Ingestion Of Erythrocytes Research Articles

NCOA4 Regulates Iron Recycling and Responds to Hepcidin Activity and Lipopolysaccharide in Macrophages.

Macrophages, via erythrophagocytosis, recycle iron from effete erythrocytes to newly developing red blood cells. Conversion of potentially cytotoxic levels of iron from its heme into nonheme form during iron recycling is safely accomplished via coordinated regulations of cellular iron transport and homeostasis. Herein, we demonstrate the roles and regulation of NCOA4 (nuclear receptor coactivator 4)-mediated ferritinophagy in macrophages after erythrophagocytosis using the mouse macrophage cell line J774 cells. Ferritin in J774 cells increased with the rise of nonheme iron by erythrocyte ingestion and declined when total cellular iron contents subsequently decreased. NCOA4, a selective autophagic cargo receptor for ferritin, was responsible for the control of cellular ferritin and total iron contents at the later stage of erythrophagocytosis. A hepcidin analog, which limits the flux of iron through iron-recycling by inhibiting iron export at the plasma membrane, repressed NCOA4 expression and led to accumulation of ferritin in the mouse macrophages. Transcriptome analyses revealed a functional association of immune response with NCOA4-dependent gene expressions, and we confirmed repression of Ncoa4 by lipopolysaccharide (LPS) in J774 cells and the spleen of mice. Collectively, our studies indicate that NCOA4 facilitates cellular ferritin turnover and the release of iron by macrophages after erythrophagocytosis and functions as a regulatory target for molecular signals of systemic iron overload and inflammation. These identify macrophage NCOA4 as a potential therapeutic target for disorders of systemic iron dysregulation, including anemia of inflammation and hemochromatosis.

Significance of higher drug concentration in erythrocytes of mice infected with Schistosoma japonicum and treated orally with mefloquine at single doses.

The purpose of the present study is to understand the pharmacokinetic feature of mefloquine measured by erythrocytes and plasma in Schistosoma japonicum (S. j.)-infected mice and non-infected mice after oral administration of the drug at single doses. A high-performance liquid chromatography (HPLC) method was used to measure the plasma and erythrocyte concentrations of mefloquine at varying intervals posttreatment. Our results demonstrated that in non-infected mice treated orally with mefloquine at an ineffective dose of 50mg/kg or effective dose of 200mg/kg for 2-72h, the erythrocyte-to-plasma ratios of mefloquine were 5.8-11.2 or 2-14.2. On the other hand, in S. j.-infected mice treated with the same single doses of the drug, the erythrocyte and plasma drug concentration ratios were 3.1-4.6 or 2.9-8.5, manifesting that either in infected mice or in non-infected mice that received oral mefloquine resulted in higher concentration of mefloquine in erythrocytes than that in plasma. Unexpectedly, under oral administration of mefloquine at a higher single dose of 200mg/kg, the pharmacokinetic parameter C max values for plasma from S. j.-infected and non-infected mice were 1.6 ± 0.3 and 2.0 ± 0.4μg/mL, respectively, which were below the determined in vitro LC50 (50% lethal concentration) value of 4.93μg/mL. Therefore, the plasma concentration of mefloquine may display a little effect against schistosomes during the treatment. Although the values of T 1/2 and AUC0-∞ for erythrocytes were significantly longer and higher in infected mice than those of corresponding non-infect mice that received the same single mefloqine dose of 50mg/kg, the C max value was only 2.6 ± 0.4μg/mL lower than the determined in vitro LC50, which may explain why this low single dose is ineffective against schistosomes in vivo. After administration of higher mefloquine dose of 200mg/kg, the C max value for erythrocytes in infected mice was 30% (7.4 ± 0.7 versus 10.7 ± 2.7μg/mL) lower than that in the corresponding non-infected mice, but its level was above the determined in vitro LC95 (95% lethal concentration) value of 6.12μg/mL. Meanwhile, longer T 1/2 value of 159.2 ± 129.3h in infected mice led to significant increase in AUC0-∞ value (1969.3 ± 1057.7 vs 486.4 ± 53.0μg/mL·h), relative to corresponding non-infected mice. In addition, the mean residence time (MRT0-∞) in infected mice was also significantly longer than that in non-infected mice. All these results may beneficial for the treatment. According to the results, we suggest that higher ratios of mefloquine concentration in erythrocytes to plasma may offer a way to transport mefloquine to the worm gut through ingestion of erythrocytes by the worms, where the gut is the site for displaying the effect by mefloquine.

