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The Journal of Immunology, 2001, 167: 6510-6517.
Copyright © 2001 by The American Association of Immunologists

Cytoadherence of Plasmodium falciparum-Infected Erythrocytes Is Mediated by a Redox-Dependent Conformational Fraction of CD361

Paola Gruarin*, Luca Primo{dagger}, Chiara Ferrandi{dagger}, Federico Bussolino{dagger}, Narendra N. Tandon{ddagger}, Paolo Arese§, Daniela Ulliers§ and Massimo Alessio2,*

* DIBIT, San Raffaele Scientific Institute, Milan, Italy; {dagger} Molecular Angiogenesis Unit, Institute for Cancer Research and Treatment, Candiolo, Italy; {ddagger} Otsuka America Pharmaceutical, Inc., Maryland Research Laboratories, Rockville, MD 20850; and § Department of Genetics, Biology, and Biochemistry, University of Torino Medical School, Torino, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260–279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Plasmodium falciparum malaria is the most important parasitic infection world-wide, with yearly 350–500 million new cases and between 1.5 and 2.7 million deaths, mainly children under 5 yr (1). P. falciparum-infected erythrocytes (IRBC)3 may adhere to postcapillary venular endothelium and be sequestered in peripheral tissues. Sequestration of IRBC avoids clearance in spleen, liver, and bone marrow and contributes to the pathogenesis of severe complications (Ref. 2 and references therein). Cytoadherence of IRBC involves specific interactions between surface ligands on the IRBC (mainly the P. falciparum erythrocyte membrane protein 1 (PfEMP1) (3, 4) and host endothelial receptors (reviewed in Refs. 2 and 5). Cytoadhesion is the result of a synergistic action between receptors such as ICAM-1 and P-selectin that favor the rolling of IRBC on endothelial cells and the CD36 receptor that mediates firm adhesion (reviewed in Refs. 2 and 6). Understanding the molecular basis of cytoadherence may provide insights into pathogenesis of severe malaria complications and suggest new therapeutic interventions.

CD36 appears to be the major surface receptor for adhesion of IRBC to endothelial cells (2, 3, 7, 8, 9, 10, 11). In fact, the majority of IRBC isolated from malaria patients bind to CD36 in vitro, while a smaller subpopulation from the same isolates binds to ICAM-1 or to both molecules (2, 7, 8). Purified CD36, synthetic CD36 peptides, as well as murine mAbs inhibit both in vitro and in vivo binding of IRBC to endothelial cells and cell lines bearing surface CD36 (12, 13, 14, 15). However, the nature of the IRBC binding site on CD36 is not yet fully understood.

In a previous work we generated a soluble CD36-IgG chimeric molecule that failed to bind IRBC (16). Using mAbs specific for conformational or structural epitopes, we have shown that failure to bind IRBC was due to incorrect folding of the soluble chimeric molecule (16). The CD36-IgG chimeric molecule expressed the Mo91 mAb epitope that is present on the intracellular precursor but absent on the correctly folded mature CD36 and did not express the mature CD36 epitope recognized by the NL07 mAb (16, 17). These results are in favor of a conformational nature of the CD36-IRBC binding site.

Since the disappearance of the Mo91 epitope during CD36 maturation correlates with the formation of disulfide bonds (18), and treatment with reducing agents has been reported to inhibit IRBC cytoadherence (19), we tried to identify structural changes of CD36 that affect its binding to IRBC. Our results indicate that treatment of CD36-expressing cells (U937, CD36-transfected COS-7, and HUVEC (CD36-EC)) with the reducing agents DTT or N-acetylcysteine (Nac) induced the reappearance of the CD36 epitope Mo91 on a fraction of mature molecules, inhibited the CD36-mediated cytoadherence of IRBC to human endothelial cells, and dissolved pre-existent IRBC-endothelial cell aggregates. Reduction of CD36 did not affect the reactivity of CD36 with other specific mAbs, nor did it abolish the binding of oxidized low density lipoproteins (oxLDL) to CD36. Reduced CD36 spontaneously reoxidized 14 h after treatment, and the IRBC binding activity was restored. These results suggest the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or specifically treat malaria complications due to IRBC cytoadhesion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Agarose anti-mouse IgG or IgM, bicinchoninic acid solution, DTT, PMA, N-ethylmaleimide (NEM), Nac, luminol, polybrene, trypsin, Triton X-100 (TX100), {beta}-octylglucothiopyranoside ({beta}-OGTP), FCS, and culture media, DMEM and medium 199, were obtained from Sigma (St. Louis, MO); protein A-Sepharose CL-4B and Percoll from Pharmacia (Uppsala, Sweden); ECL Western blotting detection reagent from Amersham (Milan, Italy). The mAbs specific for CD36 used in this study were NL07 (20), OKM5 (provided by Ortho, Raritan, NJ), Mo91 and Mo25 (21), 131.1 and 131.2 (22), SMØ (Sigma), and JCD36P (Ortho, Milan, Italy). HRP-conjugated rabbit anti-mouse Ig was obtained from Dako (Glostrup, Denmark). F(ab')2 goat anti-mouse Ig labeled with FITC was purchased from Technogenetics (Milan, Italy). Controls were class-matched irrelevant mAbs. Human LDL labeled with 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine (LDL) were purchased from Molecular Probes (Leiden, The Netherlands).

