Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.     

Biochemical analysis and subcellular localization of a neutrophil-specific antigen, PMN-7, involved in the respiratory burst

The adherence of serum-opsonized yeast to neutrophils results in phagocytosis of these particulate stimuli and activation of the respiratory burst. Both events are mediated or modulated in part by the surface receptors for IgG and complement. The link between the binding of complex particulate stimuli to the cell surface, and the triggering of these neutrophil functions, is not completely understood. We have previously described an anti-human neutrophil, murine monoclonal antibody PMN7C3, which specifically inhibits the respiratory burst of neutrophils stimulated with serum-opsonized yeast. In the present study, we show that the antigen recognized by PMN7C3 (PMN7 antigen) is present on a number of neutrophil proteins, including the recently described group of related leukocyte membrane glycoproteins CR3, LFA-1, and p150,95. The PMN-7 antigen differs from other antigens associated with the C3bi receptor complex (MAC 1, MO 1, OKM1, OKM10) in that it is present only on neutrophils among peripheral blood cells. Furthermore, the binding of PMN7C3 to the neutrophil surface inhibits the activation of the respiratory burst by serum opsonized zymosan without affecting phagocytosis of these particulate stimuli. The cross-linking of cell surface PMN7 antigen by multivalent antibody is associated with the capping and internalization of antigen-antibody complexes, and appears to be necessary for the expression of maximum inhibition of opsonized zymosan-triggered respiratory burst activity. PMN7C3 also binds to a group of granule-associated proteins biochemically distinct from CR3, LFA-1, and p150,95. These granule-associated proteins containing PMN7 antigen can be mobilized to the cell surface with secretion. PMN7 antigen-bearing proteins may play a role in modulating the activation of the respiratory burst associated with phagocytosis of serum-opsonize zymosan.     

The interaction of lymphocyte membrane proteins with the lymphocyte cytoskeletal matrix

Lymphocyte membrane proteins are important in the transduction of signals across the plasma membrane. Visual and biophysical studies have shown that after ligand binding, membrane proteins may become immobile in the plane of the membrane and may cap. In intact cells, binding of cross-linking ligands to surface immunoglobulin converts it to a detergent-insoluble state (77% insoluble). This conversion is positively correlated with the transmission of a mitogenic signal. Class II histocompatibility proteins (Ia) and thy-1 remain predominantly detergent soluble (60 to 97% soluble). Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons with 0.34 M sucrose, 0.5 mM ATP, 0.5 mM dithiothreitol, 1 mM EDTA, or 3 X 10(-5) M DNAase I, 1 mM EDTA. To determine if the membrane-associated cytoskeleton contains the sufficient components for ligand-induced receptor insolubilization, experiments were done with a crude plasma membrane fraction. The results with whole cells or crude plasma membranes were comparable. These studies support the view that ligand-induced insolubilization of membrane proteins is due to their interaction with cytoskeletal structures.     

Hemolysis of human erythrocytes under hydrostatic pressure is suppressed by cross-linking of membrane proteins

The effects of cross-linking of membrane proteins on hemolysis of human erythrocytes under high pressure (2.0 kbar) were examined. The membrane proteins were cross-linked by oxidation of their SH-groups with diamide (0.05-0.5 mM) under different pressures (1-1,000 bar) at which no hemolysis occurs. As the pressure during diamide treatment was raised, the degree of hemolysis under 2.0 kbar and the quantity of cytoskeletal proteins extracted in a low ionic strength medium were gradually decreased. However, both values were increased by reduction with dithiothreitol. From the determination of membrane SH-groups, it was found that cross-linking of membrane proteins by diamide was accelerated under pressure. Only in erythrocytes treated with diamide under pressure were parts of spectrin and ankyrin, in addition to band 3 and band 4.2 proteins, extracted by using Triton X-100. One- and two-dimensional SDS-PAGE of membrane proteins showed that cross-linking of the membrane with cytoskeletal meshwork through linking proteins, in addition to that of membrane proteins themselves, was formed only in the diamide treatment under pressure. These results indicate that pressure-induced hemolysis is greatly suppressed by the supramolecular-weight polymers formed among membrane proteins, and that the high pressure technique is useful for cross-linking membrane proteins with diamide.     

Photoactivated cross-linking of proteins within the erythrocyte membrane core.