Mechanisms of Adherence, Cytotoxicity and Phagocytosis Modulate the Pathogenesis of Entamoeba Histolytica

The unicellular parasite Entamoeba histolytica, the causative agent of the human disease amebiasis, has traditionally been distinguished from its nonpathogenic cousin Entamoeba dispar by its propensity for the ingestion of erythrocytes. This classic feature, along with the parasite's ability to cause extensive host cell death, are critical mechanisms of pathogenesis during human infection. Recent advances have led to a greater understanding of the molecular components that allow E. histolytica to kill and phagocytose extracellular targets during human infection and include detailed studies of the role of the parasite's cysteine proteinases and other effectors of cytotoxicity, as well as the mechanisms of ligand recognition, signaling and intracellular trafficking during phagocytosis.

Entamoeba histolyticaandEntamoeba disparUtilize Externalized Phosphatidylserine for Recognition and Phagocytosis of Erythrocytes

Amebic erythrophagocytosis is characteristic of invasive amebiasis, and mutants deficient in erythrocyte ingestion are avirulent. We sought to understand the molecular mechanisms underlying erythrocyte phagocytosis by Entamoeba histolytica. Following adherence to amebae, erythrocytes became round and crenulated, and phosphatidylserine (PS) was exposed on their outer membrane leaflets. These changes were similar to the effects of calcium treatment on erythrocytes, which we utilized to separate ameba-induced exposure of erythrocyte PS from the process of phagocytosis. The adherence and phagocytosis of calcium-treated erythrocytes were less inhibited by galactose than were those of healthy erythrocytes, suggesting the existence of an amebic coreceptor specific for PS. To test whether PS was recognized by amebae, calcium-treated cells were incubated with annexin V prior to adherence to or ingestion by E. histolytica. Annexin V blocked both adherence (50% +/- 12% inhibition; P < 0.05) and phagocytosis (65% +/- 10%; P < 0.05), providing evidence that at least one galactose-independent coreceptor was involved in the adherence and ingestion of red blood cells. The coreceptor was inhibited by phospho-l-serine and to a lesser extent by phospho-d-serine but not by phospho-l-threonine, which is consistent with the coreceptor functioning in the adherence and ingestion of erythrocytes via recognition of PS. We expanded our investigations to the highly related but noninvasive parasite Entamoeba dispar and demonstrated that it was deficient in red-blood-cell adherence, induction of PS exposure, and phagocytosis. These findings establish phosphatidylserine involvement in erythrophagocytosis by amebae and suggest the existence of a PS receptor on the surfaces of both E. histolytica and E. dispar.

Human low-affinity IgG receptor Fc gamma RIIa (CD32) introduced into mouse fibroblasts mediates phagocytosis of sensitized erythrocytes

