Receptor specificity of clinical Plasmodium falciparum isolates: nonadherence to cell-bound E-selectin and vascular cell adhesion molecule-1

The pathogenicity of Plasmodium falciparum is due largely to the parasite's unique ability to adhere to capillary and postcapillary venular endothelium during the second-half of the 48-hour life cycle. The resulting sequestration of infected erythrocytes (IRBC) in deep vascular beds leads to tissue hypoxia, metabolic disturbances, and organ dysfunction which characterize severe falciparum malaria. Several endothelial receptors of cytoadherence have been identified, but their clinical relevance remains controversial. In the present report, the receptor specificity of 60 clinical P falciparum isolates was determined using transfectants each expressing one of CD36, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). All isolates tested adhered to CD36 and ICAM-1, but the adherence to CD36 was at least 10-fold higher. Seven isolates adhered to E-selectin whereas none of 19 isolates adhered to VCAM-1. From a population standpoint, about 30% of IRBC in each isolate adhered to CD36, and 2% to 3% adhered to ICAM-1. The percentage adherent to E-selectin and VCAM-1 was negligible. IRBC selected on CD36 adhered almost exclusively to CD36 whereas 80% to 90% of IRBC selected on ICAM-1 could also adhere to CD36. Selected IRBC did not adhere to E-selectin or VCAM-1. These findings indicate that cytoadherence to multiple endothelial receptors is a rare occurrence with natural P falciparum isolates, but do not exclude a role for the adhesion molecules in promoting other IRBC-endothelial interactions such as rolling under flow conditions. Receptor specificity in vivo may be dictated by the ligand-receptor combination which provides the best survival potential for the parasite.     

Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1.

The CD36 and ICAM-1 glycoproteins on vascular endothelial cells have been implicated as cytoadherence receptors for Plasmodium falciparum-infected erythrocytes (IRBC). Adhesion of IRBC from Thai patients with uncomplicated and severe falciparum malaria to purified CD36 or ICAM-1 and to C32 melanoma cells was compared. All malaria isolates bound to solid phase-adsorbed CD36 and to fluid-phase 125I-labeled CD36. IRBC adhesion to purified ICAM-1 varied widely, and no correlation with clinical severity of disease was observed. The cytoadherent phenotype of IRBC was modulated by selective panning on plates coated with purified CD36 or ICAM-1. IRBC selected by panning on CD36+, ICAM-1+ melanoma cells bound to cells that express surface CD36 but not to CD36-deficient cells, indicating that CD36 exerts a strong selective pressure on the IRBC cytoadherent phenotype. IRBC adhesion to CD36 and ICAM-1 suggests that P. falciparum parasites may use these receptors in vivo to promote parasite survival and immune evasion.     

Receptor-binding residues lie in central regions of Duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites

Erythrocyte invasion by malaria parasites and cytoadherence of Plasmodium falciparum-infected erythrocytes to host capillaries are 2 key pathogenic mechanisms in malaria. The receptor-binding domains of erythrocyte-binding proteins (EBPs) such as Plasmodium falciparum EBA-175, which mediate invasion, and P falciparum erythrocyte membrane protein 1 (PfEMP-1) family members, which are encoded by var genes and mediate cytoadherence, have been mapped to conserved cysteine-rich domains referred to as Duffy-binding-like (DBL) domains. Here, we have mapped regions within DBL domains from EBPs and PfEMP-1 that contain receptor-binding residues. Using biochemical and molecular methods we demonstrate that the receptor-binding residues of parasite ligands that bind sialic acid on glycophorin A for invasion as well as complement receptor-1 and chondroitin sulfate A for cytoadherence map to central regions of DBL domains. In contrast, binding to intercellular adhesion molecule 1 (ICAM-1) requires both the central and terminal regions of DBLbetaC2 domains. Determination of functional regions within DBL domains is the first step toward understanding the structure-function bases for their interaction with diverse host receptors.     

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.     

In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.     

