Membrane proteins involved in the adherence of Plasmodium falciparum-infected erythrocytes to the endothelium

Biol Cell (1992) 74, 161-178

161

© Elsevier, Paris

Review

Membrane proteins involved in the adherence of Plasmodium falciparum-infected erythrocytes to the endothelium Irwin W Sherman, Ian Crandall, Heidi Smith Department of Biology, University of California Riverside, CA 92521, USA (Received 25 November 1991; accepted 7 January 1992)

Plasmodiumfalciparum (human malaria) infections are characterized by the attachment of erythrocytes infected with mature stage parasites to endothelial ceils lining the post-capillary venules, a phenomenon known as sequestration. In the human body, the microvessels of the heart, lungs, kidneys, small intestine, and liver are the principal sites of sequestration. Sequestered cells that clog the brain capillaries may reduce blood flow sufficiently so that there is confusion, lethargy, and unarousable comacerebral malaria. This review considers what is known about the molecular characteristics of the surface proteins, that is, the red cell receptors and the endothelial cell ligands, involved in sequestration. Recent work from our laboratory on the characterization of the adhesive proteins on the surface of the Pfalciparum-infected red cell, and the ligands to which they bind on human brain endothelial cells is also discussed. Finally, consideration is given to the multifactor processes involved in sequestration and cerebral malaria, as well as the possible role of 'anti-adhesion therapy' in the management of severe malaria. Summary -

Plasmodium faleiparum I malaria / sequestration I endothelial cell / cerebral malaria

Introduction

Worldwide, it has been estimated, there are more than 250 million humans infected with malaria. The most malignant of the four human malarias, Plasmodiumfalciparum, in Africa alone contributes to 1 - 2 million deaths annually (Bull W H O 68,667-673, 1990). In more graphic terms, each minute of every day, three people die from malaria. Severe malaria is characterized by the blood containing a high proportion of infected erythrocytes, as well as pathophysiologic changes in the renal, pulmonary and neurologic systems [90, 119]. The hallmark of P falciparum infections is sequestration, that is, the attachment of erythrocytes infected with mature stage parasites to endothelial cells (ECs) lining the post-capillary venules. In humans, the principal organs in which sequestration takes place are the heart, lung, kidney and liver [5, 120]. Sequestration in the brain microvessels - a special pathology of Pfalciparum infections called cerebral malaria - may totally occlude blood flow, and result in confusion, lethargy and deep coma [55, 89, 158]. Patients may regain consciousness within 1 - 3 days with effective antimalarial therapy, however, about one-third of the patients die in the comatose state. It should be emphasized that only a minority of infections, perhaps less than 1°70, progress to cerebral malaria. What is the biological significance of sequestration ? It has been suggested that by sequestration, the red cells containing the more mature stages of Pfalciparum do not pass through the spleen and, in this way, the parasite is able to evade destruction by the spleen filtering mechanisms [11, 52, 53]; as a consequence, the 'spleen-protected' schizont is able to release its daughter merozoites and these invade other red cells to initiate a new erythrocytic cycle of development. In addition, since sequestration occurs in the microvasculture where oxygen tensions are low and

carbon dioxide levels high, environmental conditions beneficial to the in vitro growth of P falciparum [127], the withdrawal of trophozoite-infected cells from the peripheral circulation may also favor parasite growth and reproduction in vivo. In addition, sequestration also occurs in the rodent malaria P chabaudi [25]. However, sequestration is not a universal requirement for the successful asexual reproduction of Plasmodium: there are malarias (eg, P malariae and P brasilianum) morphologically similar to P falciparum [3] in which all stages are found in the peripheral circulation and trophozoite- and schizontinfected red cells can and do survive passage through the spleen. And, in the owl monkey, Aotus trivirgatus [100] or the squirrel monkey, Saimiri sciureus [36], sequestration of P falciparum-infected red cells occurs chiefly in the spleen (and the liver and heart), not in the brain. Howard [52], Barnwell [11], Hommel [49] and Sharma [131] have previously reviewed the state of our knowledge concerning cytoadherence. Here we will survey what is known about the molecular characteristics of the surface proteins, that is, the red cell receptors and the EC ligands, involved in sequestration. We will summarize recent work from our laboratory investigating the environmental factors that contribute to cytoadherence, as well as a characterization of the adhesive proteins on the surface of the Pfalciparum-infected red cell. Finally, we will discuss the possible role for 'anti-adhesion therapy' in the management of severe malaria. T h e Plasmodium falciparum-infected red cell

Surface morphology Within the red cell, Pfalciparum undergoes a 48-h cycle of asexual reproduction. The first 18-24 h are spent first

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162

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X,-~. ;'dZ ,:.. '~~ ~,~~5 4 ~.;9

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D

Fig 1. The knobby surface of the Pfalciparum-infected erythrocyte. A. Scanning electron microscope image• B. Transmission electron microscope image. C. Freeze fracture of the knobs (K). Note the clustering of intramembranous particles in the knob region• D. High magnification of the surface of a knobby red cell ( × 28000) showing electron dense plaque below membrane elevation.

as a ring form, and later as a young feeding stage, the early trophozoite. The last 24 h involve further growth and differentiation of the trophozoite to give a multinucleate plasmodlum, the schizont. The products of schizont division, merozoites, are the invasive forms. Merozoites recognize specific receptors on the surface of the red cell, and by triggering invagination of the red cell membrane, come to lie within a space, the parasitophorous vacuole [102, 116, 162]. The membrane lining the vacuole persists throughout the maturation of the intracellular parasite and, though initially derived from the erythrocyte, this membrane is enlarged and modified during parasite growth [10, 132, 133]. By merozoite dedifferentiation, the ringstage is formed. Several changes occur, both within and on the surface of the host red cell, and both kinds of alterations are related to the intracellular development of P falciparum. Membrane-bound clefts, called Maurer's clefts, occur in the cytoplasm of the erythrocyte [3]. These clefts, which connect to the membrane of the parasitophorous vacuole, appear to increase in number as the parasite matures [88]. A second, and more noticeable change, occurs on the surface of the infected cell. Protrusions of the erythrocyte membrane, called knobs (fig 1A), begin to appear approx-

imately 24 h post-invasion [76, 148]. As much as 5°/o of the surface of an infected red cell may be associated with knobs [41]. And, development of knobs is correlated precisely with the sequestrating stages (ie red cells bearing ringstage parasites lack knobs and do not sequester, whereas trophozoite- and schizont-containing red cells have knobs and sequester). By transmission electron microscopy, knobs are conoid in shape in cross-section (fig IB, D); directly apposed to the cytoplasmic surface of the elevated plasma membrane is an a m o r p h o u s electron dense material shaped in the form of a cup [3]. By scanning electron microscopy, knobs were shown to vary, both in size and density; in trophozoite-containing cells, knobs were sparse and their diameter ranged from 110-160 nm, whereas in erythrocytes bearing schizonts, the knobs were more numerous and smaller in diameter, ranging from 7 0 - 1 1 0 nm [41]. The base-to-peak height of a knob was approximately 40 nm or less. Knob formation appears to be a dynamic process, with increased production during the later stages of parasite growth. Since knobs are not randomly distributed on the red cell surface, but display discrete patterns (such as rows, circles, etc), it appears that specific membrane domains are involved in their formation and

Malaria sequestration that cytoskeletal elements may play a role in their arrangement. Isolates of Pfalciparum, when maintained continuously in culture, sometimes lose their capacity to form knobs [76] ; however, the knobless (K - ) phenotype may not occur if, at regular intervals, laboratory selection for knobbearing cells is carried out using gelatin flotation [134]. Knoblessness is not an artifact of in vitro cultivation. The K - phenotype has been observed in fresh field isolates [19], however, the major phenotype in vivo is K + , suggesting'that the spleen exerts extreme selective pressure for maintenance of this phenotype. Knoblessness, an irreversible condition, is due to the spontaneous loss of a subtelomeric portion of chromosome 2 [70] that results in partial or complete deletion of the gene encoding the knob associated histidine rich protein (KAHRP). K - lines do not sequester and are usually non-cytoadherent, however, by in vitro selection, it has been possible to obtain knobless cytoadherent ( K - C +) lines from the Uganda-Palo Alto [153], Ituxi [17], and Malayan Camp [109] isolates. The spleen has been shown to modulate sequestration [13]. Knobby, cytoadherent (K + C +) red cells introduced into splenectomized squirrel monkeys (Saimiri sciureus) did not sequester and became non-cytoadherent after eight days, whereas in animals with an intact spleen, trophozoite- and shizont-infected cells sequestered and remained cytoadherent [30] ; reversal of the non-adherent phenotype to an adherent one occurred 20 days postinfection in animals with an intact spleen. In these studies, both the non-sequestering and sequestering forms retained the K + phenotype. The loss of sequestration after splenectomy has also been described in humans [61]. The specific role of the spleen in the maintenance of cytoadherence and sequestration has not been identified, but selection by the spleen against non-adhesive infected red cells is most probable responsible.