Multiple isoforms of human (h) Fc gamma RII are currently recognized. We set up experiments to evaluate the significance of different hFc gamma RII (CD32) isoforms in the uptake of IgG-coated particles. Therefore, a panel of stable hFc gamma RII-transfected fibroblasts was constructed to study phagocytosis. It was observed that over 40% of 3T6 fibroblasts transfected with hFc gamma RIIa cDNA phagocytose human erythrocytes (E) coated with mouse (m) IgG1 or mIgG2a (EA). This was in contrast to cells expressing a tail-minus variant of Fc gamma RII (hFc gamma RIItail-). Fibroblasts transfected with hFc gamma RIIb1 cDNA infrequently internalized EA. There was no difference in EA binding by these transfectants; all three clones formed more than 80% EA-rosettes. Uptake of particles was dependent on the degree of E sensitization, and was inhibitable by CD32 monoclonal antibody AT10. Preincubation of cells with cytochalasin D blocked internalization completely, with no effect on binding. This shows that phagocytosis underlies the observed E internalization. Immunofluorescence studies using an anticathepsin D antiserum showed the maturation of phagosomes, containing antibody- coated E, into lysosomes. In conclusion, our studies show, for the first time, that fibroblasts possess the basic machinery for ingestion of erythrocytes, and are capable of phagocytosis only when equipped with an appropriate receptor. hFc gamma RIIa and, to a lesser extent hFc gamma RIIb1, are suited for this task, which seems dictated by the cytoplasmic domain.

Human low-affinity IgG receptor Fc gamma RIIa (CD32) introduced into mouse fibroblasts mediates phagocytosis of sensitized erythrocytes

Multiple isoforms of human (h) Fc gamma RII are currently recognized. We set up experiments to evaluate the significance of different hFc gamma RII (CD32) isoforms in the uptake of IgG-coated particles. Therefore, a panel of stable hFc gamma RII-transfected fibroblasts was constructed to study phagocytosis. It was observed that over 40% of 3T6 fibroblasts transfected with hFc gamma RIIa cDNA phagocytose human erythrocytes (E) coated with mouse (m) IgG1 or mIgG2a (EA). This was in contrast to cells expressing a tail-minus variant of Fc gamma RII (hFc gamma RIItail-). Fibroblasts transfected with hFc gamma RIIb1 cDNA infrequently internalized EA. There was no difference in EA binding by these transfectants; all three clones formed more than 80% EA-rosettes. Uptake of particles was dependent on the degree of E sensitization, and was inhibitable by CD32 monoclonal antibody AT10. Preincubation of cells with cytochalasin D blocked internalization completely, with no effect on binding. This shows that phagocytosis underlies the observed E internalization. Immunofluorescence studies using an anticathepsin D antiserum showed the maturation of phagosomes, containing antibody-coated E, into lysosomes. In conclusion, our studies show, for the first time, that fibroblasts possess the basic machinery for ingestion of erythrocytes, and are capable of phagocytosis only when equipped with an appropriate receptor. hFc gamma RIIa and, to a lesser extent hFc gamma RIIb1, are suited for this task, which seems dictated by the cytoplasmic domain.

Inhibition of hydrogen peroxide release from activated macrophages by prior ingestion of erythrocytes or haemoglobin.

Production of hydrogen peroxide by mouse peritoneal macrophages activated with Corynebacterium parvum was induced by incubating the cells with opsonised zymosan. H2O2 release was reduced by 47% when macrophages were preincubated with opsonised sheep erythrocytes. A significant decrease also occurred when the cells were preincubated with heat-denatured haemoglobin, but not when preincubated with opsonised erythrocyte ghosts, even though the latter were taken up by the macrophages. The ability of macrophages in an infected lesion to destroy microorganisms may therefore be impaired by ingestion of extravasated erythrocytes.

Inhibition of hydrogen peroxide release from activated macrophages by prior ingestion of erythrocytes or haemoglobin

Production of hydrogen peroxide by mouse peritoneal macrophages activated with Corynebacterium parvum was induced by incubating the cells with opsonised zymosan. H2O2 release was reduced by 47% when macrophages were preincubated with opsonised sheep erythrocytes. A significant decrease also occurred when the cells were preincubated with heat-denatured haemoglobin, but not when preincubated with opsonised erythrocyte ghosts, even though the latter were taken up by the macrophages. The ability of macrophages in an infected lesion to destroy microorganisms may therefore be impaired by ingestion of extravasated erythrocytes.

