Cytoadherence characteristics of rosette-forming Plasmodium falciparum.

Sequestration of Plasmodium falciparum-infected erythrocytes to the capillary endothelium can cause obstruction and localized tissue damage. Occlusion of vessels in falciparum malaria infection has been related to two properties of the parasite: adhesion to endothelial cells and rosette formation. Our study on P. falciparum isolates from Thailand producing variable numbers of rosettes suggests the involvement of rosettes in capillary blockage caused by direct adhesion of the rosette-forming infected erythrocytes to various target cells, e.g., live human umbilical vein endothelial cells, monocytes, and platelets. These rosettes did not bind Formalin-fixed target cells, nor did they bind to live or fixed C32 or G361 melanoma cells. Classification of the receptors involved in cytoadherence of endothelial cells and monocytes by specific antibody blocking and flow cytometry indicated that CD36 was involved in the adherence of monocytes but that other receptors besides CD36 may be involved in parasite adherence to endothelial cells. The cytoadherence of infected erythrocytes to monocytes was also associated with CD54 (ICAM-1). Further, differentiation of adherent monocytes resulted in an inversion of CD36 and CD54 levels on the cell surface which correlated with a decrease in surface binding of infected erythrocytes. This observation suggests that the state of cell activation and differentiation may also contribute to sequestration of parasites and to the pathogenesis of malaria.     

Clinical correlates of in vitro Plasmodium falciparum cytoadherence.

To determine whether isolates of Plasmodium falciparum have intrinsically different cytoadherent properties and whether these differences contribute to the clinical severity of human falciparum malaria, we studied the cytoadherence to C32 melanoma cells in vitro of 59 parasite isolates from patients with naturally acquired infections in Thailand. Parasitized erythrocytes adhere to these melanoma cells principally via the glycoprotein CD36, which is also expressed on most vascular endothelium. In vitro cytoadherence was significantly greater for isolates from patients with biochemical evidence of severe malaria. The cytoadherent properties of P. falciparum parasites may thus be a virulence factor in human falciparum malaria. However, there was no correlation between the degree of in vitro cytoadherence and cerebral symptoms, which suggests that other receptors and/or host factors may be important in the adherence of malaria parasites to cerebral vascular endothelium. The cytokines tumor necrosis factor, interleukin-1, and gamma interferon, which have been implicated in the pathogenesis of cerebral malaria and are known to promote intercellular adhesion in other systems, did not enhance the cytoadherence of P. falciparum isolates to C32 melanoma cells.     

CD36-mediated nonopsonic phagocytosis of erythrocytes infected with stage I and IIA gametocytes of Plasmodium falciparum

Gametocytes, the sexual stages of malaria parasites (Plasmodium spp.) that are transmissible to mosquitoes, have been the focus of much recent research as potential targets for novel drug and vaccine therapies. However, little is known about the host clearance of gametocyte-infected erythrocytes (GEs). Using a number of experimental strategies, we found that the scavenger receptor CD36 mediates the uptake of nonopsonized erythrocytes infected with stage I and IIA gametocytes of Plasmodium falciparum by monocytes and culture-derived macrophages (Mphis). Light microscopy and immunofluorescence assays revealed that stage I and IIA gametocytes were readily internalized by monocytes and Mphis. Pretreating monocytes and Mphis with a monoclonal antibody that blocked CD36 resulted in a significant reduction in phagocytosis, as did treating GEs with low concentrations of trypsin to remove P. falciparum erythrocyte membrane protein 1 (PfEMP-1), a parasite ligand for CD36. Pretreating monocytes and Mphis with peroxisome proliferator-activated receptor gamma-retinoid X receptor agonists, which specifically upregulate CD36, resulted in a significant increase in the phagocytosis of GEs. Murine CD36 on mouse Mphis also mediated the phagocytosis of P. falciparum stage I and IIA gametocytes, as determined by receptor blockade with anti-murine CD36 monoclonal antibodies and the lack of uptake by CD36-null Mphis. These results indicate that phagocytosis of stage I and IIA gametocytes by monocytes and Mphis appears to be mediated to a large extent by the interaction of PfEMP-1 and CD36, suggesting that CD36 may play a role in innate clearance of these early sexual stages.     

Plasmodium falciparum gametocyte adhesion to C32 cells via CD36 is inhibited by antibodies to modified band 3.