Surface antigens Erythrocytes infected with Pfalciparum express new and extremely diverse antigens on their surface [35, 48, 50, 51, 94, 96, 141]. Gambian children naturally infected with P falciparum developed isolate-specific antibodies during convalescence, whereas sera from adults (who presumably were immune to malaria) reacted with several isolates [94, 155]. A correlation was found between agglutination of infected red cells and surface fluorescence titers, and between surface fluorescence and immunoelectron microscopic-localized antigen. Surface iodination of infected cells identified a trypsin-sensitive, high molecular weight antigen that could be immunoprecipitated by the homologous convalescent sera as well as with adult sera. Inhibition of cytoadherence was observed in some cases with convalescent, but not with acute homologous sera. Whether these antigens were of host or parasite origin was not determined in these studies. However, it was clearly shown that the knobs were antigenically distinct from the rest of the surface of the infected red cell [155]. Indeed, in an earlier study [75] when antisera were obtained from owl monkeys (/lotus trivirgatus) resistant to reinfection with two different strains antibody bound only to the knobby surface of red cells infected with that particular strain whether of human or monkey origin. Thus, parasiteinduced surface alterations are antigenically similar despite differences in host cells, and such antigenic changes occur whether the parasites are derived from animals or in vitro cultures.

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Parasite-encoded antigens At least six malarial antigens have been identified on the surface of, or in association with, the cytoskeleton of erythrocytes infected with P falciparum [52, 53, 59]: histidine-rich proteins 1 and 2 (HRP 1 = KAHRP), erythrocyte membrane proteins 1 and 2 (EMP 1 and EMP 2, MESA or mature erythrocyte surface antigen), protein 11.1, and RESA (ring-infected erythrocyte surface antigen) also called P f 155. HRP 1, a 90-kDa water-insoluble protein, is essential for the formation of knobs. Immunoelectronmicroscopy, however, suggests that the bulk of HRP 1 is in the red cell cytoplasm, and only a minor amount is associated with the knob [4, 58, 147]. HRP 2, a water soluble 70-kDa protein, occurs within the erythrocyte cytoplasm; it is associated with Maurer's clefts as well as the red cell membrane, and is secreted from infected cells [4, 58, 115]. Using mouse monoclonal antibodies (mAbs), a strong positive staining of both HRP 1 and HRP 2 was found along the cerebral microvessels packed with parasitized cells [5]. The mechanism of deposition is not known. HRP 2 is found in knobless, non-cytoadherent ( K - C - ) lines and, therefore, is unlikely to be directly involved in adherence [118]. Since all K + strains express HRP 1, whether cytoadherent or not, and because it is not expressed on the surface of the falciparum-infected red cell [147], HRP 1 is probably not directly involved in sequestration. EMP 1 and EMP 2 are not histidine-rich proteins. EMP 2, a phosphoprotein synthesized by mature stages of the parasite, is polymorphic in size, varying from 250-300 kDa in different isolates. It has been immunolocalized to the parasitophorous vacuolar membrane, Maurer's clefts, and the inner face of the plasma membrane in the region of the knob [4]. EMP 2 is associated wffh the 80-kDa erythrocyte membrane skeletal phosphoprotein band 4.1 [84]. EMP 2 is neither necessary for knob formation nor cytoadherence [57, 59]. EMP 1 is an antigenically diverse > 240-kDa surface protein correlated with the cytoadherence phenotype [56, 59]. However, to date, immunoelectronmicroscopy has failed to demonstrate its presence on the surface of the knob [4]. Protein 11.1 is a 1000-kDa surface iodinatable, Triton X-100-insoluble protein; antibodies to this protein cross-react with RESA, and another 260-kDa protein [117]. The presence of protein 11.1 is not correlated with cytoadherence. RESA is localized in small dense granules of merozoites. At, or after, merozoite entry, the granule contents are released and subsequently RESA is found in the parasitophorous vacuolar space surrounding the newly invaded parasite [28]. Later, it comes to be associated with the membrane of the infected red cell by an undefined mechanism. In mature parasites, RESA is Triton X-100-soluble, but once associated with the red cell membrane it becomes Triton-insoluble. RESA is a spectrinbinding protein that forms a ternary complex with actin, spectrin and band 4.1 [34, 124]. Since sequences in the Cterminal repeat of RESA are homologous with parts of the N-terminal of band 3, it is not entirely unexpected that a mAb against mouse band 3 reacted with RESA [47]. It is possible that RESA interacts and interferes with the function of band 3. However, the RESA-specific human mAb 33G2, which recognizes a family of cross-reacting P falciparum antigens including a protein of molecular mass similar to EMP 1 [1] and which inhibits cytoadherence [153], did not bind to band 3 [47]. It is unlikely that RESA is a receptor for adhesion.

164

lW Sherman et al MEMBRANOUS DOMAIN = 55 kDa

I

CH 35

CH 17

I

5

6

" "1

CYTOPLASMIC DOMAIN =43kOa

l

600

amino acid TR 41

(~

l" """"

COOH

INSIDE ( ~ = chymotrypsins~le NH2

= tr/psin sile

Fig 2. The major domains of the erythrocyte membrane protein, band 3 (diagrammatic).

Modified red cell antigens: altered f o r m s o f band 3 The erythrocyte membrane, when studied by freeze fracture electron microscopy, reveals arrays of intramembranous particles (IMPs) in both membrane leaflets. IMPs, composed primarily of band 3 protein, are more numerous on the protoplasmic (P face) leaflet. Examination of Pfalciparum-infected red cells by freeze fracture electron microscopy demonstrated a change in distribution (fig 1C), but not in density, of P face IMPs during knob development [9]. Prior to membrane protrusion, the IMPs of the knobby areas were clustered in discrete foci. With elevation of the plasma membrane, the central IMP cluster was surrounded by a series of IMP rings [6, 9], and with maximal elevation of the membrane, there was dispersal of the central4MPs forming the cluster as well as the rings. Such studies suggest that reorganization of band 3, in addition to parasite-encoded proteins such as H R P 1 and EMP 1, plays a role in knob formation. (Knobless cytoadherent lines have not, as yet, been studied by freeze fracture electron microscopy.) Band 3, an " 95-kDa glycoprotein, is the most abundant membrane protein of the red cell. In a single red cell, there are a minion copies of band 3, and these are presumed to exist as monomers or are self-associated as dimers and tetramers [81, 125]. Band 3 is a chimeric molecule, consisting of two dissimilar and functionally distinct domains (fig 2). The N-terminal, a 43-kDa water soluble cytoplasmic domain, has multiple binding sites for hemoglobin, hemichrome, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphofructokinase, as well as several membrane-associated proteins such as ankyrin (band 2.1) and band 4.1, both of which modulate the interaction of band 3 with the cytoskeleton and band 4.2 [81]. The C-terminal, the hydrophobic transmembrane domain of band 3 with an M r of 55-kDa, functions in an ion exchange. Based on the complete amino acid sequence [86,

146], it has been proposed that band 3 protein crosses the membrane 14 times. Kay et al [68] reported that aged red cells show autologous binding of immunoglobulin, and that these were antiband 3 IgG autoantibodies. Kay [67] suggested that the neo-antigen to which the antibody binds was a 62-kDa fragment of band 3. Using synthetic peptides, Kay et al [69] identified the antigenic sites on band 3 to be the extracellular amino acid residues 538-554 and 812-827. The carbohydrate moieties of band 3 appeared not to be required for antigenicity, since the peptides on their own blocked IgG binding. The naturally occurring anti-band 3 antibodies stimulate alternative pathway complement C3b deposition, presumably due to a high affinity for the Fc region [85]. Deposition of anti-band 3 IgG also occurs when red cells are treated with oxidizing agents (eg, A D P / F e 3+ or X / X O / F e 3+ or diamide) or when exposed to phenylhydrazine or acridine orange [15, 82], agents that cluster band 3. According to Waugh et al [159], this clustering of band 3 (with concomitant binding of IgG) occurs naturally in a variety of red cell disorders (eg, ctthalassemia, Koln hemoglobin, and sickle cell anemia) where Heinz bodies and other hemichrome aggregates are formed. Since clustering is restricted to the regions where the hemichrome deposits or Heinz bodies are attached to the underside of the membrane, it appears that copolymerization of band 3 with hemichrome forces the clustering of band 3 in the plane of the membrane. (But, see Biochim Biophys Acta 945, 105-110 (1988) and Acta Histochem Suppl Band XXXIX, $423-434 (1990), for evidence against colocalization of Heinz bodies and band 3 clustering.) Winograd, Greenan, and Sherman [162] found an increased level of binding of autologous IgG to K + erythrocytes. IgG binding was 30 times greater than it was for uninfected red cells. Binding was correlated with parasite development, and by immunoelectronmicroscopy, the mem-

