Altered phagocytosis by monocytes from HLA-DR2 and DR3-positive healthy adults is Fc gamma receptor specific

Fc gamma receptor-dependent mononuclear phagocyte system (MPS) clearance of opsonized erythrocytes is prolonged in healthy adults with the class II alloantigens HLA-DR2, DR3, or DQw1, despite normal receptor-specific Fc ligand binding by monocytes in these groups. To investigate the basis for the MPS dysfunction, we determined the phagocytic capacity of blood monocytes from 66 disease-free adults and analyzed the data according to the HLA type of the subjects. The data demonstrate decreased phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes from HLA-DR2, DR3, or DQw1-positive subjects compared with normals without these B cell alloantigens (2.87 +/- 0.83 erythrocytes/monocyte vs 3.87 +/- 1.05, p less than 0.004; 3.01 +/- 0.94 vs 3.87 +/- 1.05, p less than 0.02; 3.18 +/- 0.89 vs 3.87 +/- 1.05, p less than 0.02, respectively). Because HLA-DR2 and DQw1 are in linkage disequilibrium, we compared EA phagocytosis in subjects with DQw1 alone to normals without HLA-DR2, DR3, or DQw1. Among subjects positive only for DQw1, no significant decrease in phagocytosis could be seen (3.46 +/- 0.95 vs 3.87 +/- 1.05, p = NS). To determine whether these differences represented an Fc receptor-specific dysfunction or a more generalized decrease in phagocytic activity, we compared the quantitative phagocytosis of EA with that of neuraminidase-treated erythrocytes (EN), which is Fc receptor independent and beta-glucan receptor mediated. No segregation of phagocytic capacity for EN by HLA class II phenotypes could be demonstrated (DR2, 2.68 +/- 1.30 erythrocytes/monocyte; DR3, 2.95 +/- 1.30; DQw1, 2.84 +/- 1.15; others, 3.06 +/- 1.14). Our data indicate that the decrease in phagocytosis by blood monocytes from normal individuals with HLA-DR2 or DR3, the class II alloantigens associated with systemic lupus erythematosus and other autoimmune diseases, is specific for the Fc receptor-mediated process. This alteration of Fc receptor function among immunogenetically defined individuals may contribute to their predisposition to autoimmune disease. These differences may also apply to other Fc receptor-initiated cellular functions.     

Phagocytosis of concanavalin A-treated erythrocytes is mediated by the Fc gamma receptor

Impaired Fc gamma receptor-mediated phagocytosis has been reported in monocytes from HLA-DR2- and -DR3-positive disease-free individuals compared to normals without these B cell alloantigens. We have noted, however, a decrease in the ingestion of concanavalin A (Con A)-treated rabbit erythrocytes (E-Con A) in the same immunogenetically defined groups (DR2 vs Other: 2.94 +/- 0.84 erythrocytes/monocyte vs 4.16 +/- 1.37, p less than 0.003; DR3 vs Other: 3.35 +/- 1.51 vs 4.16 +/- 1.37, p less than 0.04). These data raised the possibility that carbohydrate-lectin interactions might trigger ingestion mediated by the Fc gamma receptor. To test this hypothesis, we performed receptor modulation and monosaccharide blocking experiments. Modulation of the Fc gamma receptor off the apical cell surface of monocytes by adherence to solid-phase IgG aggregates specifically reduced internalization of E-Con A and IgG-sensitized erythrocytes (EA) to 9.1% and 10.6% of control, respectively (p less than 0.001). Internalization of wheat germ agglutinin-treated erythrocytes, tannic acid-treated erythrocytes, and zymosan was not inhibited. In reciprocal modulation experiments using solid-phase Con A, no effects on phagocytosis of any particle was observed. alpha-Methyl mannoside, 0.1 M in PBS, did not inhibit the internalization of EA but blocked ingestion of E-Con A by 97% (p less than 0.001). Other monosaccharides had little or no effect on the ingestion of any of the phagocytic probes. These data demonstrate that a mechanism integrally involving the Fc gamma receptor mediates the ingestion of E-Con A by human monocytes. This Fc receptor has an oligosaccharide(s) with an exposed mannose which may be functionally significant. Whereas the mannose moiety does not play a crucial role in the interaction of the Fc gamma receptor with the Fc portion of IgG, engagement of the receptor via mannose can initiate internalization. Our findings raise the possibility that nonimmune functions may utilize classical immune system receptors through carbohydrate interactions. Furthermore, the ability of the Fc gamma receptor to trigger internalization is defective in HLA-DR2 and -DR3 normals, whether the receptor is ligated at its classical ligand-binding site or by way of its carbohydrate moieties.     

