ICAM-1 can play a major role in mediating P. falciparum adhesion to endothelium under flow

Molecular & Biochemical Parasitology 128 (2003) 187–193

ICAM-1 can play a major role in mediating P. falciparum adhesion to endothelium under flow Carolyn Gray a , Christopher McCormick b , Gareth Turner c , Alister Craig a,∗ a

Molecular Biochemistry and Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK b School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, West Yorkshire, UK c Division of Molecular Histopathology, Addenbrooke’s Hospital, University Department of Pathology, Box 231, Level 3, Cambridge CB2 2QQ, UK Received 3 January 2003; received in revised form 25 February 2003; accepted 2 March 2003

Abstract We have investigated the importance of adhesion molecule co-operation in mediating Plasmodium falciparum adhesion to endothelial cells under flow conditions. Using three laboratory parasite lines and a patient isolate which differ in their ICAM-1 and CD36-binding avidity, we found that blockade of ICAM-1 and CD36 separately reduced IRBC adhesion by up to 95 and 50%, respectively. These results confirm previous data showing that ICAM-1 and CD36 synergize to mediate adhesion, but differ in demonstrating that without ICAM-1, binding under flow conditions is severely impaired. Thus, in this system, ICAM-1 is critical for P. falciparum adhesion to activated endothelium and once bound, synergy with CD36 mediates the majority (≥98%) of adhesion. © 2003 Elsevier Science B.V. All rights reserved. Keywords: ICAM-1; CD36; Plasmodium falciparum; Adhesion; HDMEC; HUVEC

1. Introduction Severe malaria is thought to be due to the unique ability of Plasmodium falciparum-infected red blood cells (IRBC) to adhere in the small blood vessels of major organs (for a review see [1]). Several cell adhesion molecules of clinical interest involved in this process have been identified, including intercellular adhesion molecule-1 (ICAM-1) [2], CD36 [3–5], CD31 [6] and P-selectin [7], as well as receptors implicated in plactental malaria, for example, chondroitin sulfate A [8]; for a review see [9]. Interestingly, research showing that parasite isolates from children with severe malaria bind several receptors [10], suggests that additive and/or synergistic effects between adhesion molecules may contribute to cytopathology.

Abbreviations: HDMEC, human dermal microvascular endothelial cells; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; IRBC, infected red blood cells; mAb, monoclonal antibody; TNF-␣, tissue necrosis factor-␣ ∗ Corresponding author. Tel.: +44-151-705-3161; fax: +44-151-705-3371. E-mail address: [email protected] (A. Craig).

Of the endothelial adhesion molecules which bind IRBC, ICAM-1 is of interest because its expression is up-regulated in severe malaria [2,11] and a natural polymorphism of ICAM-1, termed ICAM-1Kilifi , affects disease severity in parts of Africa [12,13]. Research using patient isolates has found that ICAM-1 mediated rolling adhesion while CD36 mediated stationary adhesion [14], which suggests that ICAM-1 capture and CD36 stationary adhesion may co-operate to promote efficient sequestration. Indeed, synergy between ICAM-1 and CD36 has been shown under static binding assay conditions on human dermal microvascular cells (HDMEC) [15]. In this case receptor mobility was critical, because synergy could not be reproduced using purified receptors or formalin-fixed HDMEC. Interactions with multiple receptors, including CD36 and ICAM-1 have also been demonstrated under flow conditions both on activated HDMEC [16], with CD36 being responsible for the majority of binding, and in a SCID mouse/human skin graft model [17]. As adhesion to ICAM-1 tends to be higher in parasites taken from cerebral malaria patients compared to asymptomatic controls [18], we wanted to determine how critical ICAM-1 is for IRBC binding to endothelium. To investigate this, we have used three parasite lines which vary in

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ICAM-1-binding avidity, and an ICAM-1-binding isolate from a patient who died from cerebral malaria. We show that ICAM-1 and CD36 synergize to mediate IRBC adhesion on HDMEC, but that without binding to ICAM-1, subsequent adhesion is severely reduced. Thus, in this experimental system, ICAM-1 is critical for initiation of IRBC adhesion under flow conditions.