Malaria sequestration brane bound IgG was localized to the region of the knob. Purified anti-band 3 antibody specifically bound to K + cells. And, Kannan, Labotka and Low [64] and Kannan, Yuan and Low [65] reported that in sickle cells and thalassemic cells autologous IgG was bound specifically to the region of hemichrome-induced membrane reorganization ; the surface-exposed IgG, by promoting phagocytosis, could contribute to the shortened life span of such cells. Thalassemic cells infected with Pfalciparum also bind greater amounts of IgG than do normal or uninfected thalassemic cells, and an exponential increase in antibody binding was associated with parasite development [87]. In this study, the antigen(s) responsible for the greater binding of antibody were not identified, however, based on the aforementioned observations, it seems likely that band 3 protein was involved. We produced several mouse mAbs against the surface of the live Pfalciparum-infected red cell [26, 165]. One of these, mAb 4A3, immunoprecipitated an 85-kDa protein from infected red cells that had been previously surface-iodinated and extracted with SDS. This antigen was not found in Triton X-100 extracts of surfaceiodinated infected red cells, nor uninfected red cells. The 85-kDa antigen could not be metabolically labelled with radioactive amino acids, suggesting that it was not a parasite-encoded protein. Furthermore, the observation that mAb 4A3 recognized band 3 in Triton X-100 extracts made from uninfected erythrocytes, and the fact that the 85-kDa antigen bound to concanavalin A (Winograd', unpublished) indicated the possibility that this antigen might be structurally related to band 3. To explore this, twodimensional peptide maps of the two proteins were prepared. Comparisons of these iodopeptide maps revealed a striking similarity between the two proteins. However, despite the 85-kDa protein having approximately 40% fewer peptide spots than band 3 protein, all of the spots in the 85-kDa map had a corresponding spot in the band 3 map. This result suggested that either the 85-kDa antigen was derived from band 3, or that its amino acid sequence was very similar to that of band 3. To gain a better understanding of the structural differences between the 85-kDa antigen and band 3, each of the major band 3 fragments (fig 2) - cytoplasmic (TR-41),and the two membraneassociated domains (CH-17 and CH-35) - were isolated and two-dimensional peptide maps of each were prepared. These maps were used to determine the correspondence of each peptide spot in the band 3 map to band 3 fragments. With few exceptions, all of the peptide spots could be related to one of the band 3 fragments. However, a comparison of the peptide map of the 85-kDa antigen with the band 3 map showed that most or all of the TR-41 cytoplasmic domain was absent from the 85-kDa antigen ; two spots that corresponded to the CH-35 fragment were also missing. Therefore, it appeared that a small deletion at the carboxy-terminal end of band 3, in addition to the larger deletion of the cytoplasmic domain, had occurred. mAb 4A3 which recognizes a newly exposed band 3-related antigen on the surface of the infected red cell, blocked cytoadherence [165]. Another mAb, 1C4, recognizes a band 3-related 65-kDa antigen in P falciparum-infected erythrocytes [26]. By immunofluorescence, the antigen appears as dots on the surface of trophozoite- and schizont-infected cells. Ringinfected or- uninfected red cells were unreactive with 1C4. The 65-kDa antigen was insoluble in Triton X-100 but was soluble in SDS, and was present in several strains, including K - lines. By probing immunoblots of malaria-

165

infected red cells with mAbs (IVF12, IIE1) directed against two domains of band 3 [62], the 65-kDa antigen appeared to be derived from the amino-terminal of band 3, that is, from the amino terminus to approximately amino acid 600 (fig 2). This was confirmed by using a polyclonal antibody against the cytoplasmic domain of human band 3 [81] (ie, on Western blots, this antiserum recognized the 65-kDa antigen). Neither chymotrypsin nor trypsin treatment of intact parasitized red cells removed the 65-kDa antigen. This antigen, as well as the 85-kDa antigen, are parasite-induced, truncated, and covalently modified band 3 molecules. The mAb 1C4 blocked cytoadherence in a dose-dependent fashion. Red cells infected with mature forms of a K + line bound significantly greater amounts of IgG from normal human sera than did uninfected or ring-infected red cells, and naturally-occurring anti-band 3 auto-antibodies bound to the surface of these K + cells [163]. To determine the relationship of the membrane-bound immunoglobulins to antiband 3 antibody infected red cells were surface-iodinated, sequentially extracted with Triton X-100 and SDS, and immunoprecipitated. As with naturally occurring antiband 3 antibody, the endogenous immunoglobulins bound to the surface membranes of infected red cells recognized a > 240-kDa antigen in the Triton X-100 insoluble extract. This antigen was found only in K + , not K - lines, and was removed by trypsin treatment. Two-dimensional peptide maps of the > 240 kDa antigen were near identical to those of the 85-kDa antigen. It was suggested that the > 240 kDa antigen could arise by the molecular association of several modified forms of band 3 [164]. Thus, in Pfalcipan~m parasite-induced modifications in band 3 result in neo-antigen expression. In addition, the altered and clustered forms of band 3 of the Pfalciparuminfected cell through their affinity for IgG and C3b [85] could elicit macrophage recognition, and result in the selective removal of infected cells.

Sequestration In vitro models using endothelial and amelanotic melanoma cells In 1981, Udeinya et al [152] reported the development of an in vitro model for sequestration. Using in vitro cultured human umbilical vein endothelial cells (HUVECs) overlain by a suspension of Pfalciparum-infected red ceils, they showed specific adherence of infected red cells to the ECs. They also noted that not all HUVECs bound infected red cells (the basis for this still remains to be described). The lack of availability of a continuous HUVEC line, and the high degree of variability in HUVEC adhesiveness, presumably due to differences in individual donors as well as changes associated with the number of passages in culture, prompted a search for other cytoadherent target cells. Schmidt et al [128] screened 18 cell types and found the C32 amelanotic melanoma cell (ATCC CRL1585), a continuous cell line, to be more cytoadherent than HUVECs. They also found that human amnion cells and human aortic ECs were adhesive, but bovine ECs, as well as other cell lines of human origin such as vascular smooth muscle cells, lung fibroblasts, choriocarcinoma cells, colonic adenocarcinoma cells, colonic mucosal cells, and cervical epithelioid carcinoma cells, were not. Stage specificity for in vitro adherence to melanoma cells and HUVECs matched that observed in vivo: red cells bearing mature trophozoites

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IW Sherman et al as lactic acidosis [103, 104, 160] and possibly in the post capillary venules occluded by parasitized red cells, the pH may be depressed even further due to the accumulation of parasite-produced lactic acid. The presence of high (50 mM) levels of Ca 2+ in the medium, as well as desialylation of infected red cells enhanced binding [27,134], whereas high levels of amino sugars in the medium blocked adhesion [140]. These in vitro results suggest that in addition to pH, sequestration may be sensitive to cell surface charge (ie, removal of sialic acid residues and high levels of Ca 2+ reduce the surface charge of red cells and ECs I156], thereby permitting cells to come closer to one another, and conversely, the presence of positively charged amino sugars in the medium may inhibit binding by electrostatic repulsion). We studied the adhesive properties of two kinds of HUVECs, as well as human brain capillary ECs (HBECs) and found that these ECs were not entirely equivalent in their capacity for binding infected red cells (table I). Although all of the ECs had approximately the same p H o p t i m u m for adhesion, only the H B E C s (cytokinestimulated or unstimulated) bound large numbers of infected red cells. The addition of calcium ions to the medium did not significantly affect the binding of infected red cells to H U V E C s or HBECs, whereas it did increase the binding of C32 amelanotic melanoma cells. By scanning and transmission electron microscopy, the appearance of amelanotic m e l a n o m a cells and the H U V E C s are quite different: the former show numerous elongate microvilli, whereas the latter do not [33]. In vitro cultures of I-IBECs demonstrate very different growth properties and have a m o r p h o l o g y quite distinct from H U V E C s [78] (table I). Thus, despite the m a n y conveniences associated with the use of C32 amelanotic m e l a n o m a cells and H U V E C s , these cells may not serve as accurate models for sequestration and cerebral malaria.