Alteration in cytoadherence and rosetting of Plasmodium falciparum- infected thalassemic red blood cells

Hemoglobinopathies have a protective role in malaria that appears to be related to alterations in red blood cell (RBC) properties. Thalassemic RBCs infected with Plasmodium falciparum showed greatly reduced cytoadherence and rosetting properties as well as impaired growth and multiplication. A significant decrease in the levels of falciparum antigens associated with the membrane of infected beta-thalassemic RBCs was observed at trophozoite/schizont stage, but not young ring stage. This reduction was shown when a cytoadherence inhibitory monoclonal antibody, but not a noninhibitory pooled immune serum, was used. These observations suggest that protection against malaria in thalassemia is caused by both reduced parasitemias and altered adherence properties of the infected thalassemic RBCs that promote enhanced clearance of the parasite from the circulation.     

CD36 deficiency protects against malarial anaemia in children by reducing Plasmodium falciparum-infected red blood cell adherence to vascular endothelium

Objective  CD36 is a receptor that occurs on the surface of activated immune cells, vascular endothelial cells and participates in phagocytosis and lipid metabolism. CD36 is known to be the major endothelial receptor molecule for field isolates of Plasmodium falciparum. A T1264G mutation in exon X of the gene leads to deficiency of CD36. This study aimed at determining associations between CD36 deficiency, P. falciparum in vitro adherence on purified CD36 and anaemia among children in an endemic area.
Methods  Genotypes were determined by nested polymerase chain reaction of isolated DNA from filter blood spots followed by Restriction Fragment Length Polymorphism (RFLP). Plasmodium falciparum adherence assays were performed on immobilized purified CD36.
Results  The data indicate that CD36 is an important cytoadherence receptor that mediates adherence to most P. falciparum field isolates. Our findings also suggest that mutations causing CD36 deficiency may confer significant protection against malarial anaemia (MA) in children (χ² = 8.58, P  < 0.01).
Conclusion  That the protective role that CD36 deficiency may confer against MA in children seems to be mediated through reduced cytoadherence of infected red blood cells to vascular endothelium.     

恶性疟原虫PfEMP-1的研究进展

目的 恶性疟原虫红细胞表面蛋白-1(PfEMP-1)是由var基因家族编码的一种蛋白质,它是疟原虫在宿主体内存活的重要蛋白。不但参与疟原虫的抗原变异,调节人的免疫应答,而且还是重要的粘附分子。本文对PfEMP-1的基础结构特征及其与临床的病理联系,及以其为基础的抗疟疫苗研发方面的研究进展作一综述。     

A primate model for human cerebral malaria: Plasmodium coatneyi-infected rhesus monkeys.

A major factor in the pathogenesis of human cerebral malaria is blockage of cerebral microvessels by the sequestration of parasitized human red blood cells (PRBC). In vitro studies indicate that sequestration of PRBC in the microvessels is mediated by the attachment of knobs on PRBC to receptors on the endothelial cell surface such as CD36, thrombospondin (TSP), and intercellular adhesion molecule-1 (ICAM-1). However, it is difficult to test this theory in vivo because fresh human brain tissues from cerebral malarial autopsy cases are not easy to obtain. Although several animal models for human cerebral malaria have been proposed, none have shown pathologic findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied brains of rhesus monkeys infected with the primate malaria parasite, Plasmodium coatneyi. Our study demonstrated PRBC sequestration and cytoadherence of knobs on PRBC to endothelial cells in the cerebral microvessels of these monkeys. Cerebral microvessels with sequestered PRBC were shown by immunohistochemical analysis to possess CD36, TSP, and ICAM-1. These proteins were not evident in the cerebral microvessels of uninfected control monkeys. Thus, our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria. By using this animal model, we may be able to evaluate strategies for the development of vaccines to prevent human cerebral malaria.     

Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by anti-idiotype antibodies.

The CD36 molecule expressed by human endothelial cells is a receptor for the adhesion of erythrocytes infected with the human malaria parasite Plasmodium falciparum. A CD36-specific monoclonal antibody, OKM8, inhibits the adhesion of malaria-infected erythrocytes (IRBC) to purified CD36 and cells expressing CD36. Monospecific polyclonal anti-idiotype (anti-Id) antibodies, raised against monoclonal antibody OKM8, expressed determinants molecularly mimicking the CD36 binding domain for the adhesion of IRBC. Purified rabbit anti-Id antibodies reacted with the surface of IRBC by immunofluorescence, directly supported the adhesion of wild-type P. falciparum malaria isolates, and inhibited IRBC cytoadherence to melanoma cells. An approximately 270-kDa protein was immunoprecipitated by the anti-Id antibodies from surface-labeled and metabolically labeled IRBC and was competitively inhibited by soluble CD36. These results support the hypothesis that CD36 is a receptor and the approximately 270-kDa protein, sequestrin, is a complementary ligand involved in the adhesion of IRBC to host-cell endothelium. Sequestrin is a candidate malaria vaccine antigen, and anti-Id antibodies that recognize this molecule may be useful for passive immunotherapy of cerebral and severe P. falciparum malaria.     