2. Materials and methods 2.1. Parasite culture We have used three parasite lines, A4 [19], ItG [20] and C24 [19]. JDP8 (kindly provided by Dr C. Chitnis) is an ICAM-1-binding isolate from a patient who died from cerebral malaria. Parasites were cultured in RPMI 1640 medium (supplemented with 37.5 mM HEPES, 7 mM d-glucose, 6 mM NaOH, 25 ␮g ml−1 gentamicin sulphate, 2 mM l-glutamine and 10% human serum) at a pH of 7.2, in a gas mixture of 96% nitrogen, 3% carbon dioxide and 1% oxygen. Parasites were synchronised using 5% sorbitol. Prior to use, parasites were washed twice in binding buffer (RPMI 1640 medium, supplemented with 6 mM glucose, pH 7.2) and re-suspended at 1% haematocrit (Coulter counter) and 3% parasitaemia (Giemsa staining). All parasite lines are subject to antigenic switching and so were used up to 3 weeks post-selection (where possible) to minimise the effect of mixed populations. 2.2. Endothelial cells Characterised human umbilical vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HDMEC) were obtained from Clonetics (BioWhittaker). Cells were grown in Clonetics BulletKit medium and then frozen down in 7.5% DMSO/92.5% FCS. On thawing, 6th passage HUVEC and HDMEC were found to express the constitutive endothelial cell markers CD31 and von Willebrand Factor, with TNF-␣ activation overnight (0.5 ng ml−1 ) increasing ICAM-1 cell surface expression (data not shown). 2.3. Static protein assay Static protein binding assays were carried out as previously described [15]. Briefly, 2 ␮l of ICAM-1-Fc (25, 50 or 100 ␮g ml−1 ) [21] or CD36 (2 ␮g ml−1 ) [22] were spotted onto 60 mm diameter bacteriological Petri dishes and incubated in a humidified chamber for 2 h at 37 ◦ C. Proteins were aspirated off and dishes blocked overnight at 4 ◦ C in 1% BSA/PBS. Blocking solution was removed, the dish washed in binding buffer and 2 ml of parasite suspension at 3% parasitaemia and 1% haematocrit added to each dish. Dishes were incubated at 37 ◦ C for 1 h, with re-suspension for every 10 min. Unbound RBC and IRBC were removed

by repeated washing, bound cells fixed with 1% glutaraldehyde for 1 h and then stained with 5% Giemsa for 20 min. Levels of adhesion were quantitated microscopically. 2.4. Static cell assay Static cell binding assays were carried out using a modified version of a previously described method [2]. HUVEC or HDMEC (6th passage) were seeded onto 1% gelatincoated 13 mm Thermanox coverslips (Nalgene, Nunc). Once confluent, they were incubated overnight at 37 ◦ C with or without 0.5 ng ml−1 rTNF-␣ (Biosource International). Cells were washed with binding buffer and incubated with 0.5 ml of parasite suspension (3% parasitaemia, 1% haematocrit) for 1 h at 37 ◦ C, with gentle resuspension every 10 min. A 1 h gravity wash removed unbound cells, adherent cells were fixed using 2% glutaraldehyde for 1 h and then stained with 5% Giemsa for 20 min. Coverslips were dried and mounted on slides using DPX mounting buffer (BDH Lab Supplies). Levels of adhesion were quantitated microscopically. 2.5. Flow assay Microslides were coated for 2 h at 37 ◦ C with ICAM-1-Fc (25 ␮g ml−1 ) and blocked overnight at 4 ◦ C. For cell assays, HUVEC or HDMEC (6th passage) were seeded onto 1% gelatin-coated microslides (Camlab, UK). Confluent microslides were incubated overnight at 37 ◦ C with or without 0.5 ng ml−1 rTNF-␣, using intermittent flow to exchange the TC medium in the microslides. Confluent microslides were incubated for 1 h with TC medium alone (no antibody), an isotype matched control antibody (HD37, 4 ␮g ml−1 , ␣CD19, DAKO), ␣ICAM-1 (15.2, 4 ␮g ml−1 , Serotec) or ␣CD36 (FA6, 4 ␮g ml−1 , AbCam) prior to use. IRBC suspensions were flowed over for a total of 5 min, and then binding buffer flowed over to remove unbound cells. The flow rate yielded a wall shear stress of 0.05 Pa (calculated based on the formula in [14]), which has been used widely to mimic wall shear stresses in the microvasculature. The number of stationary IRBC was counted in six separate areas on the microslide, from which the number of stationary IRBC per mm2 was calculated. Cells moving at less than 15 ␮m s−1 during this time (1 min) were deemed to be stationary. Examination at 2000× magnification revealed that >99% of all adherent cells were parasitised. IRBC velocity was calculated and analysed from video recordings using a program written by Sigmapi Systems Ltd. (UK) and only IRBC showing some rolling behaviour were included in this part of the analysis. 2.6. Statistical significance Statistical significance was determined using the Tamhane T-2 test, and differences regarded as significant if P < 0.05.