(24+ h) and early schizonts (36 h) were the most adherent [95,128]. Uninfected red cells and erythrocytes containing ring-stage parasites were non-adhesive. Udeinya et a! [149] found that continuous in vitro culture of several parasite lines resulted in their loss of adhesiveness, despite retention o f the K + phenotype. Thus, cytoadherence appeared to require, in addition to the presence of knobs, expression of an adherence molecule. An exceptional case was a Brazilian isolate, Ituxi (It), which remained adhesive even after 43 days in culture. In this study, it was claimed that the binding of infected red cells to EC and amelanotic melanoma cells was similar, however, the ratio o f infected cells bound to melanoma vs HUVECs was not constant for all isolates as it should have been if the target cells were similar. The cytoadherence assay originally described by Udeinya et al [152] has been modified by several workers [27, 30, 95, 134, 150, 166] and used in the hope of better defining the causes of cerebral malaria. However, with this in vitro assay, considerable variation in the degree of binding was observed both in laboratory cultures and with blood obtained from malaria patients; this variability was neither related to the initial parasitemia nor to adaptation to culture conditions. In a study carried out in the Gambia using amelanotic melanoma cells as the targets, no correlation was found between the binding capacity of infected cells from subjects with cerebral malaria and those with uncomplicated malaria [95]. A second study with Thai patients [46] also found no correlation between the degree of adherence and cerebral malaria, although cytoadherence was greater for isolates from individuals with severe malaria. Using amelanotic melanoma cells, we found the optim u m p H for adherence to be 6 . 6 - 6 . 8 , and the best buffers for cytoadherence to be those with a p K a close to the optimum pH for adhesion, ie, Bis Tris [26]. It is of interest to note that individuals infected with Pfalciparum frequently have abnormally low levels of blood pH, as well

Table I. A comparison of the adhesive and other properties of target cells : properties of human umbilical vein endothelial cell (HUVEC), continuous line of human umbilical vein endothelial cell (EA hy926), human brain endothelial cell (HBEC), and amelanotic melanoma cell lifie (C32). Target cql

HUVEC

EA hy926

HBEC

C32

Endotech/ Dr F Hofman

Dr CJ Edgell

Dr Jay Nelson

ATCC

Cobblestone

Spindle shaped

Ameboid, forms 'vessels'

Spindle shaped

Suhstrate preference

Plastic and glass

Plastic and glass

Plastic only

Plastic and glass

Effect of trypsin

Slow detachment

Slow detachment

Rapid detachment

Moderate detachment

Rate of growth

Moderate

Rapid

Moderate

Rapid

Subculture period

5-7 days

3 - 4 days

4 - 6 days

3 - 4 days

Adhesiveness (passage number)

1-4

70-80

1-13

indefinite

1.22 _ 0.21 1.08 + 0.17 1.20 _+ 0.21

0.19 +_ 0.15 0.16 _+ 0.07 0.07 + 0.03

14.0 + 1.7 17.8 + 2.7 15.7 + 3.3

3.65 _+ 0.71 3.05 _ 0.38 2.15 :t: 0.27

Source Morphology

Adhesion a pH 6.4 pH 6.6 pH 6.8

a Mean number of infected red cells per target cell + SE.

Malaria sequestration

The ex vivo rat mesocaecum model An ex vivo model that uses the artificially perfused microvasculature of the isolated rat mesocaecum has been used to study sequestration under rheologic conditions approximating those found in the human host. Using this system, only K + lines bound to the post-capillary venules and produced blood vessel occlusion [121] (these findings parallel those observed in humans infected with Pfalciparum). K - lines did not adhere to the endothelium of the mesocaecum during shear flow conditions. Preincubation of infected red cells with soluble thrombospondin (TSP) inhibited adherence, whereas other adhesive proteins such as fibronectin and fibrinogen were without effect [123]. Preinfusion with an anti-TSP rabbit antibody, as well as serum from adult human subjects living in an endemic region (and, therefore presumed to be immune to malaria), also abolished adherence. Such studies indicate that by TSP binding to infected cells, adhesion can be blocked (could this be through a steric effect ?). Although the isolated mesocaecum does model the in vivo shear forces encountered by red cells in the microcirculation, and does maintain the in vivo EC architecture, its principal disadvantage may lie in its use of a rodent host. Are the adhesion properties of the EC mesocaecum microvasculature identical to those human organs in which sequestration takes place ? And, could the binding of K + Pfalciparuminfected cells in this system be a rheological artifact? The chemical basis for adhesion

Ligands of the endothelial cell and other cytoadherent target cells Electron microscopic studies have shown that attachment of the Pfalciparum-infected red cell to the endothelium occurs via knobs [83, 101]. On occasion strands of material are seen connecting the EC to the knob [33, 89, 161]. Thus, there appear to be specific ligands on the surface of the EC to which the receptor of the infected cell binds. The molecular characterization of the ligands on the EC that mediate adhesion is basic to an understanding of the mechanisms of sequestration. Defining such ligands can, however, pose problems. The ECs that line the entire inner surface of the cardiovascular system are heterogeneous, both among species and within the tissues of a species [32, 138]. Differentiated microdomains in microvascular beds have been identified by lectin binding, cationized ferritin labelling, and immunocytochemistry [138]. ECs from different organs show distinct preferences in their adherence to inert substrates ([78] ; Smith, personal communication). Some of these differences may result from adaptation of otherwise.identical ECs to different environmental conditions. Indeed, it is generally stated that many of the surface and metabolic properties of the endothelium are inducible rather than constitutive [38]. A variety of well-defined stimuli (lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon-gamma (IFN-y), thrombin, and phorbol diesters) have been shown to modulate various functions of the EC, especially those derived from the human umbilical vein and grown in vitro [38]. This modulation of cell function (which has also been observed in vivo) has been termed 'EC activation'. At sites of chronic inflammation, ECs have been observed to change shape (elongated and flattened cells become tall, cuboidal or columnar ceils);

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in addition, the chromatin in the EC nucleus becomes dispersed, and there are increased numbers of intracellular organelles. ECs produce mediators of procoagulant and proinflammatory activity. Proinflammatory activity includes the synthesis and expression at the cell surface of adhesive ligands, such as GP IV (CD 36), thrombospondin, intercellular adhesion molecules (ICAM-1 = CD 54 and ICAM-2), ELAM-1 (endothelial-leukocyte adhesion molecule), vascular cell adhesion molecule (VCAM-I = inducible cell adhesion molecule = INCAM-110), GMP-140 (granule membrane protein-140 = PADGEM = CD 62), as well as the synthesis and release of IL-1, prostacyclin, and tissue plasminogen activator. Some of these EC activation responses have been observed with monocytes [14] and the myelomonocytic line U937 [108], but not with the C32 amelanotic melanoma cell (Smith, unpublished).

The selectin family Three structurally related ligands of the selectin family have been identified: LAM-1, GMP-140, and ELAM-1 (reviewed in [77, 98]). It is believed that the calciumdependent, carbohydrate-rich, lectin-like domains of these molecules mediate adhesion. Both ELAM-1 and GMP-140 are found on activated ECs. ELAM-1 mediates the adhesion of neutrophils, monocytes and related cells (HL-60 and U937) to cytokine (TNF and IL-1) stimulated ECs; it does not bind lymphocytes. There are no reports of ELAM-I binding P falciparum-infected erythrocytes. GMP-140, a component of the Weibel-Palade bodies of ECs, is also found in platelets. It mediates the interaction of stimulated platelets with monocytes and neutrophils, and is also involved in the binding of these ceils to the endothelium. GMP-140 appears not to be involved in the adhesion of Pfalciparum-infected red cells (Smith, unpublished).

The immunoglobulin superfamily ICAM-1, ICAM-2, and VCAM-1 are members of the immunoglobulin superfamily [142]. ICAM-I (CD54) is a 90-kDa single chain, heavily glycosylated protein expressed on only a few cell types (eg, fibroblasts, epithelial cells, some leukocytes, and ECs). LPS, interferon-y, IL-1 and TNF cause its strong induction in ECs and this results in a great increase in the binding of lymphocytes and monocytes to the endothelium. ICAM-1 has five immunoglobulinlike domains, whereas ICAM-2, a truncated form of ICAM-I, has two immunoglobulin domains, and 35°70 of these are identical to the two N-terminal domains of ICAM-1. Increased expression of ICAM-1 after cytokine exposure is detectable after 4 - 6 h and is maximal by 9-24 h. Unlike ICAM-1, ICAM-2 is well expressed basally on ECs, and its expression is usually not upregulated by cytokines [37, 107]. The binding sites for LFA-1 (lymphocyte function-associated antigen) and rhinoviruses have been localized to the two outermost immunoglobulin domains of ICAM-1 [143]. ICAM-1 has been implicated in the adhesion of P falciparum-infected red cells (see below); the adhesiveness of ICAM-2 for malariainfected eythrocytes is presently under investigation in our laboratory. VCAM has 6 - 7 immunoglobulin domains, is expressed on the surface of IL-1 or TNF stimulated HUVECs, and serves to bind lymphocytes, monocytes, and a number of melanoma cell lines [21, 113] ; the adhesiveness of

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VCAM for Pfalciparum-infected red cells has not been reported.

