Expression of a repeating phosphorylated disaccharide lipophosphoglycan epitope on the surface of macrophages infected with Leishmania donovani.

Murine peritoneal macrophages were infected with living, virulent Leishmania donovani promastigotes. At intervals after infection, the macrophage surfaces were probed for the expression of lipophosphoglycan (LPG) epitopes by immunofluorescence with anti-LPG monoclonal antibodies. A repeating phosphorylated disaccharide epitope of LPG was detected as early as 5 to 10 min postinfection and was initially localized to the immediate area of internalization of the promastigote into the macrophage. The epitopes were evenly distributed over the entire macrophage surface by 25 min postinfection. Treatments which inhibited macrophage phagolysosomal degradation processes had no effect on epitope expression, whereas reagents that affected macrophage membrane flow and, thus, phagocytosis drastically reduced or abolished expression. Purified LPG or phosphoglycan, the delipidated form of the LPG molecule, was also shown to bind to a variety of different cell types in a temperature-independent manner. Since LPG has been implicated as having an immunoprotective role in leishmaniasis, these results suggest a further mechanism(s) by which Leishmania LPG might be involved in parasite pathogenicity and virulence.     

Inhibition of in vivo and in vitro infectivity of Leishmania donovani by tunicamycin

Leishmania donovani 2S strain promastigotes were rendered non-infectious to mice and mouse peritoneal macrophages by treatment with tunicamycin, an inhibitor of N-linked protein glycosylation. Concentrations of tunicamycin (1-10 micrograms ml-1) that reduced promastigote infectivity to 2% or less of control levels had little or no measurable effect on the in vitro growth of the promastigotes. Tunicamycin has no apparent effect on the entry of promastigotes into macrophages. These results indicate that the sugar residues of glycoproteins are important to the promastigote during the early stages of macrophage infection.     

Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands.

Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 micrograms/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 micrograms of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes.     

Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis

Significant differences were found in the ability of resident mouse peritoneal macrophages to ingest amastigote and promastigote forms of Leishmania mexicana amazonensis. Differences in the association index of the parasites to the macrophages were also found between infective and non-infective promastigotes. Evidence was obtained suggesting that the macrophage receptor, which recognizes mannose-6-phosphate-containing units found in lysosomal enzymes, is involved in the association with the macrophage of promastigotes, but not of amastigotes. Addition of mannose-6-phosphate, its structural analogue fructose-1-phosphate, Hansenula holstii phosphomannan or the mannose-6-phosphate-containing lysosomal enzyme -D-mannosidase to the interaction medium, markedly inhibits the association of the parasites with macrophages.     

Vaccine-induced immunity against cutaneous leishmaniasis in BALB/c mice.

Partially purified antigens, derived from Leishmania infantum or L. major promastigotes and isolated under reducing conditions, were used to immunize BALB/c mice. Three subcutaneous injections of the 64- to 97-kilodalton preparation in conjunction with muramyl dipeptide conferred long-lasting immunity against L. mexicana subsp. mexicana and L. major infection; they led to the development of antibodies neutralizing the infectiousness of promastigotes, induced specific delayed-hypersensitivity reactions, and generated populations of peritoneal macrophages capable of killing amastigotes. Vaccination resulted in no harmful effects, since these antigen neither exacerbated preexisting Leishmania infection nor impeded the formation of antibodies to other antigens administered concomitantly.     

Flow cytometric analysis of the effects exerted by monoclonal antibodies on binding and uptake of Leishmania mexicana subsp. mexicana promastigotes by murine peritoneal macrophages.

A flow cytometric assay was developed to quantitate the binding kinetics of Leishmania mexicana subsp. mexicana promastigotes to murine peritoneal macrophages and to determine if selected membrane-specific monoclonal antibodies would exert an effect on the binding process. A total of three monoclonal antibodies, all reactive with a similar 42-kilodalton surface membrane component by Western blot analysis, enhanced parasite-macrophage binding at levels greater than 45%. An additional three monoclonal antibodies that identified low-molecular-weight antigens of the promastigote (15 to 20 kilodaltons) had no effect on the binding process. Of these antibodies, however, one did appear to inhibit the internalization of the parasites after it attached to the macrophage membrane.     

Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies.

Leishmania major infection of macrophages is followed by a time-dependent appearance of lipophosphoglycan (LPG) that can be detected on the surface of infected cells by monoclonal antibodies. The origin of these LPG epitopes is probably the intracellular amastigote. LPG epitopes could be detected on the amastigote and the infected macrophage by a number of monoclonal antibodies directed to several distinct determinants on the phosphoglycan moiety. The macrophage-expressed LPG may be modified because, unlike the parasite LPG as expressed on promastigotes or amastigotes, it could not be radiolabeled by galactose oxidase or periodate treatment of infected cells followed by reduction with 3H-labeled sodium borohydride. Some LPG epitopes displayed on the macrophage may be anchored with glycosylphosphatidylinositol, and some may be in the water-soluble phosphoglycan form bound to macrophage integrins involved in its specific recognition. The water-soluble population could be released from the infected macrophage by gentle protease treatment.     

免疫小鼠及正常小鼠巨噬细胞对利什曼无鞭毛期的吞噬作用

内脏利什曼原虫主要寄生在巨噬细胞系统的单核吞噬细胞内,在一般情况下其无鞭毛期能抵抗巨噬细胞的杀灭作用。 为了观察经杜氏利什曼原虫免疫后的小鼠其巨噬细胞的作用,我们采用了CFW纯系小鼠,经不同免疫方法于免疫后不同时间观察了体外培养中巨噬细胞的吞噬功能。实验采用的巨噬细胞与杜氏利什曼原虫前鞭毛期的比例为1:4。从每24小时吞噬功能的结果表明,经利什曼鞭毛体纯抗原免疫及福氏佐剂加利什曼抗原免疫的两组小鼠,均以免疫后3周的吞噬率最高,分别为72％及96％;两组吞噬指数的均值±SD(4.46±1.72,6.99±4.36)亦较正常组小鼠(1.68±1.25,1.72±1.15)为高,并具有显著差异(P<0.05)。提示了特异性抗原以及与佐剂合并具有对吞噬功能的激活作用。实验并观察了巨噬细胞内利什曼原虫无鞭毛期的活力作用,从吞噬原虫后20小时开始至 144小时,正常小鼠巨噬细胞内的无鞭毛期再经三恩氏培养基培养后均能恢复为前鞭毛期,而经免疫小鼠巨噬细胞内的利什曼原虫无鞭毛期在72小时后即消失活力。 另外,对小鼠腹腔巨噬细胞吞噬利什曼原虫的动态亦作了仔细观察。 实验结果说明了经过免疫的小鼠,由于被淋巴细胞激活后的巨噬细胞能杀死利什曼原虫,巨噬细胞在宿主对感染应答中是一个重要部分,对于探索黑热病的免疫机理具有一     

Role of beta-D-galactofuranose in Leishmania major macrophage invasion

The role of glycosylinositol phospholipid 1 (GIPL-1) of Leishmania (Leishmania) major in the interaction of promastigotes and amastigotes with macrophages was analyzed. Monoclonal antibody MEST-1, which recognizes glycolipids containing terminal galactofuranose (Galf) residues (E. Suzuki, M. S. Toledo, H. K. Takahashi, and A. H. Straus, Glycobiology 7:463-468, 1997), was used to detect GIPL-1 in Leishmania by indirect immunofluorescence and to analyze its role in macrophage infectivity. L. major promastigotes showed intense fluorescence with MEST-1, and GIPL-1 was detected in both amastigote and promastigote forms by high-performance thin-layer chromatography immunostaining by using MEST-1. Delipidation of L. major promastigotes with isopropanol-hexane-water eliminated the MEST-1 reactivity, confirming that only GIPL-1 is recognized in either amastigotes or promastigotes of this species. The biological role of GIPL-1 in the ability of L. major to invade macrophages was studied by using either Fab fragments of MEST-1 or methylglycosides. Preincubation of parasites with Fab fragments reduced macrophage infectivity in about 80% of the promastigotes and 30% of the amastigotes. Preincubation of peritoneal macrophages with p-nitrophenyl-beta-galactofuranoside (10 mM) led to significant ( approximately 80%) inhibition of promastigote infectivity. These data suggest that a putative new receptor recognizing beta-D-Galf is associated with L. major macrophage infectivity and that GIPL-1 containing a terminal Galf residue is involved in the L. major-macrophage interaction.     

