Fcgamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro

Although convincing evidence exists for the role of immunoglobulin G (IgG) antibodies in immunity to malaria, antibody titres do not usually predict protection. In this study we have assessed the interaction between Plasmodium falciparum-infected erythrocytes (PE), opsonized with immune serum containing different amounts of IgG antibody isotypes, with either THP-1 cells, ex-vivo human monocytes or IIAI.6 transfectant cells expressing Fc(gamma)RIIa-Arg/Arg131 or -His/His131 allotypes. Our results show that PMA-treated THP-1 cells were capable of phagocytosing serum-opsonized PE by Fc(gamma)RI (CD64) and Fc(gamma)RIIa (CD32), acting synergistically. The known Fc(gamma)RIIa polymorphism motivated us to examine its influence on IgG isotype-mediated phagocytosis of opsonized PE with human monocytes and the IIAI.6 transfectant cells expressing either allelic forms. Regardless of the cell type, PE phagocytosis with Fc(gamma)RIIa-His/His131 was highest following opsonization with a predominantly IgG3-containing immune serum pool. In contrast, PE phagocytosis with Fc(gamma)RIIa-Arg/Arg131 tended to be higher with an IgG1-containing pool. These results suggest a genetically determined influence of effector cell phenotype on IgG antibody-pathogen interaction in P. falciparum malaria.     

Direct activation of human endothelial cells by Plasmodium falciparum-infected erythrocytes

Cytoadherence of Plasmodium falciparum-infected erythrocytes (PRBC) to endothelial cells causes severe clinical disease, presumably as a of result perfusion failure and tissue hypoxia. Cytoadherence to endothelial cells is increased by endothelial cell activation, which is believed to occur in a paracrine fashion by mediators such as tumor necrosis factor alpha (TNF-alpha) released from macrophages that initially recognize PRBC. Here we provide evidence that PRBC directly stimulate human endothelial cells in the absence of macrophages, leading to increased expression of adhesion-promoting molecules, such as intercellular adhesion molecule 1. Endothelial cell stimulation by PRBC required direct physical contact for a short time (30 to 60 min) and was correlated with parasitemia. Gene expression profiling of endothelial cells stimulated by PRBC revealed increased expression levels of chemokine and adhesion molecule genes. PRBC-stimulated endothelial cells especially showed increased expression of molecules involved in parasite adhesion but failed to express molecules promoting leukocyte adhesion, such as E-selectin and vascular cell adhesion molecule 1, even after challenge with TNF-alpha. Collectively, our data suggest that stimulation of endothelial cells by PRBC may have two effects: prevention of parasite clearance through increased cytoadherence and attenuation of leukocyte binding to endothelial cells, thereby preventing deleterious immune reactivity.     

p-Aminobenzoic acid transport by normal and Plasmodium falciparum-infected erythrocytes.

De novo folate biosynthesis is required for the growth of malarial parasites and is inhibited by several important antimalarial agents. We show here that exogenous p-aminobenzoic acid (pABA) can be utilized by malaria parasites to synthesize folates. The transport of pABA into parasite infected red cells was therefore characterized. Normal red cells transport pABA in a saturable and energy-dependent manner, with a dissociation constant of 83 nM. pABA transport in parasite-infected red cells may use the same mechanism, as demonstrated by similarities in time course, concentration-response, and dissociation constant (111 nM). The transport capacity of red cells is temperature-, energy- and pH-dependent. It is inhibited by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), but not by the sodium ionophores nigericin and monensin. p-Aminosalicylic acid (PAS) inhibits pABA transport competitively, with a inhibition constant of 378 nM. Phloritin, flufanamic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DITS), which are inhibitors of the anion transporter (band 3), and oxalic acid, a substrate of this transporter, partially inhibit pABA transport into both normal and infected red cells. Interestingly, in both normal and infected red cells, the inhibitory effects of PAS and the anion transport inhibitors are additive, suggesting the involvement of 2 independent mechanisms.     

Rosetting Plasmodium falciparum-infected erythrocytes express unique strain-specific antigens on their surface.