Thrombospondin Thrombospondin (thrombin sensitive protein, TSP), a 420-kDa glycoprotein composed of three identical subunits [79], is synthesized by ECs, monocytes [129], and fibroblasts. It is stored in resting platelets and released following platelet activation [60]. The platelet and cell-derived forms are similar in molecular mass, amino acid composition and antigenicity. TSP interacts with numerous macromolecules. TSP (but not Von Willebrand factor/Factor VIII, fibronectin, laminin, or vitronectin), when adsorbed onto plastic, specifically bound K + PfalCiparum-infected cells, and soluble TSP inhibited such binding [122]. In addition, soluble TSP inhibited the binding of K + cells to amelanotic melanoma cells [135]. Binding of TSP to K + red cells is calcium-dependent, and the red cell receptor for TSP is trypsin-sensitive [135]. The COOH-terminal fragment of TSP contains the binding site for infected ceils [136]. However, not all anti-TSP antibodies were capable of blocking the adhesion of K + cells to immobilized TSP [12, 108]. And, TSP was detected (by an anti-TSP mAb) on the surface of several melanoma cell lines irrespective of whether they were cytoadherent; TSP was not detected on the highly cytoadherent C32 arnelanotic melanoma line [114] and no differences were found in the amount of TSP secreted or expressed on the surface of binding and nonbinding melanoma cells [137]. K + cells bound to transfected COS cells expressing CD36, but such cells did not support increased binding of TSP [112]. This observation, plus the lack of correlation between the presence of TSP and cell cytoadherence, suggests that TSP may not be a primary ligand for adherence. Still, the possibility remains that localized expression and concentrations of TSP could be involved in adhesion. The proposal that TSP may serve to stabilize cell-cell binding by its acting as a bridging molecule to link the parasite surface receptor to the OKM5 ligand on the amelanotic melanoma cell or the EC [24] is presently without experimental foundation.

Glycoprotein I V (CD36) and ICAM-1 GPIV 4also called GP IIIb) is an 88-kDa sialylated glycoprotein found on the surface of platelets, U937 myelomonocytic cells, monocytes-macrophages, human erythroleukemia cells, fetal erythrocytes, C32 amelanotic melanoma cells and ECs [145]. Based on its reactivity with mAb OKM5 platelet GPIV is immunologically related to the leukocyte differentiation antigen CD36. Phenotypic heterogeneity in the expression of OKM5 antigen has been demonstrated for the vascular endothelial cells of the human liver, kidney [72], colon [97], and lung [169], though opinions differ as to whether it is present in brain [2, 5, 46]. CD36 is a receptor for TSP and, by its interaction with cell surface CD36 platelets and monocytes, can cross-link; CD36 is involved in platelet adherenceto type I collagen. CD36 from bovine ECs is distinct from that obtained from human sources in molecular mass, primary structure, and antigenicity (ie, the mAbs OKM5 and OKM8 do not react with bovine CD36 [40]). Such differences may contribute, in part, to the adherence of P falciparum-infected erythrocytes exclusively to human CD36. The CD36 specific monoclonal antibody OKM5 inhi-

bits the adhesion of falciparum-infected erythrocytes to amelanotic melanoma cells [12, 14]. The presence of OKM5 antigen is correlated directly with the ability of melanoma cells to bind infected red cells [114]. And, specific interference of melanoma cell glycoprotein synthesis (especially of CD36 expression) by tunicamycin or castanospermine-6-butyrate was shown to disrupt cytoadherence [167]. Only malaria-infected red cells bound to purified CD36 immobilized onto plastic [110, 111], or to COS cells transfected with a cDNA encoding CD36 [112]. The binding to CD36 was calcium independent. Based on these findings, it appeared that CD36 was the ligand for adhesion. However, the report by Berendt et al [16] suggested that there may be adhesive ligands other than CD36. In their work, a cloned parasite line derived from the Ituxi isolate, designated It04, was selected for its adherence to HUVECs. Adhesion of this line was not blocked by the anti-CD36 antibody OKM5, or by a polyclonal antiserum against TSP. An anti-ICAM-1 mAb (7F7) blocked the binding of this line to COS cells transfected with a cDNA encoding ICAM-1, as well as to HUVECs. The It04 line also bound to COS cells transfected with cDNA encoding CD36, as well as amelanotic melanoma cells suggesting that for this line at least two EC ligands were involved in adhesion (ie, ICAM-1 and CD36). Stimulation of HUVECs with the cytokines TNF and IL-1 increased binding of the It04 clone. It was not determined, however, whether this increase in adherence was specifically due to enhanced expression of ICAM-1. Another parasite line, FCR-3, that bound neither to HUVECs nor to ICAM-1, did bind to immobilized CD36 and amelanotic melanoma cells. Thus, for FCR-3, CD36 alone appeared to serve as the ligand. (Unexplained is why in this study the FCR-3 line did not bind to HUVECs which do express CD36.) Our results differ from those of Berendt et al in a number of ways. In our hands, the parasite line FCR-3 bound to both amelanotic melanoma cells and HUVECs; the adherence of FCR-3 infected red cel)s to both these target cells was greater than that of the ItG2 line. The adhesion of FCR-3 infected red cells to melanoma cells was inhibited (z 60°70) by OKM5, whereas antibodies to ICAM-1 had a lesser effect (z 25070 inhibition) on adhesion. Although cytoadherence to HUVECs was blocked by the same antibodies, the degree of inhibition of binding to HUVECs differed from that found for the C32 amelanotic melanoma cell : OKM5 antibody strongly blocked the adherence o f the FCR-3 line (z 85070), whereas with ItG2 inhibition was somewhat lower (z 70070). These results suggest that for the FCR-3 and the ItG2 lines the major adhesive ligand on the melanoma cell is CD36. Biggs et al [18] and Ruangjirachuporn et al [124] have shown that for the cytoadherent knobless lines, B8B6 + and BPA the principal ligand for binding to melanoma cells is CD36. (Adherence to the melanoma cells for these lines could not be inhibited by anti-ICAM or anti-TSP antibodies.) The binding of these.knobless lines was Ca2+-independent, and was stable in the presence of 1 mM EDTA. The affinity of binding by K + C + cells was higher than for the K - lines. Ultrastructurally, the interaction of the two lines with melanoma cells differed: K + C + lines had intimate interdigitations with microvilli on the melanoma cells, whereas the K - C + lines showed no interdigitations and fewer sites of contact. It would appear that such differences in melanoma cell morphology reflect an alteration in its properties as a result of interaction with different parasite lines. A recent investigation on the binding of infected eryth-

Malaria sequestration rocytes from Thai patients with uncomplicated or severe malaria to immobilized ICAM-I or CD36 showed four times more parasitized cells bound to CD36 than ICAM-I [110]; with ICAM-I no difference in the number of bound cells was evident between the two kinds of patients, however, greater numbers of infected cells did adhere to CD36 when the blood came from patients with severe malaria.

The adhesion receptors on the P falciparum infected red cell EMP 1 Antibody can affect both sequestration and cytoadherence. Serum obtained from squirrel monkeys or humans immune to malaria can reverse the adherence of infected cells to amelanotic melanoma cells [30, 139]. When immune monkey serum was given intravenously to P falciparuminfected squirrel monkeys, the number of trophozoite/schizont-infected cells in the peripheral blood rose sharply over a 30-min period and then declined slowly [30]. These experiments reveal that cytoadherence is reversible both in vitro and in vivo. A secondary decline in parasitemia occurred 24 h after passive transfer of immune serum to an infected squirrel monkey, suggesting that the interaction of antibody with sequestered cells may represent an initial step in a chain of events that eventually destroys the parasite. Indeed, it has been observed that the incidence of cerebral malaria is higher in children and in nonimmunes recently exposed to malaria. The demonstration that cytoadherence can be blocked or reversed in a strain-specific manner by serum obtained from immune subjects [151], as well as the fact that treatment of intact infected red cells with proteases (eg, trypsin, chymotrypsin and pronase) abolishes adherence [30, 80, 134] led to the suggestion [80] that the binding of K + cells was mediated by a proteinaceous molecule on the surface of the infected erythrocyte. Other work [7, 8, 59, 80] demonstrated that the cytoadherence phenotype was correlated with the presence of a large > 240-kDa molecule ( = EMP 1); this protein could be labelled with radioactive iodine under conditions that produced minimal labelling of cytoskeletal proteins and hemoglobin. Furthermore, since the > 240-kDa molecule was also labelled with several radioactive amino acids, and mature red cells do not synthesize protein, it was concluded that this molecule was a plasmodial protein. This protein, soluble in the anionic detergent SDS, and insoluble in the non-ionic detergent Triton X-100, was removed from intact cells by low concentrations of trypsin. The antigen, immunoprecipitated from different strains with strain-specific sera, varied in molecular size, and was generally absent from knobless lines, the exception being a K - - C + line, B8B6 [18]. Magowan et al [91] developed a highly cytoadherent line by selecting adherent cells of the cloned Ituxi isolate ItG2. The derived line, ItG2F6, had not one but several radioiodinatable high molecular weight proteins. Removal of these proteins by trypsin abolished adherence. When selection was relaxed, the infected cells reverted to the parental phenotype, and a sfngle protein with the original molecular size was found. It was concluded that a family of proteins, probably of parasite origin, was responsible for adherence. Howard et al [59] have suggested that the > 240-kDa antigen, EMP I, is a parasite-encoded chimeric protein with a variable epitope responsible for antigenic diversity and an invariant epitope involved in sequestration. The fine characterization of EMP l and a-definition of its pre-

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cise role in sequestration has not been achieved because no monospecific polyclonal antiserum or mAbs to it have been developed nor has the gene for this protein been cloned and sequenced.