Plasmodium falciparum: an invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen

A Plasmodium falciparum malaria blood stage antigen was detected using a human monoclonal antibody (MAb A52A6) obtained from a clinically immune donor. Immunofluorescence analysis showed that the MAb reacted with the intracellular parasite throughout the asexual blood stage cycle as well as with gametocytes. The MAb also reacted with the surface of erythrocytes containing late stage P. falciparum parasites. The antigen seen by the MAb was species- but not strain- or isolate-specific. At rupture of the infected erythrocytes, antigenic material was deposited on the membrane of uninfected cells surrounding the parasite. At merozoite invasion MAb reactive material was present on the invaginating erythrocyte membrane, indicating an involvement of the antigen in the invasion process. This was also indicated by the high capacity of the MAb to inhibit merozoite invasion in vitro. The antigen appears to be a phosphoglycolipid, sensitive to phospholipase and present in lipid extracts of P. falciparum-infected erythrocytes.     

Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors

Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytes by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis.     

Monokines released during short-term Fc gamma receptor phagocytosis up-regulate polymorphonuclear leukocytes and monocyte-phagocytic function.

Freshly explanted monocytes phagocytosing IgG antibody-coated erythrocyte targets (EIgG) release a factor(s) that stimulates phagocytosis by neighboring monocytes and polymorphonuclear leukocytes (PMN). Culture supernatants obtained after 30-min incubation of adherent monocytes with EIgG, but not unopsonized sheep erythrocytes, markedly up-regulated the extent of PMN phagocytosis and enhanced the rate at which monocytes ingested EIgG. The presence of this factor(s) was first evident in phagocytic studies in which monocytes were prepared by a colloidal silica-based continuous gradient technique (Sepracell-Mn). After introduction of erythrocyte targets, there was a 20- to 30-min delay before initiation of phagocytosis that was not observed with monocytes prepared by the standard Percoll-gradient technique. Experiments suggest that, when compared with monocytes prepared by the Percoll-gradient method, Sepracell-Mn monocytes are closer to a base line state of activation with regard to the expression of Fc gamma RI and the ability to ingest EIgG. The mechanism of PMN upregulation by the monocyte factor(s) was explored. Monocyte supernatants did not induce an increase in the surface expression of PMN Fc gamma RI, II, or III. Neither anti-TNF, anti-IL-2, nor anti-GM-CSF had any significant effect on monocyte supernatant activity. Neutrophil activating protein-1 was not detected by ELISA. In contrast, anti-IL-1 completely blocked the effect of the supernatant on subsequent monocyte phagocytosis, and partially inhibited its effect on PMN phagocytosis. Furthermore, it was shown that RIL-1 as well as TNF markedly enhanced monocyte and PMN ingestion of EIgG. These results suggest that monocytes, after Fc gamma R-mediated phagocytosis, release monokines, including at least IL-1, which enhance the phagocytic function of neighboring PMN and monocytes to augment the host defense process.     

Defective mononuclear phagocyte function in systemic lupus erythematosus: dissociation of Fc receptor-ligand binding and internalization

Fc receptor-mediated mononuclear phagocyte system (MPS) clearance is impaired in systemic lupus erythematosus (SLE) and may contribute to the pathogenesis of the immune complex disease. To investigate the basis of MPS dysfunction, we have examined concurrent in vivo and in vitro Fc receptor function in 22 patients with SLE and 23 disease-free adults. Blood monocyte Fc receptor binding was increased rather than decreased as predicted by the saturation hypothesis of MPS blockade. Rosette formation of IgG-sensitized bovine erythrocytes (EA) with monocytes demonstrated increased Fc receptor-ligand binding in SLE (percent rosettes: 40 +/- 12 vs 27 +/- 8, p less than 0.001). Scatchard analysis of the binding of radiolabeled IgG oligomers to SLE monocytes indicated a mean receptor number 30% higher than control, although this did not reach statistical significance. Despite enhanced Fc receptor-ligand (EA) binding, Fc-mediated phagocytosis of EA was decreased in SLE (1.7 +/- 0.7 erythrocytes/monocytes/hour vs 2.6 +/- 1.0, p less than 0.004). This decrease in phagocytosis by blood monocytes from SLE patients was significantly greater than that attributable to the predominance in SLE of individuals with certain HLA B cell alloantigens and intrinsically lower phagocytic rates (p less than 0.05 for all groups). This decrease therefore represents a disease-acquired characteristic. Furthermore, the phagocytic rate of the four SLE patients with marked prolongation in MPS clearance was significantly lower than that of the eight patients with near normal clearance values (p less than 0.01). Saturation of Fc receptors by immune complexes does not explain impaired immune clearance in SLE. Our results indicate that despite increased binding of the EA ligand, Fc receptor-mediated phagocytosis is markedly impaired in SLE monocytes. This impairment cannot be explained on the basis of HLA-related differences in phagocytosis among lupus patients. The defect in phagocytosis of EA is most profound in those patients with the most significantly impaired MPS clearance. Thus, the dissociation of receptor-ligand binding and receptor-mediated internalization may contribute significantly to the in vivo clearance defect in SLE.     