Cell treatments

U937 cells made adherent by PMA treatment (23); COS-7 cells and HUVEC, both transfected to express CD36 (CD36-COS-7 and CD36-EC, respectively); and mock-transfected cells (see below) were grown in 24-well culture plates and kept in culture as indicated previously (16). Cells were washed twice with PBS and incubated for 40 min at 37°C in DMEM medium containing either 1–20 mM DTT or 1–100 mM Nac. At the end of the treatment cells were washed twice with DMEM supplemented with 10% FCS and used for subsequent assays. Control cells were treated as before, but DTT and Nac were omitted. To block thiol reoxidation, in selected experiments after reducing agent treatment cells were treated for 10 min at 4°C with 10 mM NEM, a thiol-alkylating agent. In the reoxidation experiments, treated cells were washed twice and left in the DMEM supplemented with 10% FCS for 24 h at 37 or 4°C. At 2, 4, 6, 14, and 24 h, cell aliquots were collected and used to evaluate surface CD36 reactivity with different mAbs by flow cytometry. In selected experiments, 14 h after treatment cells were used for cytoadherence assay of IRBC (see below).

Retrovirus-mediated gene transfer

CD36 human gene was subcloned in the EcoRI site of the retroviral expression vector pBABE-puro (24) under control of the long terminal repeat promoter. The amphotropic cell line Phoenix (25) was transfected with pBABE and pBABE-CD36 to produce retroviral supernatant with high viral titer. The virus-containing medium was collected 48 h after transfection, filtered (0.45-µm pore filters, Millipore, Milan, Italy), and supplemented with 4 mg/ml polybrene. EC from umbilical cord veins (26) were used at first passage and plated in 100-mm tissue culture dishes in medium 199 supplemented with 20% FCS. For infections the culture medium was replaced by the appropriate viral supernatants, and cells were incubated for 5 h at 37°C in 5% CO2. Then the supernatant was replaced by 10 ml medium 199 supplemented with 20% (v/v) FCS. After 48 h puromycin (1.5 mg/ml) was added to the cells for 96 h for selection. CD36 expression was assessed by immunofluorescence with anti-CD36 mAb, followed by flow cytometric analysis.

Flow cytometry

Cells were incubated with appropriate dilutions of primary Abs as previously described (23) and bound Abs were revealed by F(ab')2 goat anti-mouse Ig labeled with FITC before analysis by flow cytometry on a FACScan cytofluorograph (BD Biosciences, San Jose, CA). Adherent cells were detached by trypsin-EDTA treatment and washed twice with PBS before incubation with mAbs.

Immunoprecipitation, SDS-PAGE, and Western blotting

Cell lysates were generated by using the detergent {beta}-OGTP as described previously (18). After preclearing with class-matched irrelevant mAb adsorbed to protein A-Sepharose, cell lysates were immunoprecipitated with protein A-Sepharose beads coated or not with the relevant Abs. In the sequential immunoprecipitations, the immunocomplexes from the first immunoprecipitation were spun down in the Eppendorf centrifuge, and the remaining lysate was incubated with the second mAb conjugated with protein A-Sepharose for the following immunoprecipitation. The immunocomplexes were eluted in sample buffer and resolved by SDS-PAGE. Proteins were electrotransferred (2 h at 60 V) to nitrocellulose filters. Filters were incubated with purified Mo91 mAbs specific for CD36 (final dilution, 1/400). Bound Abs were revealed with HRP-conjugated rabbit anti-mouse Ig (1/1000) followed by ECL processing and exposure to Kodak BioMax MR1 films (Eastman Kodak, Rochester, NY).

