Sequestration and its discontents: infected erythrocyte-endothelial cell interactions in Plasmodium falciparum malaria

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54th FORUM

IN IMMUNOLOGY

Sequestration and its discontents : infected erythrocyte-endothelial cell interactions in Plasmodium falciparum malaria A.R. Molecular

Parasitology

Berendt

Group, Institute of Molecular Medicine, Headington, Oxford OX3 9DU (UK)

Introduction

“Those of them (the erythrocytes) which have been invaded by the parasite offer greater resistance to the circulation than the normal ones, whence it happens that they accumulate towards the circumference of the larger vessels, and their circulation is either stopped or retarded in certain portions of the capillaries, in which the degenerative changes in the endothelium, induced by the defective circulation, constitute a fresh cause for sragnation” (Bignami and Bastianelli, 1889). With these words, the Italian physicians BastianelIi and Bignami were to anticipate by a century a pathogenetic concept held today in which adhesion to vascular endothelium, a critical factor in the virulence of the malarial parasite Plasnzodiurn falciparum, is modulated by changes in the host induced in the course of the infection. Bastianelli and Bignami had already observed that in the more malignant form of “estivo-autumnal fever” (due to P. falciparum), the infected red cells do not circulate for the entire asexual parasite life cycle, but withdraw from the circulation when they reach a certain stage of maturation. They speculated that this process might in some circumstances be harmful and also suggested that the cyclical paroxysms of malaria fever were due to the release of pyrogenic substances from these infected red cells, the molecular basis of which is now known to be induction of macrophage tumour necrosis factor (TNF) synthesis by parasite-derived “toxins” released at schizogeny (Kwiatkowski et al., 1989). Faced with a worldwide burden of morbidity and mortality as great today as a hundred years ago and the rapid rise of multidrug resistance (Newbold, 1990), an inevitable focus of research for modern scientists is the nature and potential manipulation of protective immune responses. Increasing our understanding of pathogenetic mechanisms, however, is also a critical area of malarial research and one which, in the area of infected cell adhesion, is bear-

John Radcliffe

Hospital,

ing fruit. This review sets out to outline our current understanding of the molecular events surrounding this process and the likely directions that future work will take. The sequestration of erythrocytes containing mature forms of P. falciparum in deep vascular beds is a consequence of the adhesion of these infected cells to post-capillary venular endothelium (Miller, 1969). As with leukocyte adhesion, this process is the result of specific interactions between receptors and counter-receptors borne on the apposing cell surfaces. Although our understanding of this leaves much to be desired, it is clear from what we do know that parallels exist with leukocyte adhesion and it is likely that such parallels will be strengthened as our knowledge increases. At the same time there are areas of similarity with numerous microbial pathogens that have evolved successfully to exploit a range of microenvironments within the host by using adhesion as a first step. In the case of P. falciparum, adhesion to endothelium may play a role in immune evasion (by avoiding passage through the spleen), in red cell invasion (since successive generations of parasites are released to invade new erythrocytes in the crowded microenvironment of the venule) or in parasite development (if specific nutritional factors are released by endothelium or present in high concentrations in the venule). In any event it is clearly a phenomenon that confers biological fitness on parasites possessing it, for despite rapid loss of the adhesive phenotype on culture in vitro (Udeinya et al., 1981), sequestration is a universal feature of clinical malaria. Despite this, the organ-specific patterns of infected cell localization can vary considerably from patient to patient (Pongponratn et al., 1991), although in fatal cases the heart, gut, liver and lungs are usually major sites of sequestration (MacPherson et al., 1985). In addition to these, the brain is a favoured site in fatal cases of cerebral malaria (MacPherson

ADHESlON

MOLECULES

IN LEUKOCYTE-ENDOTHELIUM

el al., 1985), an acute encephalopathy characterized by coma, convulsions and a high mortality (Molyneux et a/., 1989). Thus in the same way that subsets of lymphocytes have tissue-specific patterns of homing dictated in part by lymphocyte adhesive heterogeneity (in the form of the homing receptors) and in part by endothelial adhesive heterogeneity (in the form of the vascular addressins), the parasite populations between and within individuals appear to contain subpopulations with specific tissue tropisms, at least in the context of the particular host. It is probable that this appearance of tissue specificity is, in fact, due not only to parasite adhesive heterogeneity (see below) but also to variations in the site and degree of endothelial activation. Furthermore, since adhesion of infected cells occurs in vivo under flow conditions, there will be important effects of local microvascular architecture, blood flow and viscosity (an important determinant of which is the anaemia which is often a direct consequence of malarial infection). It is clear, then, that a similar set of issues concerns us is studying the adhesion to endothelial cells of infected erythrocytes and leukocytes. We need to define the adhesion pathways used, to examine the affinities, avidities and kinetics of the various interactions, to understand how the different receptors are regulated and expressed, to examine the consequences of adhesion receptor engagement and to identify those receptors playing a critical role in adhesion in severe disease. Having identified these, strategies for therapeutic intervention could be explored along similar lines to the anti-leukocyte-adhesion programmes being pursued by biotechnology and pharmaceutical companies, given the commercial will. Whether the inhabitants of the developing world offer tempting enough markets for such a dream to become a reality is open to question.