Plasmodium falciparum: association with erythrocytic superoxide dismutase

Levels of superoxide dismutase (SOD) activity and its properties in Plasmodium falciparum-infected erythrocytes, isolated parasites, and noninfected erythrocytes were studied. A higher specific activity was found in P. falciparum-infected erythrocytes compared to noninfected erythrocytes, resulting from the lower protein content of infected cells and not enzyme synthesis by the parasite, as the superoxide dismutase activity expressed per number of cells was decreased. Superoxide dismutase from noninfected erythrocytes and isolated P. falciparum parasites showed similar sensitivities to various inhibitors and had identical molecular weights and electrophoretic mobilities. These results support the hypothesis of uptake and use of the erythrocytic SOD enzyme by the parasite as a possible mechanism of defense against oxidative stress.     

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.     

Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously been shown to recognise Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This protein is inserted by the parasite into the host cell membrane and mediates the adhesion to the venular endothelium of the host organism in vivo. METHODS: Erythrocytes infected at high parasitaemias with ethidium-bromide-labelled mature forms of P. falciparum parasites were sequentially exposed to immune plasma, goat anti-human immunoglobulin (Ig) G, and fluorescein-isothiocyanate-conjugated rabbit anti-goat Ig. Plasma antibodies recognising antigens exposed on the surface of parasitised erythrocytes were subsequently detected by two-colour flow cytometry. RESULTS: Binding of human antibodies to the surface of erythrocytes infected with adhesive strains of Plasmodium falciparum can be measured by the two-colour flow cytometry (FCM) assay described. In addition, we demonstrate that the adhesive capacity of a parasite isolate correlates with the capacity of human immune plasmas to label the isolate as detected by FCM. We also show that the antigens recognised by the labelling antibodies are strain specific and that their molecular weights are in the range previously described for PfEMP1 antigens. CONCLUSIONS: Our FCM assay predominantly detects antibodies that recognise PfEMP1 and thus constitutes a convenient assay for the analysis of acquisition, maintenance, and diversity of anti-PfEMP1-specific antibodies and for the examination of class and subclass characteristics.     

Defective Fc-mediated phagocytosis in C3H/HeJ macrophages. II. Correction by cAMP agonists

Peritoneal macrophages from LPS hyporesponsive C3H/HeJ mice lose the capacity to bind and phagocytose opsonized sheep erythrocytes (EA) over a 48-hr culture period. This loss in Fc receptor capacity is markedly different from the progressive increase in phagocytic ability exhibited by cultured macrophages derived from LPS-responsive C3H/HeN mice. Since dibutyryl-cyclic adenosine monophosphate (DBcAMP) has previously been reported to modulate membrane receptor expression in lymphocytes and certain macrophage-like cell lines, we examined its effects on EA binding and phagocytosis by C3H/HeJ macrophages. DBcAMP not only reverses the binding defect in C3H/HeJ macrophages but also restores EA phagocytosis to the level of control C3H/HeN cultures. 8-Bromo-cAMP, as well as other agents known to elevate intracellular cAMP (i.e., isoproterenol plus isobutylmethylxanthine or prostaglandin E2) also corrected the phagocytic defect. Since the C3H/HeJ macrophage phagocytic defect can also be reversed by in vitro stimulation with a lymphokine-rich culture supernatant, we examined the effect of this treatment on intracellular cAMP levels. Lymphokine treatment produced a 60% increase in the levels of macrophage intracellular cAMP. These findings suggest that the C3H/HeJ differentiation defect may be secondary to some abnormality in a cAMP dependent pathway.     