Plasmodium falciparum gametocyte-infected erythrocytes are characterized by their ability to sequester in the microvasculature of various organs, primarily the spleen and bone marrow. This phenomenon is thought to play a critical role in the development and survival of the sexual stages. Little is known, however, about ligands on the gametocyte-infected erythrocyte. Infection of erythrocytes with mature asexual stages of P. falciparum (trophozoites and schizonts) has been shown to induce modification of the erythrocyte anion transporter, band 3, and this has been linked to the acquisition of an adherent phenotype. Here, we demonstrate for the first time that immature gametocyte-infected erythrocytes also express modified band 3. In vitro binding assays demonstrate that gametocyte-infected erythrocytes of the 3D7 strain utilize this surface receptor for adhesion to C32 amelanotic melanoma cells via the host cell receptor CD36 (platelet glycoprotein IIIb). Adhesion of gametocyte-infected erythrocytes to CD36-transfected CHO cells is also dependent on modified band 3. However, modified band 3 does not mediate adhesion of gametocyte-infected erythrocytes to intercellular adhesion molecule 1, a second host receptor for gametocytes expressed on C32 cells.     

Does the shape of Plasmodium falciparum gametocytes have a function?

The gametocytes of Plasmodium falciparum have a characteristic banana-shape. One could wonder if this shape has been retained by selection forces because it favored transmission. By increasing the surface to volume ratio, the elongation that takes place during the maturation of P. falciparum gametocytes increases the susceptibility of gametocytes to rheological forces. This may facilitate their exit from the bone marrow and their subsequent impaction in the cutaneous capillaries that are lined with sequestered asexual parasites. The asymmetric curvature of gametocytes may tend to present gametocytes perpendicularly to the axis of the blood flow, thus increasing their chance to be retained and accumulate where they can be absorbed by the vector.     

The mature erythrocyte surface antigen of Plasmodium falciparum is not required for knobs or cytoadherence

Intraerythrocytic Plasmodium falciparum parasites at the trophozoite and schizont stages synthesize a greater than 200-kDa protein, the mature erythrocyte surface antigen (MESA), that is localized at the membrane of infected red blood cells and manifests size polymorphism and antigenic diversity among parasite isolates. Because MESA is localized in the host cell membrane, we examined parasites with differing knob and cytoadherence phenotypes to determine whether MESA expression correlated with knob formation and cytoadherence. A cloned line of P. falciparum that was cultured with repeated selection for the knobbed and cytoadherent phenotypes did not express MESA, due to at least partial deletion of the single-copy MESA gene. In contrast, parasites from the same clone that were cultured without this selection lost the knobbed and cytoadherent phenotypes, but continued to express MESA. These results indicate that MESA is apparently not required for differentiation and multiplication of erythrocyte stage P. falciparum parasites in vitro, or for knob formation and cytoadherence. We speculate that MESA may have a role in evasion of the host immune response by P. falciparum.     

The C-terminal segment of the cysteine-rich interdomain of Plasmodium falciparum erythrocyte membrane protein 1 determines CD36 binding and elicits antibodies that inhibit adhesion of parasite-infected erythrocytes

Attachment of erythrocytes infected by Plasmodium falciparum to receptors of the microvasculature is a major contributor to the pathology and morbidity associated with malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP-1), which is expressed at the surface of infected erythrocytes and is linked to both antigenic variation and cytoadherence. PfEMP-1 contains multiple adhesive modules, including the Duffy binding-like domain and the cysteine-rich interdomain region (CIDR). The interaction between CIDRalpha and CD36 promotes stable adherence of parasitized erythrocytes to endothelial cells. Here we show that a segment within the C-terminal region of CIDRalpha determines CD36 binding specificity. Antibodies raised against this segment can specifically block the adhesion to CD36 of erythrocytes infected with various parasite strains. Thus, small regions of PfEMP-1 that determine binding specificity could form suitable components of an antisequestration malaria vaccine effective against different parasite strains.     

B-cell chronic lymphocytic leukemia: correlation of clinical stages with angiogenic cytokine expression.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of non-cycling B cells in lymphatic and extralymphatic tissues. Earlier studies had validated that angiogenesis was increased in B-CLL. Increased serum concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) connote a poor prognosis in early-stage B-CLL. Early progression is also related to transforming growth factor-beta (TGF-beta), which inhibits B-cell proliferation and immunoglobulin production. The authors investigated the expression of CD34, VEGF, bFGF, and TGF-beta and their receptors in different stages of B-CLL by analyzing bone marrow samples from 23 patients (11 with Rai stages 0-II; 12 with stages III or IV). TGF-beta2 was expressed more strongly in stages 0 to II than in stages III or IV (P=0.03). There was no significant difference in the intensity of CD34, TGF-beta1, VEGF, and bFGF and their receptors between stages 0 to II and stages III or IV. Staining showed bFGF expression to be stronger than VEGF expression (P=0.001). Results did not confirm an association between the intensity of angiogenesis and B-CLL stage. The expression of TGF-beta2 was stronger in early-stage disease and may help slow disease progression.     

Detection of a LFA-1-like epitope on the surface of erythrocytes infected with a strain of Plasmodium falciparum.