Spontaneous binding of uninfected erythrocytes to Plasmodium falciparum-infected erythrocytes (rosetting) has been suggested to have a critical role in the induction of cerebral malaria. We report here that rosetting can be mediated by several molecular mechanisms involving parasite polypeptides with M(r)s of 22,000 or 28,000, termed rosettins. Antibodies to either polypeptide disrupt rosettes in a strain-specific fashion. Rosettes of five of the seven isolates examined thus far are more easily disrpted by anti-22,000-M(r) rosettin antibodies than by anti-28,000-M(r) rosettin antibodies. Polyclonal anti-22,000-M(r) rosettin antibodies raised in mice or rabbits strongly and strain specifically stain the surface of nonfixed erythrocytes infected with late asexual stages of rosetting P. falciparum. Simultaneous antibody staining and rosetting are seen when the anti-22,000-M(r) rosettin antiserum is diluted so that only partial disruption of rosettes is obtained, confirming that the fluorescence-labelled infected erythrocytes are involved in rosetting. The 22,000-M(r) rosettin is accessible for surface iodination on erythrocytes infected with strains of rosetting parasites sensitive to anti-22,000-M(r) rosettin antibodies, whereas no labelling occurred on either normal erythrocytes or nonrosetting-P. falciparum-infected erythrocytes. Purified anti-22,000-M(r) rosettin serum immunoglobulin G immunoprecipitated three parasite-derived polypeptides with M(r)s of 22,000, 45,000 (doublet), and 50,000 from lysates of [35S]methionine-labelled, parasite-infected erythrocytes. Our results suggest that rosetting is mediated by strain-specific, antigenically distinct, P. falciparum-derived polypeptides.     

The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes

Intra-erythrocytic Plasmodium parasites digest host cell haemoglobin and use the liberated amino acids for protein synthesis. Although several endoproteases (aspartic, cysteine, and metallo-) have been shown to be involved in the initial stages of haemoglobin degradation, little is known about the steps immediately before amino acid release. In our studies, fluorometric enzyme assays indicated that the stage of the P. falciparum erythrocytic cycle with highest aminopeptidase activity was the stage at which most haemoglobin degradation occurs, i.e. the trophozoite. Consistent with these results, metabolic growth assays indicated that the late ring/trophozoite stage was most susceptible to aminopeptidase inhibitors. To reconstitute the terminal stages of haemoglobin breakdown in vitro, we synthesised three peptides with amino acid sequences corresponding to known products of the endoproteolytic digestion of haemoglobin and employed them as substrates for aminopeptidases. Both trophozoite cytosolic extract, and partially-purified aminopeptidase, hydrolysed these peptide fragments to amino acids. Hydrolysis appeared to occur sequentially from the amino-termini of the peptides, and was inhibited in a concentration-dependent manner by the aminopeptidase-specific inhibitor nitrobestatin. The results suggest that P. falciparum aminopeptidases could be the enzymes responsible for the hydrolysis of haemoglobin-derived peptides to free amino acids. Lack of ultrastructural change in parasites treated with relevant concentrations of aminopeptidase-specific inhibitors, however, indicated that little feedback exists whereby the inhibition of cytosolic aminopeptidases results in obvious inhibition of initial haemoglobin degradation in the digestive vacuole.     

Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria.

Noninfected erythrocytes form rosettes around those infected with trophozoites and schizonts of Plasmodium falciparum in vitro. These rosettes are thought to contribute to the microvascular obstruction which underlies the pathophysiology of severe falciparum malaria. To determine whether the percentage of infected erythrocytes forming rosettes for a parasite isolates in vitro correlates with the in vivo severity of disease, we studied the rosette formation behavior of 35 isolates of P. falciparum from patients with uncomplicated, severe, and cerebral malaria. There was a wide variation in the degree of rosette formation (0 to 53%). Four parasite isolates formed rosettes well (30 to 53%), and seven isolates formed rosettes poorly or not at all (0 to 5%), while the majority of the isolates formed rosettes to various degrees between these two extremes. In this relatively small sample of patients, we were unable to demonstrate a significant association between in vitro rosette formation and patients with cerebral malaria or conscious patients with significant renal (serum creatinine greater than 200 mumol/liter) or hepatic dysfunction (serum bilirubin greater than 50 mumol/liter and aspartate aminotransferase greater than 50 Reitman-Frankel units). However, there was an inverse relationship between rosette formation and cytoadherence (r = -0.575, P less than 0.01) which could not be explained on the basis of steric hindrance. This finding suggests that cytoadherence and rosette formation properties are intrinsic to the parasites, with isolates having a greater propensity for one or the other but not both. Further studies are required to establish the occurrence and pathophysiological role of rosette formation in vivo.     

