Recognition of oxidatively damaged erythrocytes by a macrophage receptor with specificity for oxidized low density lipoprotein.

Macrophages specifically bind and internalize oxidatively modified low density lipoprotein (LDL) via the acetyl-LDL receptor and possibly one or more additional receptors jointly designated here as scavenger receptors. It is well accepted that these receptors are intimately involved in the formation of foam cells during atherogenesis. However, the normal physiological or pathophysiological role for these receptors has not been established. Oxidation of plasma membranes is a common accompaniment of cell damage and senescence. In particular, aged erythrocytes demonstrate peroxidation of their cell membrane lipids. In the present studies we show that oxidized human erythrocytes (treated with copper plus ascorbate or hydrogen peroxide) are bound and phagocytosed by mouse peritoneal macrophages in the absence of opsonizing antibodies. There was little or no binding of untreated erythrocytes. Oxidized LDL, but not acetylated or native LDL, inhibited this binding and uptake of oxidized erythrocytes. Inhibitors of scavenger receptor binding, including polyinosinic acid and fucoidin, also prevented binding of the oxidized red blood cells. We suggest that oxidative damage of erythrocytes results in the formation of lipid-protein conjugate(s) closely related to some of the conjugates found in oxidized LDL, making the oxidized erythrocyte a ligand for the macrophage scavenger receptors, apparently at a site distinct from that responsible for the binding of acetylated LDL. Oxidative modification of plasma membranes may represent a general mechanism that marks damaged cells for phagocytosis by macrophages.     

The mechanism behind the antileishmanial effect of zinc sulphate. I. An in-vitro study

In an attempt to determine the possible mechanism(s) behind the antileishmanial activity of zinc sulphate, promastigotes, axenic amastigotes and intracellular amastigotes of both Leishmania major and L. tropica were incubated with different concentrations of the compound. For each of the two Leishmania species, all three forms were found to be inhibited by the zinc sulphate, in a dose-dependent manner, the promastigotes being the most resistant form, followed by the axenic amastigotes. These results indicate that zinc sulphate has a direct antileishmanial effect. Compared with macrophages from starch-treated mice, the macrophages recovered from mice that had been injected intraperitoneally with zinc sulphate (daily for the 4 days prior to the macrophage collection) or BCG (once, 4 days before the cell collection) showed increased phagocytosis and increased killing of L. major and L. tropica. As the effects of the zinc sulphate were not statistically different from those of the known immunomodulating agent BCG, zinc sulphate appears to have an immunomodulating effect, in addition to its direct antileishmanial effect.     

Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes.

Leishmania are obligatory intracellular parasites in mammalian macrophages that gain entry by receptor-mediated phagocytosis. Their major cell surface glycoconjugate, lipophosphoglycan (LPG), has been implicated in this process. A monoclonal antibody specific for Leishmania major LPG (WIC 79.3), which has been shown to block promastigote attachment to macrophages, was used to identify a macrophage-binding determinant of LPG. WIC 79.3 bound exclusively to the phosphorylated repeats of LPG and not to the saccharide core or lipid anchor. Furthermore, the epitope recognized by WIC 79.3 mapped to the phosphorylated oligosaccharide P5b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-, which is unique to the LPG of promastigotes of L.major. Phosphorylated oligosaccharides P3, PO4-6[Gal(beta 1-3)[Gal(beta 1-4) Man(alpha 1-, and P4b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)] Gal(beta 1-4)Man(alpha 1-, were also recognized by WIC 79.3 but with considerably lower (approximately 100-fold) affinities. The phosphorylated oligosaccharide P5b inhibited attachment of promastigotes of L. major to the macrophage cell line J774 to the same degree as phosphoglycan (derived from LPG) and Fab fragments of WIC 79.3, suggesting that P5b is a site of L. major LPG that is recognized by macrophage receptor(s) and is an important determinant in the attachment of promastigotes to host macrophages and initiation of infection.     

