- Email: [email protected]
Cytoadhesion and Falciparum Malaria: Going with the flow
Reviews to designing not only protease inhibitors, but inhibitors of other aspects of T. cruzi metabolism that would be promising chemotherapeutic leads.
extracts of parasites; and (2) the fluorescent micrograph image confirmed the selectivity of the inhibitor for the enzyme in infected mammalian cells. Apparently, the inhibitor is efficiently taken up by the parasite, and insignificant quantities enter the lysosomal compartment where homologous host enzymes are present. Thus, even before we began seeking an inhibitor selective for cruzain, an irreversible cysteine protease inhibitor proved efficacious in blocking parasite replication because the parasite took it up selectively. In retrospect, this may not be surprising, considering the adaptation of T. cruzi to a life within a mammalian cell. Undoubtedly, it has evolved very effective scavenger mechanisms for taking up small molecules from host cell cytoplasm. If we can learn more about this pathway of uptake, perhaps this may be another route
References 1 Webber, S.E. et al. (1993) 1. Med. Chem. 36, 733-746 2 van Itzstein, M. et al. (1993) Nature 363,418-423 3 ,Bontempi, E. et al. (1984) Camp. Biochem. Physio2. 77B, 599-604 4 Cazzulo, J.J. et al. (1989) Mol. Biochem. Parasitol. 33, 33-42 5 Scharfstein, J. et al. (1986) J, Immuno2. 137, 1336-1341 6 Meirelles, M.N. et al. (1992) Mol. B&hem. Purusitol. 52, 175-184 7 Harth, G. et al. (1993) Mol. Biochem. Durusifol. 58,17-24 8 Sakanari, J.A. et al. (1989) Proc. Nutl Acad. Sci. USA 86,4863-4867 9 Eakin, A.E. et al. (1993) J. Biol. Chem. 268,6115-6118 10 McGrath, M.E. et al. J. Mol. Biol. (in press) 11 Meng, E.C. et al. (1992) J. Camp. Gem. 13,505-524 12 Ring, C.S. et al. (1993) Proc. Nutl Acud. Sci. USA 90, 3583-3587 13 McGinty, A. et al. (1993) Purasito2ogy 106,487-493 14 Campetella, 0. et al. (1992) Mol. Biochem. Parasitol. 50, 225-234
Cytoadhesion and Falciparum Malaria: Going with the Flow B.M. Cooke
and R.L. Coppel
Sequestration of parasitized red blood cells in the cerebral vasculature is the predisposing event to the development of cerebral malaria during infection with Plasmodium falciparum. The adhesive interaction between these cells and receptors on the endothelial cell (cytoadhesion) occurs in the dynamic environment of the microcirculation, but most studies have neglected this factor and have concentrated on measuring adhesion in static (nojlow) assays. Such studies ignore the markedly difieren t rheological properties of parasitized red blood cells that become apparent when adhesion is examined under dynamic, flow conditions that resemble those of the circulation in vivo. Here, Brian Cooke and Ross Coppel review a number of novel aspects of cytoadhesion that have been identified usingjow-based assays, and discuss their relevance to the pathophysiology, investigation and clinical management offalciparum malaria. It is only in the past 100 years that scientists have become aware that red blood cells parasitized by mature, pigmented forms (trophozoites and schizonts) of Plasmodium falciparum do not circulate in the peripheral blood, but become localized in the microvasculature of a variety of organs l. This phenomenon, known as sequestration, is believed to be critically important in the pathophysiology of falciparum malarial-5. The distribution of sequestered parasitized red blood cells (PRBC) correlates well with the type of severe malaria, so that histological examination of individuals with cerebral malaria, for example, reveals that the blood vessels in the brain of these individuals are preferentially packed with PRBC. Closer examination using electron microscopy shows that PRBC are directly attached to the vascular endothelial cells via minute, electron-dense
Brian Cooke and Ross Coppel are at the Department of MicrobIology, Monash Unlverslty. C ayton, Vlctorla 3 168, Australia. Tel: +6 I 3 990 54827, Fax: +61 3 990 5481 I, e-mail: [email protected]
protuberances called knobs, present on the surface of the PRBC. Over the past decade, a variety of molecules to which PRBC can adhere have been identified (Table 1). Moreover, these molecules have been detected on the surface of vascular endothelial cells in brains of humans who died from cerebral malariabJ, consistent with the idea that these receptors may bind to parasitized cells in the circulation, in vivo. Experimental systems Static assays. The occurrence of cytoadhesion in infected humans is not directly amenable to study and the interaction has, therefore, been examined using in z&o, in vivo and ex vivo modelss,9. Each model system attempts to simulate the interaction between PRBC and the endothelium (cerebral endothelial cells in particular), but none is a perfect model of the circulation in every respect. The in vivo and ex tiivo assays use either a cross-species mixture of parasite and host animal, or examine non-human malarias. In contrast, the in vitro assays have almost exclusively examined P. falciparum adhering to various human cell lines, most commonly C32 melanoma cells, human umbilical vein endothelial cells (HUVEC) or purified human endothelial cellexpressed proteins. It should be stressed that the majority of studies of adhesion have used a limited number of different laboratory-adapted parasite isolates. Studies of two strains in particular, FCR3 and ItG2 (which may even be the same strain) dominate the published literature and this may distort our understanding of the adhesive process. Adhesion has been most commonly investigated in static assays by allowing suspensions of PRBC to settle onto sub-confluent monolayers of cultured cells or purified, immobilized proteins in Petri dishes. After a period of incubation (typically 30-60min) during which the dishes are gently agitated (either continuously or periodically), non-adherent cells are removed by a ,54 .! d’:$:v )_
Porositology Today, voi. I I, no. 8, I995
Reviews Table I. Cells and proteins to which PRBC adhere in cytoadhesion assays” Static assay C32 melanoma cells Platelets Purified CD36 Transfected COS-7 cells Transfected CHO cells Microvascular endothelial cells U937 myelomonocytic cells
Refs 3 I ,35,36,57-62 63 34.42.47.58 I6,65 66 67 69.70
Flow-based assay C32 melanoma cells Platelets Purified CD36
Refs I2 28,33,64 28
Microvascular endothelial cells
26.68
HUVEC Purified/recombinant ICAM- I Transfected COS-7 cells Transfected CHO cells Human aortic endothelial cells Microvascular endothelial cells
16,35.59,61.71,72 6,34,73 I6 66 59 67
HUVEC Recombinant ICAM- I
I2,26,28,32,33 28
Microvascular endothelial cells
26.68
TSP
Purified TSP
47.74
Purified TSP
28
VCAM- I (CD 106)
Recombinant VCAM- I
6
Not tested
E-selectinb (CD62E)
Recombinant E-selectin
6
Not tested
Receptor CD36
ICAM- I (CD54)
’ Abbreviations: Cl-IO cells, Chinese hamster ovary cells; HUVEC,
human umbilical vein endothelial cells; ICAM- I, intercellular adhesion molecule(CD54); TSP, thrombospondin; VCAM- I, vascular cell adhesion molecule- I (CD 106) (CD numbers have only recently become available). b Formerly endothelial l&kocyte adhesion molecule- I (ELAM- I).
