Sequestration Of Erythrocytes Research Articles

Fresh isolates from children with severe Plasmodium falciparum malaria bind to multiple receptors.

The sequestration of Plasmodium falciparum-infected erythrocytes (pRBC) away from the peripheral circulation is a property of all field isolates. Here we have examined the pRBC of 111 fresh clinical isolates from children with malaria for a number of adhesive features in order to study their possible coexpression and association with severity of disease. A large number of adhesion assays were performed studying rosetting, giant rosetting, and binding to CD36, intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, thrombospondin, heparin, blood group A, and immunoglobulins. Suspension assays were performed at the actual parasitemia of the isolate, while all the static adhesion assays were carried out at an equal adjusted parasitemia. The ability to bind to multiple receptors, as well as the ability to form rosettes and giant rosettes, was found to be more frequent among isolates from children with severe versus mild malaria (P = 0.0015). Rosettes and giant rosettes were more frequent for children with severe malaria, and the cell aggregates were larger and tighter, than for those with mild disease (P = 0.0023). Binding of immunoglobulins (97% of isolates) and of heparin (81% of isolates) to infected erythrocytes was common, and binding to heparin and blood group A was associated with severity of disease (P = 0.011 and P = 0.031, respectively). These results support the idea that isolates that bind to multiple receptors are involved in the causation of severe malaria and that several receptor-ligand interactions work synergistically in bringing about severe disease.

Expression of Intercellular Adhesion Molecule 1 (ICAM-1) in Plasmodium falciparum -infected Placenta

We investigated the expression of intercellular adhesion molecule 1 (ICAM-1) in malarial placenta and related histological changes. Thirty-two malarial and 40 control term placentae were collected at Tanga, Tanzania and examined histologically and immunohistochemically. Malaria infected placentae were further divided into acute (15) and chronic (17) cases according to the presence of malarial pigment. The expression of ICAM-1 on monocyte, syncytio- and cytotrophoblasts, endothelial and stromal cells was assessed. Birthweight was lower and leukocyte counts higher in placentae with chronic infection. Many monocytes were present within the intervillous spaces, especially in placentae with chronic infection, and aggregated with parasitized erythrocytes. Some monocytes were adhesive to the surface of fibrinoid deposits. ICAM-1 expression on monocytes of malarial placentae was significantly conspicuous and correlated to the degree of intervillous leukocyte infiltration. Syncytiotrophoblasts often did not show ICAM-1, even though ICAM-1 was expressed by endothelium and weakly by cytotrophoblasts and stromal cells in both infected and control placentae. These results suggest that the expression of ICAM-1 on monocytes contributes to sequestration of infected erythrocytes within the intervillous spaces and their adhesion to fibrin masses and that ICAM-1 is unlikely to be associated with the direct adhesion of infected erythrocytes to the syncytiotrophoblasts.

Platelets in malaria pathology

Platelets might play a hitherto unrecognized role in the clinical features of malarial disease. A recent study [Pain, A. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 1805–1810] presents the novel finding that clumping of Plasmodium falciparum-infected erythrocytes often involves platelets. This was unexpected, because agglutination of infected erythrocytes was previously thought to be a result of the autoadhesive nature of these cells alone. Instead, now it appears that CD36-expressing platelets can be responsible for this phenomenon. Clumping and sequestration of malaria-infected erythrocytes in the peripheral circulation is thought to be a major contributing factor to the virulence of P. falciparum. SHK