Host cell factor CD59 restricts complement lysis of Plasmodium falciparum-infected erythrocytes

Here we demonstrate that components of the entire complement cascade are fixed on the surface of erythrocytes infected with the human malarial parasite Plasmodium falciparum. Despite the activation of lytic complement factors, no complement-mediated lysis of parasitized erythrocytes was observed. Complement-mediated lysis of P. falciparum-infected erythrocytes occurred only in the absence of functional intrinsic CD59. These data suggest that the restriction of the complement attack of P. falciparum-infected erythrocytes is principally mediated by intrinsic host cell factors, in particular CD59.     

Band 3 clustering promotes the exposure of neoantigens in Plasmodium falciparum-infected erythrocytes

Erythrocytes infected with the human malaria parasite Plasmodium falciparum become structurally and antigenically modified as a consequence of intracellular parasite development. The new antigens that appear on the surface of the infected erythrocyte originate from parasite-encoded proteins and by modification of the erythrocyte membrane protein band 3. Here, we show that anti-peptide antibodies generated against an amino acid sequence (YETFSKLIKIFQDH) of human band 3, and previously identified as mediating adhesion of infected erythrocytes to CD36, recognized P. falciparum-infected erythrocytes. In addition, sera from individuals living in a malaria endemic area (and who are presumably immune) contained immunoglobulins specific for this region of band 3. The anti-peptide antibodies reacted with the surface excrescences (knobs) on falciparum-infected erythrocytes. In uninfected erythrocytes, the band 3 region was cryptic and its exposure on the falciparum-infected erythrocyte surface required clustering of band 3 protein. Thus, a parasite-induced modification of band 3 promotes adhesion and induces antigenic changes in the P. falciparum-infected erythrocyte.     

Cytoadherence and ultrastructure of Plasmodium falciparum-infected erythrocytes from a splenectomized patient.

In malarial infections of primates, the spleen has been shown to modulate parasite antigen expression on the surfaces of infected erythrocytes. The processes affected include cytoadherence, which is central to the pathophysiology of severe falciparum malaria, and the related phenomenon of rosette formation. In this study, the cytoadherence and rosette formation behaviors of Plasmodium falciparum-infected erythrocytes from a splenectomized patient were examined during the first erythrocytic cycle in vitro. Ultrastructural studies were also performed. Infected erythrocytes were found to cytoadhere to C32 melanoma cells via leukocyte differentiation antigen CD36 but not intercellular adhesion molecule 1. They also displayed on their surfaces electron-dense knobs similar in structure and density to those on infected erythrocytes from intact hosts. These findings may reflect a stable cytoadherent phenotype of the parasite isolate that is unaffected by the absence of the spleen. Alternatively, the modulating role of the spleen may have been assumed by other organs of the mononuclear phagocytic system in a previously infected individual. No rosette formation was observed, but as not all natural isolates form rosettes, this observation may or may not be related to the asplenic status of the patient. Parasite and host factors appear to be important in determining the effect of splenectomy on cytoadherence and rosette formation in human falciparum malaria.     

A transferrin-independent iron uptake activity in Plasmodium falciparum-infected and uninfected erythrocytes.

Non-heme iron is essential for the asexual growth of the human malaria parasite Plasmodium falciparum in mature erythrocytes. Utilization of iron bound to serum transferrin by the parasitized cells has been postulated, but direct evidence for its specific delivery has not been reported. Here we demonstrate that normal levels of transferrin in human serum are not required for intraerythrocytic P. falciparum growth: culture medium immunodepleted 500-1000 fold in human transferrin was capable of supporting parasitemias and rates of invasion comparable to those observed in non-depleted medium. 55Fe bound to transferrin was not taken up by infected cells. A transferrin-independent non-heme iron uptake activity was, however, detected in both infected and uninfected erythrocytes when iron was presented to the cells as 55Fe-NTA or 55Fe-citrate. Although the uptake activity was not parasite specific, the radiolabel was found in association with parasites mechanically released from the infected erythrocytes, indicating that it is delivered to the intracellular organism. Evidence is presented that the transferrin-independent iron uptake activity is time-, temperature- and concentration-dependent, but apparently not energy-dependent.     