Host

receptors

for sequestration

Five molecules have so far been identified as receptors for infected erythrocytes; the platelet and endothelial antigen CD36 (Barnwell et al., 1989; Ockenhouse et al., 1989; Oquendo er al., 1989), the secreted glycoprotein thrombospondin (Roberts et al., 1985), intercellular adhesion molecule-l (ICAM-I) (Berendt et al., 1989), vascular cell adhesion molecule-l (VCAM-1) and E-selectin (Ockenhouse et a/., 1992). All except thrombospondin are integral membrane glycoproteins expressedat the surface of endothelial cells; thrombospondin associates with the extracellular matrix (Lawler, 1986) and is turned over rapidly, but may exist at the luminal surface of endothelium in amounts and conformations that allow it to play a role on intact cells. If such a

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role exists, however, it has yet to be demonstrated and the best evidence for its ability to act as a receptor comes from studies of the purified material immobilized on plastic (Roberts et al., 1985). By contrast, both CD36 and ICAM- have been shown to mediate adhesion of a range of parasite strains either as cell surface molecules (on endothelium, platelets, monocytes, melanoma cells and transfectants) or as purified native or recombinant proteins (Berendt et ol., 1989; Ockenhouse et al., 1989). Adhesion is inhibited by appropriate monoclonal antibodies and in the case of ICAM-1, techniques of monoclonal epitope mapping, domain deletion, mouse-human chimera construction and site-directed mutagenesiswere usedto localize the binding site for the infected cell to a region of domain 1 distinct from the LFA-1 or rhinovirus binding sites(Berendt ef al., 1992; Ockenhouse et al., 1992). Rather lessis known about the interaction with CD36, partly becausethe molecule has until recently stood alone, with no clear relationship to a family or superfamily of defined structure. The CD36 antigen has unusual characteristics; very short cytoplasmic domains at both the N- and C-termini, an apparently uncleaved signal sequencethat probably acts as an N-terminal transmembrane anchor (but which is rather short), a cysteine-rich region which forms part of a central, probably extracellular domain and a predicted C-terminal transmembrane anchor which is again rather short, but probably retained in the mature protein (Oquendo ef al., 1989). These unusual structural features have made the molecule more difficult to work with than members of the immunoglobulin superfamily such as ICAM-1, but some of these difficulties may easenow that two human CD36-related geneshave been cloned; the human homologue of the rat lysosomal integral membrane protein II (LIMPHII) (Vega et al., 1991) and a molecule designated CD36 and LIMP11 analogous-l (CLA-1) (Calve and Vega, 1993). The functions of these new members of the CD36 gene family are unclear and differences in their subcellular localization (LIMP11 is targetted to lysosomesby a specific sequence in the cytoplasmic C-terminus) make it likely that they have varying functions depending upon site. CD36 itself has now been identified as a receptor for thrombospondin (Asch et a/., 1987), type I collagen (Tandon el al., 1989) and most recently, oxidized low-density lipoprotein (Endemann el al., 1993). It many play an early role in platelet aggregation and in the ingestion of apoptotic neutrophils by macrophages(Savill et al., I992), but clear evidence of the physiological role this promiscuous receptor plays is lacking. Although individuals have been identified who apparently lack CD36 and generate anti-CD36 antibodies in responseto platelet transfusions (the Nak” phenotype), they do not have an obvious diseaseor syndrome related to their deficien-