CD36 associates with CD9 and integrins on human blood platelets

The membrane glycoprotein CD36 is involved in platelet aggregation, inhibition of angiogenesis, atherosclerosis, and sequestration of malaria-parasitized erythrocytes. In this study, immunoprecipitations with anti-CD36 antibodies were performed to identify proteins that associate with CD36 in the platelet membrane. Platelets were solubilized in 1% Triton X-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Brij 96, or Brij 99, and the proteins that coprecipitated with CD36 were identified by peptide mass spectrometry and Western blotting. The tetraspanin protein CD9 and the integrins alphaII(b)beta3 and alpha6beta1 specifically coprecipitated with CD36 from platelets that were solubilized in CHAPS and Brij 99 but not from platelets that were solubilized in Triton X-100. Only CD9 is coprecipitated with CD36 from platelets that were solubilized in Brij 96. Reciprocal immunoprecipitations with antibodies to CD9, alpha6, alphaIIb, or beta3 from Brij 99-solubilized platelets coprecipitated CD36. Coprecipitation of CD36, CD9, and alpha6beta1 was also observed on platelets from a patient with Glanzmann thrombasthenia, indicating that alphaII(b)beta3 is not required for the other proteins to associate. Colocalization of alpha6 and CD36, of CD9 and CD36, and of alpha6 and CD9 was observed on intact platelets prior to solubilization, using double immunofluorescence microscopy. These data indicate that CD36 associates with CD9 and integrins on human blood platelets. These associated proteins may mediate or participate in some of the diverse biological functions of CD36.

Platelet-mediated clumping of Plasmodium falciparum -infected erythrocytes is a common adhesive phenotype and is associated with severe malaria

Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.

The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors.

Erythrocytes infected with mature forms of Plasmodium falciparum do not circulate but are withdrawn from the peripheral circulation; they are bound to the endothelial lining and to uninfected erythrocytes in the microvasculature. Blockage of the blood flow, hampered oxygen delivery, and severe malaria may follow if binding is excessive. The NH(2)-terminal head structure (Duffy binding-like domain 1 [DBL1alpha]-cysteine-rich interdomain region [CIDR1alpha]) of a single species of P. falciparum erythrocyte membrane protein 1 (PfEMP1) is here shown to mediate adherence to multiple host receptors including platelet-endothelial cell adhesion molecule 1 (PECAM-1)/CD31, the blood group A antigen, normal nonimmune immunoglobulin M, three virulence-associated receptor proteins, a heparan sulfate-like glucosaminoglycan, and CD36. DBL2delta was found to mediate additional binding to PECAM-1/CD31. The exceptional binding activity of the PfEMP1 head structure and its relatively conserved nature argues that it holds an important role in erythrocyte sequestration and therefore in the virulence of the malaria parasite.

The ves Multigene Family of B. bovis Encodes Components of Rapid Antigenic Variation at the Infected Erythrocyte Surface

B. bovis, an intraerythrocytic protozoal parasite, establishes chronic infections in cattle in part through rapid variation of the polymorphic, heterodimeric VESA1 protein on the infected erythrocyte surface and sequestration of mature parasites. We describe the characterization of the ves 1α gene encoding the VESA1a subunit, thus providing a description of a gene whose product is involved in rapid antigenic variation in a babesial parasite. This three-exon gene, a member of a multigene family ( ves), encodes a polypeptide with no cleavable signal sequence, a single predicted transmembrane segment, and a cysteine/lysine-rich domain. Variation appears to involve creation and modification or loss of a novel, transcribed copy of the gene.

Rouleaux-Forming Serum Proteins Are Involved in the Rosetting of Plasmodium falciparum-Infected Erythrocytes

Treutiger, C. J., Scholander, C., Carlson, J., McAdam, K. P., Raynes, J. G., Falksveden, L., and Wahlgren, M. 1999. Rouleaux-forming serum proteins are involved in the rosetting of Plasmodium falciparum-infected erythrocytes. Experimental Parasitology93, 215–224. Excessive sequestration of Plasmodium falciparum-infected (pRBC) and uninfected erythrocytes (RBC) in the microvasculature, cytoadherence, and rosetting, have been suggested to be correlated with the development of cerebral malaria. P. falciparum erythrocyte membrane protein-1 (PfEMP1) is the parasite-derived adhesin which mediates rosetting. Herein we show that serum proteins are crucial for the rosette formation of four strains of parasites (FCR3S1, TM284, TM180, and R29), whereas the rosettes of a fifth strain (DD2) are serum independent. Some parasites, e.g., FCR3S1, can be depleted of all rosettes by washes in heparin and Na citrate and none of the rosettes remain when the parasite is grown in foetal calf serum or ALBUMAX. Rosettes of other parasites are less sensitive; e.g., 20% of TM180 and R29 and 70% of TM284 rosettes still prevail after cultivation. A serum fraction generated by ion-exchange chromatography and poly-ethylene-glycol precipitation restored 50% of FCR3S1 and approx 40 to 100% of TM180 rosettes. In FCR3S1, antibodies to fibrinogen reverted the effect of the serum fraction and stained fibrinogen bound to the pRBC surface in transmission electron microscopy. Normal, nonimmune IgM and/or IgG was also found attached to the pRBC of the four serum-dependent strains as seen by surface immunofluorescens. Our results suggest that serum proteins, known to participate in rouleaux formation of normal erythrocytes, produce stable rosettes in conjunction with the recently identified parasite-derived rosetting ligand PfEMP1.