Baculovirus-expressed constructs induce immunoglobulin G that recognizes VAR2CSA on Plasmodium falciparum-infected erythrocytes

We raised specific antisera against recombinant VAR2CSA domains produced in Escherichia coli and in insect cells. All were reactive in enzyme-linked immunosorbent assay, but only insect cell-derived constructs induced immunoglobulin G (IgG) that was reactive with native VAR2CSA on the surface of infected erythrocytes. Our data show that five of the six VAR2CSA Duffy-binding-like domains are surface exposed and that induction of surface-reactive VAR2CSA-specific IgG depends critically upon antigen conformation. These findings have implications for the development of vaccines against pregnancy-associated Plasmodium falciparum malaria.     

Inhibition of chondroitin-4-sulfate-specific adhesion of Plasmodium falciparum-infected erythrocytes by sulfated polysaccharides

Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin 4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Soluble polysaccharides that release mature-stage parasitized erythrocytes into the peripheral circulation may help elucidate these interactions and have the potential to aid in developing therapeutic strategies. We have screened a panel of 11 sulfated polysaccharides for their capacities to inhibit adhesion of infected erythrocytes to CSA expressed on CHO-K1 cells and ex vivo human placental tissue. Two carrageenans and a cellulose sulfate (CS10) were able to inhibit adhesion to CSA and to cause already bound infected erythrocytes to de-adhere in a dose-dependent manner. CS10, like CSA and in contrast to all other compounds tested, remained bound to infected erythrocytes after washing and continued to inhibit binding. Both carrageenans and CS10 inhibited adhesion to placental tissue. Although highly sulfated dextran sulfate can inhibit CSA-mediated adhesion to CHO cells, this polysaccharide amplified adhesion to placental tissue severalfold, demonstrating the importance of evaluating inhibitory compounds in systems as close to in vivo as possible. Interestingly, and in contrast to all other compounds tested, which had a random distribution of sulfate groups, CS10 exhibited a clustered sulfate pattern along the polymer chain, similar to that of the undersulfated placental CSA preferred by placental-tissue-binding infected erythrocytes. Therefore, the specific anti-adhesive capacity observed here seems to depend not only on the degree of charge and sulfation but also on a particular pattern of sulfation.     

Amino-sugars inhibit the in vitro cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells

Twenty-two sugars and related compounds, nine neoglycoproteins, dopamine, four polyamines and oligomers of glucosamine were examined for their effect on the cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells. Inhibition of cytoadherence was high in the presence of the amino-sugars, glucosamine, galactosamine and mannosamine, and dopamine, and significant, although lower, in the presence of the polyamines, spermine, spermidine and putrescine. N-acetylated amino-sugars and the other compounds were not significant inhibitors of cytoadherence.     

Selective accumulation of mature asexual stages of Plasmodium falciparum-infected erythrocytes in the placenta

A feature of malaria in pregnancy is accumulation of P. falciparum-infected erythrocytes (IEs) in the placenta, which is associated with adverse outcomes for mothers and infants. Infection appears to involve parasite adhesion to molecules such as chondroitin sulfate A, hyaluronic acid, and immunoglobulins. In vitro, adhesion is predominantly a property of mature asexual forms of IEs; however, adhesion of immature or ring forms has recently been reported. We have assessed the parasitemia and developmental stages of IEs in the placenta by examination of placental blood and histological sections with comparison to parasites in the peripheral blood from the same individuals. Approximately 90% of IEs in the placenta were mature forms. Compared to peripheral blood, the placental parasitemia was 10-fold higher and the density of mature IEs was over 200-fold higher. By contrast, the average peripheral and placental ring-stage parasitemias were not significantly different. In 2 of 14 cases, the density of ring forms was higher in placental than in peripheral blood. These findings demonstrate prominent selective accumulation of mature asexual-stage IEs but infrequent accumulation of ring stages in the placental blood spaces, consistent with an important role for mature-stage IE adhesion.     