Fc receptor-independent phagocytosis by mouse macrophages of erythrocytes sensitized with a lectin from a marine invertebrate.

A lactose-specific lectin present in the haemolymph of the colonial ascidian Botrylloides leachii was tested for its ability to act as an opsonin for the phagocytosis of erythrocytes by mouse macrophages. Sensitization of sheep or mouse erythrocytes with one agglutinating dose of the invertebrate agglutinin enhanced significantly the clearance of these cells from the circulation of mice, as did treatment with specific antibody. By measuring the adhesion and phagocytosis of 51Cr-labelled erythrocytes by monolayers of macrophages in vitro, it was shown that the B. leachii agglutinin was highly opsonic, promoting the ingestion of erythrocytes by the macrophages with an initial efficiency similar to that of specific antibody. The adherence and ingestion of antibody-coated erythrocytes was severely inhibited by blocking Fc receptors on the macrophages with soluble immune complexes, whereas the binding and ingestion of lectin-coated erythrocytes was unaffected by this treatment. The results demonstrate that invertebrate lectins can act as efficient opsonins for the uptake of particles by vertebrate phagocytes and that it is not necessary for particles to bind to Fc receptors for phagocytosis to be induced.

Blood destruction in pulmonary alveoli: signs of vitality and determination of survival time

Lungs from 26 cases were examined in which blood was present as a result of a gunshot wound, a stab wound, or aspiration. Signs of vitality and of a time-dependent reaction sequence were evaluated to determine survival time. Only those morphologic criteria were considered that could be obtained on paraffin sections. In addition to H & E staining, siderin was identified with the Prussian blue reaction and the activities were determined of tartrate-resistant acid phosphatase, as macrophage marker, and naphthol AS-D chloracetate esterase, as granulocyte marker. The following criteria were evaluated: granulocyte emigration, erythrocyte adherence to the surface of macrophages, macrophage ingestion of erythrocytes, and determination of siderin as indicator of intracellular erythrocyte digestion. Adherence was also observed in those cases that did not survive. The initial sign of vitality was granulocyte emigration, which was observed for the first time after a survival time of 5 min. Erythrophages were found after a survival time of 30 min at least, siderophages after 17 h at the earliest. Literature dealing with vitality and age determination, as well as the pathogenesis of the reaction sequence, is discussed.

Effect of erythrocyte ingestion on macrophage antibacterial function.

Individuals with sickle cell anemia are subject to serious infections caused by a number of bacteria, including Salmonella species and Staphylococcus aureus. It has been suggested that in sickle cell anemia, extensive erythrophagocytosis by macrophages may interfere with their antibacterial function and thereby predispose to infection. As a means of investigating this possibility, we evaluated the effects of erythrocyte ingestion on the Killing of Salmonella typhimurium by peritoneal macrophages and of S. aureus by alveolar macrophages. Monolayers of rabbit macrophages were exposed to erythrocytes or latex particles immediately before and during bacterial challenge. Erythrophagocytosis markedly inhibited intracellular killing of S. typhimurium by peritoneal macrophages (bacterial survival was 181% of control) and of staphylococci by alveolar macrophages (bacterial survival was greater than 200% of control). Exposure to latex particles depressed the bactericidal activity of alveolar macrophages to a lesser degree. Next we investigated the possibility that erythrophagocytosis inhibits oxidative bactericidal mechanisms in macrophages. Hexose monophosphate shunt activity was stimulated by erythrocyte ingestion. However, zymosan-induced superoxide generation and chemiluminescence were suppressed by erythrocytes. Furthermore, a cell-free (hypoxanthine-xanthine oxidase) system for chemiluminescence generation was also depressed in the presence of erythrocytes (intact or lysate) or by purified hemoglobin. These studies reveal that erythrophagocytosis inhibits macrophage antibacterial function, probably because of interactions between erythrocyte components and reactive products of phagocyte oxygen metabolism. This host defense abnormality may predispose to bacterial infection in certain hemolytic anemias.