standardized washing procedure. Adherent cells are then counted, most commonly by direct microscopic observation of the dishes after staining with Giemsa. This type of assay is technically easy and inexpensive, and allows a relatively large number of assays to be carried out simultaneously. However, it is performed in a static environment that ignores the shear forces exerted on adherent cells by circulating blood, and which presumably affect the propensity of PRBC to localize in the venular circulation, in oivo~~‘~~~. An alternative form of static assav has been developed using small (2-6 km diameter) single or dual glass micropipettes, which allow individual cells to be manipulated into contact with other cells or proteinsI*. Once contact has been made between the PRBC and its target, the PRBC can subsequently be detached, permitting quantification of not only the proportion of cells in a population which possess the potential to adhere, but also the strength of the adhesive interaction once formed. Micromanipulative technology, however, is expensive to establish and the experimental techniques demand technical excellence from the operator. Measurements are tedious and time-consuming and, consequently, only a small number of parasitized cells in any one sample is usually measured. Flow-bused assays. While there is little doubt that the above-mentioned assays have made an important contribution to our understanding of cytoadhesion, they do not model the dynamic nature of the circulation, in viva, and they ignore numerous important questions concerning the qualitative and quantitative aspects of adhesion. The influence of the shear forces generated by the flow of blood in the vasculature must be considered in any attempt to assess the pathological importance of cytoadhesion. Attempts to solve the shortcomings of static assays have resulted in the development of assays using laboratory animals or in vitro systems that attempt to mimic in zko blood flow. We will briefly discuss the animal models of adhesion before focusing on an in rlifro flow model. Animal models. Natural infection by P. falciyarutn is unique to humans. Plasmodi~~m_falciparumartificially inPormtolo~
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oculated into New World monkeys such as Aotus and Saimiri has been shown to cytoadhere via knobs to monkey endothelium in various organs, but sequestration in the brain and associated cerebral pathology do not occur to the same extent as in human cerebral malarial3,*4. The best available primate model of human cerebral malaria appears to be P. fragile-infected rhesus monkeys 15, but no specific data on adhesive interactions are yet available. An ex zlizv model comprising an isolated, acutely drnervated rat mesoappendix that can be artificially perfused with PRBC is an animal model in which the interaction between P. falcipavum-parasitized cells and the vascular intima has been visualized under flowlo. In this system, P. falciparum-infected PRBC do adhere to rat microvascular endothelium, suggesting some commonality of structure of endothelial cell-expressed receptors. Increased flow resistance correlated well with the occurrence of sequestration of knob-positive PRBC and consequent microvasculature occlusion. Adhesion occurred predominantly in post-capillary venules in keeping with post-mortem findings from humans who died from falciparum malaria, although capillary obstruction was secondary to occlusion of venules. It is likely that adhesion was mediated via receptors other than intercellular adhesion molecule-l (ICAM-1) in this model, since the parasite strain used (FCR3A2) cannot bind to this receptor, at least when tested several years after the original study (Ref. 16, and B.M. Cooke and G.B. Nash, unpublished). The number of adherent cells was not quantitated in these studies, nor were local flow conditions known or varied systematically. Soluble thrombospondin (TSP) and rabbit anti-T%’ antibodies were shown to inhibit adhesion of P. falciparum-infected PRBC to the rat venular endothelium and reduced the effect on flow resistanceIT. These results are at variance with those performed using in zlitvo flow-based systems in which TSP is a poor receptor for immobilization of PRBC under flow conditions (see below). The cheek pouches of golden hamsters infected with P. beqhei can also be studied in a similar manner but the sequence of events and cell types adhering is 283
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Fig. I. Bright-field video-photomicrograph of human red blood cells, parasitized by mature forms of Plasmodium fikiparum, adhering to cultured endothelial cells under flow conditions. The arrow indicates the direction of flow. Scale bar = IO Fm.
different to that seen in humans, limiting the utility of this system as a model for human falciparum malaria*s. Although interesting results have been obtained with these animal models, they have their inadequacies, such as the inability to achieve complete control of the flow conditions and the inability to modulate expression of endothelial cell-expressed receptors. In vitro j7ow-based assays. In vitro flow-based assays permit the qualitative and quantitative aspects of adhesive interactions to be studied over a range of wall shear stresses that reflect the levels proposed to exist in the microcirculation in vivo*%20. For example, it is possible to observe flowing cells directIy and determine the range of wall shear stresses over which PRBC are capable of forming an adhesive contact with endothelial cells or a variety of immobilized proteins. Similarly, once cells have adhered, it is possible to test their ability to withstand detachment by exposing them to stepwise increases in shear stress, and thereby derive a measure of the strength of the adhesive bonds, allowing the quantitative estimation of adhesive force.+. Adhesion of flowing cells to the endothelium depends not only on the affinity/avidity between a receptor and its cognate ligand, but also on the kinetics of the interaction, ie. receptor/ligand on/off rates22. Additionally, the ability of a flowing cell to form an adhesive contact and its ability to withstand detachment once formed are likely to be critically important parameters, in vizTo, but they may have little influence on adhesion under static conditions. Surprisingly, flow284
based investigations had been restricted to the study of adhesion of white blood cells, particularly neutrophils23, and sickle red blood cells?,~, and have only recently been extended to include PRBC (see Fig. 1 and Box 1). Under carefully controlled laminar flow conditions using cell-coated chambers with parallel-plate geometry, investigators have shown that PRBC are able to adhere to endothelial cells at wall shear stresses within the physiological range for venulesQ26. If efficiency of adhesion is defined as the percentage of PRBC that adhere during passage over the endothelial cells, then the maximum efficiency observed for PRBC is 1.5%: over 1000 times greater than for non-parasitized cellsu. Once bound, a proportion of the PRBC could subsequently resist detachment when exposed to stresses one order of magnitude higher than those encountered in the venular circulation. Probably the best-understood cellular adhesive process is the way in which leukocytes emigrate from the circulation as part of the inflammatory response. Different stages of this well-orchestrated, stepwise process are mediated by different receptor-ligand combinations and the adhesive phenomena are strongly influenced by the level of applied flow stress2sJr. After making contact with the endothelium, leukocytes first roll, mediated by vascular and leukocyte selectins until they become activated by chemotactic factors or cytokines such as interleukin 8 (IL-8) or platelet-activating factor. Once activated, the leukocytes become immobilized and form stable and stationary interactions with molecules such as ICAM- before moving out into the tissues to execute their function. When PRBC are flowed over either cells or purified proteins, the different endothelial cell-expressed receptors for PRBC also operate with different efficiencies and mediate characteristically different types of adhesion*s. When adhesion to ICAM-1, CD36 or TSI? was measured over a range of physiologically relevant wall shear stresses, the number of PRBC adhering decreased with increasing shear stress but with consistent differences in adhesion to the different receptors (Fig. 2). In terms of the absolute number of PRBC binding at a single stress, adhesion was in the order CD36>ICAM-l>TSP. Adhesion via ICAM- was less sensitive to increasing stress than adhesion to CD36 while TSP was the most shear sensitive and least efficient receptor. Furthermore, in contrast with ICAM- or CD36 mediated binding, adhesion to TSP was not long-lasting, but transient. Taken together, these results raise doubt about the relevance of TSP as a receptor for PRBC, in vivo. There were also striking differences in the nature of the interaction between PRBC and different endothelial cell-expressed receptors. While the majority of adherent PRBC that adhered to CD36 remained stationary, those adhering to ICAMrolled continuously. The velocity at which PRBC rolled appeared to be inversely related to the concentration of ICAM-1, an observation that may be of pathophysiological significance. Increased expression of ICAMinduced by inflammatory cytokines has previously been shown to increase endothelial adhesion of PRBC in vitrol6, and thus it has been suggested that tumor necrosis factor alpha (TNF-o) may promote cytoadhesion in viz+. Serum levels of TNF-ol are consistently elevated during acute malaria infection and correlate with the level of peripheral parasitaemia and severity Porasrtology Today, vol.
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Reviews Box 1. Assays that Measure Adhesion in vitro Schematic representation (Fig. right) of an in vitro assay to Video visualize and quantify adhesion of flowing PRBC to endom,croscope thelial cells, platelets or purified, immobilized proteins. Cell-free Cell A prerequisite for accurate quantification of adhesion buffer suspension under flow conditions is well-defined, reproducible flow. Laminar flow is perhaps the only state of flow that can be produced in a controlled manner with any accuracy within cylindrical tubes or between two parallel plates. Fluid flowing between two such plates moves as a series of thin layers or laminae. The laminae alone the central axis of the channel MIcroslide 01 move with a greater veloci”9 than those near the walls, flow-chamber thus the leading edge of the flowing fluid assumes a para\ bolic profile. Each individual lamina slides or shears over the ones juxtaposing and is, therefore, subjected to a tangential force called the shear stress. When flow occurs in such parallel laminae that do not mix it is termed laminar flowis. I Harvard The flow assembly is either a parallel-plate flow chamsyringepump Microscope stage beG** or a flat, glass microcapillary tube (microslide)“h with rectangular cross-section and well-defined dimensions. The flow chamber described here comprises two flat plates separated by a thin (-150 pm) plastic gasket into which a rectangular channel (50mm x 90 mm) has been cut. The lower of the two plates is a glass or plastic slide onto which monolayers of HUVEC or C32 melanoma cells can be cultured, or a plastic slide onto which discrete spots of purified proteins such as CD36, ICAM- or TSP can be adsorbed 1228.The upper plate is a glass slide or piece of perspex into which inlet and outlet ports have been drilled to direct flow into and out of the channel. The plates are clamped together, and then placed on the stage of a microscope. Alternatively, the flow assembly may be a microslide with one internal surface pre-coated with confluent monolayers of cultured HUVEC or human blood platelet+%. Microslides have several advantages over parallel-plate flow chambers. They are simpler to assemble and ideal for field studies of adhesion of clinical isolates32,3-?. The flow chamber, or microslide, is placed on the stage of a microscope and is observed under video-microscopy whilst a suspension of PRBC of known concentration or cell-free buffer is flowed over at controlled flow rate (Q), chosen to obtain a desired wall shear stress (r), calculated from the width (~1) and the height (h) of the flow channel and the viscosity of the flowing medium (77)using the equation%
Adhesion can be quantified by counting adherent PRBC by direct microscopic observation. Alternatively, video recordings of individual experiments can be made and analysed retrospectively with the aid of computerized-image processing (Fig. 1). Adhesion can be characterized as rolling or stationary and the proportion of cells which remain stationary or which roll, along with their velocities, can also be calculated. The cells commonly used in flow-based adhesion studies are HUVEC (ICAM-1) and platelets (CD36), as C32 melanoma cells show a tendeticy to ‘pile-up’ in some areas, causing perturbation in the laminar flow profile.
