Phagocytosis of senescent erythrocytes by autologous monocytes: requirement of membrane-specific autologous IgG for immune elimination of aging red blood cells

The role of membrane-bound IgG present on the membrane of senescent erythrocytes in immune eliminations of aging red cells was investigated. Phagocytosis of populations of red blood cells (RBC) of different ages by autologous monocytes was assessed both by direct phagocytosis and by induction of microsomal heme oxygenase. Removal of IgG from older RBCs inhibited their phagocytosis; in contrast, preincubation of neuraminidase-treated young or in vitro aged RBCs with IgG eluted from old cells led to phagocytosis of RBCs treated by autologous monocytes. It was also found that the Fc portion of membrane-bound IgG is essential for the elimination of senescent cells; less than 15% of old heat-inactivated RBCs coated with F(ab)2 fragment of membrane-bound IgG were phagocytosed. In contrast, more than 50% of old heat-inactivated RBCs coated with heat-eluted IgG were phagocytosed by autologous monocytes. A possible mechanism of elimination of aged cells is discussed.     

Effect of serum on autologous cell recognition by macrophages

The non-immune mechanisms of recognition of self and non-self substances by macrophages has not yet been clarified. In this work, we report the ability of mouse peritoneal macrophages to attach to and phagocytize in vitro autologous and homologous erythrocytes in proportions as high as those for certain heterologous red blood cells. This ability was abrogated by autologous or homologous serum but not by heterologous serum or a serum-free supplement. This effect of serum was dose dependent and did not affect the phagocytosis of homologous "old" red cells. Procedures for the identification of this serum factor indicated that it was dialyzable (10 kD cut off) and was excluded by filtration in Sephadex G-25. We conclude that this finding supports the possibility that macrophages do not selectively phagocytize foreign particles or senescent cells but, rather, that they do phagocytize all particles or cells indiscriminately and this serum factor may prevent phagocytosis of normal self cells.     

Receptors of human red blood cells for anti-TAH and anti-AHP in the course of intravascular aging

Human o-erythrocytes aged in situ do not expose free receptors for anti-T agglutinin of Arachis hypogea. New receptors for concentrated anti-A agglutinin of Helix pomatia are manifested but are lost again in the course of further ageing of the cells in situ. A remasking of exposed receptors for anti-TAH and anti-AHP byautologous, strongly binding globulins is supposed. These globulins could constitute the decisive signal for the autologous phagocytosis of senescent red blood cells.     

Comparative analysis of agglutinins from hemolymph and albumin gland ofHelix pomatia

Summary The hemolymph ofHelix pomatia contains a weak agglutinating activity. This lectin concentration was calculated to be about 1.8 g·ml^–1. Among the different red blood cells tested, pronase-treated sheep erythrocytes were found to be the most suitable indicator cells. Their agglutination could be inhibited by GalNAc and GlcNAc. The serum agglutinin was isolated by affinity chromatography using Sephadex G-200 as the matrix. It exhibited a single band in discontinuous PAGE. In the presence of SDS, subunits of 27000 daltons were obtained which, after addition of 2-mercaptoethanol, partly dissociated into 13000-dalton subunits. The biochemical properties observed were compared with those of the well-known blood group A-specific lectin from the albumin gland ofH. pomatia.Abbreviations GalNac N-acetyl-galactosamine - GlcNAc N-acetyl-glucosamine - SDS sodium dodecylsulfate     

Impaired monocyte function in liver cirrhosis.

Monocyte function in patients with cirrhosis of the liver was measured by phagocytosis and killing of Candida pseudotropicalis and C albicans. Both variables were significantly decreased in the patients compared with controls. Control monocytes exposed for two hours to patients'' serum showed a significant decrease in intracellular killing compared with control monocytes incubated in autologous serum. This suggests the presence of an inhibiting factor in the patients'' serum. This inhibitory factor passed through a dialysis membrane that permitted the passage of molecules of less than 12 000 daltons. Treating monocytes from patients with trypsin significantly increased phagocytosis, indicating that the possible inhibitory factor was attached to the monocyte surface. Metabolism of monocytes during phagocytosis as determined by chemoluminescence was significantly lower in monocytes from patients compared with controls, indicating metabolic impairment. Monocytes are a component of the monocyte-macrophage system, which includes Kupffer''s cells. Impairment of the function of these cells, which have a pivotal role in clearing portal blood, might well be extremely important in the development of chronic liver disease.     

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.     