Peptides and Mo91 epitope identification

CD36-derived peptides of 20 residues overlapping by five residues that included the cysteine-rich region (M1, residues 230–249; M2, residues 245–264; M3, residues 260–279; M4, residues 275–294; M5, residues 290–309; M6, residues 305–324; M7, residues 320–339; M8, residues 335–354) were custom synthesized by PRIMM (Milan, Italy). The amino acid sequences of the peptides are described in Fig. 2Go. All peptides were synthesized by the F-moc method, purified to homogeneity by HPLC, and confirmed structurally by mass spectrometry. Peptide concentration was determined by bicinchoninic acid protein assay (Sigma). Due to partial insolubility, peptides M3, M4, and M8 were resuspended in 50% DMSO.



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  		FIGURE 2. Mo91 epitope is localized on the CD36 cysteine-rich region (residues 260–279). Twenty micrograms of CD36-derived 20-mer synthetic peptides M1–M8 overlapping by five residues and including the cysteine-rich region were spotted on nitrocellulose. After saturation with 5% BSA the filter was incubated with the Mo91 mAb, and reactivity detected as described in Materials and Methods. Fifty micrograms of the total lysate obtained in the presence of 10 mM 2-ME and 1% {beta}-OGTP of U937 cells expressing CD36 was used as a positive control (U937), while 20 µg BSA was used as a negative control.

 
Twenty micrograms of each peptide was spotted on nitrocellulose filters in triplicate using the Bio-dot apparatus (Bio-Rad, Hercules, CA). Lysates of U937 cells obtained in the presence of 10 mM 2-ME and 1% {beta}-OGTP, and 20 µg BSA were used as positive and negative controls, respectively. Filters were saturated with 5% BSA in PBS, incubated with purified Mo91 mAbs (final dilution, 1/400), and processed as for Western blot (see below).

Cytoadherence assay of IRBC

IRBC (strain FCR3, mycoplasma-free) were kept in culture as previously described (27). IRBC parasitized with different stages of parasite maturation were isolated from nonsynchronized cultures by the Percoll-mannitol method (28). The trophozoite-infected RBC fraction contained up to 5% ring-stage infected RBC and no noninfected RBC. Fractionated cells were washed three times in PBS supplemented with 10 mM glucose and used for binding assay. Adherent U937, CD36-COS-7, CD36-EC, and mock-transfected cells were grown in 24-well culture plates. IRBC were added at a 50/1 ratio to the wells, then the plates were centrifuged for 3 min at 60 x g and incubated for 20 min at 37°C. After two washes with PBS at 37°C, cells were lysed with lysis solution (0.1 M NaOH, 0.05% (w/v) EDTA, and 0.5% (v/v) TX100), and the number of adherent IRBC or RBC was evaluated by quantitative determination of hemoglobin by luminescence (29). When the expression of CD36 was not homogeneous, as in the case of transfected cells, the number of adherent cells was normalized for the percentage of expressing cells as determined by immunofluorescence and FACS analysis. In the case of DTT and Nac treatment, cells were incubated with reducing agents and washed twice before the addition of IRBC, or after cytoadhesion in the case of the aggregate reversion experiments. Triplicate experiments were performed for each experimental point. The values obtained in each experiment were normalized, taking into account the percentage of CD36-expressing cells in each experiment. Noninfected RBC were used as a control.

Binding and uptake of LDL and oxLDL

OxLDL were prepared from LDL. A 200 µg/ml solution was dialyzed against 5 µM CuSO4 for 24 h at 4°C before further dialysis against 150 mM NaCl and 0.01% (w/v) EDTA (pH 7.2) for 24 h at 4°C. OxLDL were sterile-filtered before addition to cell cultures. OxLDL binding was conducted using CD36-COS-7 cells plated on six-well plates. Cells (2 x 105/well) were incubated for 20 min at 37°C in PBS containing 1 mM CaCl2, 1 mM MgCl2, and 5 µg/ml oxLDL. Cells were washed with PBS containing 1 mM CaCl2 and 1 mM MgCl2 and left in this buffer for 30 min at 37°C to allow internalization. Cells were detached by trypsin/EDTA treatment and fixed with 3% (v/v) paraformaldehyde. Fixed cells were then stained with CD36-specific mAb JCD36P as described above, and binding was revealed with FITC-conjugated goat anti-mouse IgG. Stained cells were then analyzed by flow cytometry.