of disease, and high levels have been associated with a poor prognosism. The precise role of TNF-cy in the pathogenesis of cerebral malaria is not known-5. However, the increased level of expression of ICAMdetected on endothelial cells in the cerebral microvasculature of individuals with cerebral malaria6 would be expected to increase levels of PRBC adhesion and reduce the velocity at which PRBC roll along vessel walls. Both these effects would tend to promote sequestration in uivo, either through ICAM- alone or in concert with other receptors. Rolling of PRBC can, therefore, be likened to the rolling adhesion seen when marginating leukocytes first interact with stimulated endothelium. The fact that neutrophils form stationary interactions with ICAM-I (Ref. 27) (whereas PRBC roll on this molecule) suggests that rolling is a function of the nature of a specific receptor-ligand pair, and not peculiar to ICAM- or the vascular selectins which mediate neutrophil rolling. These findings are consistent with the idea that adhesion of PRBC may be a multi-step process, analogous with neutrophil adhesion, in which initial adhesion to ICAMat high wall shear stress adequately slows down PRBC so that they can become immobilized via CD36 and possibly other receptors. At lower wall shear stresses, however, it appears that CD36 alone is capable ParasrtoioS~v To&v.
LOI
1: ‘-1:3
2’
Range proposed to exist in venules
) Increasing
wall shear stress
Fig. 2. Flow curves showing the relative ability of different receptors to bind PRBC at different wall shear stresse9. The y axis, percentage of adherent PRBC. is that relative to the percentage of PRBC adhering at the lowest wall shear stress tested. The range of wall shear stress proposed to exist in venules’9,20 is indicated.
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Reviews of immobilizing flowing PRBC without the assistance of ICAM-1. Flowbased studies of clitlical isolates. It is essential to know whether or -not clinical isolates act in the same manner as long-term laboratory-cultured isolates. Furthermore, the study of clinical isolates may provide information as to whether the severity of falciparum malaria is related to the capacity of PRBC to cytoadhere3i. The adhesive properties of PRBC taken from individuals with mild (uncomplicated) or severe malaria have been tested using static assays with both cells and immobilized proteins as the adhesive target, yet no correlation has ever been detected between the degree of adhesiveness of PRBC and the occurrence of cerebra1 malaria. Using flow assays, field studies have clearly shown that red blood cells parasitized by wild-type P. falciparum (obtained directly from patients and cultured in zCtro to the trophozoite stage) were able to adhere to HUVEC and platelets at physiologically relevant wall shear stress and that the adhesive interaction to the two cell types was mediated predominantly by ICAM- and CD36, respectivelyQ33. The rolling-type interaction with ICAMalso occurs and is, therefore, not an artefact induced bv in vitro parasite culture. The inherent adhesiveness of wild isolates was highly variable, even after correction for differences in parasitaemia, and there was no significant correlation in the capacity of individual isolates to bind to these two receptors. However, in keeping with previous studies using static assay+Js, PRBC bound to CD36 at a greater level, on average, than to ICAM-1. From the studies reported to date, there appears to be no simple relationship between cerebral malaria and adhesion of PRBC to ICAMor CD36, although the ability to bind to the latter might correlate with major organ pathology in more severe disease36. Inhibition and reversal of adhesion underjlow. The relative avirulence of non-cytoadherent strains of parasitesJ7 suggests that inhibition of binding, in viva, mav be a useful therapeutic intervention to prevent or assuage the severe complications of the disease. Cytoadhesion can be inhibited in vitro by immune serum or antibody38mQ. Furthermore, the dramatic demonstration that immune serum41 or, more recently, synthetic peptides”3 could reverse cytoadhesion, in uivo, when infused into monkeys suggests that the administration of various agents, such as anti-adhesion antibodies, soluble adhesion receptors or rheologically active drugs could also be used. The in z!itro flow assay is a particularly valuable method for assessing potential efficacy of new therapies, as it provides quantitative data about the level of shear stress at which adhesion may be lost or reversed. For example, compounds that interfere with adhesion only at high shear stress would be expected to be of little value. The data available on physiological relevance of receptors are also crucial in determining the potential therapeutic targets, as is the case for TSP. Monoclonal antibodies and antibody agents would appear to be most profitably directed against the receptor on the endothelial cell, as the structure is apparently highly conserved between individual humans. Such antibodies could bind directly to the receptor itself, or may bind to a site nearby and inhibit cytoadhesion by steric hindrance. Antibodies against several receptors may be required for full therapeutic effect. The parasite-encoded ligand on the PRBC, currently believed to 286
be P. falcipamrn erythrocyte membrane protein-l (PfEMP-I), is a highly variable molecule capable of antigenic variation and, as such, is an unfavourable target for antibody therapy. The ability of antigenically distinct forms of PfEMP-1 to bind a single receptor would imply that there are conserved regions of the protein that could be potential targets for antibody, but there are no reports of monospecific antibody reagents capable of recognizing many distinct antigenic forms of PfEMP-1. Flow-based studies have examined the effect of antiCD36 monoclonal antibody on adhesion of PRBC to activated platelets 28. Pre-incubation of the platelet monolayer with the antibody reduced adhesion by greater than 98%. This occurred even though activated platelets express large amounts of surface available TSP, confirming the relative unimportance of TSP as a receptor under flow conditions. It has been suggested that parasite-induced modifications to native red blood cell surface proteins, such as Band 3, may enhance the ability of PRBC to cytoadhere44. It is not clear whether such changes increase the adhesiveness of PRBC nonspecifically or whether a specific receptor-ligand interaction is involved; however, the capacity of different PRBC to bind specifically to different receptors may favour the former possibility 45. Using a static assay, Crandall and co-workers have reported the inhibition of binding of PRBC to melanoma cells, by the addition of synthetic peptides based on the Band 3 sequence43. Such peptides may be useful in decreasing adhesiveness of PRBC and perhaps lessening the severity of disease caused by sequestration. It is also possible that compounds that have been shown to be of benefit in decreasing adhesive interactions in other conditions such as sickle cell disease may have a place in malaria therapy. No such data from flow-based assays are yet published. However, preliminary studies (performed under flow conditions) have indicated that two such compounds, a non-ionic co-polymer surfactant and a dimethyl xanthine derivative, oxpentifylline, are unlikely to have any antiadhesive effect on PRBC at pharmacological concentrations (B.M. Cooke and G.B. Nash, unpublished). Rosetting PRBC can also adhere strongly to one or more nonparasitized red blood cells, forming rosette+. After a brief period of iu vitro culture, clinical isolates show a varying degree of rosetting, with zero to 70% of trophozoites forming rosettes 47-49.The level of rosette formation iiz zlitro has been shown to be associated with the occurrence of cerebral malaria in studies performed in The Gambia4yJ0, but not in Thailand48. Certainly, aggregates of parasitized and non-parasitized cells, hypothesized to be rosettes, have been reported to be present in the cerebra1 vasculature of individuals who had died from cerebra1 malariasi. The precise importance of rosetting in the pathophysiology of malaria remains unclear, but will probably depend on the phenotype of the parasite and the density and nature of endothelial cell-expressed receptors. Perfusion of a rosetting strain of P. falciparum through the ex vivo rat mesoappendix preparation indicated that rosettes localize preferentially in the venules where they could occlude flowj2. It has recently been shown that rosettes can pass into glass micropipettes with a hiJS!tOiogy
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Reviews diameter similar to that of small capillaries without disruption of the rosettes”. Moreover, when sheared in a cone-plate viscometer, rosettes were able to withstand stresses typical of those in arteries for minutes without becoming disaggregated, suggesting that rosettes could survive intact in the circulation in vivu. In a flow-based study of clinical isolates, rosettes were found attached to cultured HUVEC, suggesting that either pre-formed rosettes can adhere or that rosettes can form under flow32. In either case, these studies support the hypothesis that rosetting could occur in ZI~PO and contribute to vascular obstruction in malaria. Does rosetting interfere with flow-based assays and thereby invalidate our interpretation of the adhesive properties of PRBC? Using a static assay, it has been noted that the level of adhesion of clinical isolates to C32 melanoma cells was inversely related to the level of rosettinp. Similarly, the level of adhesion of a laboratory-adapted strain of P. falciparum to C32 melanoma cells or CD36 was higher if rosettes were first disrupted with heparin 9. Such a reduction in the level of adhesion may be explained by a decrease in PRBC available to cytoadhere or physical hindrance by non-parasitized cells and is also likely to occur in flow-based assays. However, there is no evidence to suggest that rosetting alters parameters such as the strength or the nature of the adhesive interaction. Future directions The availability of flow-based assays for use in the laboratory and in the field now allows a large number of questions about cytoadhesion in malaria to be studied quantitatively. For example, there are now cytoadherent parasites available that do not express some of the accessory red blood cell membrane, parasite-encoded molecules such as knob-associated histidine-rich protein (KAHRP) or mature erythrocyte surface antigen (MESA). The contribution of these molecules to strengthening the adhesive interaction can now be assessed. Similarly, there are parasite lines that express multiple ligands or that have been selected for high levels of adhesion. Quantification of binding of these lines to individual receptors or receptor mixtures would be of great interest. Questions concerning the endothelial cell such as the relative binding efficiencies of microvascular endothelial cells from a variety of different human organs or the physiological importance of other potential receptors such as vascular cell adhesion molecule-l (VCAM-1) or E-selectin can also now be studied. Studies that mimic the combinations of acute- or chronic-phase reactants likely to be present in individuals infected with malaria have also not yet been performed. Finally, in terms of clinical intervention and disease management using anti-adhesion therapy, flow-based methodology is well suited for investigation of agents with putative anti-adhesive potential and can be adapted to screen for ability to inhibit or reverse adhesion. Acknowledgements This work IS supported by the Natlonal lnstltutes of Health grant number DK32094-I 0 and the NatIonal Health and MedIcal Researcl Council. The authon would llke to acknowledge the guidance and expert advlce of Gerard Nash at the Unlverslty of Blrmngham, UK V. the development of the IR vrtro flow-based assays and analysts of cell adhesion revlewed In this paper.