Deletion of the gene for the membrane-bound acid phosphatase of Leishmania mexicana

The membrane-bound acid phosphatase of Leishmania mexicana (LmxMBAP) has been shown to be a heterogeneously N-glycosylated type I transmembrane protein, which is localized predominantly in vesicular structures close to the flagellar pocket in promastigotes and amastigotes. Its expression in both life stages prompted us to analyse its function by performing deletion analysis. Both alleles of the single copy gene were sequentially replaced by resistance marker genes and the resulting deletion mutant was tested for its potential to infect Balb/c mice and peritoneal macrophages. There was no obvious difference detectable between the mutant and the wild-type. Therefore, we conclude that LmxMBAP is neither involved in the infection process nor required for amastigote survival in the infected host cell. LmxMBAP null mutant promastigotes were used to establish a system for homogeneous overexpression of LmxMBAP which will be useful to investigate protein sorting in L. mexicana.     

Differential production of granulocyte-macrophage colony-stimulating factor by macrophages from mice susceptible and resistant to Leishmania mexicana amazonensis.

The present results demonstrate that macrophages from mice susceptible to infection with Leishmania mexicana amazonensis sustain a higher production of granulocyte-macrophage colony-stimulating factor (GM-CSF) throughout the in vitro infection than macrophages from a resistant strain. Resident macrophages from BALB/c and C57B1/10 mice were infected with promastigotes of L. mexicana amazonensis and the amount of biologically active GM-CSF was measured in the supernatants collected at different times of infection. Measurements were made by bone marrow and GM-CSF/interleukin-3 addicted cell proliferation. Because GM-CSF is a disease-exacerbating cytokine, its differential production by infected macrophages may be one of the mechanisms defining resistance or susceptibility to a leishmanial infection.     

Leishmania mexicana promastigotes inhibit macrophage IL-12 production via TLR-4 dependent COX-2, iNOS and arginase-1 expression

The effects of Leishmania mexicana metacyclic promastigotes upon MAP kinase signalling in mouse bone marrow macrophages and subsequent expression of the disease regulatory proteins iNOS and COX-2 were studied. At a ratio of 5:1, promastigotes caused a marked increase in phosphorylation of the three major MAP kinases, ERK, p38 and JNK. MAP kinase signalling was substantially reduced in TLR-4(-/-) but not TLR-2(-/-) deficient macrophages and completely abolished in double TLR-2/4(-/-) macrophages. A similar outcome was observed using cysteine peptidase B deficient amastigotes. Furthermore, whilst promastigotes had no independent effect on iNOS or COX-2 expression, they prolonged the induction of these proteins stimulated by LPS and enhanced PGE(2) and NO production. Induction of COX-2 and iNOS was also TLR-4 dependent. Blockade of either PGE(2) or NO production with indomethacin or l-NAME reversed promastigote inhibition of LPS induced IL-12 production. Promastigotes also increased macrophage arginase-1 expression and enhanced arginase activity, both of which were substantially reduced in TLR-4 but not TLR-2 deficient macrophages. Surprisingly, arginase inhibition by Nor-NOHA also caused a reversal of promastigote mediated inhibition of macrophage IL-12 production. These data demonstrate for the first time the role of TLR-4 in mediating the effects of L. mexicana promastigotes on MAP kinase activation, up-regulation of COX-2, iNOS as well as arginase-1 expression in macrophages and further shows that PGE(2), NO and arginase activity all contribute substantially to the inhibition of host cell IL-12 production.     