Cytoadherence in human falciparum malaria as a cause of respiratory distress

The ultrastructure of three cases of fatal human falciparum malaria was studied in order to identify the cytoadherence of the endothelial cells in relation to parasitized red blood cells and septal interstitial changes which could be related to respiratory distress. Two cases showed marked endothelial oedema narrowing the capillary lumen with areas of adherence preferentially related to knobs, accompanied by septal interstitial oedema. One case showed no endothelial cells oedema, no knobs in parasitized red blood cells with no cytoadherence, no septal interstitial oedema and no respiratory distress. Cytoadherence seems to be the mechanism responsible for the septal pulmonary changes in severe falciparum malaria.     

Uninfected erythrocytes form "rosettes" around Plasmodium falciparum infected erythrocytes

The human malaria parasite, P. falciparum, exhibits cytoadherence properties whereby infected erythrocytes containing mature parasite stages bind to endothelial cells both in vivo and in vitro. Another property of cytoadherence, "rosetting," or the binding of uninfected erythrocytes around an infected erythrocyte, has been demonstrated with a simian malaria parasite P. fragile which is sequestered in vivo in its natural host, Macaca sinica. In the present study we demonstrate that rosetting occurs in P. falciparum. Rosetting in P. falciparum is abolished by protease treatment and reappears on further parasite growth indicating that, as in P. fragile, it is mediated by parasite induced molecules which are protein in nature. P. vivax and P. cynomolgi, which are not sequestered in the host, did not exhibit rosetting. Rosetting thus appears to be a specific property of cytoadherence in malaria parasites.     

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: a parasite adhesion trait implicated in cerebral malaria

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated alpha, beta, gamma, delta, and epsilon. The DBL domain from the A4tres that binds ICAM-1 is DBLbeta type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLbeta domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLbeta domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.     

Plasmodium falciparum intercellular adhesion molecule-1-based cytoadherence-related signaling in human endothelial cells

BACKGROUND: Cytoadherence of Plasmodium falciparum-infected erythrocytes to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying disease are poorly understood. METHODS: We compared parasite lines varying in their binding properties to human endothelial cells for their ability to stimulate signaling activity. RESULTS: In human umbilical vein endothelial cells (HUVECs), which rely on adhesion to intercellular adhesion molecule (ICAM)-1 for binding, signaling is related to the avidity of the parasite line for ICAM-1 and can be blocked either through the use of anti-ICAM-1 monoclonal antibodies or HUVECs with altered ICAM-1 binding properties (i.e., ICAM-1(Kilifi)). Human dermal microvascular endothelial cells (HDMECs), which can bind infected erythrocytes via ICAM-1 and CD36, have a more complex pattern of signaling behavior, but this is also dependent on adhesive interactions rather than merely contact between cells. CONCLUSIONS: Signaling via apposition of P. falciparum-infected erythrocytes with host endothelium is dependent, at least in part, on the cytoadherence characteristics of the invading isolate. An understanding of the postadhesive processes produced by cytoadherence may help us to understand the variable pathologies seen in malaria disease.     

CD4 T cell responses to a variant antigen of the malaria parasite Plasmodium falciparum, erythrocyte membrane protein-1, in individuals living in malaria-endemic areas

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variant antigen on the surface of malaria-infected red blood cells. Antibody responses to PfEMP-1 correlate with immunity, and, therefore, PfEMP-1 may be a good candidate for a malaria vaccine. However, the specificity of CD4 T cells required for a protective variant-specific antibody response is not known. We have measured the CD4 T cell response to 3 different regions that are relatively homologous among different PfEMP-1 variants. The response to the cysteine-rich interdomain region was unusual in that the majority of donors, whether malaria exposed or not, had positive CD4 T cell, interleukin-10, and interferon-gamma responses. The CD4 T cell response to the exon 2 and duffy binding-like domain proteins was significantly greater in malaria-exposed donors than in unexposed Europeans, which suggests that these regions contain peptides recognized by T cells, which thus may be useful as components of a vaccine.     

Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein bind to separate domains of CD36.

The cytoadherence of erythrocytes (red blood cells) infected with Plasmodium falciparum (pRBCs) to endothelial cells and the uptake of oxidized low-density lipoprotein (oxLDL) by macrophages are both mediated, in part, by the glycoprotein receptor CD36. The interaction of lipoproteins and pRBCs competing for the human CD36 receptor was examined by use of Chinese hamster ovary cells expressing human CD36. OxLDL competitively inhibits the adherence of pRBCs to CD36, but native LDL and high-density lipoprotein do not. Modification of Lys residues in CD36 inhibits both oxLDL and pRBC binding; however, only oxLDL binding is inhibited by receptor iodination, and only pRBC binding is influenced by pH variations and receptor reduction. Furthermore, peptide inhibitors of the pRBC/CD36 interaction do not influence oxLDL binding. These results suggest that, although oxLDL competitively inhibits the adherence of pRBCs, these ligands interact with distinct domains on the CD36 receptor.     

Multiple ligands for cytoadherence can be present simultaneously on the surface of Plasmodium falciparum-infected erythrocytes.

A major virulence factor of Plasmodium falciparum is the adherence of parasitized erythrocytes to the wall of postcapillary venules via a specific interaction between parasite-derived erythrocyte surface ligands and receptors on endothelial cells. To study this phenomenon in vitro, we selected a parasite population that expressed at least two different ligands and demonstrated that parasitized cells may coexpress ligands with specificity for multiple receptors. This selected parasite line had several antigenic and cytoadherence characteristics that were different from those of the parent line. Single parasitized erythrocytes were able to adhere to three distinct receptors via at least two separate ligands; a trypsin-sensitive molecule mediated cytoadherence to CD36 and intercellular adhesion molecule 1 and a trypsin-insensitive molecule(s) was responsible for adherence to a third receptor on the surface of melanoma cells. We present evidence that this newly discovered receptor for cytoadherence is an N-linked glycosaminoglycan, as treatment of melanoma cells with endoglycosidase H abolished cytoadherence. These observations emphasize the adaptability of P. falciparum and the complexity of the cytoadherence phenomenon.     

Modifications in the CD36 binding domain of the Plasmodium falciparum variant antigen are responsible for the inability of chondroitin sulfate A adherent parasites to bind CD36

Adhesion of mature Plasmodium falciparum parasitized erythrocytes to microvascular endothelial cells or to placenta contributes directly to the virulence and severe pathology of P falciparum malaria. Whereas CD36 is the major endothelial receptor for microvasculature sequestration, infected erythrocytes adhering in the placenta bind chondroitin sulfate A (CSA) but not CD36. Binding to both receptors is mediated by different members of the large and diverse protein family P falciparum erythrocyte membrane protein-1 (PfEMP-1) and involves different regions of the molecule. The PfEMP-1-binding domain for CD36 resides in the cysteine-rich interdomain region 1 (CIDR-1). To explore why CSA-binding parasites do not bind CD36, CIDR-1 domains from CD36- or CSA-binding parasites were expressed in mammalian cells and tested for adhesion. Although CIDR-1 domains from CD36-adherent strains strongly bound CD36, those from CSA-adherent parasites did not. The CIDR-1 domain has also been reported to bind CSA. However, none of the CIDR-1 domains tested bound CSA. Chimeric proteins between CIDR-1 domains that bind or do not bind CD36 and mutagenesis experiments revealed that modifications in the minimal CD36-binding region (M2 region) are responsible for the inability of CSA-selected parasites to bind CD36. One of these modifications, mapped to a 3-amino acid substitution in the M2 region, ablated binding in one variant and largely reduced binding of another. These findings provide a molecular explanation for the inability of placental sequestered parasites to bind CD36 and provide additional insight into critical residues for the CIDR-1/CD36 interaction.     

Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes.

Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. falciparum malaria.