C-reactive protein reactivity with complement and effects on phagocytosis.

In the studies described here we have attempted to evaluate the hypothesis that CRP may function in host defense using two systems in which CRP in the presence of C appears to have opsonic properties. In the first, CRP and C were found to stimulate ingestion of erythrocytes by human monocyte or mouse macrophages in vitro, and to alter clearance patterns in vivo. In the second, we have studied opsonization of S. pneumoniae by CRP and C. Experiments with human neutrophils indicate that although CRP and C can enhance opsonization of S. pneumoniae, this effect is more pronounced in the absence of antibody. In vivo CRP was found to protect mice against intravenous infection with S. pneumoniae.

Lectin-Induced Phagocytosis of Erythrocytes by Guinea Pig and Mouse Macrophages

Various lectins were examined for their ability to induce phagocytosis of sheep erythrocytes by peritoneal macrophages of the guinea pig and mouse. Phaseorous vulgaris lectin-L (PHA(L)) induced the phago-cytosis of erythrocytes by guinea pig macrophages and wheat germ agglutinin (WGA) that of erythrocytes by mouse macrophages. Concanavalin A (Con A) induced little ingestion by either macrophage, although it induced much attachment of the erythrocytes to both. The PHA(L)-induced phagocytosis of erythrocytes by guinea pig macrophages was inhibited specifically by N-acetyl-D-galactosamine. PHA(L) linked the guinea pig macrophages and sheep erythrocytes. Con A did not suppress the PHA(L)-induced phagocytosis of erythrocytes by guinea pig macrophages. These results suggest that the attach-ment of erythrocytes to macrophages is not sufficient for phagocytosis and that certain sites on the surface of the macrophages must be stimulated for phago-cytosis to occur. Soluble antigen-antibody complexes, which block the Fc receptors of macrophage and other cell types, inhibited both the antibody-induced and the PHA(L)-induced ingestion of erythrocytes by guinea pig macrophages. A prior incubation of guinea pig macrophages with high con-centrations of PHA(L) suppressed the ingestion of erythrocytes which was induced by PHA(L), but not that induced by antibody. This is evidence that some relation exists between the Fc receptors and the PHA(L) binding site that is responsible for the induction of phagocytosis.

Ingestion of Erythrocytes Containing 51Cr-Labeled Hemoglobin by Psoroptes Cuniculi (Acari: Psoroptidae)

Washed rabbit erythrocytes labeled with radioactive chromium in vitro were resuspended in plasma and injected into 2 adult female rabbits that were heavily infested with ear canker mites, Psoroptes cuniculi . After 24 h the mites were harvested and the radioactivity was measured. The mites (850 mg) contained approximately 77 μl of blood and had digested almost ½ the hemoglobin. The data suggest that the mites actually penetrate the capillaries and ingest intact erythrocytes during feeding.

Inhibition by amoeba-specific antiserum and by cytochalasin B of the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures.

Inhibitors of trophozoite motility and phagocytosis were used to investigate the mechanism of Naegleria fowleri cytopathogenicity in mouse-embryo (ME)-cell cultures. Amoebae that were immobilised and agglutinated by specific antiserum exhibited no cytopathic activity, although they remained alive and were in constant contact with the ME cells. Mammalian-cell damage occurred only when the organisms recovered pseudopodium function and began to migrate over the monolayers as they overcame the inhibitory effects of the antiserum. Cytochalasin B at a concentration of 10 microgram/ml, shown to prevent the engulfment of chick erythrocytes by amoebae, also inhibited the cytopathogenicity of Naegleria when incorporated in ME-cell culture medium. Despite repeated contact with active trophozoites, the ME cells showed only those morphological changes characteristically induced by cytochalasin B itself. The amoebae in turn showed signs of starvation after 3 or 4 days' incubation, suggesting that the feeding activity of trophozoites was suppressed. Colchicine, on the other hand, inhibited neither the ingestion of erythrocytes nor the destruction of ME cells by amoebae. It was concluded that the cytopathogenicity of N. fowleri in ME-cell cultures was due to physical rather than biochemical or cytotoxic mechanisms and was associated with the phagocytic activity of trophozoites.

