The kinetics of colloid osmotic hemolysis. II. Photohemolysis

Many of the known features of photohemolysis have been organized in a kinetic model that simulates the lytic time-course in a variety of conditions. The model combines Nernst-Planck flux principles, the osmotic equilibrium model of Freedman and Hoffman, equations relating illumination parameters to ion permeability, and an empirical relation between cell volume and lysis. Model simulations are compared with experiments showing the dependence of lysis kinetics on sensitizer concentration and on the osmotic content of the reaction medium. Additional experiments demonstrate that the inherent osmotic fragility of erythrocytes is not altered by illumination conditions that cause major delayed lysis 23 h later. The successful simulations support the hypothesis that photohemolysis is a colloid osmotic lysis occurring in cells behaving as imperfect osmometers.     

Effects of monoclonal antibodies on α-staphylotoxin action against erythrocytes and model phospholipid membranes

It was found that one of twenty tested monoclonal antibodies (MABs) existed which drastically enhanced ability of Staphylococcus aureus α-tosin (ST) to both lysis of human erythrocytes and increase of planar phospholipid bilayer conductance more than 10 and 1000 times respectively. Other 19 MABs possessed only neutralized effect. The activation could only be observed if the activating MAB (AMAB) interacted with ST in solution but not in membrane. The one molecule of AMAB was able to activate approximately 2–4 molecules of ST. It was assumed that this activation was a result of the AMAB-induced transition of ST from a hydrophilic to an amphiphilic form. The activation could not be observed when the activity of AMAB/ST mixtures was tested on highly sensitive rabbit erythrocytes. All the tested MABs (including AMAB) were able to inhibit the ST-induced lysis of rabbit erythrocytes. The activating effects of AMAB on ST action in BLM and in human erythrocytes as well as their inhibiting influence on the ability of toxin to cause a lysis of rabbit erythrocytes indicate the presence of an ST-specific receptor on the membrane of rabbit erythrocytes.     

Streptavidin-induced lysis of homologous biotinylated erythrocytes. Evidence against the key role of the avidin charge in complement activation via the alternative pathway

It is shown that non-covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non-immune rabbit serum. Efficiency of lysis inhibition is greater for avidin- and streptavidin-induced lysis of biotinylated human erythrocytes than for antibody-sensitized sheep erythrocytes. In contrast to positively charged avidin (pI 11), streptavidin is a neutral protein. Hence, hemolysis of streptavidin-carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross-linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.     

Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes.

Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of > or = 2.1 nm were present in the extracellular space. (2) Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximately 2 nm.     

Bacteriocin (hemolysin) of Streptococcus zymogenes

The sensitivity of Streptococcus faecalis (ATTC 8043) to S. zymogenes X-14 bacteriocin depends greatly on its physiological age. Sensitivity decreases from the mid-log phase on and is completely lost in the stationary phase. The sensitivity of erythrocytes to the hemolytic capacity of the bacteriocin showed considerable species variation. The order of increasing sensitivity was goose < sheep < dog < horse < human < rabbit. However, when red cell stromata were used as inhibitors of hemolysis in a standard system employing rabbit erythrocytes the order of increasing effectiveness was sheep < rabbit < human < horse < goose. When rabbit cells were used in varying concentrations with a constant hemolysin concentration, there was a lag of about 30 min, which for a given hemolysin preparation was constant for all red cell concentrations. Furthermore, the rate of hemolysis increased with increasing red cell concentration. If red cells are held constant and lysin varied, the time to reach half-maximal lysis varies directly with lysin but is not strictly proportional. Bacterial membranes were one to three orders of magnitude more effective than red cell stromata as inhibitors. The order of increasing effectiveness seems to be Escherichia coli < Bacillus megaterium < S. faecalis < Micrococcus lysodeikticus. In addition to membranes, a d-alanine containing glycerol teichoic acid, trypsin in high concentration, and deoxyribonuclease also inhibited hemolysis. Ribonuclease, d-alanine, l-alanine, dl-alanyl-dl-alanine, N-acetyl-d-alanine, N-acetyl-l-alanine did not inhibit hemolysis.     