Inhibition of adhesion of Plasmodium falciparum-infected erythrocytes by structurally defined hyaluronic acid dodecasaccharides

We recently reported that Plasmodium falciparum-infected erythrocytes (IRBCs) can adhere to hyaluronic acid (HA), which appears to be a receptor, in addition to chondroitin sulfate A (CSA), for parasite sequestration in the placenta. Further investigations of the nature and specificity of this interaction indicate that HA oligosaccharide fragments competitively inhibit parasite adhesion to immobilized purified HA in a size-dependent manner, with dodecasaccharides being the minimum size for maximum inhibition. Rigorously purified and structurally defined HA dodecasaccharides, free of contamination by CSA or other glycosaminoglycans, effectively inhibited IRBC adhesion to HA but not CSA, providing compelling evidence of a specific interaction between IRBCs and HA.     

A biotin derivative blocks parasite induced novel permeation pathways in Plasmodium falciparum-infected erythrocytes

The malaria parasite Plasmodium falciparum infects human erythrocytes, and it induces an increased rate of uptake into the infected cell of a range of solutes, including essential nutrients required for parasite development. Several models have been proposed for the mechanism(s) underlying parasite-induced solute uptake, each differing with respect to the site of entry into infected cells. We show that a biotin derivative that is excluded from non-infected erythrocytes gains access to infected erythrocytes via a pathway that is inhibited by compounds shown previously to block the pathways responsible for the increased uptake of solutes. The derivative was found to bind erythrocyte cytoskeletal proteins and to hemoglobin, providing evidence that the novel pathways are in the erythrocyte membrane and allow direct access of solutes to the erythrocyte cytosol. The derivative inhibited its own uptake and blocked the parasite-induced transport of other solutes. In whole-cell patch-clamp analyses, biotinylation of infected erythrocytes caused significant decrease in a parasite-induced outward rectifying conductance. In vitro, biotinylation of trophozoite-stage parasitized erythrocytes delayed parasite development. Treatment of infected cells in the final developmental stage abrogated the parasite's ability to complete development. The data are consistent with the novel pathways playing an important role in parasite growth.     

Release of merozoites from Plasmodium falciparum-infected erythrocytes could be mediated by a non-explosive event

Little is known about the molecular mechanism underlying the release of merozoites from malaria-infected erythrocytes. In the present study, video microscopy was carried out, and images throughout the process of merozoite release from Plasmodium falciparum-infected erythrocytes were digitized and analyzed. Merozoites were shown to escape from the infected host cell in about 1 s through a single site of the infected erythrocyte membrane, whose dimension was estimated to be 2.5 μm. Merozoites were released together with the residual body containing hemozoin, leaving behind a membranous structure that persisted even after an extended period of observation. Densitometric measurements showed that the cytoplasmic content of the infected erythrocyte did not diffuse out as parasites were released, but was gradually lost thereafter. This would indicate that the release of merozoites from infected erythrocytes is not mediated by an explosive event. Received: 22 December 1998 / Accepted: 20 January 1999     

A high capacity in vitro assay for measuring the cytoadherence of Plasmodium falciparum-infected erythrocytes

A novel flow cytometric technique was developed to determine the absolute numbers of leukocytes of specific phenotypes in whole blood from two lines of inbred chickens (line 7(2) and line 6(1)). This single step method is rapid, accurate, repeatable, can be used in the presence of nucleated erythrocytes and addresses the problems encountered when electronically counting the numbers of leukocytes in specific subpopulations in the blood of non-mammalian species. It is superior to previous methods in that (1) peripheral blood leukocytes (PBL) do not need to be separated by density gradient centrifugation, (2) erythrocyte lysis is not necessary and (3) absolute numbers of specific phenotypes of cells are determined directly. A standard volume of diluted whole blood was added to a standard number of fluorescent beads before incubation with fluorescently-conjugated monoclonal antibodies recognising specific PBL surface antigens. Samples were analysed by flow cytometry and electronic gates were set to count a standard number of beads and the concomitant fluorescently-labelled cells. Absolute numbers of B, CD4+ and CD8+ PBL were determined. Since the bead fluorescence is constant, it was also possible to measure relative MHC class I expression using fluorescence intensity. In both lines of chickens absolute numbers of all of the phenotypes of PBL measured increased with age. Although line 7(2) chickens had greater numbers of B, CD4+, and CD8+ PBL than line 6(1) chickens, there was no significant difference in the CD4+:CD8+ PBL ratios, the T:B PBL ratios or relative MHC class I expression between the two lines. Relative MHC class I expression increased with age in both lines.