References 1 MacPherson, G.G. et al. (1985) Am. I. Puthol. 119,385-401 2 Berendt, A.R. ef al. (1994) Parhitolo~y 108, S19-S28 3 Grau, GE. and Dekossodo, S. (1994) Parasitology Today 10,408-409 4 Berendt, A.R. et al. (1994) Parasitology Today 10,412-414 5 Clark, LA. and Rockett, K.A. (1994) Parasitology Today 10,410-412 CF. et al. (1992) 1. Exp. Med. 176,1183-1189 6 Ockenhouse, 7 Turner, G.D.H. et ~1.(1994) Am. 1. Pathol.145,1057-1069 A.D. (1989) Blood 74,2603-2618 8 Howard, R.J. and Gilladoga, 9 Udeinya, I.J. (1990) Am. J. Trap. Med. Hyg. 43, 2-5 C. et al. (1985) Proc. Nutl Acad. Sci. USA 82, 10 Raventos-Suarez, 3829-3833 11 Kaul, D.K. et al. (1994) Am. J. Trap. Med. Hyg. 50,512-521 12 Nash, G.B. ef al. (1992) Blood 79,798-807 13 Miller, L.H. (1969) Am. J. Trap. Med. Hyg. l&860-865 14 Whiteley, H.E. et al. (1987) Am. J. Trop. Med. Hyg. 37, l-8 15 Fujioka, H. et nl. (1994) Exp. Purasitol. 78,371-376 16 Berendt, A.R. et al. (1989) Nature 341, 57-59 17 Rock, E.P. et ul. (1988) Blood 71, 71-75 18 Franz, D.R. et al. (1987) Am. J. Trap. Med. Hyg. 36,474-480 19 Chien, S. (1972) in Hemodi2ution: Theoreticnl Basis and C2inicul Application (Messmer, K. and Schmid-Schonbein, H., eds), pp l-45, Karger 20 Turitto, V.T. (1982) Prog. Hemostasis Thromb. 6, 139-177 21 Forrester, J.V. and Lackie, J.M. (1984) 1. Cell Sci. 70, 93-110 22 Williams, A.F. (1991) Nature 352,473-474 23 Godin, C. et al. (1993) J. Cell Sri. 106,441-452 24 Barabino, G.A. et al. (1987) Blood 70,152-157 23 Rowland, P.G. et al. (1993) 1. Lab. C/in. Med. 121, 706-713 26 Wick, T.M. and Louis, V. (1991) Am. 1. Trap. Med. Hyg. 45,578-586 27 Kishimoto, T.K. (1991) J. NZH Res. 3, 75-77 78 Cooke, B.M. et al. (1994) Br. J. Huemutol. 87, 162-170 29 Berendt, A.R. et al. (1990) Purusitoloa Today 6,247-254 D. (1990) Zmmunol. Left. 25,213-216 30 Kwiatkowski, 71 Marsh, K. et al. (1988) Exp. Purusitol. 65, 202-208 107,359-368 32 Cooke, B.M. et al. (1993) hrusifology 33 Cooke, B.M. et al. Am. 1. Troa. Med. Hva. (in Dress) 34 Ockenhouse, C.F. et ul.‘(199i) J. Infect:&. l&, 183-169 I’. et al. (1993) Infect. Immun. 61,5198-5204 35 Ringwald, 36 Ho, M. ef n[. (1991) Infect. Immun. 59, 873-878 37 Langreth, S.G. and Peterson, E. (1985) Infect. Immun. 47, 760-766 38 Udeinva, I.J. et al. (1983) Nature 303, 429-431 39 Leech;J.H. et al. (l&34) i. Exp. Med. 159, 1567-1575 40 Sineh. B. et al. (1988) Cliri. Exa. Immunol. 72, 145-150 31 Da%, P.H. et al. (1983) Proc.‘Nutl Acud. SCZ:USA 80,5075-5079 CF. et al. (1989) Science 243, 1469-1471 42 Ockenhouse, -13 Crandall, I. et al. (1993) Proc. Nutl Acud. Sci. USA 90,4703-4707 14 Crandall, I. and Sherman, I.W. (1994) Purusitology 108,257-267 45 Crandall, I. ef al. (1994) Exp. Purusitol. 78,203-209 M. et al. (1994) Purusitology Today 10, 73-79 -lb Wahlgren, 47 Hasler, T. et al. (1990) Blood 76, 1845-1852 48 Ho, M. et al. (1991) Infect. Immun. 59, 2135-2139 4’) Treutiger, C.J. ef al. (1992) Am. 1. Trap. Med. Hy,q 46, 503-510 50 Carlson, J. et al. (1996) Lancet 336.1457-1460 __ 51 Rieanti, M. et al. (1990) Immunol. Left. 25, 199-205 78,812-819 52 K&l, d.K. et al. (i99ljBlood 53 Nash, G.B. et al. (1992) Br. J. Huemafol. 82, 757-763 54 Handunnetti, S.M. et al. (1992) Infect. Immun. 60, 928-932 55 Matrai, A. et ul. (1987) in Clinical Hemorheology. Applications in Curdiovuscular and Hemafologicul Disease, Diabetes, Surgery and Gynecology (Chien, S. et al., eds), pp 9-71, Martinus Nijhoff 56 Cooke, B.M. et al. (1993) Microvusc. Res. 45,33-45 J.W. ef al. (1985) J. Immunol. 135, 3494-3497 57 Barnwell, J.W. ct al. (1989) J. C/in. best. 84, 765-772 58 Barnwell, 59 Schmidt, J.A. ef al. (1982) J. Clin. Invest. 70,379-386 R. et al. (1989) Nufure 338, 763-765 60 Udomsangpetch, 61 Udeinya, I.J. et al. (1983) Exp. Purusifol. 56, 207-214 62 Sherman, I.W. and Valdez,‘E. (1989) Pmasitology 98,359-369 CF. ef al. (19891 1. Clin. Invest. 84,468-475 63 Ockenhouse, 64 Cooke, B.M..and Nash, ‘G.B. ii995) Exp. Purasitol. 80, 116-123 6.5 Oquendo, I’. et al. (1989) Cell 58, 95-101 66 Hasler. T. et al. (1993) Am. J. Trap. Med. Hyg. 48, 332-347 67 Smith, H. et al. (1992) Exp. Purusitol. 75,269-280 hX Johnson, J.K. et al. (1993) J. Infect. Dis. 167, 698-703 69 Ockenhouse, C.F. and Chulay; J.D. (1988) J. Infecf. Dis. 157,584-588 70 Goldrine. I.D. ef al. (1992) Br. I. Huernutol. 3,413-418 71 Udeinya, I.J. et al. (1$81) icier&e 213, 555-557 72 Udeinya, I.J. ef al. (1985) J. Protozool. 32,88-90 73 Ockenhouse, C.F. et al. (1992) Cell 68, 63-69 74 Jioberts, D.D. of al. (1985) Nature 318, 64-66
extracts of parasites; and (2) the fluorescent micrograph image confirmed the selectivity of the inhibitor for the enzyme in infected mammalian cells. Apparently, the inhibitor is efficiently taken up by the parasite, and insignificant quantities enter the lysosomal compartment where homologous host enzymes are present. Thus, even before we began seeking an inhibitor selective for cruzain, an irreversible cysteine protease inhibitor proved efficacious in blocking parasite replication because the parasite took it up selectively. In retrospect, this may not be surprising, considering the adaptation of T. cruzi to a life within a mammalian cell. Undoubtedly, it has evolved very effective scavenger mechanisms for taking up small molecules from host cell cytoplasm. If we can learn more about this pathway of uptake, perhaps this may be another route