Characteristics ofPseudomonas aeruginosa strains isolated from urinary tract infections

Thirty-three uropathogenic strains ofPseudomonas aeruginosa were investigated for hemolytic activity in both bacterial broth culture filtrates and isolate lyzates, resistance to bactericidal activity of fresh human serum, resistance to six antibiotics and plasmid DNA profile. Twenty-four of the 33 (73%) bacterial filtrates showed lysis of rabbit erythrocytes, as did the three after guinea-pig erythrocyte treatment. Twelve of 33 isolate lysates showed in parallel lysis of both types of erythrocytes used. Serum resistance was found in 17 (52%) isolates, intermediate resistance in 15 (45%) isolates and only one isolate showed serum sensitivity. Resistance to antibiotics was detected as follows (in %): tetracycline 94, kanamycin 79, chloramphenicol 76, septrin 73, ampicillin 64, streptomycin 45, gentamicin 18. None of the isolates investigated showed resistance to colistine. With the exception of one isolate, plasmid DNA was detected in allP. aeruginosa strains.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Regulation of the alternative pathway of complement by pH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia. The abnormal PNH erythrocytes are highly susceptible to complement-mediated lysis in vitro, especially at pH 6.4. Lysis has been shown to be due to alternative pathway activation. The purpose of this study was to determine why lysis of PNH erythrocytes is increased at acidic pH. The results presented demonstrate that at pH 6.4: binding of C5 and Factor B to C3b deposited on human erythrocytes is markedly enhanced; generation of the two C3 convertases, C3(H2O), Bb and C3b,Bb is increased; and control of C3b on human erythrocytes by CR1 and Factor I is diminished. In addition, it was found that rabbit erythrocytes, which activate the human alternative pathway, are also lysed much better at pH 6.4 than at pH 7.4. These results indicate that the optimal pH for the initiation and amplification of the alternative complement pathway, and probably also for the activation of the membrane attack complex, is 6.4.     

Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages

We reconfirmed that the LD₅₀S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.     

Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.     

A reexamination of the role of clusterin as a complement regulator.

Clusterin is a highly conserved glycoprotein which has been proposed to protect host cells against complement-mediated cytolysis. We tested the hypothesis that clusterin is a complement regulator using erythrocytes and cells which had been stably transfected with a membrane-anchored form of clusterin as targets for complement-mediated cytolysis. Clusterin gave dose-dependent protection of antibody-coated sheep erythrocytes against complement-mediated lysis by diluted normal human serum. There was a linear relationship between the concentration of clusterin giving 50% protection and the concentration of serum; extrapolation of this to the case of undiluted human serum showed that a clusterin concentration at least two orders of magnitude greater than its physiological plasma concentration would be needed to confer protection against complement-mediated cytolysis under physiological conditions. Physiological concentrations of clusterin did not protect rabbit erythrocytes against alternative complement pathway-mediated lysis using dilute human serum. Exogenous clusterin had no effect on lysis of human erythrocytes triggered by the addition of inulin to autologous human serum. Induction of cell-surface clusterin expression by L929 (murine fibroblast) cells which had been stably transfected with cDNA for human clusterin linked to DNA coding for the 44 C-terminal amino acid residues of CD55 did not protect the cells against complement-mediated lysis by either normal or clusterin-depleted human serum. These data suggest that clusterin may not be a physiologically relevant regulator of complement activation.     

Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Ca2+.

The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.     

Heterotrimeric G protein Gi is involved in a signal transduction pathway for ATP release from erythrocytes

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein G(s) resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the G(i) subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins G(alphai1), G(alphai2), and G(alphai3) but not G(alphao) were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples G(i/o) from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 microM) or Mas-7 (1 microM), which are compounds that directly activate G(i) proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 microM), which is an inactive Mas analog. In separate experiments, Mas (10 microM) but not Mas-17 (10 microM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein G(i) is a component of a signal transduction pathway for ATP release from erythrocytes.     

Hemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

We demonstrated that Klebsiella pneumoniae and Klebsiella oxytoca possess a selective haemolytic activity on rabbit erythrocytes. Thirty one Klebsiella strains (18 strains of K. pneumoniae and 13 strains of K. oxytoca) were isolated from hospitalized patients. The liquid (Trypcase-soy broth--TSB) and solid (Trypcase-soy agar--TSA) medium, containing the red cells were used for the tests. All the screened strains showed a haemolytic effect on rabbit erythrocytes, provided that the supernatants of the cultures were preincubated with beta-mercaptoethanol or calcium chloride. There was no human and sheep erythrocyte lysis.     