Sequestrin Recently, a monospecific immunologic probe has been used to characterize the adhesion receptor on the surface of the P falciparum-infected erythrocyte [109]. mAb OKM8 which, like OKM5, reacts with CD36 and blocks cytoadherence, was used to prepare a polyclonal antiidiotype rabbit antibody. This antibody, which was neither strain nor knob-specific, reacted (by immunofluorescence) only with the surface of cytoadherent lines (K + C + or K - C + ) and blocked their adhesion. The anti-idiotype antibody which did not react with K - C - lines immunoprecipitated a z 270-kDa radioiodinatable protein from Triton X-100 soluble lysates of a knobless, cytoadherent ( K - C + ) line. This protein could be labelled' with radioactive isoleucine. It was not immunoprecipitated from uninfected cells, or non-cytoadherent lines, nor was it present in extracts of infected red cells if they had been treated as intact cells with trypsin. This plasmodial protein which mediates binding to CD36 was named sequestrin. Sequestrin could be a member of the EMP 1 family of proteins that are both trypsin-sensitive and antigenically diverse (see above), but this is far from certain, since the identification of sequestrin was based entirely on one cytoadherent-selected knobless line derived from one culture-adapted knobless clone (Malayan Camp). If sequestrin is identical to EMP 1, then the anti-idiotype antibody should immunoprecipitate proteins of different molecular size from different strains as was demonstrated with strain-specific sera. Ockenhouse et al [109] did not report whether sequestrin could be isolated from naturally occurring sequestering lines (ie, those that are phenotypically knobby). And there was no indication whether the anti-idiotype antibody blocked the binding of infected cells to ECs. Further, since the anti-idiotype antibody did not completely block the adhesion of infected cells to amelanotic melanoma cells, it suggests that other adhesive ligands may be present. AItered forms of band 3 We attempted to define the receptors on the surface of the Pfalciparum-infected red cell through the use of adhesionblocking mAbs prepared against live infected red cells. Several murine monoclonal antibodies (mAbs, 4A3, 1C4, 2B3, 3E12, 4F4, 3H3, 5H12) were developed [26, 165]. None of the mAbs reacted (by surface immunofluorescence) with either uninfected red cells or red cells bearing ring-stage parasites. The membrane antigens, with Mrs of > 240, 85, 65 and 55 kDa, by peptide mapping and immunoblotting were shown to be modified forms of band 3. These parasite-induced neoantigens were not found in uninfected erythrocytes, aged red cells, or ring-infected erythrocytes. The neoantigens are exposed on the outer surface of the infected red cell since they are both trypsin and iodination sensitive, and live cells bind antibody. The band 3-related neoantigens appear to result from truncation and covalent modification of the native form of the protein. The immunoblot patterns allowed the mAbs to be assigned to two regions of the band 3 molecule: amino and carboxyl. The mAbs 1C4, 2B3, and 3H3 comprise the amino group, whereas 4A3, 3E12, 4F4 and 5H12 belong to the carboxyl group. The mAbs 4A3, 1C4, and 3H3 inhibited cytoadherence in a dose-responsive fashion. The loca-

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~G

Fig 3. Flow chart depicting the various factors involved in adherence of the Pfalciparum-infected red cell to the endothelium.

tion of the epitopes to which the mAbs bind were deduced from the sites of chymotrypsin and calpain cleavage. The epitope of 1C4 is located on external loop 3 between residues 553 and 575, whereas 3H3 is located between 541 and 553, and the epitope of 4A3 is located on external loop 7 between amino acids 807 and 860. In the primary structure of band 3, loops 3 and 7 are distant from one another, however, in the tertiary structure of band 3, these loops may be in close proximity; therefore, the inhibitory effect of 1C4 or 3H3 on adhesion may be due to a steric effect on an adhesive receptor sequence on external loop 7, or conversely, the blocking of adhesion by 4A3 could be due to steric hindrance by it on external loop 3. Studies with 5H12 make it probable that adhesion primarily involves sequences found in the external loop 3 of band 3. Base~l on the epitope mapping studies, three peptides were prepared by solid-phase synthesis. All of the peptides were water soluble, and > 95% pure, as judged by H P L C , amino acid analysis, and mass spectrometry. Two of the peptides containing the amino acid residues in extracellular loops 3 and 7 (fig 2) inhibited cytoadherence in a dose-dependent manner. The third peptide, containing external loop 4 sequences had little effect. Synthetic peptides were able to inhibit cytoadherence in the presence of serum. Thus, the altered forms of the erythrocyte membrane protein band 3 are directly involved in Pfalciparum cytoadherence. These parasite-induced antigens are variable in M r, and are expressed only on the surface of red cells parasitized by trophozoites and schizonts - the plasmodial stages involved in cytoadherence/sequestration. The epitopes recognized by the cytoadherence-inhibiting mAbs are not strain-specific. And, just as cytoadherence is trypsin-sensitive, these band 3-related neoantigens are altered by trypsin treatment of intact infected cells. These neoantigens, related to but not identical with band 3, fulfill the criteria for the cytoadherence protein.

Sequestration and cerebral malaria: multifactor processes The attachment of Pfalciparum-infected red cells to the endothelium cannot be explained by a single factor. Indeed, sequestration and cerebral malaria probably involve several interacting elements, including the properties of the erythrocyte and the endothelial cell, as well as the environment that surrounds them (fig 3).

Environmental factors Rheologic characteristics of the blood vessel Pfalciparum infections are characterized by sequestered infected red cells being restricted to certain sites in the microvasculature: capillaries and post-capillary venules [120]. In these vessels, red cell velocity and wall shear forces are lowest and consequently this provides for prolonged contact of the infected erythrocyte with the surface of the EC. And, once the microvessels are partially occluded a further drop in blood flow may contribute to the formation of aggregates of infected cells and rosettes (see below). Blood p H and levels of CO 2 In vitro cytoadherence studies clearly show that adhesion can be enhanced by. either a slightly acidic pH (eg z pH 6.6), or elevation of the level of carbon dioxide [27]. In patients with severe malaria, lactic acidosis is frequently observed [103, 104, 160] ; in the microenvironment of the post-capillary venules, the pH of the blood plasma may be further depressed by the increased production of lactic acid by trophozoite- and schizont-infected red cells and accumulation in the partially occluded blood vessels. The stasis of blood in the occluded vessels would also increase local levels of pCO 2. A lowered pH and an elevation of CO 2, in microvessels would contribute to increased adhesiveness of infected red cells and promote sequestration and cerebral malaria.

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Immune status o f the host Immune serum obtained from humans or monkeys is capable of reversing cytoadherence [30, 139, 151], sequestration [30], and rosetting (see below). It has been reported that anti-rosetting capacity was highest in the sera of patients who were immune or who were without cerebral malaria [22]. In addition, cerebral malaria is more frequently observed amongst children and non-immunes recently exposed to malaria. In a study in the Gambia, inhibition of cytoadherence was observed both with sera obtained from children who were convalescing or from immune adults, but not with sera obtained from children in the acute phase of the disease [155].

sequestration and cerebral malaria. It should be noted, however, that in unselected lines, and in fresh isolates, binding to cytokine-stimulated HUVECs or to ICAM-1 immobilized to plastic was always lower than to ceils expressing CD36 (eg, amelanotic melanoma cells) or to immobilized CD36 [24, 110]. These findings suggest that for recent field isolates CD36 is the principal adhesive ligand. However, there remains the possibility that sequestration and cerebral malaria could result from organspecific, cytokine-induced, up-regulation of ligands such as CD36, ICAM, etc.

Spleen The spleen exerts a powerful selective force for the adhesive phenotype [13] ; in splenectomized squirrel monkeys, both cytoadherence and sequestration were reduced [30]. And, sequestration was not observed in a splenectomized human [61]. Therefore, in splenectomized individuals, the occurrence of cerebral malaria would be expected to be exceedingly rare.

Genotypic variation We (Smith, Crandall, Prudhomme and Sherman, unpublished) have observed that considerable differences in cytoadherence can occur with a single parasite line (FCR-3) growing in red cells obtained from different donors. The molecular basis for this difference in red cell adhesiveness induced by a single parasite line remains unknown.