54th FORUM

IN tMA4UNOLOG

cy. Finally, a single parasite strain has been shown to adhere in vitro to both VCAM-I and E-selectin (Ockenhouse et al., 1992), though further details of the interaction are lacking. Although low levels of adhesion to these proteins are seen in a small number of Kenyan field isolates (A.B., unpublished data), they appear very much to be minority receptors. Very little data exists on the kinetics, affinities or avidities of interaction of these various receptor pathways. This is in large part due to the fact that the counterreceptors on the infected cell surface are undefined. However it is clear from the study of field isolates that dramatic differences exist between isolates in their levels of adhesion to the different receptors (Ockenhouse et al., 1991). Whether these represent differences in the absolute affinities of the counterreceptors, perhaps varying on a strain to strain basis, or avidity differences due to variations in copy number or presentation of the counterreceptors remains obscure. Studies have begun to address the kinetic issue, however, by studying adhesion under shear flow conditions. Cellular targets commonly used in static assays, such as human umbilical vein endothelial cells (HUVEC), immobilized platelets and human dermal microvascular endothelium (HDMEC) will also support adhesion under flow (Wick and Louis, 1991 ; Nash et al., 1992; Swerlick et al., 1992). Interesting differences between receptors emerge, however, in a manner reminiscent of, but also contrasting with, leukocyte adhesion. While adhesion to immobilized platelets (and in subsequent experiments, immobilized purified CD36) was of a stationary nature, adhesion to HUVEC and to immobilized purified recombinant ICAM-I-Fc was of a rolling character. Unlike the selectin/integrin paradigm for leukocyte adhesion (see other papers in this Forum), however, an additional complexity is that CD36 can mediate its immobilized adhesion without a prior rolling phase (Cooke et al., manuscript submitted). Differences in the relative efficiencies of the two receptors are difficult to assess without determination of site numbers in the various experimental systems used, but over a range of coating densities (for purified proteins) and on the semi-physiological environment of the cell surface in vitro, ICAM-l-bearing surfaces are more efficient at capturing cells from flow than CD36-bearing surfaces. It may therefore be that the forward binding kinetics of the infected cell-ICAMinteraction are more rapid than that with CD36, but that the off-rate is very much greater for the ICAM-l-infected cell complex than for the CD36-infected cell complex, especially when the receptor-ligand bond is stressed under flow conditions. These findings raise a possibility obvious to leukocyte biologists; that initial rolling interactions with ICAM- may facilitate subsequent immobilized adhesion to CD36, in beds where it is expressed or with other receptors in CD36-negative vasculature.

Sequestration

Y receptors

in disease

While such studies are crucial in our understanding of the way the different receptors may operate in vivo, they do not provide data on the relationship of adhesion to a given receptor and the clinical outcome. To identify receptors as critical in disease, additional criteria must be fulfilled beyond the experimental data that define a given protein as a receptor able to operate under physiologically relevant shear flow forces. Receptors must be expressed in vascular beds where sequestration occurs and the expression of the receptor should correlate with the accumulation of infected cells. Proof of these criteria requires the examination of human tissue, for most organs only available from fatal cases. In addition to evidence of receptor expression, at least some of the parasites from individuals with the complication in question (ideally from the same patient whose tissues are available for receptor expression studies) must adhere to the candidate receptor. In cases where massive selective localization of infected cells has occurred in a particular organ, it would also be reassuring (for believers in the hypothesis) to detect preferential adhesion to a receptor strongly expressed in the target tissue. This last may not be an absolute requirement, however ; strong expression of a receptor in a particular site may be enough to localize parasites selectively despite wide variation in the adhesive ability of infected cells from different parasite strains. Studies to address these issues have either been inconclusive to date or are still in progress and for these reasons no single receptor has yet been clearly implicated in the pathogenesis of severe disease. Post-mortem studies on fatal cases of cerebral and non-cerebral malaria support the concept of widespread endothelial activation which includes the induction of potential receptors on the cerebral microvasculature (Ockenhouse et a/., 1992; and Turner et al., in preparation). Studies on the adhesion of field isolates have not shown preferential adhesion to CD36 or ICAM-I, either as purified proteins or when expressed on a melanoma cell line, in severe compared to non-severe disease (Marsh et al., 1988; Ockenhouse et al., 1991). Preliminary analysis of more recent data in African children (Newbold et al., in preparation) suggests that mean levels of adhesion to CD36 are comparable in a range of different clinical groups including asymptomatic parasitization, whereas adhesion to ICAM- is greater in some groups of clinically unwell individuals. Since TNF levels are known to correlate with severity, however (Grau et al., 1989; Kwiatkowski et al., 1990), it may be that individuals with severe disease are distinguished not by their parasites but by their levels of cytokine-induced receptor expression. Further studies, ultimately including the ability to assess the adhesive phenotype of sequestered infected cells in post-mortem material, are required.