The adhesion of erythrocytes infected with Plasmodium falciparum (P. falciparum) is one of the major pathological features of severe malaria. Several potential receptors to endothelium for falciparum-infected erythrocyte on endothelium have been described. Recently, the malaria binding site on ICAM-1(CD54) has been mapped to a site distinct but overlapping with the LFA-1 (CD11a/CD18) site. We detected by flow cytometry, confocal laser microscopy and immunoprecipitation, a molecule expressed at the surface of erythrocytes infected with mature stages of the M96 strain of P. falciparum that was recognized by a monoclonal antibody (mAb) (TS1/22) directed against an LFA-1 epitope. However, this molecule was not recognized by mAbs directed against other epitopes of LFA-1 or against other integrins. Furthermore, the mAb TS1/22 partially inhibited cytoadherence of parasitized red blood cells to human-brain microvascular endothelial cells. The expression of a molecule sharing an epitope with human LFA-1 integrin on the parasitized erythrocyte surface could be involved in the sequestration of these cells and thus in the pathogenesis of severe disease.     

Cytoadherence of Plasmodium falciparum to intercellular adhesion molecule 1 and chondroitin-4-sulfate expressed by the syncytiotrophoblast in the human placenta.

Late stages of Plasmodium falciparum-infected erythrocytes (IRBCs) frequently sequester in the placentas of pregnant women, a phenomenon associated with low birth weight of the offspring. To investigate the physiological mechanism of this sequestration, we developed an in vitro assay for studying the cytoadherence of IRBCs to cultured term human trophoblasts. The capacity for binding to the syncytiotrophoblast varied greatly among P. falciparum isolates and was mediated by intercellular adhesion molecule 1 (ICAM-1), as binding was totally inhibited by 84H10, a monoclonal antibody specific for ICAM-1. Binding of the P. falciparum line RP5 to the syncytiotrophoblast involves chondroitin-4-sulfate (CSA), as this binding was dramatically impaired by addition of free CSA to the binding medium or by preincubation of the syncytiotrophoblast with chondroitinase ABC. ICAM-1 and CSA were visualized on the syncytiotrophoblast by immunofluorescence, while CD36, E-selectin, and vascular cell adhesion molecule 1 were not expressed even on tumor necrosis factor alpha (TNF-alpha)-stimulated syncytiotrophoblast tissue, and monoclonal antibodies against these cell adhesion molecules did not inhibit cytoadherence. ICAM-1 expression and cytoadherence of wild isolates was upregulated by TNF-alpha, a cytokine that can be secreted by the numerous mononuclear phagocytes present in malaria-infected placentas. These results suggest that cytoadherence may be involved in the placental sequestration and broaden the understanding of the physiopathology of the malaria-infected placenta.     

Molecular analysis of the cytoadherence phenotype of a Plasmodium falciparum field isolate that binds intercellular adhesion molecule-1

The ability of Plasmodium falciparum-infected erythrocytes to adhere to endothelial receptors and sequester in diverse host organs is an important pathogenic mechanism. Cytoadherence is mediated by variant surface antigens, which are referred to as PfEMP-1 and are encoded by var genes. The extracellular regions of PfEMP-1 contain multiple conserved cysteine-rich domains that are referred to as Duffy-binding-like (DBL) domains. Here, we analyze the adhesive phenotype of an Indian P. falciparum field isolate, JDP8, which binds ICAM-1 but does not bind CD36. This is a unique cytoadherence phenotype because P. falciparum strains that bind ICAM-1 described thus far usually also bind CD36. Moreover, binding to both receptors is thought to be important for static adhesion under flow. The ICAM-1 binding population of P. falciparum JDP8 adheres to endothelial cells under flow despite poor binding to CD36. We have also identified an expressed var gene, JDP8Icvar, which mediates the ICAM-1 binding phenotype of JDP8. Expression of different regions of JDP8Icvar on the surface of COS-7 cells followed by binding assays demonstrates that the ICAM-1 binding domain maps to the DBL2betaC2 domain of JDP8Icvar. Sequence comparison with two previously identified ICAM-1 binding domains of PfEMP-1, which also map to DBLbetaC2 domains, suggests that diverse P. falciparum isolates use a structurally conserved domain to bind ICAM-1. It thus appears that functional constraints may place limits on the extent of sequence diversity in receptor-binding domains of PfEMP-1.     

A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain.