Target cell factors Phenotypic variation In humans, the preferential localization of infected red cells to the vascular endothelium of organs such as the kidney, brain, lung, small intestine, heart and placenta [120] may result from a phenotypic heterogeneity of endothelial cell ligands. It has been shown that the distribution of two putative ligands, CD36 and ICAM-1, is not uniform throughout the organs of the body [2, 14]. Indeed, even in a single organ there may be differences in ligand expression. Although the distribution of CD36 generally follows the sites o f sequestration, the correlation is by no means exact. For example, platelets and monocytes express CD36 [108, 145], but only rarely are such cells attached to infected red cells. And, CD36 is reported to occur in the human spleen, yet there is no evidence for sequestration here [120]. Such tissue distinctions could be due to differences in ligand density, or to a diminished capacity for activation, but as yet there is no evidence for either of these occurring in human malaria infections. And there is no in vivo evidence for the preferential activation of brain ECs in humans with cerebral malaria. Cytokine activation It is well-established that both in vitro and in vivo, the expression of adhesive ligands on the surface of ECs may be up-regulated by cytokines. Elevated levels of interferony, and TNF-~ have been found in the sera of patients infected with Pfalciparum [39, 71, 73, 130]. High levels of IL-6 were correlated with high levels of TNF-0t. And parasitized red cells or products of parasites have been shown to stimulate mononuclear phagocytes to produce soluble mediators such as TNF-a and IL-I [144]. Up-regulation of HUVECs by T N F and IL-1 resulted in increased adhesion of a line (It04) selected for adhesion to HUVECs [16]. The binding of this line to HUVECs or to COS cells transfected with a cDNA encoding ICAM-1 was inhibited by an anti-ICAM-1 antibody. Since the transfected cells and the cytokine-stimulated HUVECs expressed increased amounts of ICAM-I, it was presumed that ICAM-1 alone was responsible for the binding of infected cells. Thus, it may be that by the differential action of cytokines on ECs surface levels of ICAM-1 would be increased leading to

Red cell factors

Parasite isolate A common observation with field isolates is that different lines show different propensities for adherence to melanoma cells even when similar numbers of parasitized cells are used [46, 95]. Although much speculation has surrounded this observation, there has been no clear correlation of adhesiveness with the presence or absence of plasmodialspecific receptors such as sequestrin and EMP 1, nor has there been a correlation of specific isolates with a greater frequency of cerebral malaria. Rosette formation In 1988, David et al [29] described the spontaneous in vitro adherence of uninfected red cells to Plasmodium fragileinfected red cells, a phenomenon they called rosetting. Rosette formation is not an exclusive property of P fragile infections in the toque monkey (Macaca sinica); other sequestering species such as Pfalciparum [42] and P coatneyi [154] also form rosettes in vitro. (Non-sequestering malarias, eg, P cynomolgi and P vivax, do not form rosettes.) In P falciparum, only trophozoite- and schizontinfected cells are involved in rosetting, and a single infected red cell may be surrounded by as many as ten uninfected red ceils, and occasionally clumps of twenty infected red ceils and 4 0 - 5 0 infected red ceils form. Rosetting occurs both with knobby and knobless lines [153] of Pfalciparum, and can be modulated by the spleen (ie, parasite lines derived from splenectomized animals lose the capacity to rosette). Preincubation of a rosetting isolate with the monoclonal antibody OKM5 blocked cytoadherence, but rosette formation was unaffected. And, an inverse relationship between rosette formation and cytoadherence was demonstrated. Such observations have prompted the suggestion that, although cytoadherence and rosette formation are intrinsic parasite properties, isolates have a propensity for one or the other, but not for both [157]. This view of mutual exclusion of cytoadherence and rosetting has been disputed by Howard et al [59]. Indeed, Hasler et al [43] showed that an individual infected cell can express receptors both for cytoadherence and rosetting. In a study in the Gambia, all isolates from children with cerebral malaria were able to form rosettes, whereas those having a milder form of the disease usually did not [22]. In addition, infusion of a bolus of cells containing a rosetting line resulted in endothelial sequestration and vaso-

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occlusion in the post-capillary venules of the denervated artificially perfused rat mesocaecum vasculature [66]. Rosettes can be disrupted by heparin, calcium chelators, and monoclonai and polyclonai antibody against the histidinerich protein, HRP1 [23]. And although anti-rosetting activity has been demonstrated in the sera of individuals living in endemic areas, non-endemic areas, and in people infected with other parasites, the highest degree of anti-rosetting activity was found in the sera of children with non-cerebral malaria. Since rosetting can be abolished by treatment of infected red cells with trypsin, it would appear that rosette formation involves a receptor protein on the surface of the infected red cell. Such a protein, however, has neither been identified on infected red cells nor on uninfected ones. Rosetting may contribute to microvascular obstruction, and lead to the development of cerebral malaria. However, rosettes have never been observed in post-mortem tissues, and with Thai patients Ho et al [45] were unable to demonstrate a correlation between in vitro rosette formation and cerebral malaria.

Erythrocyte membrane lipids P falciparum-infected red cells show reduced levels of erythrocyte membrane cholesterol, and an increased surface exposure of phosphatidylserine (PS) [92, 93]. Reductions in cholesterol result in an increase in fluidity of the inner leaflet of the red cell membrane. Since clustering of membrane adhesins (eg, EMP 1 and the neantigens related to band 3) would be favored by a more fluid membrane this would lead to an enhancement of the cell's adhesiveness. Phosphatidylserine exposure in reversibly sickled cells, restricted to those areas of the membrane in spicular form, has been shown to be involved in increased EC adherence; the in vitro adherence of sickle cells to ECs was inhibited by the addition of phosphatidylserine, but not other phospholipids [63]. In vitro manipulation of red cell membrane lipid asymmetry to produce increased amounts of PS in the outer monolayer (ie, symmetric ghosts or oxidant treated red cells), resulted in greater adherence of these cells. Therefore, exposure of PS as well as reductions in m e m b r a n e cholesterol in the malariainfected red cell could contribute to adhesion. Sialic acid Pfalciparum-infected red ceils preferentially bound to agarose b ~ d s conjugated to the lectins conA, ricin, peanut agglutinin and soybean agglutinin, suggesting that during the development of the parasite, there are changes in the exposure or expression of surface galactosyl residues [31]. O f particular interest is the enhanced agglutination of P falciparum-infected red cells, but not uninfected human red cells, with concanavalin A (conA) (Sherman, unpublished), whose receptor is band 3 [132]. Sialic acid residues, primarily located on glycophorin, are the principal contributors to the surface charge of the red cell. A consistent observation for malaria-infected and aged red cells is a reduction in the amount of surface sialic acid ([54] ; Sherman, unpublished). Loss of sialic acid from the oligosaccharide chains of glycophorin results in an unmasking of/3-galactosyl residues. Such residues may interact directly with lectin-like receptors on macrophages or indirectly with macrophage Fc receptors due to bound IgG. Treatment of Pfalciparum-infected cells with neuraminidase or uninfected red ceils depleted of surface sialic acid are more adherent [134]. Infected red cells, but not uninfected red cells, are also more adherent after treatment with fl-galactosidase [134]. The enhanced binding of sialic acid

depleted cells may simply be due to a reduction in electrostatic repulsion between the charged surfaces of red cells and ECs, nevertheless it could contribute to a preferential binding of infected red cells to the endothelium.

Shape and deformability It has been observed with sickle cells that the densest and the most irregularly shaped cells are the least adherent, yet these cells, because of their reduced deformability, are more effectively trapped and contribute to microcirculatory occlusion. In Pfalciparum-infected red cells bearing trophozoites or schizonts, the normal biconcave shape of the red cell is lost [41] ; infected cells are less dense (presumably because of the higher water content of the plasmodium) and less deformable. In red cells bearing the more mature stages of the parasite, the erythrocyte membrane is slightly more rigid than that of uninfected red cells or erythrocytes containing ring-stage parasites [105]. Changes in red cell deformability concomitant with parasite growth would contribute to the trapping of trophozoiteand schizont-infected cells and lead to the blocking of microvessels. Receptors E M P 1, sequestrin, and H R P 1 A strong correlation has been found between the presence of the polymorphic, high molecular weight, parasiteencoded, protease-sensitive, surface molecule EMP 1 and cytoadherence [52, 55, 80]. Polyclonal antisera immunoprecipitate EMP 1 only from surface iodinated or metabolically labelled infected red cells, either K + or K - , that are cytoadherent [18]. Treatment of intact infected red cells with low levels o f trypsin removes EMP 1 and abolishes cytoadherence [18, 80, 91]. Convalescent sera obtained from naturally infected children agglutinated infected red cells, and reacted with the surface of infected red cells by immunofluorescence in an isolate specific manner; on occasion, immunoglobulin binding to the knobs was evident by transmission electronmicroscopy [59]. Because the sera used in this and all other studies with EMP 1 were polyclonal, and had broad reactivity with many antigens, it is not clear whether the cytoadherence-inhibiting antibody reacted with an antigen identical to that which resulted in the immunoprecipitation of EMP 1. Since there are no monospecific antisera to EMP 1 and the gene encoding this protein has not been cloned, the evidence for it being the adhesive receptor remains circumstantial. The recently described parasite-encoded CD36-binding protein, sequestrin, may be identical to EMP 1, and could be an adhesive receptor on the surface of the Pfalciparuminfected red cell [109]. However, this protein has not been demonstrated in any K + C + line, nor has it been shown to be strain-specific - characteristics of EMP 1. Once the genes for these proteins have been cloned and sequenced, we will have a better understanding of their relationship to one another, as well as a molecular basis for their adhesive properties. The parasite-encoded protein H R P 1, localized to the electron dense plaque below the elevated red cell membrane at the knob [147], is not exposed on the surface of the infected red cell, and therefore cannot be directly involved in adhesion [4]. It is conceivable however, that its contribution to binding may be indirect. K - C + lines have been produced by in vitro selection, and these lack H R P 1 ; however, such lines do not adhere to target cells to the same degree as K + C + lines [124]. Therefore, enhanced