C. Gray et al. / Molecular & Biochemical Parasitology 128 (2003) 187–193 Table 1 Adhesion of A4, ItG, JDP8 and C24 IRBC per mm2 to purified ICAM-1 and CD36 ICAM-1 (␮g ml−1 ) 100 A4 ItG JDP8 C24

2082 5615 6701 291

50 ± ± ± ±

421 738 1148 85

547 4331 5511 213

± ± ± ±

169 615 1107 82

32 738 1901 87

Table 2 Adhesion of A4, ItG, JDP8 and C24 IRBC per mm2 to HUVEC and HDMEC

CD36 (2 ␮g ml−1 )

25 ± ± ± ±

21 451 616 48

1655 2589 407 2797

± ± ± ±

408 326 122 740

Static assays were carried out as described in the methods. Briefly, IRBC (3% parasitaemia, 1% haematocrit) were incubated for 1 h with re-suspension every 10 min. Data shown is the mean number of IRBC per mm2 ± S.E. of the means of 3–5 experiments.

3. Results 3.1. Characterisation of ICAM-1 and CD36 binding Adhesion of IRBC varies both qualitatively and quantitatively between different strains, which may explain the variation seen between different studies. Therefore the adhesive behaviour, with respect to CD36 and ICAM-1, was determined primarily using purified proteins for all four parasite lines used in this study. Both ItG and JDP8 bound strongly to ICAM-1, and while A4 also bound ICAM-1 levels of adhesion were significantly lower at all ICAM-1 concentrations (P < 0.00001 for both, Table 1). In contrast, the parasite line C24 was unable to adhere to ICAM-1 efficiently. All three ICAM-1-binding parasite lines bound CD36, however, JDP8 adhesion to CD36 was significantly lower than A4, ItG and C24 (P < 0.0015 for all, Table 1). In contrast to static assays, under flow conditions there was little difference between A4, ItG and JDP8 stationary adhesion on ICAM-1 (25 ␮g ml−1 ), although rolling velocities were slightly lower for ItG and JDP8 (Fig. 1A and B). Thus, avidity may not be the sole determining factor in IRBC binding under flow. Unsurprisingly, C24 binding to ICAM-1 under flow conditions is minimal.

189

HUVEC Unactivated A4 ItG JDP8 C24

10 18 11 3

± ± ± ±

1 1 1 0

HDMEC Activated 37 224 231 19

± ± ± ±

2 12 23 4

Unactivated 56 25 35 6

± ± ± ±

7 2 5 0

Activated 707 1521 1134 73

± ± ± ±

57 124 65 14

Static assays were carried out as described in the methods. Briefly, IRBC (3% parasitaemia, 1% haematocrit) were incubated for 1 h with re-suspension every 10 min. Data shown is the mean number of IRBC per mm2 ± S.E. of the means of 3–5 experiments.