References 1 Webber, S.E. et al. (1993) 1. Med. Chem. 36, 733-746 2 van Itzstein, M. et al. (1993) Nature 363,418-423 3 ,Bontempi, E. et al. (1984) Camp. Biochem. Physio2. 77B, 599-604 4 Cazzulo, J.J. et al. (1989) Mol. Biochem. Parasitol. 33, 33-42 5 Scharfstein, J. et al. (1986) J, Immuno2. 137, 1336-1341 6 Meirelles, M.N. et al. (1992) Mol. B&hem. Purusitol. 52, 175-184 7 Harth, G. et al. (1993) Mol. Biochem. Durusifol. 58,17-24 8 Sakanari, J.A. et al. (1989) Proc. Nutl Acad. Sci. USA 86,4863-4867 9 Eakin, A.E. et al. (1993) J. Biol. Chem. 268,6115-6118 10 McGrath, M.E. et al. J. Mol. Biol. (in press) 11 Meng, E.C. et al. (1992) J. Camp. Gem. 13,505-524 12 Ring, C.S. et al. (1993) Proc. Nutl Acud. Sci. USA 90, 3583-3587 13 McGinty, A. et al. (1993) Purasito2ogy 106,487-493 14 Campetella, 0. et al. (1992) Mol. Biochem. Parasitol. 50, 225-234
Cytoadhesion and Falciparum Malaria: Going with the Flow B.M. Cooke
and R.L. Coppel
Sequestration of parasitized red blood cells in the cerebral vasculature is the predisposing event to the development of cerebral malaria during infection with Plasmodium falciparum. The adhesive interaction between these cells and receptors on the endothelial cell (cytoadhesion) occurs in the dynamic environment of the microcirculation, but most studies have neglected this factor and have concentrated on measuring adhesion in static (nojlow) assays. Such studies ignore the markedly difieren t rheological properties of parasitized red blood cells that become apparent when adhesion is examined under dynamic, flow conditions that resemble those of the circulation in vivo. Here, Brian Cooke and Ross Coppel review a number of novel aspects of cytoadhesion that have been identified usingjow-based assays, and discuss their relevance to the pathophysiology, investigation and clinical management offalciparum malaria. It is only in the past 100 years that scientists have become aware that red blood cells parasitized by mature, pigmented forms (trophozoites and schizonts) of Plasmodium falciparum do not circulate in the peripheral blood, but become localized in the microvasculature of a variety of organs l. This phenomenon, known as sequestration, is believed to be critically important in the pathophysiology of falciparum malarial-5. The distribution of sequestered parasitized red blood cells (PRBC) correlates well with the type of severe malaria, so that histological examination of individuals with cerebral malaria, for example, reveals that the blood vessels in the brain of these individuals are preferentially packed with PRBC. Closer examination using electron microscopy shows that PRBC are directly attached to the vascular endothelial cells via minute, electron-dense
Brian Cooke and Ross Coppel are at the Department of MicrobIology, Monash Unlverslty. C ayton, Vlctorla 3 168, Australia. Tel: +6 I 3 990 54827, Fax: +61 3 990 5481 I, e-mail: [email protected]
protuberances called knobs, present on the surface of the PRBC. Over the past decade, a variety of molecules to which PRBC can adhere have been identified (Table 1). Moreover, these molecules have been detected on the surface of vascular endothelial cells in brains of humans who died from cerebral malariabJ, consistent with the idea that these receptors may bind to parasitized cells in the circulation, in vivo. Experimental systems Static assays. The occurrence of cytoadhesion in infected humans is not directly amenable to study and the interaction has, therefore, been examined using in z&o, in vivo and ex vivo modelss,9. Each model system attempts to simulate the interaction between PRBC and the endothelium (cerebral endothelial cells in particular), but none is a perfect model of the circulation in every respect. The in vivo and ex tiivo assays use either a cross-species mixture of parasite and host animal, or examine non-human malarias. In contrast, the in vitro assays have almost exclusively examined P. falciparum adhering to various human cell lines, most commonly C32 melanoma cells, human umbilical vein endothelial cells (HUVEC) or purified human endothelial cellexpressed proteins. It should be stressed that the majority of studies of adhesion have used a limited number of different laboratory-adapted parasite isolates. Studies of two strains in particular, FCR3 and ItG2 (which may even be the same strain) dominate the published literature and this may distort our understanding of the adhesive process. Adhesion has been most commonly investigated in static assays by allowing suspensions of PRBC to settle onto sub-confluent monolayers of cultured cells or purified, immobilized proteins in Petri dishes. After a period of incubation (typically 30-60min) during which the dishes are gently agitated (either continuously or periodically), non-adherent cells are removed by a ,54 .! d’:$:v )_
Porositology Today, voi. I I, no. 8, I995
Reviews Table I. Cells and proteins to which PRBC adhere in cytoadhesion assays” Static assay C32 melanoma cells Platelets Purified CD36 Transfected COS-7 cells Transfected CHO cells Microvascular endothelial cells U937 myelomonocytic cells
Refs 3 I ,35,36,57-62 63 34.42.47.58 I6,65 66 67 69.70
Flow-based assay C32 melanoma cells Platelets Purified CD36
Refs I2 28,33,64 28
Microvascular endothelial cells
26.68
HUVEC Purified/recombinant ICAM- I Transfected COS-7 cells Transfected CHO cells Human aortic endothelial cells Microvascular endothelial cells
16,35.59,61.71,72 6,34,73 I6 66 59 67
HUVEC Recombinant ICAM- I
I2,26,28,32,33 28
Microvascular endothelial cells
26.68
TSP
Purified TSP
47.74
Purified TSP
28
VCAM- I (CD 106)
Recombinant VCAM- I
6
Not tested
E-selectinb (CD62E)
Recombinant E-selectin
6
Not tested
Receptor CD36
ICAM- I (CD54)
’ Abbreviations: Cl-IO cells, Chinese hamster ovary cells; HUVEC,
human umbilical vein endothelial cells; ICAM- I, intercellular adhesion molecule(CD54); TSP, thrombospondin; VCAM- I, vascular cell adhesion molecule- I (CD 106) (CD numbers have only recently become available). b Formerly endothelial l&kocyte adhesion molecule- I (ELAM- I).