Microvascular sequestration was assessed in the brains of 50 Thai and Vietnamese patients who died from severe malaria (Plasmodium falciparum, 49; P. vivax, 1). Malaria parasites were sequestered in 46 cases; in 3 intravascular malaria pigment but no parasites were evident; and in the P. vivax case there was no sequestration. Cerebrovascular endothelial expression of the putative cytoadherence receptors ICAM-1, VCAM-1, E-selectin, and chondroitin sulfate and also HLA class II was increased. The median (range) ratio of cerebral to peripheral blood parasitemia was 40 (1.8 to 1500). Within the same brain different vessels had discrete but different populations of parasites, indicating that the adhesion characteristics of cerebrovascular endothelium change asynchronously during malaria and also that significant recirculation of parasitized erythrocytes following sequestration is unlikely. The median (range) ratio of schizonts to trophozoites (0.15:1; 0.0 to 11.7) was significantly lower than predicted from the parasite life cycle (P < 0.001). Antimalarial treatment arrests development at the trophozoite stages which remain sequestered in the brain. There were significantly more ring form parasites (age < 26 hours) in the cerebral microvasculature (median range: 19%; 0-90%) than expected from free mixing of these cells in the systemic circulation (median range ring parasitemia: 1.8%; 0-36.2%). All developmental stages of P. falciparum are sequestered in the brain in severe malaria.     

Parasite virulence factors during falciparum malaria: rosetting, cytoadherence, and modulation of cytoadherence by cytokines.

To determine virulence factors of isolates of Plasmodium falciparum and the potential role of cytokines in cerebral malaria, 46 Malagasy patients presenting with cerebral (n = 10), severe (n = 10), and uncomplicated (n = 26) malaria were enrolled in a study. The capacity of 21 of 46 P. falciparum isolates to form rosettes in vitro and to adhere to human umbilical vein endothelial cells (HUVECs) that express intercellular adhesion molecule-1 receptors and to C32 amelanotic melanoma cells that express mainly CD36 receptors was investigated together with the effects of tumor necrosis factor alpha (TNF-alpha), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-6 alone and in two-by-two combinations on the cytoadherence of infected erythrocytes to HUVECs. Plasma levels of these cytokines were also measured in the patients at admission. The percentage of rosette formation was higher for the isolates from patients with cerebral (n = 6; 19.5%) and severe (n = 6; 30.5%) malaria than for those from patients with uncomplicated malaria (n = 9; 5%) (P < 0.002). The cytoadherence properties of the isolates did not differ among the three groups whatever the target cell used, but adherence to melanoma cells was systematically higher than that to HUVECs. Adhesion to HUVECs was increased more after TNF-alpha stimulation than after GM-CSF, IL-3, or IL-6 stimulation (P < 0.01). Only the combination of TNF-alpha and IL-3 enhanced cytoadherence more than TNF-alpha used alone (P < 0.02). No difference in the modulation of cytoadherence by cytokines was found in relation to the severity of the disease. TNF-alpha and IL-6 levels in peripheral blood were higher in the patients with cerebral and severe malaria than in the patients with uncomplicated malaria (P < 0.005). Most of the patients' sera contained little or no IL-3 or GM-CSF. Our results challenge the role of intercellular adhesion molecule-1 as the principal receptor mediating the cytoadherence of P. falciparum-infected erythrocytes and contrast with data obtained in the murine model.     

Improved synchronous production of Plasmodium falciparum gametocytes in vitro

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis.     

In vivo developmental stages in murine natural killer cell maturation

Natural killer (NK) cells develop in the bone marrow, but their in vivo stages of maturation, expansion and acquisition of receptors that guide target cell specificity are not well defined. We describe here such stages of development. We also show that developing NK cells actively proliferate at a phenotypically distinguishable immature stage after they have acquired expression of Ly49 and CD94-NKG2 receptors. These studies provide a developmental framework for NK cell maturation in vivo and suggest the possible involvement of the Ly49 and CD94-NKG2 receptors themselves in modulating expansion of NK cell populations with a given NK cell receptor repertoire.     

A regulatory role for Fc gamma receptors (CD16 and CD32) in hematopoiesis.

Progenitor cells of the T- and B-lineages in mice express (CD32) and Fc gamma RIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for Fc gamma R occur on thymic and bone marrow stromal cells, the possibility exists that Fc gamma R might participate in some aspect of T- and B-lineage development prior to the stage of antigen receptor expression. Previous studies provided evidence that Fc gamma R can influence murine T-lineage development. In the present studies we found that anti-Fc gamma RII/III mAb accelerated B-lineage development in bone marrow cultures from normal mice, but not in cultures from CD16-/- or CD32-/- mice. Similar results were observed when FACS-purified B-progenitor cells were co-cultured with BMS2, a bone marrow stromal cell line. Fresh bone marrow from CD32-/- mice contained about two-fold more B-lineage cells compared to bone marrow from normal or CD16-/- mice. These studies indicate that the Fc gamma R on B-lineage progenitor cells can influence their further development and add to a growing body of evidence that implicates Fc gamma R as regulatory elements in hematopoiesis.     

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.