Failure of hamster macrophages to discriminate between infective and noninfective promastigotes of Leishmania donovani during attachment in vitro.

The attachment of infective and noninfective promastigotes of Leishmania donovani to hamster macrophages derived from spleen, lymph nodes, and peritoneal exudate was studied in vitro. Regardless of macrophage origin, no significant differences were observed between attachment of promastigotes that were infective and those that were noninfective for hamsters.     

Treatment of murine macrophages with interferon-gamma inhibits their ability to bind leishmania promastigotes.

The binding of leishmania promastigotes to macrophages pretreated with interferon-gamma (IFN-gamma) was compared to binding to untreated (resident) cells. IFN-gamma-treated macrophages bound fewer leishmania promastigotes than did untreated cells. The decreased binding was apparent over a wide dose range of parasite inocula when the assays were performed in the absence of exogenous complement. This decrease was specific to leishmania, since treated and untreated macrophages bound comparable amounts of immunoglobulin G- and complement-coated sheep red blood cells. Decreased parasite binding occurred early in the macrophage activation pathway. Pretreatment of macrophages with IFN-gamma for as little as 6 h, a time insufficient to induce other macrophage activation parameters, significantly reduced their ability to bind leishmania promastigotes. To determine the mechanism of this decreased phagocytosis by activated cells, macrophages were pretreated with specific inhibitors before the addition of leishmania. The binding of promastigotes to untreated (resident) macrophages was inhibited by approximately 50% by reagents that blocked either of two macrophage receptors, complement receptor type 3 (Mac-1) or a leishmania species-specific lectin-like receptor. Binding to IFN-gamma-treated macrophage populations, in contrast, was substantially inhibited only by antibody to Mac-1. Saccharides that were 50% inhibitory in the resident cell population, decreased binding by less than 10% in activated cells. The lack of saccharide inhibition by IFN-gamma-treated cells was also reflected in an inability of activated macrophages to bind to beads coated with purified leishmania lipophosphoglycan (LPG). These LPG-coated beads bound well to resident macrophages but poorly to activated cells. Thus, leishmania bind to macrophages by two distinct mechanisms, one that utilizes Mac-1 and a second mechanism that does not depend on complement and is saccharide inhibitable. These two binding mechanisms are distinct and differentially regulated in resident and activated cells.     

Freeze-fracture and cytochemistry study of the interaction between Leishmania mexicana amazonensis and macrophages

The process of interaction between macrophages and promastigote and amastigote forms of Leishmania mexicana amazonensis was analyzed using freeze fracture and cytochemistry. The promastigotes inside endocytic vacuoles of macrophages presented an altered distribution of intramembranous particles and a wavy aspect of the plasma membrane. However, amastigotes did not show such alterations. The membrane alterations are probably caused by intracellular cell lysis of the promastigotes by the macrophages. An accumulation of intramembranous particles was seen in the plasma membrane of amastigote forms in the area of adhesion to the macrophages. The parasitophorous vacuole membrane had intramembranous particles randomly distributed. The enzyme activity of Mg++-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected, at the ultrastructural level, in normal mouse peritoneal macrophages and in macrophages infected with Leishmania mexicana amazonensis. Mg++-ATPase and 5'-nucleotidase are uniformly distributed throughout the macrophage's plasma membrane but were not detected in the membrane lining endocytic vacuoles containing ingested parasites (parasitophorous vacuole). NAD(P)H-oxidase activity was seen in those portions of the macrophage's plasma membrane which enter in direct contact with parasites and also in association with the membrane of the parasitophorous vacuole. The amount of reaction product, indicative of NAD(P)H-oxidase activity, was larger in macrophages which interacted with the promastigote than in those which interacted with the amastigote form of L. mexicana amazonensis. Concanavalin A binding sites and anionic sites of the macrophage's surface, labeled before the interaction, are not interiorized together with the parasites, however, are observed in endocytic vacuoles which do not contain parasites.     