Leishmania (Viannia) braziliensis: interaction of mannose-binding lectin with surface glycoconjugates and complement activation. An antibody-independent defence mechanism

The activation of complement on the surface of Leishmania promastigotes appears to be an important factor for parasite infectivity in the mammalian host, allowing their attachment and the invasion of macrophages via complement receptors. Mannose-binding lectin (MBL) is a well-known complement activator and an efficient opsonine. We have investigated here whether serum and purified MBL bind to and promote lysis of live promastigotes of L. braziliensis; and evaluated the deposition of MBL, C1q, C4 and C3 on the parasite surface after interaction with non-immune normal human serum (NHS). We observed that both serum MBL and the purified MBL-MASP complex bind to the surface of L. braziliensis and that this binding occurred via the carbohydrate recognition domains of MBL. The binding of MBL, however, did not affect the lytic effect of complement on the parasites. The deposition of C1q, C4, C3 and parasite lysis was observed after incubation with NHS. EDTA but not EGTA abolished C3 deposition on the parasite surface, indicating the involvement of the alternative pathway in this process. Our results indicate that MBL binds to L. braziliensis and that this is mediated by a specific carbohydrate on the surface of parasites and provides evidence for antibody-independent mechanisms that complement activation on the parasite surface.     

Exogenous interferon administration in experimental leishmaniasis: in vivo and in vitro studies

The potential feasibility of using exogenously administered human interferon for the treatment of selected cases of leishmaniasis prompted us to study the effects of murine interferon on the course of Leishmania tropica infection in C57Bl/6 mice. L cell-derived mouse interferon was administered daily by intraperitoneal (1,000 and 10,000 U) or intralesional (100 U) injection in mice inoculated into footpads with L. tropica amastigotes. Footpad swelling and tissue parasite density were assessed over the course of infection. Interferon treatment did not significantly affect these clinical and parasitological parameters. Furthermore, addition of interferon (100--100,000 U) to cultures of amastigote-infected mouse peritoneal macrophages or to axenic cultures of promastigotes did not affect replication. We conclude that interferon lacks intrinsic antileishmanial activity and does not significantly enhance host defense against Leishmania.     

C-reactive protein-mediated phagocytosis of Leishmania donovani promastigotes does not alter parasite survival or macrophage responses

C-reactive protein (CRP) is an acute phase protein that binds to surface structures of a number of different organisms. Leishmania donovani express CRP ligand when first entering the mammalian host and CRP has been shown to alter macrophage function. The aim of this study was to investigate the functional significance of CRP-mediated uptake of L. donovani on survival of the parasite within human macrophages and macrophage cell responses to the infection. CRP opsonized L. donovani uptake was inhibitable by including excess CRP in the fluid phase, suggesting Fc receptor usage rather than indirect complement-mediated uptake. Comparing equivalent initial infection loads, parasite survival over 72 h within peripheral blood derived macrophages (PBMs) and differentiated U937 cells was unaltered by CRP. Whereas CRP increased macrophage responses to phosphorylcholine coated erythrocytes, no significant alteration in tumour necrosis factor-alpha, interleukin (IL)-10 or IL-12 production from PBMs was observed between CRP opsonized or unopsonized L. donovani promastigotes. Thus, in contrast to other systems, where CRP opsonization results in macrophage activation, Leishmania can use CRP to improve infection without inducing detrimental macrophage activation.     

Fibronectin dependent macrophage fibrin binding

Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.     

Leishmania immune adherence reaction in vertebrates

In normal human blood, C3-opsonized Leishmania promastigotes immune adhere to erythrocytes, a mechanism believed to enhance their clearance from blood and phagocytosis. Given the potential importance of this reaction in host defence against infection, the promastigote-erythrocyte interaction was studied in blood of individuals from one avian and 12 mammalian genera; [111In]-labelled promastigotes were found to bind only to primate erythrocytes. Nevertheless, previous experiments coincubating platelets isolated from nonprimate mammals with C3-opsonized promastigotes led to promastigote-platelet adherence. To ascertain whether this is a natural mechanism in nonprimate Leishmania infection, normal blood from members of Leishmania animal models of interest, dog, guinea-pig, hamster, mouse and rabbit, was infected ex vivo with promastigotes. Within 1 min of blood contact, the promastigote surface was loaded with platelets, rapidly evolving into large aggregates. These results confirm the physiological nature of the reaction and demonstrate that promastigote-erythrocyte and promastigote-platelet binding are the first parasite-host cell encounters after Leishmania invasion of primates and nonprimate mammals, respectively. Leishmania immune adherence shares the characteristics of the nonanticipatory immune systems, and we consider it should be viewed as an innate vertebrate host effector mechanism.     