Statistical analysis

Data were analyzed by t test of paired data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DTT treatment induces expression of the Mo91 epitope on a fraction of mature CD36

The reactivity of the Mo91 mAb under native conditions is restricted to the intracellular precursor of CD36 (16, 17) and is dependent on the absence of disulfide bonds that are formed during CD36 processing and intracellular transport (18). In U937 cells expressing CD36, reduction of disulfide bonds by DTT treatment resulted in the ability of Mo91 to react with a fraction of the mature form of CD36 expressed on the cell surface (Fig. 1GoA). The reactivity of the NL07 mAb, specific for mature CD36, was not affected by DTT (Fig. 1GoA). Since DTT was dose-dependently effective at concentrations between 3 and 10 mM (data not shown), it was consistently used at 7 mM.



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  		FIGURE 1. Reactivity of Mo91 mAb on mature CD36 is dependent on disulfide bond reduction. A, Adherent U937 cells expressing CD36, untreated (solid line) or after treatment with DTT (broken line), were analyzed by flow cytometric analysis using Mo91 and NL07 conformation-specific mAbs or control (ctrl) mouse Ig. The Abs were detected with goat anti-mouse Ig-FITC. The ordinate shows cell numbers, and the abscissa shows fluorescence intensity. B, Sequential immunoprecipitation of CD36 with Mo91 and 131.1 from U937 cells, untreated or after treatment with DTT, followed by alkylation with NEM (10 min at 37°C), to avoid reoxidation. Cell lysates were obtained by using the {beta}-OGTP detergent. Sequential immunoprecipitations were performed with the first mAb, and the remainder were sequentially immunoprecipitated with the second mAb (indicated by the arrows). Immune complexes were resolved on 10% SDS-PAGE under reducing conditions. Resolved proteins were electrotransferred to nitrocellulose and then immunoblotted for CD36 using Mo91 as described in Materials and Methods. The total amount of precipitable CD36 present in the cell lysate is shown on Western blot as (tot ip). Arrowheads indicate precursor and mature CD36.

 
Immunoprecipitation experiments indicate that DTT treatment induced the expression of the Mo91 epitope on mature CD36 and not the surface expression of CD36 precursors (Fig. 1GoB, lanes 2 and 3). Mo91 reacted with only a fraction of mature CD36 as indicated by sequential immunoprecipitation. The 131.1 mAb that recognizes all mature and precursor CD36 molecules precipitated a reduced amount of mature CD36 from the leftovers of the Mo91 immunoprecipitation in the samples treated with DTT (Fig. 1GoB, lanes 4 and 5). Moreover, Mo91 was not able to precipitate mature CD36 in the sample treated with DTT when the immunoprecipitation was performed after the 131.1 immunoprecipitation (Fig. 1GoB, lanes 6–9). The amount of CD36 molecules that became Mo91 positive after DTT treatment ranged between 15 and 50% of the total mature CD36, as evaluated by densitometric analyses of different gels. These data suggest the existence of two different conformers of CD36 on the cell surface characterized either by different sensitivity to reduction or by different physical localization.

Mo91 epitope is localized in the CD36 cysteine-rich region

Since Mo91 reactivity was influenced by disulfide bond reduction, the most likely region where epitope might map to was the CD36 C-terminal, cysteine-rich region (residues 230–354). By using 20-mer peptides covering the entire region, we were able to demonstrate that Mo91 epitope is localized in the peptide M3 that includes residues 260–279 (Fig. 2Go). The presence of DMSO in the solubilization solution did not cause the reactivity of Mo91 to the M3 peptide, since unreactive peptides such as M4 and M8 were also resuspended in 50% DMSO.

DTT treatment inhibits cytoadherence of IRBC on different cell types expressing CD36

Treatment with DTT inhibited the binding of IRBC to U937 cells expressing CD36 by 61 ± 4.3% (Fig. 3GoA). To rule out interference of other adhesion receptors expressed by U937 cells, the same experiments were performed on COS-7 cells stably transfected with CD36 (CD36-COS-7) (30). Also in these cells, DTT treatment induced the expression of the Mo91 epitope without affecting the binding of NL07 (Fig. 3GoC) and almost completely inhibited the CD36-specific cytoadherence of IRBC (Fig. 3GoB). Finally, the effect of DTT was tested on CD36-transfected HUVEC (CD36-EC), a cellular model closer to the microvascular EC responsible for IRBC binding in severe malaria (31). The percentage of cells expressing CD36 ranged from 46–62%, as tested by immunofluorescence (Fig. 4Go, A and B) and Western blot (not shown). IRBC were cytoadherent only to cells expressing CD36 (Fig. 4GoB, a and b). DTT treatment also induced the expression of the Mo91 epitope (Fig. 4GoA) and inhibited cytoadherence of IRBC by 53 ± 14% (Fig. 4GoC, CD36/DTT + IRBC). DTT treatment had only a small effect when performed on IRBC before coincubation with CD36-EC (Fig. 4GoC, IRBC/DTT + CD36). Taken together, these results indicate that both expression of the Mo91 epitope and inhibition of cytoadherence were exclusively dependent on conformational changes elicited by DTT reduction on a fraction of surface-expressed CD36.