Modulation of phagolysosome biogenesis by the lipophosphoglycan of Leishmania

Promastigotes of the protozoan parasite Leishmania are inoculated into the mammalian host by an infected sandfly and are phagocytosed by macrophages. There, they differentiate into amastigotes, which replicate in phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs), plays an important role in the ability of promastigotes to survive the potentially microbicidal consequences of phagocytosis. Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, has received considerable attention over the past several years. Of interest for this review, lipophosphoglycan confers upon Leishmania donovani promastigotes the ability to inhibit phagolysosome biogenesis. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-harboring phagosomes from interacting with late endosomes and lysosomes. Thus, similar to several other pathogens, Leishmania promastigotes hijack the host cell's cytoskeleton early during the infection process. Here, we review this phenomenon and discuss the potential underlying mechanisms.     

Leishmania braziliensis promastigotes and amastigotes interact differently with host macrophages

In vitro and in vivo ultrastructral studies reveal that the parasite entrance into the macrophage occurs by phagocytosis. The early stage of phagocytosis exhibited different ultrastructural characteristics in both forms of the parasite. Long and prominent projections from peritoneal exudate macrophages made focal contacts with the promastigote surface. The amastigotes, in turn, laid on cup-shaped extensions of the macrophage membrane. Later stages of the phagocytosis are characterized by progressive and complete engulfment of both promastigotes and amastigotes.     

An experimental model system for leishmaniasis. An ultrastructural study on cultured macrophages exposed to Leishmania parasites and sodium stibogluconate

To facilitate studies on the effect of chemotherapeutic agents on the host-parasite interaction in leishmaniasis, we have developed an experimental model for infecting mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigotes. As the drug action is often dependent on concentration, the distribution of sodium stibogluconate, which is the commonly used drug for treatment of leishmaniasis, was studied in various parts of the macrophages by energy dispersive X-ray microanalysis. The drug was found to accumulate in secondary lysosomes. The ultrastructural examination, using TEM and SEM, of macrophages, whose secondary lysosomes had been preloaded with gold particles, showed that leishmania parasites are phagocytosed and finally located in secondary lysosomes. Using flameless atomic absorption spectrophotometry, the concentration of Mn, Fe and Cu in promastigotes of Leishmania donovani, Leishmania aethiopica, Leishmania crithidia, Leishmania major and their culture media was estimated. Of the three transition metals, the parasites accumulated only Mn from the medium, which they may use in a primitive defense mechanism against reactive oxygen metabolites produced by macrophages during the respiratory burst associated with phagocytosis.     

Comparisons of mutants lacking the Golgi UDP-galactose or GDP-mannose transporters establish that phosphoglycans are important for promastigote but not amastigote virulence in Leishmania major

Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(beta1,4)Man(alpha1-PO(4))]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2(-) L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2(-), instead resembling the L. major lipophosphoglycan-deficient lpg1(-) mutant. Metacyclic lpg5A(-)/lpg5B(-) promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A(-)/lpg5B(-) mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2(-) Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.     

Expression of Leishmania antigen on the surface membrane of infected human macrophages in vitro.

Resolution of leishmaniasis is associated with host immunological responsiveness to parasite antigens. In clinical disease, leishmania are found as amastigotes contained with macrophages. We investigated the possibility that Leishmania antigens are expressed on the infected macrophage surface by reacting infected macrophages with antibody to Leishmania. In vitro-infected human monocyte-derived macrophages were labelled with antibody to amastigotes when examined with immunofluorescent or immunoelectron microscopic techniques. Infected macrophages were poorly labelled by antibody to promastigotes (insect forms of Leishmania). Certain antisera that reacted with the surface membranes of amastigotes did not label the infected macrophage surface. These results indicate that human macrophages infected in vitro express Leishmania amastigote antigen(s) on their surface membranes, that such antigen(s) may not be present in large quantities in promastigotes, and that certain antigen(s) on the amastigote surface are not expressed on the surface membranes of infected macrophages.