3.2. Characterisation of endothelial cell binding Similarly to previous studies [16] TNF-␣ activation up-regulated ICAM-1 expression on HUVEC (CD36− ) and HDMEC (CD36+ ) (data not shown), which correlated with significantly increased adhesion of all ICAM-1-binding parasite lines to activated HUVEC and HDMEC (P < 0.0001 for all, Table 2). In contrast, binding to CD36 alone, shown using C24, did not promote adhesion on HDMEC nor HUVEC, although a slight but significant increase in adhesion to HDMEC was observed on TNF-␣ stimulation. Therefore, under static binding assay conditions, strong ICAM-1 binders like ItG and JDP8 are more efficient at binding activated endothelial cells. 3.3. Importance of endothelial cell ICAM-1 and CD36 under flow conditions 3.3.1. HUVEC Despite comparable A4, ItG and JDP8 stationary adhesion to ICAM-1 protein under flow conditions (Fig. 1), A4 stationary binding to TNF-␣-activated HUVEC was significantly greater than ItG and JDP8 levels (P < 0.00001 for both, Fig. 2A–C). Blockade of ICAM-1 significantly reduced A4 stationary adhesion, and produced a reduction, albeit not statistically significant, in the low levels of ItG and

Fig. 1. ICAM-1-mediated IRBC adhesion under flow conditions. Flow assays (four experiments) were carried out as described in the methods. Briefly, IRBC (3% parasitaemia, 1% haematocrit) were flowed over ICAM-1-coated (25 ␮g ml−1 ) microslides at a wall shear stress of 0.05 Pa. (A) The mean number, and standard error of the means, of stationary A4, ItG, JDP8 and C24 IRBC per mm2 . (B) Rolling velocity (␮m s−1 ) of individual A4, ItG, JDP8 and C24 IRBC from all experiments.

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Fig. 2. IRBC adhesion to HUVEC under flow conditions. Flow assays (four experiments) were carried out as described in the methods. Briefly, confluent microslides were incubated for 1 h with no antibody (no Ab), control antibody (CIg, 4 ␮g ml−1 ) or anti-ICAM-1 mAb (␣ICAM-1, 4 ␮g ml−1 ) prior to use. IRBC (3% parasitaemia, 1% haematocrit) were flowed over microslides at a wall shear stress of 0.05 Pa. (A–C) The mean number, and standard error of the means, of stationary A4 (A), ItG (B) and JDP8 (C) IRBC per mm2 . (D–F) Rolling velocity (␮m s−1 ) of individual A4 (D), ItG (E) and JDP8 (F) IRBC from all experiments. *: Statistically significant if P < 0.05.

JDP8 stationary adhesion. Although both A4 and ItG rolled on anti-ICAM-1-treated HUVEC (Fig. 2D and E), far fewer cells adhered and a general increase in A4 rolling velocity did suggest weaker binding. In contrast, anti-ICAM-1 treatment completely abrogated JDP8 rolling adhesion (Fig. 2F). Negligible C24 stationary or rolling adhesion to activated HUVEC was observed (data not shown). 3.3.2. HDMEC Activation of HDMEC with TNF-␣ significantly increased A4, ItG and JDP8 stationary adhesion (P < 0.002 for all, Fig. 3A–C). However, this enhanced A4, ItG and JDP8 stationary adhesion was blocked potently using anti-ICAM-1 mAb (P < 0.001 for all). Furthermore, A4, ItG and JDP8 rolling adhesion was significantly reduced using anti-ICAM-1 mAb (P < 0.01 for all) but not anti-CD36 mAb (Fig. 3D–F). Although CD36 inhibition reduced A4 and ItG stationary adhesion, neither were significant (Fig. 3A and B). However, JDP8 stationary adhesion was significantly reduced (P < 0.0005, Fig. 3C), which is surprising