A mathematical model for drug administration by using the phagocytosis of red blood cells

 A mathematical model for the delivery of drug directly to the macrophages by using the phagocytosis of senescent red blood cells is proposed. The model is based on the following assumption: At time t=0 a preassigned red blood cell population n(0, a)=φ(a), a>0, loaded by the drug, is injected in the blood circulation. Among the cells of that population only those with an age a≧ā (ā=120 days) will be phagocytosed by macrophages. Of course, the lifetime of the drug must be higher than ā. Within the red blood cells it cannot be metabolized, neither can it diffuse through their membranes. The emphasis of the paper is on the mathematical properties and on the formulation of the control problem. Received 15 December 1994; received in revised form 20 July 1995     

Naturally occurring anti-band 3 antibodies and complement in phagocytosis of oxidatively-stressed and in clearance of senescent red cells

Treatment of human red blood cells with diamide and opsonization with whole serum enhanced their phagocytosis by mononuclear phagocytes. Opsonization of diamide-treated red cells with whole serum containing 20-100 times the physiologic concentration of naturally occurring anti-band 3 antibodies further increased the extent of phagocytosis. Enhanced phagocytosis was due to an anti-band 3 mediated binding of C3b to red cells via the alternative pathway. Red cell-bound anti-band 3 was slightly elevated on diamide-treated cells and elicited a C3 binding that exceeded the amount of bound antibody by two orders of magnitude. Pretreatment of red cells with a monoclonal anti-CR1 did not significantly inhibit opsonization and phagocytosis if cells were opsonized at elevated anti-band 3 concentrations. On the other hand, phagocytosis of mildly oxidized (20 microM diamide) red cells was completely inhibited by blocking CR1 if cells were opsonized with serum containing physiologic concentrations of anti-band 3. The results suggest that two types of opsonization mediate in vitro phagocytosis: one operating at physiologic anti-band 3 concentrations with mildly oxidized red cells (IC-like mechanism) and one that operates with either heavily oxidized (greater than 200 microM diamide) red cells at physiologic anti-band 3 concentrations, or with mildly oxidized cells opsonized at elevated concentration of anti-band 3. The latter mechanism is relevant in vivo. It is most likely that it starts by Fab-dependent binding of anti-band 3 to diamide-induced band 3 protein oligomers. Complement activation may occur by assembly of an alternative convertase on C3b covalently bound to red cell-associated anti-band 3. This mechanism is also likely to mediate clearance of senescent red cells, as it was primarily from senescent red cells that we could isolate complexes containing IgG covalently bound to C3b.     

Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

Chemoenzymatic synthesis and characterization of N-glycolylneuraminic acid-carrying sialoglycopolypeptides as effective inhibitors against equine influenza virus hemagglutination

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 → 3)- and α(2 → 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a–c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.     

Erythrophagocytosis in malaria: Host defence or menace to the macrophage?

Macrophages in the host's bloodstream and tissue serve as a first line of defence during infection with Plasmodium. While the killing effect of these cells on parasites has been investigated extensively, relatively little is known about the phagocytosis of infected red blood cells. In this article, Paolo Arese and Franca Turrini have joined Hagai Ginsburg to address the perplexing relationships between the macrophage and the malaria-infected red blood cell. They suggest that the same molecular mechanisms that normally operate to remove senescent or damaged red blood cells also operate during malaria, although the parasite may indirectly cause the destruction of macrophages.     

Platelet-activating factor enhances complement-dependent phagocytosis of diamide-treated erythrocytes by human monocytes through activation of protein kinase C and phosphorylation of complement receptor type one (CR1)

Oligomerization of band 3 protein has been recently indicated as an early event in senescent or damaged red cell membrane followed by specific deposition of anti-band 3 antibodies and binding of complement C3 fragments. The band 3-anti-band 3-C3b complex is recognized by homologous monocytes, and phagocytosis ensues. This study shows that recognition of the anti-band 3-C3b complex by the monocyte C3b receptor type one (CR1) plays a crucial role in the process of removal of damaged red cells. Indeed, blocking of monocyte CR1 with an anti-CR1 monoclonal antibody abrogated phagocytosis of diamide-treated red cells. Platelet-activating factor (PAF) is a phospholipid mediator involved in inflammatory processes. Nanomolar (R)-PAF enhanced the CR1-dependent phagocytosis of diamide-treated human red cell and of sheep red cells coated with C3b, induced the fast translocation of protein kinase C to monocyte membrane compartment, and stimulated the phosphorylation of monocyte CR1. The biologically inert lyso-PAF and the enantiomer (S)-PAF were inactive. PAF receptor antagonists and inhibitors of protein kinase C blocked the enhancement of phagocytosis induced by PAF. Protein kinase C translocation, phosphorylation of CR1, and stimulation of this receptor to an active state capable of mediating phagocytosis represent a novel pathway by which PAF interferes with red cell homeostasis and possibly modulates inflammatory reactions and host mechanisms against infections.     

P2X7 receptor-mediated scavenger activity of mononuclear phagocytes toward non-opsonized particles and apoptotic cells is inhibited by serum glycoproteins but remains active in cerebrospinal fluid

Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1-5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1-10 mM tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells.     