standardized washing procedure. Adherent cells are then counted, most commonly by direct microscopic observation of the dishes after staining with Giemsa. This type of assay is technically easy and inexpensive, and allows a relatively large number of assays to be carried out simultaneously. However, it is performed in a static environment that ignores the shear forces exerted on adherent cells by circulating blood, and which presumably affect the propensity of PRBC to localize in the venular circulation, in oivo~~‘~~~. An alternative form of static assav has been developed using small (2-6 km diameter) single or dual glass micropipettes, which allow individual cells to be manipulated into contact with other cells or proteinsI*. Once contact has been made between the PRBC and its target, the PRBC can subsequently be detached, permitting quantification of not only the proportion of cells in a population which possess the potential to adhere, but also the strength of the adhesive interaction once formed. Micromanipulative technology, however, is expensive to establish and the experimental techniques demand technical excellence from the operator. Measurements are tedious and time-consuming and, consequently, only a small number of parasitized cells in any one sample is usually measured. Flow-bused assays. While there is little doubt that the above-mentioned assays have made an important contribution to our understanding of cytoadhesion, they do not model the dynamic nature of the circulation, in viva, and they ignore numerous important questions concerning the qualitative and quantitative aspects of adhesion. The influence of the shear forces generated by the flow of blood in the vasculature must be considered in any attempt to assess the pathological importance of cytoadhesion. Attempts to solve the shortcomings of static assays have resulted in the development of assays using laboratory animals or in vitro systems that attempt to mimic in zko blood flow. We will briefly discuss the animal models of adhesion before focusing on an in rlifro flow model. Animal models. Natural infection by P. falciyarutn is unique to humans. Plasmodi~~m_falciparumartificially inPormtolo~
Todav ,voiI
/ ns 8. ; Wi
1
oculated into New World monkeys such as Aotus and Saimiri has been shown to cytoadhere via knobs to monkey endothelium in various organs, but sequestration in the brain and associated cerebral pathology do not occur to the same extent as in human cerebral malarial3,*4. The best available primate model of human cerebral malaria appears to be P. fragile-infected rhesus monkeys 15, but no specific data on adhesive interactions are yet available. An ex zlizv model comprising an isolated, acutely drnervated rat mesoappendix that can be artificially perfused with PRBC is an animal model in which the interaction between P. falcipavum-parasitized cells and the vascular intima has been visualized under flowlo. In this system, P. falciparum-infected PRBC do adhere to rat microvascular endothelium, suggesting some commonality of structure of endothelial cell-expressed receptors. Increased flow resistance correlated well with the occurrence of sequestration of knob-positive PRBC and consequent microvasculature occlusion. Adhesion occurred predominantly in post-capillary venules in keeping with post-mortem findings from humans who died from falciparum malaria, although capillary obstruction was secondary to occlusion of venules. It is likely that adhesion was mediated via receptors other than intercellular adhesion molecule-l (ICAM-1) in this model, since the parasite strain used (FCR3A2) cannot bind to this receptor, at least when tested several years after the original study (Ref. 16, and B.M. Cooke and G.B. Nash, unpublished). The number of adherent cells was not quantitated in these studies, nor were local flow conditions known or varied systematically. Soluble thrombospondin (TSP) and rabbit anti-T%’ antibodies were shown to inhibit adhesion of P. falciparum-infected PRBC to the rat venular endothelium and reduced the effect on flow resistanceIT. These results are at variance with those performed using in zlitvo flow-based systems in which TSP is a poor receptor for immobilization of PRBC under flow conditions (see below). The cheek pouches of golden hamsters infected with P. beqhei can also be studied in a similar manner but the sequence of events and cell types adhering is 283
Reviews
Fig. I. Bright-field video-photomicrograph of human red blood cells, parasitized by mature forms of Plasmodium fikiparum, adhering to cultured endothelial cells under flow conditions. The arrow indicates the direction of flow. Scale bar = IO Fm.
different to that seen in humans, limiting the utility of this system as a model for human falciparum malaria*s. Although interesting results have been obtained with these animal models, they have their inadequacies, such as the inability to achieve complete control of the flow conditions and the inability to modulate expression of endothelial cell-expressed receptors. In vitro j7ow-based assays. In vitro flow-based assays permit the qualitative and quantitative aspects of adhesive interactions to be studied over a range of wall shear stresses that reflect the levels proposed to exist in the microcirculation in vivo*%20. For example, it is possible to observe flowing cells directIy and determine the range of wall shear stresses over which PRBC are capable of forming an adhesive contact with endothelial cells or a variety of immobilized proteins. Similarly, once cells have adhered, it is possible to test their ability to withstand detachment by exposing them to stepwise increases in shear stress, and thereby derive a measure of the strength of the adhesive bonds, allowing the quantitative estimation of adhesive force.+. Adhesion of flowing cells to the endothelium depends not only on the affinity/avidity between a receptor and its cognate ligand, but also on the kinetics of the interaction, ie. receptor/ligand on/off rates22. Additionally, the ability of a flowing cell to form an adhesive contact and its ability to withstand detachment once formed are likely to be critically important parameters, in vizTo, but they may have little influence on adhesion under static conditions. Surprisingly, flow284
based investigations had been restricted to the study of adhesion of white blood cells, particularly neutrophils23, and sickle red blood cells?,~, and have only recently been extended to include PRBC (see Fig. 1 and Box 1). Under carefully controlled laminar flow conditions using cell-coated chambers with parallel-plate geometry, investigators have shown that PRBC are able to adhere to endothelial cells at wall shear stresses within the physiological range for venulesQ26. If efficiency of adhesion is defined as the percentage of PRBC that adhere during passage over the endothelial cells, then the maximum efficiency observed for PRBC is 1.5%: over 1000 times greater than for non-parasitized cellsu. Once bound, a proportion of the PRBC could subsequently resist detachment when exposed to stresses one order of magnitude higher than those encountered in the venular circulation. Probably the best-understood cellular adhesive process is the way in which leukocytes emigrate from the circulation as part of the inflammatory response. Different stages of this well-orchestrated, stepwise process are mediated by different receptor-ligand combinations and the adhesive phenomena are strongly influenced by the level of applied flow stress2sJr. After making contact with the endothelium, leukocytes first roll, mediated by vascular and leukocyte selectins until they become activated by chemotactic factors or cytokines such as interleukin 8 (IL-8) or platelet-activating factor. Once activated, the leukocytes become immobilized and form stable and stationary interactions with molecules such as ICAM- before moving out into the tissues to execute their function. When PRBC are flowed over either cells or purified proteins, the different endothelial cell-expressed receptors for PRBC also operate with different efficiencies and mediate characteristically different types of adhesion*s. When adhesion to ICAM-1, CD36 or TSI? was measured over a range of physiologically relevant wall shear stresses, the number of PRBC adhering decreased with increasing shear stress but with consistent differences in adhesion to the different receptors (Fig. 2). In terms of the absolute number of PRBC binding at a single stress, adhesion was in the order CD36>ICAM-l>TSP. Adhesion via ICAM- was less sensitive to increasing stress than adhesion to CD36 while TSP was the most shear sensitive and least efficient receptor. Furthermore, in contrast with ICAM- or CD36 mediated binding, adhesion to TSP was not long-lasting, but transient. Taken together, these results raise doubt about the relevance of TSP as a receptor for PRBC, in vivo. There were also striking differences in the nature of the interaction between PRBC and different endothelial cell-expressed receptors. While the majority of adherent PRBC that adhered to CD36 remained stationary, those adhering to ICAMrolled continuously. The velocity at which PRBC rolled appeared to be inversely related to the concentration of ICAM-1, an observation that may be of pathophysiological significance. Increased expression of ICAMinduced by inflammatory cytokines has previously been shown to increase endothelial adhesion of PRBC in vitrol6, and thus it has been suggested that tumor necrosis factor alpha (TNF-o) may promote cytoadhesion in viz+. Serum levels of TNF-ol are consistently elevated during acute malaria infection and correlate with the level of peripheral parasitaemia and severity Porasrtology Today, vol.
I I, no. 8, I995
Reviews Box 1. Assays that Measure Adhesion in vitro Schematic representation (Fig. right) of an in vitro assay to Video visualize and quantify adhesion of flowing PRBC to endom,croscope thelial cells, platelets or purified, immobilized proteins. Cell-free Cell A prerequisite for accurate quantification of adhesion buffer suspension under flow conditions is well-defined, reproducible flow. Laminar flow is perhaps the only state of flow that can be produced in a controlled manner with any accuracy within cylindrical tubes or between two parallel plates. Fluid flowing between two such plates moves as a series of thin layers or laminae. The laminae alone the central axis of the channel MIcroslide 01 move with a greater veloci”9 than those near the walls, flow-chamber thus the leading edge of the flowing fluid assumes a para\ bolic profile. Each individual lamina slides or shears over the ones juxtaposing and is, therefore, subjected to a tangential force called the shear stress. When flow occurs in such parallel laminae that do not mix it is termed laminar flowis. I Harvard The flow assembly is either a parallel-plate flow chamsyringepump Microscope stage beG** or a flat, glass microcapillary tube (microslide)“h with rectangular cross-section and well-defined dimensions. The flow chamber described here comprises two flat plates separated by a thin (-150 pm) plastic gasket into which a rectangular channel (50mm x 90 mm) has been cut. The lower of the two plates is a glass or plastic slide onto which monolayers of HUVEC or C32 melanoma cells can be cultured, or a plastic slide onto which discrete spots of purified proteins such as CD36, ICAM- or TSP can be adsorbed 1228.The upper plate is a glass slide or piece of perspex into which inlet and outlet ports have been drilled to direct flow into and out of the channel. The plates are clamped together, and then placed on the stage of a microscope. Alternatively, the flow assembly may be a microslide with one internal surface pre-coated with confluent monolayers of cultured HUVEC or human blood platelet+%. Microslides have several advantages over parallel-plate flow chambers. They are simpler to assemble and ideal for field studies of adhesion of clinical isolates32,3-?. The flow chamber, or microslide, is placed on the stage of a microscope and is observed under video-microscopy whilst a suspension of PRBC of known concentration or cell-free buffer is flowed over at controlled flow rate (Q), chosen to obtain a desired wall shear stress (r), calculated from the width (~1) and the height (h) of the flow channel and the viscosity of the flowing medium (77)using the equation%
Adhesion can be quantified by counting adherent PRBC by direct microscopic observation. Alternatively, video recordings of individual experiments can be made and analysed retrospectively with the aid of computerized-image processing (Fig. 1). Adhesion can be characterized as rolling or stationary and the proportion of cells which remain stationary or which roll, along with their velocities, can also be calculated. The cells commonly used in flow-based adhesion studies are HUVEC (ICAM-1) and platelets (CD36), as C32 melanoma cells show a tendeticy to ‘pile-up’ in some areas, causing perturbation in the laminar flow profile.