as JDP8 did not bind strongly to purified CD36 under static conditions (Table 1). Adhesion of C24 to activated HDMEC was minimal, although blockade of CD36 did significantly reduce stationary binding (P < 0.00001) and also appeared to increase C24 rolling adhesion (data not shown). Blockade of ICAM-1 had no effect upon C24 adhesion on activated HDMEC (data not shown). This suggests that receptor co-operation may be involved in mediating adhesion of A4, ItG and JDP8 to HDMEC under flow conditions. 3.4. Synergy between ICAM-1 and CD36 As A4, ItG, and JDP8 stationary adhesion is almost abolished in the presence of anti-CD36 and anti-ICAM-1 mAbs (Fig. 3A–C), the majority of IRBC binding can be attributed to ICAM-1 and CD36. ICAM-1-mediated adhesion may be inferred from anti-CD36 mAb binding levels, and vice versa. However, the total number of bound IRBC in the presence of anti-CD36 mAb alone (ICAM-1-mediated) plus anti-ICAM-1 mAb alone (CD36-mediated) was less

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Fig. 3. Importance of ICAM-1-mediated IRBC adhesion to HDMEC under flow conditions. Flow assays (four experiments) were carried out as described in the methods. Briefly, confluent microslides were incubated for 1 h with no antibody (no Ab), control antibody (CIg, 4 ␮g ml−1 ), anti-ICAM-1 mAb (␣ICAM-1, 4 ␮g ml−1 ) or anti-CD36 mAb (␣CD36, 4 ␮g ml−1 ) prior to use. IRBC (3% parasitaemia, 1% haematocrit) were flowed over microslides at a wall shear stress of 0.05 Pa. (A–C) The mean number, and standard error of the means, of stationary A4 (A), ItG (B) and JDP8 (C) IRBC per mm2 . (D–F) Rolling velocity (␮m s−1 ) of individual A4 (D), ItG (E) and JDP8 (F) IRBC from all experiments. *: Statistically significant if P < 0.05.

than 50%, of control levels (ICAM-1 and CD36-mediated). Therefore, synergy between ICAM-1 and CD36 augments IRBC adhesion to activated HDMEC under flow conditions.

4. Discussion We have used three different parasite lines and one patient isolate to investigate the importance of ICAM-1 and CD36 in mediating IRBC adhesion to endothelium. Characterisation of IRBC binding agrees with previous data showing that A4 binds purified ICAM-1 weakly and CD36 strongly [19], ItG is a strong ICAM-1 binder which also adheres to CD36 [20] and C24 only binds CD36 [19]. The previously uncharacterised patient isolate JDP8 adheres strongly to ICAM-1 but weakly to CD36. Our results show that ICAM-1 and CD36 synergize to mediate IRBC adhesion, and that for these parasite lines ICAM-1 is critical in capturing IRBC under flow conditions. In preliminary experiments we also neutralised the constitutive EC surface molecule CD31 using the mAb JC70A,

which has previously been shown to reduce CD31-mediated IRBC binding by up to 50% [6]. Incubation of HDMEC with anti-CD31 mAb did not affect IRBC binding on HDMEC in our model system. As CD31 is constitutively expressed, this result emphasises that the effects upon adhesion seen using anti-ICAM-1 and CD36 mAbs were specific, and not due to steric hindrance caused by antibody coating the endothelial surface. We did not investigate the contribution of P-selectin as it became clear during our initial experiments that the majority of adhesion in our model system is mediated by ICAM-1 and CD36. Synergy between ICAM-1 and CD36 has been observed before using A4 and ItG under static conditions [15], with CD36 found to mediate the majority of IRBC binding. Other studies have also shown co-operation between multiple receptors under flow conditions [16,17], with CD36 also acting as the principal receptor. However, in this study elevated IRBC adhesion to HDMEC requires ICAM-1 capture from flow, and is highlighted by the inability of the poor ICAM-1 binder C24 to roll or adhere on activated HDMEC. Therefore, it appears that in our model system