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  		FIGURE 3. DTT treatment inhibits IRBC cytoadherence to U937 and CD36-COS-7 cells. A, IRBC cytoadherence was evaluated by luminometric evaluation of hemoglobin. Adherent U937 cells expressing CD36, untreated (U937) or after treatment with DTT (U937/DTT) were incubated with IRBC or noninfected RBC (ctrl). Nonadherent IRBC or RBC were washed away, and the remaining cells were lysed. The number of adherent IRBC or RBC was evaluated by quantitative determination of hemoglobin by luminescence (see Materials and Methods). Values are the mean ± SD (error bars) of three independent experiments. *, p < 0.001). B, Adherent CD36-COS-7 cells or mock-transfected cells (mock) were incubated with IRBC or noninfected RBC. Nonadherent IRBC or RBC were washed away, and remaining cells were lysed. The number of adherent IRBC or RBC was evaluated by quantitative determination of hemoglobin by luminescence (see Materials and Methods). Values are the mean ± SD (error bars) of three independent experiments. *, p < 0.001. C, COS-7 cells transfected with CD36 (CD36-COS-7), untreated (thin line) or after treatment with DTT (thick line), were analyzed by flow cytometry using Mo91 and NL07 conformation-specific mAbs or control (ctrl) mouse Ig. The bound Abs were detected with goat anti-mouse Ig-FITC. The ordinate shows cell numbers, and the abscissa shows fluorescence intensity.

 


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  		FIGURE 4. DTT treatment inhibits IRBC cytoadherence to CD36-EC and dissolves preformed aggregates. A, CD36-EC, untreated (thin line) or after treatment with DTT (thick line), were analyzed by flow cytometry using the Mo91 and NL07 anti-CD36 mAbs, or control (ctrl) mouse Ig. The Abs were detected with goat anti-mouse Ig-FITC. The ordinate shows cell numbers, and the abscissa shows fluorescence intensity. B, After binding to CD36-EC, nonadherent IRBC were washed out, and cells were fixed with 3% paraformaldehyde and stained with JCD36P anti-CD36 mAb directly conjugated with FITC. Cells were then analyzed by epifluorescence microscopy (x200 magnification). Only CD36-EC (a) showed cytoadherence to IRBC (b). C, CD36-EC, or mock-transfected cells (mock) were incubated with IRBC or noninfected RBC. In some samples, CD36-EC, IRBC, or RBC were treated with DTT (CD36/DTT, IRBC/DTT, and RBC/DTT, respectively) and washed twice before coincubation; in other samples the treatment with DTT was performed after coincubation of EC with IRBC (CD36/IRBC + DTT). Nonadherent IRBC or RBC were washed away, and the remaining cells were lysed. The number of adherent IRBC or RBC was evaluated by quantitative determination of hemoglobin by luminescence (see Materials and Methods). Values are the mean ± SD (error bars) of five independent experiments. *, p < 0.001.

 
DTT treatment is able to dissolve pre-existent IRBC aggregates on CD36-EC

DTT treatment performed after the incubation of IRBC with CD36-EC was able to dissolve 78 ± 2.9% of the IRBC aggregates (Fig. 4GoC, CD36/IRBC + DTT). This result might indicate reducing treatment as a suitable treatment to reverse thrombus formation in microvasculature.

Reduction does not impair the functionality of the CD36 receptor

DTT treatment did not disrupt the overall configuration of the receptor, as reactivity with other mAbs, either conformationally (NL07, SMØ) (16, 17, 31) or structurally (OKM5, 131.2) (16, 22) specific, was maintained (Table IGo). In addition, DTT did not impair functionality of the CD36 receptor, since binding and internalization of another CD36 ligand, such as oxLDL, were not affected (Table IIGo). Furthermore, the specificity of the epitope induction by treatment with DTT was confirmed by the reactivity after treatment of Mo25, a mAb identical with Mo91 (17, 22).