Malaria sequestration adhesiveness of naturally occurring K + lines may occur by a stabilization of knob-associated receptors, via the H R P 1-containing plaque.

Modified forms of band 3 protein There is abundant evidence, derived from many different kinds of experiments, for the presence of band 3-related neoantigens in the Pfalciparum-infected red cell: 1) there is increased agglutination of infected red ceils with conA (Sherman, unpublished); the con A receptor is band 3; 2) con A-binding is localized to knobby regions [132]; 3) exposure of senescent antigen a n d / o r a > 240-kDa antigen, both altered forms of band 3, result in autologous IgG binding, and this is restricted to knobby regions [163, 164] ; 4) clustering of IMPs, principally composed of band 3, occurs in the region of the knob [9]; 5) neoantigens, expressed on the surface of thefalciparuminfected red cell, are recognized on Western blots by murine anti-band 3 mAbs [26, 165]; 6) mAbs prepared against live P falciparum-infected red ceils recognize parasite-induced band 3-related neoantigens, and react on Western blots with modified band 3 protein [26, 165]. We hypothesize that in the P falciparum-infected red cell alterations in band 3 occur in several distinct steps. In restricted regions of the erythrocyte membrane band 3 undergoes a structural modification, which though as yet uncharacterized, may result from an oxidative process. Following this there is degradation of band 3 protein, catalyzed by an enzyme similar to or identical with the calcium-dependent, membrane bound, neutral protease, calpain I. Following proteolysis, band 3 undergoes a covalent modification, and this leads to an altered tertiary structure with exposure of novel epitopes. How do the altered forms of band 3 contribute to the adhesiveness of the Pfalciparum-infected red cell ? It has been demonstrated that the capacity of cell surface receptors to mediate cell-cell adhesion can be regulated by changes in the distribution of receptors in the plane of the membrane [44]. Since receptors recognize clustered ligands more efficiently than those that are randomly distributed, ligand clustering may also enhance adhesion. The punctate distribution of fluorescence with the anti-band 3 mAbs specific for Pfalciparum-infected red cells [26] suggests that the altered band 3 molecules are clustered in the membrane. (Some of these altered forms of band 3 can be localized by electron microscopy [165] to the surface of the knob.) Although we do not know how a specific amino acid sequence of an altered form of band 3 results in adhesion it is clear from epitope mapping studies with the mAbs specific for the surface of the P falciparum-infected red cell that binding involves only a restricted number or regions of the band 3 molecules. The altered forms of band 3 could promote adhesion either by a conformational change in the protein itself, a n d / o r by a multivalent binding between altered band 3 clusters that serve to stabilize the interactions between the infected red cell and the EC. Whether one or both of these is operational in sequestration remains undetermined, although at present we favor the latter.

The

potential

for anti-adhesion

therapy

The antibody-mediated removal of Pfalciparum-infected red cells from the ligand to which they are bound, and the inhibition of adhesion both by excess soluble ligand (eg, CD36 and TSP) or antibody [30, 139, 151], suggest that

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cerebral malaria might be managed through anti-adhesion therapy. For example, the administration of anti-adhesion antibodies a n d / o r adhesion-inhibiting peptides to comatose cerebral malaria patients could unplug microvessels containing packed parasitized red cells and bring the patient out of coma. And, with severe malaria patients, the intravenous introduction of adhesion-blocking antibodies or peptides could lead not only to a reversal of sequestration, but to prevention of the onset of cerebral malaria as well. Anti-adhesion therapy which could reduce the severe complications offalciparum malaria would be ideal in hyperendemic areas since it would reduce both mortality and morbidity in those individuals who were most susceptible, ie young children without any natural immunity [501. At present the only available modalities for the treatment of cerebral malaria, are intravenous quinine (or quinidine), judicious fluid management and exchange transfusion [168]. However, even after treatment with quinine infusion mortality is 20-40°7o. There are no drugs or vaccines that specifically affect sequestration. Therefore, development of an anti-cytoadherence therapy could provide considerable benefit to those living in areas where malaria is endemic, as well as individuals who have no natural immunity to malaria. Anti-adhesion therapy has been tested in a number of animal models of inflammation using mAbs to adhesion proteins (reviewed in [20]). The results have been encouraging, and there is now interest in clinical trials in myocardial infarction, shock, and renal allograft rejection. But, would such a therapy work with P falciparum infections ? Several questions have been raised concerning the use of anti-adhesion therapy in falciparum malaria [50]. First, since studies with immune sera suggest that the response is strain-specific, will it be difficult to obtain a polyvalent serum or a peptide that will reverse adhesion for allfalciparum strains? Second, since administration of sera a n d / o r peptides would have to be intravenous would this pose a safety problem ? Third, if adhesion reversal were organ-specific might anti-adhesion peptides or antibodies not work in the brain ? Fourth, aren't there risks involved in the administration of large amounts of antimalarial antibodies or peptides to a heavily infected individual? Complex as these questions are, the available answers provide considerable hope for the practicality of the antiadhesion approach in the management of severe malaria as well as protection against the disease. - A pool of sera reversed/inhibited the cytoadherence of Thai isolates to amelanotic melanoma cells, and an IgG pool from the Ivory Coast was better than that from Thailand [139]. Further, the murine mAbs which recognize altered forms of band 3 and block cytoadherence were shown not to be strain-specific [26]. - Fab fragments were as effective as intact IgG from the Ivory Coast in inhibiting cytoadherence (Singh and Hommel, unpublished). - Passive transfer experiments using hyperimmune serum with humans or experimental animals have shown no side effects [30, 48, 99]. - Since the anti-adhesion therapy would involve peptides or antibodies specific to the surface of the malariainfected cell, not the EC, impairment of the host vascular tissues should not occur. The practical application of anti-adhesion therapy in extreme life-threatening malaria will require the identification of ligand- or receptor-specific antibodies, as well as the production of peptides that have a configuration

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similar to the binding region. However, since some of the adhesion ligands on ECs and leukocytes are involved in phagocyte migration to sites of infection and tissue injury, the question arises whether anti-adhesion therapy may impair host defense and repair. Long-term use of antiadhesion therapy using murine mAbs or soluble ligands could indeed result in damage, however, with an acute disorder, the administration of a single injection might not produce serious impairment of the patient. Thus, a human anti-idiotype antibody analogous to that for sequestrin [109] or human monoclonal antibodies similar to the murine m A b s t.hat recognize the band 3-related neoantigens, and which block cytoadherence, could be of considerable therapeutic value. By immunologic mapping of the cytoadherence receptor, and determination of its amino acid sequence, it should be possible to synthesize small peptides capable of adherence reversal. Such an approach, rather than administration of mAbs, would certainly be more practical for chronic malaria infections. Indeed, animal studies with a synthetic mimetic peptide to the RGD-containing peptide sequence (which is recognized by a platelet receptor) inhibited the binding of activated platelets to fibrinogen, and prevented platelet aggregation [106]; presumably such a compound, by preventing thrombus formation, would be of therapeutic value in the treatment of acute arterial thrombosis. A similar approach has been suggested for t u m o r therapy. For example, dissemination of intravenously injected tumor cells in mouse tissues could be inhibited by the simultaneous injection of an R G D peptide [125]. Thus, it is conceivable that the management of cerebral malaria and reversal of sequestration could be accomplished through the use of anti-adhesion peptides and antibodies.

Acknowledgments Support for this research was provided by the National Institutes of Health, Institute of Allergy and Infectious Diseases, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the University of California Agricultural Experiment Station.

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