Isoantibodies in Immunoglobulin for Intravenous Use May Cause Erythrocyte Sequestration

Abstract Background and Objectives: IVIg, while generally a safe therapy for a variety of immunological disorders, can have detrimental effects on erythrocyte (RBC) homeostasis. We studied the mediation of erythrophagocytosis by isoantibodies in IVIg. Materials and Methods: RBC were exposed to IVIg and binding of IgG determined by flow cytometry. In vitro phagocytosis of these RBC was assayed. Results: Anti‐A and anti‐B in IVIg mediate Fc‐dependent erythrophagocytosis even in the presence of excess IVIg. Removal of these isoantibodies from IVIg prevents IgG binding and erythrophagocytosis. Complement enhances IVIg‐mediated RBC sequestration. Conclusion: It may be desirable to remove isoantibodies from IVIg especially for anemic patients or those who respond to the IVIg with a hemolytic episode.

Isoantibodies in Immunoglobulin for Intravenous Use May Cause Erythrocyte Sequestration

Background and Objectives: IVIg, while generally a safe therapy for a variety of immunological disorders, can have detrimental effects on erythrocyte (RBC) homeostasis. We studied the mediation of erythrophagocytosis by isoantibodies in IVIg. Materials and Methods: RBC were exposed to IVIg and binding of IgG determined by flow cytometry. In vitro phagocytosis of these RBC was assayed. Results: Anti–A and anti–B in IVIg mediate Fc–dependent erythrophagocytosis even in the presence of excess IVIg. Removal of these isoantibodies from IVIg prevents IgG binding and erythrophagocytosis. Complement enhances IVIg–mediated RBC sequestration. Conclusion: It may be desirable to remove isoantibodies from IVIg especially for anemic patients or those who respond to the IVIg with a hemolytic episode.

In vivoAdministration of Intravenous Immunoglobulin (IVIg) can Lead to Enhanced Erythrocyte Sequestration

Enhanced erythrocyte sequestration is one of the very few major adverse effects of intravenous immunoglobulin (IVIg). IVIg contains high molecular weight IgG complexes (∼300kDa) which, in the presence of serum, mimic immune complexes by activating complement, binding to CR1 of red blood cells (RBC) (CD35) and mediating erythrophagocytosis. Four of seven patients undergoing IVIg therapy showed significant drops in haematocrit and haemoglobin that were not due to isoantibodies in the IVIg. Prior to treatment, patients' RBC carried IgG and complement (C′) 3d that were not bound as immune complexes via CR1 (CD35). The patients whose RBC bound immune complex-like moieties and showed drops in haematocrit and haemoglobin subsequent to IVIg were young adults (22–35 years); older patients (50–69 years) showed no ill effects. In the presence of complement, RBC of young patients bound IVIg complexes in vitro while those of older patients did not. It is not the absolute levels of erythrocyte-associated IgG or C′3 fragments, neither pre- nor post-therapy, which are predictive of IVIg associated decreases in haematocrit and haemoglobin levels. Patient age and RBC inability to bind the IVIg immune complex-like moieties in vitro both appear to be predictors of resistance to sequestration after in vivo treatment with IVIg.