Adhesion ofHelicobacter pylori strains toα-2,3-linked sialic acids

Helicobacter pylori is a human pathogen associated with gastritis and peptic ulcer. Adhesion properties ofH. pylori to various structures have been described in the literature, including evidence for sialic acid-binding. To study the specificity and frequency of sialic acid-binding, fourteenH. pylori strains were investigated using haemagglutination with derivatized erythrocytes carrying sialic acids only on defined glycans and using haemagglutination inhibition assays. From these studiesH. pylori strains can be grouped into sialic acid-dependent and sialic acid-independent classes. The sialic acid-dependent strains require -2,3-linked sialic acid for haemagglutination. The potential roles of sialic acid-dependent adhesions forH. pylori-related infections are discussed.Abbreviations Sia sialic acids - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolylneuraminic acid - Neu5Fm N-formylneuraminic acid - Neu5TFA N-trifluoroacetylneuraminic acid - RBC human red blood cells (erythrocytes)     

Target cell deformability determines the type of phagocytic mechanism used by Entamoeba histolytica-like,Laredo strain

Summary— Morphological study of red blood cell phagocytosis by Entamoeba histolytica-like (Laredo strain) has shown that this amoeba is able to ingest by two distinct mechanisms. One is classical phagocytosis and the other is by suction or microphagocytosis. Rigidification of red blood cells by treatment with glutaraldehyde shows that there is a correlation between the deformability of the ingested cell and the type of phagocytosis observed. Indeed, as the red cells become more rigid, less microphagocytosis is observed. To demonstrate that this shift in phagocytic mechanisms is not induced by the modification of a surface receptor by the glutaraldehyde treatment, the amoebas were fed with erythrocyte ghosts. Since these have lost most of their hemoglobin content, they are less rigid than the intact erythrocytes. The ghosts, even after glutaraldehyde treatment, are always ingested by microphagocytosis. These results have therefore led us to conclude that the type of erythrocyte phagocytosis used by E histolytica-like (Laredo strain) is determined by the deformability of the targetted red blood cells.     

Importance of molecular studies on major blood groups—Intercellular adhesion molecule-4, a blood group antigen involved in multiple cellular interactions

Several blood groups, including the LW-blood group were discovered in the first part of last century, but their biochemical characteristics and cellular functions have only more recently been elucidated. The LW-blood group, renamed ICAM-4 (CD242), is red cell specific and belongs to the intercellular adhesion molecule family. ICAM-4 binds to several integrin receptors on blood and endothelial cells and is thus able to form large cellular complexes containing red cells. Its physiological function(s) has remained incompletely understood, but recent work shows that macrophage integrins can bind red cells through this ligand. In this article we discuss molecular properties of major blood group antigens, describe ICAM-4 in more detail, and show that phagocytosis of senescent red cells is in part ICAM-4/β₂-integrin dependent.     

Plasmodium falciparum: Assay in vitro for inhibitors of merozoite penetration of erythrocytes

Highly synchronous cultures of the erythrocytic stages of Plasmodium falciparum were used both to assay penetration of merozoites into human red blood cells, and to subsequently study the inhibitory effects of various substances on penetration. While several sugars exhibited no inhibitory effect, fucose, glucosamine-HCl, and N-acetyl glucosamine, when added to synchronous cultures at the schizont stage, inhibited invasion. On further testing fucose and glucosamine-HCl were found to be toxic to the intracellular growth and development of the parasite; only N-acetyl glucosamine had an inhibitory effect solely related to the inhibition of merozoite penetration. Glycophorin A, the major glycoprotein of the red blood cell surface, had no inhibitory effect at low concentrations, but had a slight effect at higher (500 μg/ml) levels.     

Muramyl dipeptide bound to poly-l-lysine substituted with mannose and gluconoyl residues as macrophage activators

Poly-l-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized byN-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-l-lysine substituted with several mannose derivatives and gluconoyl residues (GlcA_x-, Man_y-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficientin vitro than free MDP in macrophage cytostasis assays.Abbreviations MDP N-acetylmuramyl dipeptide - PLK poly-l-lysine - BSA bovine serum albumin - BOC t-butyloxycarbonyl - DMF dimethylformamide - DCHU N, N-dicyclohexylurea - DCCl N, N-dicyclohexylcarbodiimide - TEA triethylamine - Su succinimidyl - DMSO dimethylsulfoxide - FITC fluoresceinyl isothiocyanate - RPMI Roswell Park Memorial Institute - PBS phosphate buffered saline - Fl fluoresceinyl - GG glycyl-glycyl     

Brief report: a new profile of terminal <Emphasis Type="Italic">N</Emphasis>-acetyllactosamines glycans on pig red blood cells and different expression of α-galactose on Sika deer red blood cells and nucleated cells

It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galβ1-4GlcNAc-R) and α-Gal epitopes (Galα1-3Galβ1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galβ1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack α-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that α-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-α-Gal epitopes. Whereas, the expression of the α-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution.     

Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC₅₀<20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC₅₀=0.4 μg/ml)) compared to colon (IC₅₀>20.0 μg/ml) and stomach (IC₅₀>20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.