Surface properties of Entamoeba: increased rates of human erythrocyte phagocytosis in pathogenic strains.

The assertion that ingestion of human erythrocytes is restricted to invasive strains of Entamoeba histolytica has not been evaluated previously by comparative studies. In this report we describe the in vitro ingestion of human erythrocytes by pathogenic and nonpathogenic Entamoeba. Microscopic evaluation of erythrophagocytosis by eight different Entamoeba grown in culture revealed that strains of E. histolytica isolated from cases of human dysentery show a much higher rate of erythrocyte ingestion than nonpathogenic strains. However, all strains are able to phagocytize erythrocytes. The extremely high rate of phagocytic activity shown by pathogenic E. histolytica could be one of the properties related to the pathogenicity of this parasitic protozoan.

Macrophage cytophilic antibodies in mice: inability to induce ingestion of sheep erythrocytes by mouse peritoneal macrophages.

By using an in vitro test for phagocytosis, it was demonstrated that mouse antisheep erythrocyte macrophage-cytophilic antibodies did not significantly contribute to erythrocyte ingestion by mouse peritoneal macrophages.

The formation of biliverdin by chicken macrophages in tissue culture observations on the effect of inhibitors

1. 1. Ingestion of erythrocytes and haemin suspensions by chicken macrophage monolayers results in the excretion of a green pigment into the medium identified as biliverdin IXα. 2. 2. Formation of biliverdin from untreated red cells is stimulated by ascorbate, H 2O 2 and aminotriazole due to increased phagocytosis. 3. 3. The above compounds have no effect on bile pigment formation from N-ethylmaleimide-treated red cells. 4. 4. In concentrations which are not inhibitory to cell growth KCN, Nan 3, phenobarbital, acetanilide and SKF-525A have no effect on biliverdin formation. CO has weak inhibitory properties consistent with substrate stabilisation rather than haemoprotein oxidase inhibition. 5. 5. Mepacrine (atebrine) and the antibiotics cycloheximide, actinomycin D and mitomycin C are potent inhibitors of bile pigment formation but chloramphenicol is without effect.

Human monocytes: distinct receptor sites for the third component of complement and for immunoglobulin G.

Human monocytes contain two distinct receptor sites, one specific for the third component of complement (C'3), the other for immunoglobulin G(gammaG). The two receptors may function either independently or cooperatively in the induction of phagocytosis. Ingestion of erythrocytes coated with immunoglobulin M antibody requires a relatively large number of bound C'3 molecules per cell. Ingestion of erythrocytes sensitized with gammaG antibody is independent of complement; however, the reaction is inhibited by concentrations of gammaG far below those in normal serum. Inhibition by gammaG-globulin is overcome by a relatively small number of bound C'3 molecules per cell. The two monocyte receptors exert a cooperative effect on ingestion by monocytes of erythrocytes coated with gammaG antibody in the presence of inhibitory amounts of free gammaG.

The Ingestion of Erythrocytes by Trichomonas hominis and Its Occurrence in the Pus of an Amoebic Liver Abscess

The question of the pathogenicity of Trichomonas in man has in the past been open to discussion and the present tendency (Hegner and Taliaferro, 1924) resulting from the observations by a number of workers on the ingestion of erythrocytes by Pentatrichomonas and its presence in cases of long standing diarrhoea (Chatterjee 1917, Haughwout and de Leon 1919, and Kofoid and Swezy 1923) is to differentiate between Trichomonas hominis which is usually described as possessing four anterior flagella and Pentatrichomonas ardin-delteli which has five anterior flagella, and to assign the role of pathogenicity to Pentatricho-