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ICAM-1 efficiently captures IRBC from the blood flow, and that ICAM-1-mediated rolling adhesion facilitates binding to CD36. Our observations are further supported by previous research showing ICAM-1-mediated rolling adhesion of patient isolates on fixed HUVEC [14]. In the same study, Cooke et al. also found that CD36-mediated stationary adhesion of patient isolates, which corroborates reduced stationary adhesion using anti-CD36 mAb in our model system. Receptor co-operation is also supported by the observation that JDP8 has a significant CD36-binding component under flow, despite having relatively low binding to CD36 under static conditions. Thus, binding to one receptor may facilitate adhesion to another, even when the apparent avidity of a parasite for this receptor is low. Co-operation between multiple adhesion receptors is not unique to malaria cytoadherence, with leukocyte recruitment to inflamed endothelium offering a paradigm of adhesion to endothelial cells. This is initiated by selectins, which support rolling adhesion, allowing subsequent firm tethering by integrins [23]. The inability of two strong ICAM-1 binders (ItG and JDP8) to adhere to activated HUVEC under flow conditions is perplexing, especially considering that the weaker ICAM-1 binder (A4) adheres in an ICAM-1-dependent fashion. Formation of adhesive contacts under flow conditions depends upon avidity and the mechanical properties of bond formation [24]. IRBC adhesion to endothelium has been shown to involve P. falciparum erythrocyte membrane protein 1 (PfEMP1) [25]; as ItG and JDP8 PfEMP1 molecules are smaller than A4 PfEMP1 (Dr. S. Kyes and Dr. C. Chitnis, personal communication) perhaps ItG and JDP8 PfEMP1 molecules are not long enough to bind efficiently to cell surface-expressed ICAM-1. This may affect bond rigidity and thereby the duration of transient adhesion during capture from flow [26]. However, ItG and JDP8 can bind ICAM-1 when presented on a cell surface, as anti-ICAM-1 mAb treatment dramatically reduced ItG and JDP8 adhesion on HDMEC, suggesting that even very short duration interactions with ICAM-1 may be enough to facilitate binding to CD36, which is present on HDMEC but not HUVEC [27]. Other differences in binding interactions with ICAM-1 may also be involved and other studies in our laboratory indicate that the contact residues on ICAM-1 for PfEMP1 may differ between the three ICAM-1-binding lines (AGC, unpublished observations). Although the molecular basis for the differences in binding to ICAM-1 protein and HUVEC remain to be defined, these results highlight the difficulties in scoring adhesive phenotypes of IRBC using simple systems. As the pattern of endothelial cell receptors expressed by HUVEC is thought to be similar to brain microvascular endothelium, with both lacking significant amounts of CD36, this raises the question of how ItG and JDP8-like parasites can bind to cerebral endothelium. Co-infection with more than one parasite strain is common in natural infections [28,29], which may enable adhesion of one parasite strain

to promote the binding of another, in a similar way to the formation of leukocyte “string” formation under flow [30]. Thus, following A4-like adhesion, induced changes in the endothelial cell surface or obstructed blood flow may enable strong-ICAM-1 binders such as ItG and JDP8 to adhere. Future experiments will investigate IRBC-induced changes in the endothelial cell surface using competitive flow studies [31], which have been shown to allow small differences in IRBC adhesion to be quantified, as well as changes in IRBC and endothelial cell-signalling following adhesion. Our work clearly shows that ICAM-1 can be a critical factor in capturing IRBC from the blood flow and enabling synergy with CD36. Although the ability of patient isolates to bind ICAM-1 is common (approximately 80% of patient isolates were able to adhere to ICAM-1 in one large study [18]) it is not universal. In contrast, sequestration in P. falciparum infection is universal. Therefore, while ICAM-1 is important, other receptors must also be able to establish sequestration foci, and other studies have identified patient isolates from Thailand that show adhesion to endothelium under flow conditions but do not bind efficiently to ICAM-1 [7,16]. Future studies on the association between cytoadherence and pathogenesis will need to take into account the very different behaviours of parasite lines in terms of efficient recruitment of IRBC from flow, as well as potential co-operation between parasite populations and endothelial cell receptors.

Acknowledgements We would like to thank Ian Hastings (Liverpool School of Tropical Medicine) for advice on statistical analysis and Chetan Chitnis and Kaushik Chakrabarti (International Centre for Genetic Engineering and Biotechnology, Delhi) for the patient isolate JDP8 and sharing their preliminary results with us. This research was funded by grants from The Wellcome Trust and European Union (FPV QLK2/CT/2000/00109).

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