Refractory barriers in the sand fly Phlebotomus papatasi (Diptera: Psychodidae) to infection with Leishmania panamensis.

The life cycle of Leishmania panamensis in Phlebotomus papatasi was studied to characterize barriers limiting parasite colonization, differentiation, migration, and attachment in an unnatural sand fly host. The insects were fed a suspension of L. panamensis-infected macrophages and human erythrocytes, and were examined up to 16 days post-infection by light and electron microscopy. Histologic examination of 401 flies showed the peritrophic membrane to be the first important barrier to parasite establishment in the gut lumen. In most flies, parasites were unable to escape from the closed peritrophic sac, which was either excreted or retained intact in the midgut. After five days, only 31% of the flies were infected; attached parasites colonized the pylorus-ileum and/or colon regions of the hindgut. Anterior migration into the cardia region of the midgut occurred in less than 1% of infected flies; no parasites colonized the foregut. In the bloodmeal and residual bloodmeal, five morphologic forms developed from ingested amastigotes: stumpy, spatulate, elongate, short nectomonad promastigotes, and paramastigotes. Abnormal retention of amastigotes in macrophages and delayed development of promastigote stages was observed. The primary form attached in the hindgut was a pear-shaped haptomonad promastigote. Differentiation of L. panamensis in Ph. papatasi appeared to be similar to that described in natural hosts, except that metacyclic infective forms were not observed, and some forms developed in unusual locations. Phlebotomus papatasi was a partly refractory biological host for L. panamensis. The peritrophic membrane adversely affected the infection rate; rare anterior migration and a lack of metacyclic promastigotes may preclude transmission by bite.     

Studies on the effect of the anti-phagocytic agent cytochalasin B on Leishmania-macrophage interaction.

Mouse peritoneal exudate cells were cultured on coverslips in Eagle's Basal Essential Medium. The adhering cells were infected with promastigotes of three different species of Leishmania. After 8 h incubation, the macrophages were fixed and stained, and a total of one hundred cells were counted. The rates of infection of macrophages were respectively 53.5 +/- 5% for L. enriettii, 52.3 +/- 5% for L. donovani and 11.7 +/- 2% for L. tropica. When cytochalasin B at concentrations of 2.5, 5 and 10 microgram/ml and Leishmania promastigotes were added to the adhering cells at the same time, the drug did not have any effect on the uptake of the organisms by the macrophages. However, when the cells were treated for a 2-h period with the drug and then were infected with the promastigotes, only 1-2% of the cells were infected. On the other hand, when cytochalasin B-treated cells which had lost their phagocytic ability were washed and then were infected with the promastigotes, some degree of cellular infection was observed. It was concluded that infection of mouse p.e.c. by three different species of Leishmania which were used in our study was by phagocytosis rather than active penetration of the organisms into the cells. It was also of interest to note that although our outbred strain of mice gets infected easily with L. tropica, the p.e.c. of these animals phagocytosed L. tropica with least efficiency in comparison with L. donovani and L. enriettii.     