Activity of <Emphasis Type="Italic">Lentinus edodes</Emphasis> intracellular lectins at various developmental stages of the fungus

We studied changes of the hemagglutinating activity of intracellular lectins of the basidiomycete Lentinus edodes (shiitake) at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree. Under certain experimental conditions, the specific lectin activity at the brown mycelium film stage exceeded the corresponding value for nonpigmented mycelium. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied. The general regularities of specific activity change did not depend on nutrient medium composition. With purification of intracellular shiitake lectins, their sensitivity to human erythrocytes decreased seventyfold or more, whereas their sensitivity to rabbit erythrocytes increased by the same factor.     

Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.     

PPIX induced photohemolysis of erythrocytes partially-depleted of cholesterol

The protoporphyrin IX (PPIX)-sensitized hemolysis of erythrocytes depleted of cholesterol was investigated. From 20% to 30% of the total membrane cholesterol was removed from cells by incubation with old autologous plasma or by means of interaction with L-alpha-phosphatidylcholine dipalmitoyl (DPPC) liposomes. As expected, after this treatment, the cells show an overall increase in membrane fluidity revealed by means of specific fluorescent probes. The same cells are more susceptible to the photohemolysis induced by PPIX excited by visible light, but gave no lysis in the absence of the sensitizer. As a consequence, the primary oxidative damage which is produced during irradiation can be possibly assigned to the phospholipidic and/or proteic moiety instead of the steroidal moiety.     

Evidence for the participation of cytosolic protein kinases in membrane phosphorylation in intact erythrocytes.

The effects of adenosine 3':5'-monophosphate (cyclic AMP) on the phosphorylation of membrane proteins in intact rabbit and human erythrocytes were investigated. The addition of cyclic AMP to intact human or rabbit erythrocytes results in an increase in the incorporation of ortho[32P]phosphate into several membrane protein components which are known to serve as substrates for the cyclic-AMP-dependent protein kinases. Thus this increase in protein phsophorylation is probably due to the activation of either soluble or membrane-bound cyclic-AMP-dependent protein kinases. Incubation of human erythrocytes in the presence of ortho [32P]phosphate and cyclic AMP also leads to the phosphorylation of a membrane protein component, band 7, which has not been previously detected in the autophosphorylation of isolated ghosts. Since rabbit erythrocyte membranes do not contain any cyclic-AMP-dependent protein kinase, the results suggest that cytoplasmic kinases also play a role in the phosphorylation of membrane proteins in intact cells.     

Hemagglutinin specificity of Cl. botulinum types A, B, and F in reaction with erythrocytes of various animals]

The following differences were revealed in the haemagglutination reaction with the erythrocytes of man, sheep, rabbit, chicks and mice between the haemagglutinins of Cl. botulinum, types A, B and F, having a close affinity with one another: haemagglutinin of type A actively reacted with the erythrocytes of man, sheep, rabbit, rats and chicks; haemagglutinin of type B reacted only with the erythrocytes of man and rabbits; haemagglutinin of type F failed to react with any of the types of the erythrocytes tested; only with the use of erythrocytes treated with neuraminidase was it possible to establish the presence of haemagglutinin fraction in Cl. botulinum, type F. Treatment of human erythrocytes with neuraminidase and proteolytic enzymes caused a marked increase in the sensitivity of the haemagglutination reaction in haemagglutinins of Cl. botulinum, types A, B and F.     

Activity of Lentinus edodes intracellular lectins at various developmental stages of the fungus

We studied changes of the hemagglutinating activity of intracellular lectins of the basidiomycete Lentinus edodes (shiitake) at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree. Under certain experimental conditions, the specific lectin activity at the brown mycelium film stage exceeded the corresponding value for nonpigmented mycelium. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied. The general regularities of specific activity change did not depend on nutrient medium composition. With purification of intracellular shiitake lectins, their sensitivity to human erythrocytes decreased seventyfold or more, whereas their sensitivity to rabbit erythrocytes increased by the same factor.