (by cell growth assays and tritiated thymidine uptake) and differentiation (by cytotoxicity assay) of human lymphocytes into killer cells. Electroporation conditions chosen for the loading of rIL-2, based upon those which provided for good recovery of carriers and minimal hemoglobin release, involved a lower field intensity (i.e., 6 kV/cm instead of 7 or 8 kV/cm) and multiple pulses (eight pulses, 5 microseconds) rather than a single pulse (40 microseconds). Human erythrocyte carriers consistently encapsulated 5-7.5% of the rIL-2 by electroporation (6 kV/cm, eight pulses, 5 microseconds duration). A rIL-2 concentration of 600,000 U/ml surrounding the erythrocytes during loading resulted in ca. 245,000 U/ml carriers, which represents a therapeutically significant quantity. Thus, rIL-2 shows potential as an encapsulated agent for slow release in the erythrocyte carrier system.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes.

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and nonagglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. This was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination. Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg2+, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca2+ (0.2 mM) had little effect on agglutinability, although high Ca2+ (5 mM) inhibited agglutinability of the resealed membranes somewhat. Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells. The agglutination of erythrocytes was not affected by cytochalasin B (40 mug/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes. It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca2+ or Mg2+ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitivie to vinblastine.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and non-agglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. this was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination.Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg²⁺, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca²⁺ (0.2 mM) had little effect on agglutinability, although high Ca²⁺ (5 mM) inhibited agglutinability of the resealed membranes somewhat.Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells.The agglutination of erythrocytes was not affected by cytochalasin B (40 μg/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes.It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca²⁺ or Mg²⁺ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitive to vinblastine.     

Haemolytic effect of the scorpion Heterometrus fulvipes venom

The haemolytic effect of the venom from scorpion Heterometrus fulvipes was studied using sheep erythrocytes. While there was no haemolytic effect seen directly by the erythrocytes, erythrocytes sensitized with homologous haemolysin were lysed by the venom. This factor in Heterometrus fulvipes venom having a 'complement like' haemolytic effect was found to be thermolabile and dialysable and sensitive to the action of 2-mercaptoethanol. It was identified as a low molecular weight peptide containing disulphide groups.     

Electrical fusion of cells and nucleiin vitro: A preliminary note

The electrical fusion of male skin fibroblasts of the Indian muntjac (Muntiacus muntjak)in vitro is described. Fusion of cell nuclei in the resulting multinucleated giant cells was observed after application of up to ten additional DC pulses. Electrical fusion of bovine erythrocytes and alveolar macrophages is also described. Cell fusion can be obtained without the use of protease pretreatment, but the yield of fused cells is about 20 times lower than with prior use of proteases.     

Two types of haemolytic activity of detergents

The nonionic detergent Triton X-100 at concentrations of about 0.003 to 0.008% causes swelling followed by the haemolysis of erythrocytes suspended in 160 mM KCl. The rate of haemolysis increases with the increase in detergent concentration. Finally all the erythrocytes are haemolysed. The resistance of erythrocytes to this detergent decreases with an increase in temperature. The effect of Triton X-100 is explained by increased membrane permeability to KCl and colloid osmotic haemolysis. The anionic detergent, sodium dodecyl sulfate (SDS), at concentrations of about 0.003 to 0.001% causes the haemolysis of a certain number of erythrocytes. This number increases with an increase in detergent concentration. The resistance of erythrocytes to SDS increases with an increase in temperature. The effect of SDS is explained by direct disruption of membranes by the detergent.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

Interaction of thermostable direct hemolysin of Vibrio parahaemolyticus with human erythrocytes.

The interaction between thermostable direct hemolysin produced by Vibrio parahaemolyticus WP-1 and human erythrocytes was studied. The lysis of human erythrocytes by the hemolysin was dependent of temperature and no hemolysis occurred at low temperature (0-4 C), but the hemolysin was adsorbed on human erythrocytes even at low temperature. No hemolysis was observed when antihemolysin antiserum was mixed with the hemolysin and human erythrocytes at zero time. On the other hand, lysis of the cells by hemolysin was not completely inhibited when the antiserum was added during the lag time and the inhibitory effect decreased with delay in the time of addition of antiserum. The inhibitory effect of the antiserum decreased with increase in the incubation temperature, increase in the concentration of divalent cations, and decrease in pH. These results suggest that lysis of human erythrocytes by the hemolysin is at least a two-step process consisting of adsorption of the hemolysin to human erythrocytes and the step(s) following adsorption.     

Tellurite effect on ATPase activity of contractile membrane protein.

The effect of tellurite on ATPase activity of the contractile membrane protein in human erythrocytes was studied. Tellurite, even at a concentration of 0.01 mM, inhibited 25 per cent of the saponin-stimulated ATPase activity of the contractile membrane protein; the inhibition increased with increasing tellurite concentration, and was reversible. On the other hand, in erythrocytes preincubated with tellurite, the ATPase activity of the membrane contractile protein, non-stimulated by saponin, increased, and the increase was further enhanced by washing the erythrocytes. The behaviour is analogous to the tellurite effect on erythrocyte morphology: incubation of erythrocytes with tellurite caused morphological changes which were more pronounced after removing the tellurite by washing. The complex effect of tellurite is discussed.     

Iron deficiency influences the course of malaria in Plasmodium berghei infected mice

Iron deficiency accelerates suicidal erythrocyte death, which is evident from phosphatidylserine exposure. The present study explored whether iron deficiency compromises intraerythrocytic growth of Plasmodium and enhances death of infected erythrocytes thus influencing the course of malaria. As a result, phosphatidylserine exposure is increased in Plasmodium falciparum infected human erythrocytes, an effect significantly more marked in iron deficiency. Moreover, iron deficiency impairs in vitro intraerythrocytic growth and infection of erythrocytes. In mice, iron-deficient erythrocytes are more rapidly cleared from circulating blood, an effect increased by infection with Plasmodium berghei. Parasitemia in P. berghei infected mice was significantly decreased (from 54% to 33% of circulating erythrocytes 20 days after infection) and mouse survival significantly enhanced (from 0% to 20% 30 days after infection) in iron-deficient mice. In conclusion, iron deficiency favourably influences the course of malaria, an effect partially due to accelerated suicidal death and subsequent clearance of infected erythrocytes.