We describe the reactions of three lipophilic, photoactivated cross-linking reagents, 1,5-diazidonapthalene, 4,4'-diazidobiphenyl, and the reversible 4,4'-dithiobisphenylazide, with erythrocyte membranes. Cross-linking occurs only upon photoactivation. At pH 7 to 8, only spectrin components are cross-linked by these reagents. At pH 5.0 to 5.5 several additional membrane proteins including the major "integral" membrane proteins are also cross-linked, despite equivalent binding of the cross-linkers at neutral and acid pH. The cross-linking rates of various membrane proteins at pH 5.0 to 5.5 depend distinctly upon duration of photoactivation. Bidimensional electrophoresis of membrane proteins after cross-linking with the reversible cross-linker, 4,4'-dithiobisphenylazide, has allowed for the identification of homopolymeric products of cross-linking (e.g. dimers and tetramers of Band 3) and heterocomplexes (spectrin plus other membrane proteins). The data suggest that at reduced pH, cross-linking can proceed not only at the membrane surface but also in the membrane core.     

Analysis of integral membrane protein contributions to the deformability and stability of the human erythrocyte membrane

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.     

Red blood cell phagocytosis following hexokinase inactivation

Hexokinase inactivating antibodies were loaded into human erythrocytes using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing. Red blood cells loaded with anti-hexokinase IgG showed 20 percent residual hexokinase activity and reduced glycolytic activity. ⁹Incubation of these cells in the presence of an oxidizing agent such as terbutyl hydroperoxide (TBH) and then in autologous plasma, promoted opsonization by autologous IgG and complement deposition, but not haemolysis. Furthermore, the antihexokinase IgG loaded cells treated with TBH were actively recognized and phagocytosed by macrophages. Thus, metabolic impairment of human erthrocytes promotes autologous IgG binding, C3 deposition and phagocytosis, a mechanism already reported for the removal of senescent erythrocytes.     

An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors

CD11b/CD18 is a heterodimeric leukocyte surface receptor which functions in both C3bi-ligand binding and homotypic and heterotypic cell adherence. We have examined the effect of several anti-CD11b/18 mAb on phagocytosis of IgG (EIgG) or complement (EC4b) opsonized erythrocytes by polymorphonuclear leukocytes (PMN) and monocytes. F(ab')2 of two mAb (IB4, an anti-beta-chain mAb and Mo-1 an anti-alpha-chain mAb), inhibited both phagocytosis of EIgG and phorbol ester-stimulated phagocytosis of EC4b by PMN and monocytes. These F(ab')2 inhibited the binding of EIgG to monocytes, but they had no effect on binding of EIgG to PMN, or EC4b to either phagocyte. In addition, IB4 inhibited phorbol-ester stimulated phagocytosis of sheep E opsonized with C component 3bi (EC3bi) without inhibiting rosetting of these same targets. These data separate the anti-phagocytic effect of these mAb from effects on phagocyte-target adherence. When PMN were adherent to an anti-CD11b/CD18 F(ab')2-coated surface, EC3bi binding was abolished, but phagocytosis of EIgG or EC4b was unaffected. Subsequent addition of fluid- phase IB4 or Mo-1 F(ab')2 inhibited phagocytosis of EIgG or EC4b by the adherent cells. This suggested that the CD11b/CD18 involved in C3bi rosetting were mobile in the membrane, whereas those involved in phagocytosis of EIgG or EC4b were not. Cytochalasin treatment of PMN during adherence to F(ab')2-coated plates decreased both apical expression of CD11b/18 and subsequent ingestion of EIgG by 70%, suggesting that microfilaments are important in maintaining immobile CD11b/18 on the apical PMN surface. We conclude that there are functionally distinct populations of CD11b/CD18 on monocytes and PMN: one involved in C3bi rosetting and another involved in the process of phagocytosis mediated via several different receptors. CD11b/18 is not required for optimal target binding in all cases, but is always required for ingestion. As with several other integrins, the CD11b/18 molecules involved in phagocytosis have a functional association with the cell cytoskeleton.     

Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes. Formation of senescent antigen on erythrocyte surface by an oxidative mechanism

Incubation of human erythrocytes oxidized by iron catalysts, ADP/Fe3+ or xanthine/xanthine oxidase/Fe3+, with autologous IgG resulted in IgG binding as detected by enzyme immunoassay using protein A-beta-galactosidase conjugate. The binding of autologous IgG to ADP/Fe3(+)-treated erythrocytes maximized when the cells were treated with 1.8:0.1 mM ADP/Fe3+, and declined when treated above this concentration, suggesting that autologous IgG binds to moderately but not to excessively oxidized erythrocytes. The antibody involved in the binding was anti-Band 3, the autoantibody known to bind to aged erythrocytes, because isolated anti-Band 3 bound to the oxidized cells, but anti-Band 3-depleted autologous IgG did not. In addition, purified Band 3 inhibited the autologous IgG binding. Anti-alpha-galactosyl IgG, another natural antibody which has been reported to bind to aged erythrocytes, did not bind to the oxidized cells. Oxidation of membrane lipids, SH-groups of membrane proteins, and Hb of these cells was slight, but the cells contained an increased amount of membrane-bound native Hb, indicating that the oxidized cell membrane has an altered property. alpha-Tocopherol prevented the lipid oxidation and the subsequent IgG binding. Reduction of the oxidized erythrocytes with dithiothreitol resulted in a loss of the IgG binding. These results suggest that anti-Band 3 binding sites (Band 3 senescent antigen) are formed on moderately oxidized erythrocytes as a result of oxidation of membrane protein SH-groups which can be mediated by the membrane lipid oxidation and that formation of the anti-Band 3 binding sites on the oxidized cells is an essentially reversible membrane event which is linked to oxidation and restoration of the protein SH-groups.     

Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.     

Cross-linking of membrane glycoconjugates is not a sufficient condition for neural induction by concanavalin A

Tetravalent native concanavalin A (Con A) has a neural inducing effect on amphibian presumptive ectoderm. The divalent dimeric form of this lectin, succinylated Con A (Succ-Con A), is devoid of neuralizing action on this target tissue in Pleurodeles waltlii. These results suggested that cross-linking of Con A receptors on the cell membrane (which is not provoked by the divalent lectin) might be required for neural induction. To test this possibility, Succ-Con A binding sites were experimentally cross-linked after binding of Succ-Con A to the target cell surface, using anti-Con A antibodies. The combination of these two agents mimics the cross-linking of Con A. The results showed that cross-linking alone, either by treatment with Succ-Con A and anti-Con A antibodies, or with the lectins WGA and PHA, which also cross-link cell surface binding sites, was not able to induce neuralization. This suggested that the inductive action of Con A cannot be explained in terms of receptor cross-linking.     

Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.     

Effect of chloroquine and methylamine on endocytosis of fluorescein-labelled controlled IgG and of anti-(plasma membrane) IgG by cultured fibroblasts

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein-labelled control immunoglobulin G (control IgG) a marker for non-specific adsorptive endocytosis and of anti-(plasma membrane) IgG (specific IgG), a specific ligand of cell-surface antigens. At 4 degrees C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture medium. They do not affect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return of the cell surface of membrane antigens and of membrane fragments bearing 5'-nucleotidase or binding sites for control IgG. We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.     

Induction of granulocyte histaminase release by particle-bound complement C3 cleavage products (C3b, C3bi) and IgG

The interaction of opsonized particles with human granulocytes promotes a number of important biologic functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have previously determined that histaminase release occurs via a C3-dependent process. Although fluid-phase C3b dimers can mediate release, the relative effects of particle-bound C3b and C3bi and of IgG have not been examined. In this report we demonstrate that particle-bound C3 deposited on activators of the alternative C pathway effected histaminase release in the absence of IgG. Particle-bound C3bi and C3b were both effective as mediators of histaminase release. The extent of release varied as a function of the activating surface on which C3 was deposited (zymosan C3b was considerably more potent than C3b bound to rabbit erythrocytes, which was slightly more potent than C3b bound to neuraminidase-treated sheep erythrocytes). In contrast, C3b or C3bi deposited on nonactivating surfaces (such as sheep erythrocytes) at inputs of up to 2,000,000 molecules per granulocyte failed to induce histaminase release unless IgG was also present. The ability of C3b bound to particles that serve as activators of the alternative pathway to induce histaminase release is apparently not the result of decreased susceptibility of C3b to proteolysis or to an increased binding affinity to the C3b receptor, but may relate to the interaction of other surface structures on activating particles with the PMN membrane.     

Effect of glycyrrhetinic acid on membrane band 3 in human erythrocytes

Glycyrrhetinic acid (GA) is a hydrolytic product of the triterpene glycoside of glycyrrhizic acid, one of the main constituents of licorice root, which has long been studied, due to its several biological and endocrine properties. In this paper, GA was tested on human erythrocytes, and GA-induced alterations were compared with those caused by diamide, a mild oxidant inducing well-characterized cell/membrane alterations, and n-ethylmaleimide (NEM), as alkylating agent. In order to verify the biochemical steps underlying the action of GA, band 3 Tyr-phosphorylation level, enzyme recruitment and band 3 clustering in cells pre-incubated with GA before diamide treatment were all examined. Results show that GA, in a dose-dependent manner, prevents both diamide and NEM-induced band 3 Tyr-phosphorylation, but not GSH decrease caused by both compounds. In addition, diamide-induced band 3 clustering and IgG binding to altered cells were also completely reversed by GA pre-treatment. Also, when membrane sensitivity toward proteolytic digestion was tested, GA-treated cells showed high resistance to proteolysis. In conclusion, in human erythrocytes, GA is proposed to strengthen membrane integrity against both oxidative and proteolytic damage.