ADHESION Adhesion surface

molecules

MOLECULES at the

infected

IN LEUKOCYTE-ENDOTHELIUM erythrocyte

It is a sad fact that we can say little more than that such molecules must exist to explain the diverse adhesive behaviours of infected cells. No monoclonal antibodies exist against these molecules, and although this, in part, reflects the number of groups active in the field, there appear to be significant difficulties in generating such reagents. Nor have promising candidate cDNA molecules been cloned, a problem worsened by the likely large size of the molecules involved, the predilection of the malarial parasite for multiple tandem repeat arrays in important antigens and the A-T richness of the genome, which combine to produce a potent recipe for DNA instability in E. coli. Current attention is focused on a family of high molecular weight proteins (200-350 kDa) that can be surface-labelled by radioiodination and that have been designated P. falciparurn erythrocyte membrane protein 1 (PfEMPl) (Leech 1984). These proteins are highly variable in molecular weight between antigenically different strains of parasite and they are defined by immunoprecipitation of labelled protein with strain-specific antisera but not with antisera raised against antigenically distinct parasites (Leech 1984). Manipulations of the adhesive properties of a parasite strain in vifro (by repeated selections, for example) are associated with alterations in the size and antigenicity of the PfEMPl associated with that strain (Magowan et al., 1988; Howard et al., 1990). Furthermore, it has recently been shown that P. falciparum, like trypanosomes, undergoes clonal antigenie variation (Biggs ef al., 1991; Roberts et al., 1992), with an individual singly-infected cell able to give rise to sub-clones expressing a different erythrocyte surface antigenic phenotype. Clonal antigenic variation is associated with changes in the adhesive phenotype (Roberts ef al., 1992; Biggs ef al., 1992). Adhesion to ICAM- is rapidly lost concomitant with antigenic variation, but can be restored by positive selection; a further antigenic variant related to the original, ICAM-l-binding parent is the result (Roberts et al., 1992). Since the process is a stochastic and rapid one (with a calculated mean switch rate of 2 Vo per generation), it follows that all bulk isolates of infected ceils are, in fact, mixtures of one or two dominant, and many minor, populations which may vary enormously amongst themselves in adhesive capacity. This introduces many possibilites for the selection of certain adhesive phenotypes in response either to the pressure of receptor availability (if the receptor in question is an efficient one) or to immune pressure, depending on which variants the host has previously encountered. It is thus possible that the bulk adhesive properties of a culture will be both misleading (if a highly adhesive subpopulation is more pathologically important than the majority population) and unstable (since in response to selec-

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tive forces, very rapid alterations could occur from one parasite generation to the next) (Roberts et al., 1993). These observations impose further limitations on the interpretation of the studies of the adhesive properties of field isolates described above. The expression of the PfEMPl moiety is not the only erythrocyte surface modification made by the parasite. Electron-dense parasite-encoded histidinerich protein is exported to submembranous locations where it associates with the cytoskeleton to form structures called knobs (Trager et al., 1982). It is generally assumed that knobs act as sites for the presentation of at least some of the PfEMPl/adhesion molecules and there is immunolocalization data with immune sera, soluble CD36 and thrombospondin to support this view (Nakamura ef al., 1992). Certainly, the presentation of clusters of adhesive molecules would be expected to increase avidity and might make a critical difference to adhesion under flow conditions. It would seem that when presented on knobs, the PfEMPl molecules are associated with the cytoskeleton, as they are Triton-X100-insoluble.

Prospects

for intervention

It is a clear theoretical possibility that antiadhesive therapy will become realistic, though it will be essential first to identify the critical targets for intervention as outlined above. The principle is indicated both by the various mAb which effectively block adhesion and by data suggesting that soluble CD36 (Ockenhouse et al., 1989), soluble ICAM- or peptides derived from the ICAM-I sequence (based on the defined binding site) (Ockenhouse et al., 1992; Staunton er al., 1992) can all inhibit adhesion of laboratory lines of parasite. An additional approach has been taken by another group who have produced evidence that alterations in erythrocyte band 3 lead to the adhesive phenotype (Winograd and Sherman, 1989) and who have therefore produced peptides based on sequences in band 3 which appear to inhibit adhesion in a monkey model (of sequestration rather than disease) (Crandall et al., 1993). However, all these possibilities are far from clinically realizable in their present forms and if targets for intervention can be identified it will be necessary to develop small molecule inhibitors. The practicalities of this will clearly depend upon the degree of heterogeneity of adhesive mechanisms. Finally, efforts in this field would be greatly accelerated by definitive characterization not only of receptors on critical and specialized sites, such as cerebral endothelium, but also of the parasite-derived adhesins themselves, the structure-function, immunological and regulatory aspects of which will be of extreme interest. Progress in these areas would open up a wide range of potential therapeutic and preventive strategies, as well as

54rh adding vaccine

to the candidate components against this ancient scourge

FORUM

for the elusive of humanity.

1 would like to thank all my colleagues and collaborators in Oxford and the tropics, in particular Chris Newbold and Alister Craig, for support and stimulating discussions ; Shona Reed-Purvis for personal support ; and the Wellcome Trust, the Medical Research Council and the Lister Institute for Preventive Medicine for financial support. A.R.B. is a Lister Institute Research Fellow.

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