of disease, and high levels have been associated with a poor prognosism. The precise role of TNF-cy in the pathogenesis of cerebral malaria is not known-5. However, the increased level of expression of ICAMdetected on endothelial cells in the cerebral microvasculature of individuals with cerebral malaria6 would be expected to increase levels of PRBC adhesion and reduce the velocity at which PRBC roll along vessel walls. Both these effects would tend to promote sequestration in uivo, either through ICAM- alone or in concert with other receptors. Rolling of PRBC can, therefore, be likened to the rolling adhesion seen when marginating leukocytes first interact with stimulated endothelium. The fact that neutrophils form stationary interactions with ICAM-I (Ref. 27) (whereas PRBC roll on this molecule) suggests that rolling is a function of the nature of a specific receptor-ligand pair, and not peculiar to ICAM- or the vascular selectins which mediate neutrophil rolling. These findings are consistent with the idea that adhesion of PRBC may be a multi-step process, analogous with neutrophil adhesion, in which initial adhesion to ICAMat high wall shear stress adequately slows down PRBC so that they can become immobilized via CD36 and possibly other receptors. At lower wall shear stresses, however, it appears that CD36 alone is capable ParasrtoioS~v To&v.
LOI
1: ‘-1:3
2’
Range proposed to exist in venules
) Increasing
wall shear stress
Fig. 2. Flow curves showing the relative ability of different receptors to bind PRBC at different wall shear stresse9. The y axis, percentage of adherent PRBC. is that relative to the percentage of PRBC adhering at the lowest wall shear stress tested. The range of wall shear stress proposed to exist in venules’9,20 is indicated.
285
Reviews of immobilizing flowing PRBC without the assistance of ICAM-1. Flowbased studies of clitlical isolates. It is essential to know whether or -not clinical isolates act in the same manner as long-term laboratory-cultured isolates. Furthermore, the study of clinical isolates may provide information as to whether the severity of falciparum malaria is related to the capacity of PRBC to cytoadhere3i. The adhesive properties of PRBC taken from individuals with mild (uncomplicated) or severe malaria have been tested using static assays with both cells and immobilized proteins as the adhesive target, yet no correlation has ever been detected between the degree of adhesiveness of PRBC and the occurrence of cerebra1 malaria. Using flow assays, field studies have clearly shown that red blood cells parasitized by wild-type P. falciparum (obtained directly from patients and cultured in zCtro to the trophozoite stage) were able to adhere to HUVEC and platelets at physiologically relevant wall shear stress and that the adhesive interaction to the two cell types was mediated predominantly by ICAM- and CD36, respectivelyQ33. The rolling-type interaction with ICAMalso occurs and is, therefore, not an artefact induced bv in vitro parasite culture. The inherent adhesiveness of wild isolates was highly variable, even after correction for differences in parasitaemia, and there was no significant correlation in the capacity of individual isolates to bind to these two receptors. However, in keeping with previous studies using static assay+Js, PRBC bound to CD36 at a greater level, on average, than to ICAM-1. From the studies reported to date, there appears to be no simple relationship between cerebral malaria and adhesion of PRBC to ICAMor CD36, although the ability to bind to the latter might correlate with major organ pathology in more severe disease36. Inhibition and reversal of adhesion underjlow. The relative avirulence of non-cytoadherent strains of parasitesJ7 suggests that inhibition of binding, in viva, mav be a useful therapeutic intervention to prevent or assuage the severe complications of the disease. Cytoadhesion can be inhibited in vitro by immune serum or antibody38mQ. Furthermore, the dramatic demonstration that immune serum41 or, more recently, synthetic peptides”3 could reverse cytoadhesion, in uivo, when infused into monkeys suggests that the administration of various agents, such as anti-adhesion antibodies, soluble adhesion receptors or rheologically active drugs could also be used. The in z!itro flow assay is a particularly valuable method for assessing potential efficacy of new therapies, as it provides quantitative data about the level of shear stress at which adhesion may be lost or reversed. For example, compounds that interfere with adhesion only at high shear stress would be expected to be of little value. The data available on physiological relevance of receptors are also crucial in determining the potential therapeutic targets, as is the case for TSP. Monoclonal antibodies and antibody agents would appear to be most profitably directed against the receptor on the endothelial cell, as the structure is apparently highly conserved between individual humans. Such antibodies could bind directly to the receptor itself, or may bind to a site nearby and inhibit cytoadhesion by steric hindrance. Antibodies against several receptors may be required for full therapeutic effect. The parasite-encoded ligand on the PRBC, currently believed to 286
be P. falcipamrn erythrocyte membrane protein-l (PfEMP-I), is a highly variable molecule capable of antigenic variation and, as such, is an unfavourable target for antibody therapy. The ability of antigenically distinct forms of PfEMP-1 to bind a single receptor would imply that there are conserved regions of the protein that could be potential targets for antibody, but there are no reports of monospecific antibody reagents capable of recognizing many distinct antigenic forms of PfEMP-1. Flow-based studies have examined the effect of antiCD36 monoclonal antibody on adhesion of PRBC to activated platelets 28. Pre-incubation of the platelet monolayer with the antibody reduced adhesion by greater than 98%. This occurred even though activated platelets express large amounts of surface available TSP, confirming the relative unimportance of TSP as a receptor under flow conditions. It has been suggested that parasite-induced modifications to native red blood cell surface proteins, such as Band 3, may enhance the ability of PRBC to cytoadhere44. It is not clear whether such changes increase the adhesiveness of PRBC nonspecifically or whether a specific receptor-ligand interaction is involved; however, the capacity of different PRBC to bind specifically to different receptors may favour the former possibility 45. Using a static assay, Crandall and co-workers have reported the inhibition of binding of PRBC to melanoma cells, by the addition of synthetic peptides based on the Band 3 sequence43. Such peptides may be useful in decreasing adhesiveness of PRBC and perhaps lessening the severity of disease caused by sequestration. It is also possible that compounds that have been shown to be of benefit in decreasing adhesive interactions in other conditions such as sickle cell disease may have a place in malaria therapy. No such data from flow-based assays are yet published. However, preliminary studies (performed under flow conditions) have indicated that two such compounds, a non-ionic co-polymer surfactant and a dimethyl xanthine derivative, oxpentifylline, are unlikely to have any antiadhesive effect on PRBC at pharmacological concentrations (B.M. Cooke and G.B. Nash, unpublished). Rosetting PRBC can also adhere strongly to one or more nonparasitized red blood cells, forming rosette+. After a brief period of iu vitro culture, clinical isolates show a varying degree of rosetting, with zero to 70% of trophozoites forming rosettes 47-49.The level of rosette formation iiz zlitro has been shown to be associated with the occurrence of cerebral malaria in studies performed in The Gambia4yJ0, but not in Thailand48. Certainly, aggregates of parasitized and non-parasitized cells, hypothesized to be rosettes, have been reported to be present in the cerebra1 vasculature of individuals who had died from cerebra1 malariasi. The precise importance of rosetting in the pathophysiology of malaria remains unclear, but will probably depend on the phenotype of the parasite and the density and nature of endothelial cell-expressed receptors. Perfusion of a rosetting strain of P. falciparum through the ex vivo rat mesoappendix preparation indicated that rosettes localize preferentially in the venules where they could occlude flowj2. It has recently been shown that rosettes can pass into glass micropipettes with a hiJS!tOiogy
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Reviews diameter similar to that of small capillaries without disruption of the rosettes”. Moreover, when sheared in a cone-plate viscometer, rosettes were able to withstand stresses typical of those in arteries for minutes without becoming disaggregated, suggesting that rosettes could survive intact in the circulation in vivu. In a flow-based study of clinical isolates, rosettes were found attached to cultured HUVEC, suggesting that either pre-formed rosettes can adhere or that rosettes can form under flow32. In either case, these studies support the hypothesis that rosetting could occur in ZI~PO and contribute to vascular obstruction in malaria. Does rosetting interfere with flow-based assays and thereby invalidate our interpretation of the adhesive properties of PRBC? Using a static assay, it has been noted that the level of adhesion of clinical isolates to C32 melanoma cells was inversely related to the level of rosettinp. Similarly, the level of adhesion of a laboratory-adapted strain of P. falciparum to C32 melanoma cells or CD36 was higher if rosettes were first disrupted with heparin 9. Such a reduction in the level of adhesion may be explained by a decrease in PRBC available to cytoadhere or physical hindrance by non-parasitized cells and is also likely to occur in flow-based assays. However, there is no evidence to suggest that rosetting alters parameters such as the strength or the nature of the adhesive interaction. Future directions The availability of flow-based assays for use in the laboratory and in the field now allows a large number of questions about cytoadhesion in malaria to be studied quantitatively. For example, there are now cytoadherent parasites available that do not express some of the accessory red blood cell membrane, parasite-encoded molecules such as knob-associated histidine-rich protein (KAHRP) or mature erythrocyte surface antigen (MESA). The contribution of these molecules to strengthening the adhesive interaction can now be assessed. Similarly, there are parasite lines that express multiple ligands or that have been selected for high levels of adhesion. Quantification of binding of these lines to individual receptors or receptor mixtures would be of great interest. Questions concerning the endothelial cell such as the relative binding efficiencies of microvascular endothelial cells from a variety of different human organs or the physiological importance of other potential receptors such as vascular cell adhesion molecule-l (VCAM-1) or E-selectin can also now be studied. Studies that mimic the combinations of acute- or chronic-phase reactants likely to be present in individuals infected with malaria have also not yet been performed. Finally, in terms of clinical intervention and disease management using anti-adhesion therapy, flow-based methodology is well suited for investigation of agents with putative anti-adhesive potential and can be adapted to screen for ability to inhibit or reverse adhesion. Acknowledgements This work IS supported by the Natlonal lnstltutes of Health grant number DK32094-I 0 and the NatIonal Health and MedIcal Researcl Council. The authon would llke to acknowledge the guidance and expert advlce of Gerard Nash at the Unlverslty of Blrmngham, UK V. the development of the IR vrtro flow-based assays and analysts of cell adhesion revlewed In this paper.