Survival of human carrier erythrocytes in vivo

Erythrocytes offer the exciting opportunity of being used as carriers of therapeutic agents. Encapsulation within erythrocytes will give the therapeutic agent a clearance equivalent to the normal life of the erythrocyte therefore maintaining therapeutic blood levels over prolonged periods and also giving a sustained delivery to the monocyte–macrophage system (reticulo-endothelial system). Both the dose and frequency of therapeutic interventions could thus be reduced. Ensuring a near-physiological survival time of carrier erythrocytes is essential to their successful use as a sustained drug delivery system, and this has not been demonstrated in man. In this study we assessed the survival in vivo of autologous unloaded energy-replete carrier erythrocytes in nine volunteers, using a standard 51Cr erythrocyte-labelling technique. Within 144 h after infusion there was a 3 to 49% fall in circulating labelled cells, followed thereafter by an almost complete return to initial circulating levels; surface counting demonstrated an initial sequestration of erythrocytes by the spleen and subsequent release. Mean cell life and cell half-life of the carrier erythrocytes were within the normal range of 89 to 131 days and 19 to 29 days respectively. These results demonstrate the viability of carrier erythrocytes as a sustained drug delivery system.

Survival of human carrier erythrocytes in vivo

Erythrocytes offer the exciting opportunity of being used as carriers of therapeutic agents. Encapsulation within erythrocytes will give the therapeutic agent a clearance equivalent to the normal life of the erythrocyte therefore maintaining therapeutic blood levels over prolonged periods and also giving a sustained delivery to the monocyte-macrophage system (reticulo-endothelial system). Both the dose and frequency of therapeutic interventions could thus be reduced. Ensuring a near-physiological survival time of carrier erythrocytes is essential to their successful use as a sustained drug delivery system, and this has not been demonstrated in man. In this study we assessed the survival in vivo of autologous unloaded energy-replete carrier erythrocytes in nine volunteers, using a standard 51Cr erythrocyte-labelling technique. Within 144 h after infusion there was a 3 to 49% fall in circulating labelled cells, followed thereafter by an almost complete return to initial circulating levels; surface counting demonstrated an initial sequestration of erythrocytes by the spleen and subsequent release. Mean cell life and cell half-life of the carrier erythrocytes were within the normal range of 89 to 131 days and 19 to 29 days respectively. These results demonstrate the viability of carrier erythrocytes as a sustained drug delivery system.

Plasmodium Falciparum-infected Erythrocytes: A Mutational Analysis of Cytoadherence via Murine Thrombomodulin

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Systemic inflammation promotes erythrocyte sequestration

Changes in red blood cell (RBC) flexibility and membrane surface properties affect their circulatory clearance. Glutaraldehyde (GLUT) stiffened RBC are cleared by about 70% within 30 min after transfusion [1]. Recently, we have shown that endotoxin (ETX) increases RBC adhesion to human vascular endothelial cells [2]. In this study we investigated the effects of endotoxin and cecal ligation and perforation induced sepsis (CLP) on RBC survival and sequestration in healthy and septic rats.

Widespread expression of MRP8 and MRP14 in human cerebral malaria by microglial cells.

Human cerebral malaria (CM) is an often fatal infection. The cascades of signaling events resulting in tissue trauma and coma are only slowly becoming unraveled. Here we report that microglial cells--sensitive cellular sensors of threats to the central nervous system--in CM express the myeloid-related proteins MRP8 (S100A8) and MRP14 (S100A9), Ca2+-binding sensor proteins of activated monocytes. Surprisingly, microglial activation was widespread throughout the brain in white and gray matter and not limited to areas of petechial bleedings or sequestration of infected erythrocytes. Further, apoptosis/necrosis is prominent in CM; not only leukocytes appeared apoptotic, neurons also appeared damaged and DNA fragmentation was revealed by in situ nick translation. Thus, a prominent feature of human CM is activation of microglia, and analysis of these reactive microglia might further promote our understanding of CM pathology and guide development of future therapeutic intervention of the local reactive processes.

Short report: Plasmodium falciparum: cytoadherence to alpha(v)beta3 on human microvascular endothelial cells.