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  		Table I. Effect of DTT treatment on reactivity of different anti-CD36 mAbs

 

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  		Table II. Binding of oxLDL by CD36+ cells after treatment with DTT

 
CD36 reduction is reversible

Mo91 reactivity decreased with time at both 37 and 4°C in cells previously treated with DTT and then incubated in DTT-free medium. No Mo91 reactivity was detected on the cell surface 14 h after DTT treatment, while reactivity of NL07 mAb was not affected in similarly treated cells (Fig. 5GoA). Since internalization and neosynthesis slow down at 4°C, we hypothesized that reoxidation was responsible for the disappearance of Mo91 reactivity. Therefore, the same experiments were performed after blockage of reoxidation by thiol alkylation with NEM. Under this condition Mo91 reactivity decreased slightly, but was still present 24 h after treatment (Fig. 5GoA). The reactivity of NL07 was not affected by NEM treatment. Reoxidized CD36 restored approximately 70% of total cytoadherence of IRBC (Fig. 5GoB, open bars, DTT + IRBC), suggesting that the six cysteines present in the extracellular domain of CD36 had re-established the physiologically correct disulfide bonds. Cytoadherence of IRBC was not restored when reoxidation was blocked by alkylation with NEM (Fig. 5BGo, open bars, DTT/NEM+IRBC). Reduction or reduction and alkylation performed just before the binding assay were similarly effective in the inhibition of IRBC cytoadherence (Fig. 5GoB, filled bars).



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  		FIGURE 5. Reduced CD36 spontaneously reoxidizes, and IRBC binding is restored. A, Adherent U937 cells, untreated (ctrl) or after treatment with DTT, were incubated for 24 h at 37 or 4°C. Some samples treated with DTT were alkylated with NEM and left for 24 h at 4°C. At scheduled times (2, 4, 6, 14, and 24 h) cells were analyzed by flow cytometry using Mo91 ({blacksquare}) and NL07 ({blacktriangleup}) anti-CD36 mAbs or control mouse Ig (bckg; {diamondsuit}). The Abs were detected with goat anti-mouse Ig-FITC. The ordinate shows the ratio between mean fluorescence peak of treated cells vs untreated cells; the abscissa shows time. Values are the mean ± SD (error bars) of three independent experiments. B, Adherent U937 cells, untreated or after treatment with DTT or with DTT followed by alkylation with NEM (DTT/NEM), were incubated with either IRBC or RBC immediately after treatment (filled bars). In a set of samples (open bars) cells were left after treatment for 14 h at 4°C before incubation with IRBC or noninfected RBC. Nonadherent IRBC or RBC were washed away, and the remaining cells were lysed. The number of adherent IRBC or RBC was evaluated by quantitative determination of hemoglobin by luminescence (see Materials and Methods). Values are the mean ± SD (error bars) of three independent experiments.

 
Nac treatment induces Mo91 epitope on CD36, inhibits IRBC cytoadherence to CD36-expressing cells, and dissolves preformed aggregates

Nac, a low toxicity reducing agent admitted for therapeutic use in humans, was tested. The compound, used at concentrations ranging between 1 and 100 mM, was able to induce Mo91 reactivity in U937 and CD36-COS-7 cells in a dose-dependent manner without affecting NL07 reactivity (not shown). Since Nac was effective in a dose-dependent manner at concentrations between 10 and 40 mM, it was consistently used at 30 mM. The treatment with Nac inhibited the binding of IRBC to U937 cells expressing CD36 to stably transfected CD36-COS-7 and CD36-EC by 92 ± 3, 88 ± 6.2, and 79 ± 6%, respectively (Fig. 6Go, CD36/Nac + IRBC). All the inhibition values have a significance of p < 0.001 compared with controls. Like DTT, Nac treatment had small nonsignificant effect when performed on IRBC before coincubation (Fig. 6Go, IRBC/Nac + CD36). Interestingly, Nac treatment performed after the incubation of IRBC with CD36-expressing cells was able to dissolve the IRBC aggregates, with a reduction of 72 ± 12% for U937 cells, 82 ± 8% for CD36-COS-7 cells, and of 83 ± 6.7% for CD36-EC, respectively (Fig. 6Go, CD36/IRBC + Nac).