The role of T lymphocytes in immunity to Plasmodium falciparum. Enhancement of neutrophil-mediated parasite killing by lymphotoxin and IFN-gamma: comparisons with tumor necrosis factor effects

Human neutrophils were treated with IFN-gamma or lymphotoxin and their interaction with the asexual blood forms of Plasmodium falciparum was studied by a radiometric assay and microscopy. The results showed that human neutrophils inhibited the growth of P. falciparum and killed the parasite via a phagocytic mechanism. The cytokines significantly augmented the neutrophil-mediated killing of the parasite. When compared with the effects of TNF-alpha similar results were seen although IFN-gamma and TNF-alpha were effective at 10-fold less the concentration of lymphotoxin. The maximum neutrophil-mediated parasite killing was seen in the presence of immune serum and the cytokines. These findings suggest that T cells regulate the antimalarial activity of neutrophils.     

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation.     

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.     

Parasite adhesion and immune evasion in placental malaria.

Parasite sequestration in the placenta is a key feature of infection by Plasmodium falciparum during pregnancy and is associated with severe adverse outcomes for both mother and baby. Here, James Beeson and colleagues draw together the findings of recent studies on parasite mechanisms that mediate this process. They review evidence for novel parasite variants that appear able to evade pre-existing immunity, for the adhesion of P. falciparum-infected erythrocytes to placental glycosaminoglycans (and the molecular basis of these parasite properties) and for the expression of var genes encoding the variant antigen and adhesive ligand P. falciparum-erythrocyte membrane protein 1 (PfEMP1).     

Staphylococcal lipase affects phagocytosis of Staphylococcus aureus by human granulocytes and monocytes.

The rate of immunological and non-immunological phagocytosis of staphylococci by lipase pre-treated human granulocytes and monocytes was compared. It was found that the effect of this enzyme on two types of cells is opposite. Lipase decreases phagocytosis by granulocytes and increases by monocytes. The revealed differences between phagocytosing cells studied prompted us to investigate the influence of lipase on Fc receptors on these cells (rosette EA test). The different susceptibility of Fc receptors on non-activated phagocytes to lipase was found. This could be at least partially responsible for the difference observed between phagocytic activity of granulocytes (decreased) and monocytes (increased) pretreated with staphylococcal lipase. Inactivated enzyme showed a similar effect as active enzyme in the case of granulocytes. However, inactivated enzyme had no effect on rosette formation by lipase pretreated monocytes, indicating an enzymatic effect.     

Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.     

Plasmodium falciparum signal sequences: simply sequences or special signals?

The malaria parasite, Plasmodium falciparum, synthesises and exports several proteins inducing morphological and biochemical modifications of erythrocytes during the erythrocytic cycle. The protein trafficking machinery of the parasite is similar to that of other eukaryotic cells in several ways. However, some unusual features are also observed. The secretion of various polypeptides was inhibited when P. falciparum-infected erythrocytes were incubated with Brefeldin A. Immunoelectron microscopy studies revealed substantial morphological changes in the endoplasmic reticulum following exposure of parasitised erythrocytes to the drug. Immunofluorescence studies of Brefeldin A-treated parasites suggest that polypeptide sorting to different intracellular destinations begins at the endoplasmic reticulum. The parasite also secretes polypeptides by a Brefeldin A-insensitive route that bypasses the classical endoplasmic reticulum-Golgi complex pathway.     

Plasmodium Falciparum: The Adherence of Erythrocytes Infected with Human Malaria Can Be Mimicked Using Pfalhesin-Coated Microspheres

Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542-555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by irnmunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells tTansfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546-553 and 820-829 and called pfalhesin) were produced. The pfalhesin-coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546-553 had little binding capacity for melanoma cells.

These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel 'artificial' P. falciparum-infected erythrocytes for use in in vitro and in vivo studies.     

Plasmodium Falciparum: The Adherence of Erythrocytes Infected with Human Malaria Can Be Mimicked Using Pfalhesin-Coated Microspheres

Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542–555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by irnmunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells tTansfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546–553 and 820–829 and called pfalhesin) were produced. The pfalhesin-coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546–553 had little binding capacity for melanoma cells.

These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel ‘artificial’ P. falciparum-infected erythrocytes for use in in vitro and in vivo studies.