Sequestration of infected erythrocytes in brain microvasculature contributes to cerebral malaria, a severe complication of Plasmodium falciparum infection. Sequestration likely involves cytoadherence of parasitized erythrocytes to cerebral microvascular endothelium. Elucidation of the receptors and ligands involved in cytoadherence will likely contribute to a more complete understanding of malaria pathophysiology. The integrin receptor alpha(v)beta3 is involved in several physiologic and pathologic adherence processes, but its role in cytoadherence has not been investigated. In this study, the ability of erythrocytes infected with P. falciparum to adhere to alpha(v)beta3 on human microvascular endothelium was investigated. Cytoadherence was quantified under continuous flow at a shear stress of 1.0 dyne/cm2 to mimic shear forces in the cerebral microcirculation. Adherence of erythrocytes infected with P. falciparum to human microvascular endothelial cells was 7-270-fold greater than uninfected erythrocytes. Pretreatment of microvascular endothelial cells with anti-alpha(v) antibody inhibited P. falciparum-infected erythrocyte adherence by 45 +/- 6% (mean +/- SEM). These data suggest that in addition to other endothelial receptors previously described, P. falciparum parasitized red blood cells may bind to the integrin alpha(v)beta3 on microvascular endothelial cells.

Multiple Adhesive Phenotypes Linked to Rosetting Binding of Erythrocytes inPlasmodium falciparumMalaria

The cerebral form of severe malaria is associated with excessive intravascular sequestration of Plasmodium falciparum-infected erythrocytes (PRBC). Retention and accumulation of PRBC may lead to occlusion of brain microvessels and direct the triggering of acute pathologic changes. Here we report that by selection, cloning, and subcloning, we have identified rare P. falciparum parasites expressing a pan-adhesive phenotype linked to erythrocyte rosetting, a previously identified correlate of cerebral malaria. Rosetting PRBC not only bound uninfected erythrocytes but also formed autoagglutinates, adhered to endothelial cells, and bound to CD36, immunoglobulins, and the blood group A antigen. The linkage of rosetting, autoagglutination, and cytoadherence involved the coexpression on a single PRBC of ligands with multiple specificities and the binding to two or more receptors on erythrocytes and to at least two other cell adhesion molecules, including a new endothelial cell receptor for P. falciparum-infected erythrocytes. Limited proteolysis that differentially cleaved the rosetting ligand PfEMP1 from the PRBC surface abrogated all the binding phenotypes of these parasites, implicating the variant antigen PfEMP1 as a carrier of multiple ligand specificities. The results encourage the further study of pan-adhesion as a potentially important parasite phenotype in the pathogenesis of severe P. falciparum malaria.

Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum.

Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.

Hydroxylamine Treatment Increases Glutathione-Protein and Protein-Protein Binding in Human Erythrocytes

ABSTRACT: Hydroxylamine is a direct-acting hematotoxic agent leading to hemolytic anemia in animals and man. The effect of hydroxylamine on the morphology, sulfhydryl status and membrane skeletal proteins of human erythrocytes were studied. Loss of reduced glutathione (GSH) from the red blood cells was directly proportional to the hydroxylamine concentration used. This loss of GSH was larger than the sum of the increase in the amounts of extracellular glutathione and intracellular oxidized glutathione (GSSG). The extracellular glutathione is mainly present as GSSG, which is in agreement with the fact that only GSSG is exported from the erythrocytes by membrane bound ATPases. Lack of GSSG export was not limited by decreased ATP levels in the erythrocytes and we concluded that the GSH that disappeared did not become available as intracellular GSSG. After reduction of the erythrocyte incubates the lost GSH was almost completely recovered indicating that the lost GSH is present in the cell as protein-glutathione mixed disulfides. Glutathione thus stored within the cell can be quickly recovered by combined thioltransferase and glutathione reductase activity when conditions become more favorable again. SDS-polyacrylamide gel electrophoresis of membrane ghosts from human red cells revealed changes in skeletal proteins with a smearing of bands 1, 2 and 3 to the higher molecular weight end of the gel and the appearance of new monomeric and dimeric hemoglobin bands at about 16 and 30 kD. The observed alterations are probably a consequence of disulfide bridge formation between cellular proteins (mainly hemoglobin) and skeletal proteins as well as between hemoglobin monomers. Exposure of hydroxylamine to erythrocytes caused severe Heinz body formation but the outside morphology of the cells was only marginally altered. The described changes in sulfhydryl status of the red blood cells are likely to play a major role in the premature splenic sequestration of hydroxylamine-damaged erythrocytes.