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  		FIGURE 6. Nac treatment induces Mo91 epitope on CD36, inhibits IRBC cytoadherence to CD36-expressing cells, and dissolves preformed aggregates. Adherent U937 cells expressing CD36, CD36-COS-7 cells, CD36-EC, or mock-transfected cells (mock) were incubated with IRBC or noninfected RBC. In some samples CD36-expressing cells or IRBC were treated with Nac (CD36/Nac and IRBC/Nac, respectively) and washed twice before coincubation; in other samples the treatment with Nac was performed after coincubation of CD36-expressing cells with IRBC (CD36/IRBC + Nac). Nonadherent IRBC or RBC were washed away, and the remaining cells were lysed. The number of adherent IRBC or RBC was evaluated by quantitative determination of hemoglobin by luminescence (see Materials and Methods). In the case of CD36-COS-7 cells and CD36-EC, the number of adherent IRBC or RBC was normalized for the percentage of CD36-positive cells as determined by immunoreactivity and FACS analysis. Values are the mean ± SD (error bars) of three independent experiments. *, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that treatment with reducing agents of CD36-EC, U937, and CD36-COS-7 cells expressing mature CD36 induced the partial reappearance of a conformational epitope of CD36 recognized by Mo91 mAb, consistently inhibited cytoadherence of IRBC, and largely dissolved pre-existent IRBC/CD36-EC aggregates. The epitope recognized by Mo91 was localized on residues 260–279 of the C-terminal, cysteine-rich region of CD36. CD36 reduction was reversible, Mo91 reactivity disappeared, and cytoadherence reappeared upon spontaneous reoxidation of CD36. Reduction of CD36 did not remarkably modify its functionality apart from disrupting its ability to bind IRBC. We assume that CD36 reduction generated reversible conformational changes accompanied by the reappearance of the Mo91 epitope and abrogation of cytoadherence. These changes occurred on a fraction of CD36 molecules, but were responsible for 53, 61, or almost 100% abrogation of cytoadherence to IRBC in CD36-EC, U937, or CD36-COS-7 cells, respectively. The formation of disulfide bonds during CD36 processing and intracellular transport seems to mask the epitope recognized by Mo91 on native CD36 (18). CD36 has six cysteine residues located in a 90-aa, cysteine-rich region (residues 243–333). These cysteines are conserved within the CD36 family, suggesting that they participate in a common three-dimensional structure. The small shift in electrophoretic mobility produced by CD36 reduction, visible only after deglycosylation of the molecule (18), suggested that the conformational changes generated by disulfide bond reduction are limited. A further indication that conformational folding masks the Mo91 epitope was its reappearance on CD36 Western blots under denaturing nonreducing conditions (17, 18). Our results indicate the conformational nature of the CD36 region involved in the IRBC binding. The inability of the M3 peptide to inhibit IRBC cytoadhesion (data not shown) seems to support this conclusion. Observations supporting the relevance of CD36 cysteines for the conformational IRBC binding region have been previously reported (19, 32), although the binding region for IRBC was suggested to be external to the cysteine-rich region (13, 14). Finally, we can exclude that cytoadherence was dependent from disulfide-mediated CD36 dimers or oligomers. These have been reported previously (33, 34), but we never observed CD36 complexes in our cells under nonreducing conditions.

We tried to better characterize the two CD36 conformers, but obtained contradictory results. It has been shown that surface-expressed CD36 segregates in both TX100-soluble and TX100-insoluble (also defined as detergent-insoluble glycocomplexes (DIG)) fractions (18, 33, 35, 36). However, it was not possible to assign the reducing agent-sensitive or -resistant form of CD36 to a specific TX100 fraction. In fact, reduction of CD36 affected segregation of native CD36 into the TX100-insoluble fraction, but it induced the Mo91 epitope in both native CD36 and a mutant CD36 that does not segregate into the TX100-insoluble fraction because the four intracellular cysteines essential for DIG localization had been replaced (M. Alessio, unpublished observations). In addition, {beta}-methyl-cyclodextrin, a drug known to disrupt DIG membrane organization by extracting cholesterol from the outer leaflet of the plasma membrane bilayer (37), slightly affected cytoadherence to IRBC (M. Alessio, unpublished observations). In conclusion, we are unable to assign the CD36 fraction that mediates IRBC binding to any TX100-insoluble or -soluble fraction.

It is known that CD36 interacts with the CIDR1{alpha} cysteine-rich domain of the PfEMP1 protein expressed on the IRBC, and that structural conformation of this domain is important for binding (38). It has also been reported that reduction and alkylation of the purified fragment of the CIDR1{alpha} affected the binding to CD36, but fragments were able to reach the correct conformation and rescue the binding if reduction was followed by oxidation (38). This suggested that the conformational structure and function of the whole PfEMP1 molecule expressed on the cell surface are only marginally affected by treatment with reducing agent, as, for example, for Ig {kappa} light chains (39). Thus, the observed small effect of reduction treatment on IRBC (Fig. 5GoC, IRBC/DTT + CD36; and Fig. 6Go, IRBC/Nac + CD36) may be due to the limited conformational changes affecting the CIDR1{alpha} cysteine-rich domain on the intact cell surface expressed PfEMP1 molecules.