References 1 MacPherson, G.G. et al. (1985) Am. I. Puthol. 119,385-401 2 Berendt, A.R. ef al. (1994) Parhitolo~y 108, S19-S28 3 Grau, GE. and Dekossodo, S. (1994) Parasitology Today 10,408-409 4 Berendt, A.R. et al. (1994) Parasitology Today 10,412-414 5 Clark, LA. and Rockett, K.A. (1994) Parasitology Today 10,410-412 CF. et al. (1992) 1. Exp. Med. 176,1183-1189 6 Ockenhouse, 7 Turner, G.D.H. et ~1.(1994) Am. 1. Pathol.145,1057-1069 A.D. (1989) Blood 74,2603-2618 8 Howard, R.J. and Gilladoga, 9 Udeinya, I.J. (1990) Am. J. Trap. Med. Hyg. 43, 2-5 C. et al. (1985) Proc. Nutl Acad. Sci. USA 82, 10 Raventos-Suarez, 3829-3833 11 Kaul, D.K. et al. (1994) Am. J. Trap. Med. Hyg. 50,512-521 12 Nash, G.B. ef al. (1992) Blood 79,798-807 13 Miller, L.H. (1969) Am. J. Trap. Med. Hyg. l&860-865 14 Whiteley, H.E. et al. (1987) Am. J. Trop. Med. Hyg. 37, l-8 15 Fujioka, H. et nl. (1994) Exp. Purasitol. 78,371-376 16 Berendt, A.R. et al. (1989) Nature 341, 57-59 17 Rock, E.P. et ul. (1988) Blood 71, 71-75 18 Franz, D.R. et al. (1987) Am. J. Trap. Med. Hyg. 36,474-480 19 Chien, S. (1972) in Hemodi2ution: Theoreticnl Basis and C2inicul Application (Messmer, K. and Schmid-Schonbein, H., eds), pp l-45, Karger 20 Turitto, V.T. (1982) Prog. Hemostasis Thromb. 6, 139-177 21 Forrester, J.V. and Lackie, J.M. (1984) 1. Cell Sci. 70, 93-110 22 Williams, A.F. (1991) Nature 352,473-474 23 Godin, C. et al. (1993) J. Cell Sri. 106,441-452 24 Barabino, G.A. et al. (1987) Blood 70,152-157 23 Rowland, P.G. et al. (1993) 1. Lab. C/in. Med. 121, 706-713 26 Wick, T.M. and Louis, V. (1991) Am. 1. Trap. Med. Hyg. 45,578-586 27 Kishimoto, T.K. (1991) J. NZH Res. 3, 75-77 78 Cooke, B.M. et al. (1994) Br. J. Huemutol. 87, 162-170 29 Berendt, A.R. et al. (1990) Purusitoloa Today 6,247-254 D. (1990) Zmmunol. Left. 25,213-216 30 Kwiatkowski, 71 Marsh, K. et al. (1988) Exp. Purusitol. 65, 202-208 107,359-368 32 Cooke, B.M. et al. (1993) hrusifology 33 Cooke, B.M. et al. Am. 1. Troa. Med. Hva. (in Dress) 34 Ockenhouse, C.F. et ul.‘(199i) J. Infect:&. l&, 183-169 I’. et al. (1993) Infect. Immun. 61,5198-5204 35 Ringwald, 36 Ho, M. ef n[. (1991) Infect. Immun. 59, 873-878 37 Langreth, S.G. and Peterson, E. (1985) Infect. Immun. 47, 760-766 38 Udeinva, I.J. et al. (1983) Nature 303, 429-431 39 Leech;J.H. et al. (l&34) i. Exp. Med. 159, 1567-1575 40 Sineh. B. et al. (1988) Cliri. Exa. Immunol. 72, 145-150 31 Da%, P.H. et al. (1983) Proc.‘Nutl Acud. SCZ:USA 80,5075-5079 CF. et al. (1989) Science 243, 1469-1471 42 Ockenhouse, -13 Crandall, I. et al. (1993) Proc. Nutl Acud. Sci. USA 90,4703-4707 14 Crandall, I. and Sherman, I.W. (1994) Purusitology 108,257-267 45 Crandall, I. ef al. (1994) Exp. Purusitol. 78,203-209 M. et al. (1994) Purusitology Today 10, 73-79 -lb Wahlgren, 47 Hasler, T. et al. (1990) Blood 76, 1845-1852 48 Ho, M. et al. (1991) Infect. Immun. 59, 2135-2139 4’) Treutiger, C.J. ef al. (1992) Am. 1. Trap. Med. Hy,q 46, 503-510 50 Carlson, J. et al. (1996) Lancet 336.1457-1460 __ 51 Rieanti, M. et al. (1990) Immunol. Left. 25, 199-205 78,812-819 52 K&l, d.K. et al. (i99ljBlood 53 Nash, G.B. et al. (1992) Br. J. Huemafol. 82, 757-763 54 Handunnetti, S.M. et al. (1992) Infect. Immun. 60, 928-932 55 Matrai, A. et ul. (1987) in Clinical Hemorheology. Applications in Curdiovuscular and Hemafologicul Disease, Diabetes, Surgery and Gynecology (Chien, S. et al., eds), pp 9-71, Martinus Nijhoff 56 Cooke, B.M. et al. (1993) Microvusc. Res. 45,33-45 J.W. ef al. (1985) J. Immunol. 135, 3494-3497 57 Barnwell, J.W. ct al. (1989) J. C/in. best. 84, 765-772 58 Barnwell, 59 Schmidt, J.A. ef al. (1982) J. Clin. Invest. 70,379-386 R. et al. (1989) Nufure 338, 763-765 60 Udomsangpetch, 61 Udeinya, I.J. et al. (1983) Exp. Purusifol. 56, 207-214 62 Sherman, I.W. and Valdez,‘E. (1989) Pmasitology 98,359-369 CF. ef al. (19891 1. Clin. Invest. 84,468-475 63 Ockenhouse, 64 Cooke, B.M..and Nash, ‘G.B. ii995) Exp. Purasitol. 80, 116-123 6.5 Oquendo, I’. et al. (1989) Cell 58, 95-101 66 Hasler. T. et al. (1993) Am. J. Trap. Med. Hyg. 48, 332-347 67 Smith, H. et al. (1992) Exp. Purusitol. 75,269-280 hX Johnson, J.K. et al. (1993) J. Infect. Dis. 167, 698-703 69 Ockenhouse, C.F. and Chulay; J.D. (1988) J. Infecf. Dis. 157,584-588 70 Goldrine. I.D. ef al. (1992) Br. I. Huernutol. 3,413-418 71 Udeinya, I.J. et al. (1$81) icier&e 213, 555-557 72 Udeinya, I.J. ef al. (1985) J. Protozool. 32,88-90 73 Ockenhouse, C.F. et al. (1992) Cell 68, 63-69 74 Jioberts, D.D. of al. (1985) Nature 318, 64-66