An in vitro model for investigation of chemotherapeutic agents in leishmaniasis

Clinically achievable concentrations of the three major antileishmanial drugs in use--pentavalent antimony, pentamidine, and amphotericin B--eliminated 90%--100% of the mammalian forms (amastigotes) of Leishmania tropica and Leishmania donovani from in vitro infected human monocyte-derived macrophages. This is apparently the first report of in vitro susceptibility of Leishmania to pentavalent antimony or to pentamidine. The insensitivity of insect forms (promastigotes) multiplying in cell-free media to thee drugs suggests that amastigotes are more sensitive than promastigotes to these antileishmanial agents. Alternatively, macrophages may concentrate or metabolize the drugs to increase their toxicity. In contrast, amphotericin B was toxic to both amastigotes and promastigotes. The sensitivities of Leishmania within human monocyte-derived macrophages in vitro to clinically achievable concentration of antileishmanial agents suggests that this model may be useful for investigation of mechanisms of sensitivity and resistance of antimicrobial agents against Leishmania.     

Lectin-like attachment sites on murine pulmonary alveolar macrophages bind Aspergillus fumigatus conidia

Murine pulmonary alveolar macrophages bound Aspergillus fumigatus conidia in vitro at 4 C and 37 C in the absence of serum or opsonins. This attachment was dependent on calcium and was sensitive to mild trypsinization and paraformaldehyde pretreatment of the macrophage membrane. Chitotriose, N-acetylglucosamine, D-mannose, alpha-methyl-mannoside, and L-fucose, but not D-galactose, were effective inhibitors of conidial binding. This pattern of reduction of conidial binding was consistent with that for the mannosylfucosyl receptor. In addition, conidial binding may be mediated by another lectin on the macrophage membrane, one that recognizes chitin components, because N-acetylglucosamine and chitotriose exhibited greater inhibition than expected for the mannosyl-fucosyl lectin.     

Receptors for human IgG subclasses on human alveolar macrophages

The biology of individual heavy chain subclasses of human IgG (IgG1-4) in lung host defenses has become important now that specific deficiencies of certain subclasses (IgG2 and IgG4) can be associated with chronic sinopulmonary infections and that IgG4 can be increased in forms of hypersensitivity lung disease. Because IgG is an important opsonic antibody that promotes attachment of bacteria or particles to phagocytes, the relative binding of IgG subclasses to membrane receptors on human alveolar macrophages might predict the efficacy of specific opsonin-mediated phagocytosis. With in vitro cultured normal alveolar macrophages, various IgG complexes were assessed for receptor binding with a rosetting assay. For respiratory cells in culture for 24 h, about 25% of the macrophages bound IgG3 and about 10% bound IgG1; binding with IgG2 and IgG4 complexes was minimal. In macrophage cultures maintained for as long as 6 days, this pattern of binding persisted. However, in very short-term cultures, 30 min and 105 min after cell adherence had occurred, binding was much greater for IgG3 complexes (about 60%); likewise IgG1 and IgG4 bound to about 20% of the cells. The IgM erythrocyte complexes, usually showing no binding at later time points in culture, bound to 20% of the cells, acutely. Therefore, our studies found that IgG3 consistently bound to more alveolar macrophages than the other subclasses, including IgG1. Also, the duration in culture of adherent cells may significantly affect the pattern of binding.     

Immune-complexes-mediated evasion of Plasmodium knowlesi from destruction by macrophages

The role of immune-complexes in the evasion of Plasmodium knowlesi from destruction by macrophages was studied in vitro. Incubation of macrophages with immune-complexes, prepared either by mixing total parasite antigens soluble in culture medium with normal or immune monkey serum, or by polyethylene glycol precipitation of serum from monkeys acutely infected with P. knowlesi, significantly reduced both the pool size of the macrophages that bound parasitized erythrocytes, and the number of parasitized erythrocytes bound per macrophage. Parasitized erythrocytes with mature schizonts were invariably preferred over those containing rings. These observations appear to indicate that during P. knowlesi infection in rhesus monkeys, immune-complexes may inhibit the binding of parasitized erythrocytes with mononuclear phagocytes and thus may enable them to evade the destructive mechanisms mounted by the host.     

Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection

The ability to infect macrophages is a common characteristic shared among many mycobacterial species. Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium kansasii enter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identify M. avium genes and host cell pathways involved in the bacterial uptake by macrophages, we screened a M. avium transposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of six M. avium clones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to the M. avium subsp. paratuberculosis MAP2446c gene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in the M. avium genome. The region is unique for M. avium and is not present in M. tuberculosis or M. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understand M. avium interaction with two evolutionarily distinct hosts, the mutants were evaluated for Acanthamoeba castellanii invasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting that M. avium might have evolved mechanisms that are used to enter amoebas and human macrophages.     

Salmonella typhimurium invasion induces apoptosis in infected macrophages.

Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.     

Development of specific surface receptors recognizing mannose-terminal glycoconjugates in cultured monocytes: a possible early marker for differentiation of monocyte into macrophage

Surface receptors for mannose-terminal glycoconjugates have been reported in various macrophage populations and are thought to be involved in specific binding and internalization of mannose-rich substances. They thereby may serve a function in such phenomena as phagocytosis of yeast and tumor cell recognition. Little is known of mannosyl receptors in blood monocytes. We synthesized a probe by covalently linking D-mannose to bovine serum albumin (BSA). Using this probe in fluoresceinated or latex minibead-derivatized forms, we searched the surface of human monocytes for the presence of mannosyl receptors. 125I-labeled probe was further used to quantify the number of receptors and the kinetics of the binding. Freshly isolated monocytes did not bind the probe, indicating the absence of mannosyl receptors. When placed in a culture system that preserves functional and morphological homogeneity of the cells, surface receptors for D-mannosyl glycoproteins developed within four days, reached a peak after one week, and then remained fairly stable. Binding parameters (Kd, Bmax, and receptor number) also remained stable and were not dissimilar to those reported for macrophages, although the Kd was consistently larger in cultured monocytes. When studied at 37 degrees C, the ligand-receptor complex was internalized through a system of coated pits and vesicles. The development of these receptors before evidence of morphological or functional differentiation suggests that these receptors may constitute an early marker for differentiation of monocytes into macrophages.     

Molecular Mechanisms of In vitro Betulin-Induced Apoptosis of Leishmania donovani

Although leishmanial infections of humans occur globally, the major health impact lies in developing nations, thus, leishmaniases remain “neglected” diseases for new drugs development. Multidrug resistance has been documented in most countries where leishmaniases is endemic. Betulin is a widely available and affordable natural product exerting leishmanicidal activity at micromolar concentration. In this study, the molecular mechanisms of death that contribute to the anti-leishmanial activity of betulin are investigated. In promastigotes, betulin stimulated reactive oxygen species generation at micromolar concentrations in Leishmania. Apoptosis was observed in betulin-treated promastigotes using flow cytometric analysis of treated cells stained with annexin V-FITC and propidium iodide. Furthermore, betulin treatment of promastigotes led to mitochondrial membrane damage, activation of caspase-like proteases, and DNA fragmentation in Leishmania donovani promastigotes. Betulin treatment of amastigotes cultured within macrophages, resulted in a reduced number of amastigotes, with no substantive cytotoxic damage to the host macrophage cells at leishmanicidal drug concentrations.     

Association state of human red blood cell band 3 and its interaction with ankyrin

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.     

Transferrin Uptake by Rabbit Alveolar Macrophages in Vitro

Rabbit alveolar macrophages were shown to bind 125I-human transferrin in vitro. The binding reaction was characterized by three stages: (1) adsorption of transferrin to the cells, followed by (2) rapid uptake of the protein to reach (3) a constant level of cell-bound transferrin. The latter two stages were dependent upon temperature and metabolic energy. Macrophages released 125I-transferrin rapidly when incubated with unlabelled transferrin. Small quantities of 125I-rabbit and 125I-bovine serum albumin, by comparison, were bound to and released by the cells; the attachment of these proteins may be solely the result of adsorption. Transferrin, 80% saturated with iron, was bound to a greater extent than 10 or 50% saturated transferrin; 10% saturated transferrin was bound more readily than the 50% saturated preparation. The findings are consistent with the presence of a transferrin receptor on the cell membrane of the alveolar macrophage and imply that transferrin may interact directly with this cell type in order to remove or donate iron.