Several features described here may offer new approaches to malaria therapy. Firstly, the reducing agents treatment almost completely inhibited IRBC binding to CD36-COS-7 cells and significantly inhibited IRBC cytoadherence to U937 cells and CD36-EC. The latter result is interesting because CD36-EC bear a close resemblance to the real target of IRBC in vivo. Most data reported in the literature were obtained with purified CD36 or transfected cells expressing CD36 that poorly reflect in vivo conditions. Although the involvement of other endothelial adhesive receptors in sustaining IRBC adhesion must be taken into account, a 50–80% reduction in IRBC binding as observed here with CD36-EC, is expected to significantly reduce the pathological effects of cytoadhesion. However, reducing agents may affect many cell molecules and cellular pathways other than CD36. Thus, it is possible that the observed effects may be also mediated by synergistic effects of reducing agents on other adhesion molecules or metabolic pathways. Secondly, reduction of CD36 was reversible, implying that of the 15 random possibilities only the physiologically relevant disulfide bonds were formed spontaneously. A similar kinetics of CD36 regeneration is expected to occur in vivo, in view of the similar redox potential of body fluids compared with the artificial culture media used here. Slow kinetics of reoxidation combined with preserved functionality of CD36 in reduced cells are favorable elements for a therapeutic application of reducing agents. Thirdly, the observation that reducing treatment was able to prevent and to reverse already formed aggregates would add a therapeutic option to the hardly tractable acute phases of organ failures due to enhanced cytoadhesion of IRBC. Additional positive effects of reducing agents may reside in their inhibition of NF-{kappa}B activity and reduced secretion of proinflammatory cytokines that induce proadhesive molecules (40, 41) and in the detoxification of malarial pigment hemozoin (42), a toxic aggregate of noncatabolized heme groups (Ref. 28 and references therein). IRBC binding to dendritic cells expressing CD36 was found to inhibit their maturation and subsequently reduce their capacity to stimulate T cells, contributing to malaria immunosuppression (43, 44). Reducing agent-elicited inhibition of CD36-mediated cell adhesion might have positive effects in this case as well, possibly counterbalancing the inhibitory effect of reducing agents on the primary immune response in dendritic cells (40). Finally, it is of extreme importance that the reducing agent Nac was found to display similar effects as DTT. Nac is a cheap, low toxicity drug without remarkable side effects and with known pharmacokinetics (45) that is used in the treatment of pulmonary disorders (46), hepatic failure (47), severe oxidative stress caused by acetaminophen (48), and AIDS (49, 50, 51). Nac was effective at dosages higher than DTT because it has a lower reducing potential due to the presence of only one thiol group. Nevertheless, concentrations of Nac similar to those used in our experimental conditions can be obtained in plasma of patients after i.v. administration to patients in several clinical trials and therapeutic protocols (47, 48, 49, 52).

Variability in CD36 binding has been described. However, >90% of field isolates of P. falciparum bind CD36 (reviewed in Ref. 2). Within the observed variability, the characteristics of CD36 binding are quite uniform and point at a highly conserved binding site restricted to the relatively conserved CIDR1 region of the molecule (53, 54, 55). Additional support for a single binding region is provided by the adhesion blockage of all strains studied to date by Abs to CD36 such as OKM5 (2, 3), by peptide inhibition studies (14), and by binding to CD36 of most clones of the variant Ag PfEMP1, a family with 50 or more genes. Thus, it can be assumed that reducing agents will affect CD36 binding in most P. falciparum strains. However, it will be interesting to assess the sensitivities of different field isolates to reducing agents to optimize their therapeutic usage.

In conclusion, our results indicate the presence of two redox-modulated conformers of CD36 on the cell surface. Characterization of the conformers will allow better understanding of the CD36-IRBC molecular interactions. The present data suggest that treatment with low toxicity reducing agents might offer a new therapeutic approach for severe malaria complications.


   Acknowledgments
 
We thank Prof. R. Pardi, Prof. R. Sitia, Dr. P. Dellabona, and Dr. E. Bianchi for helpful discussions, and S. Trinca for skillful assistance.


   Footnotes
 
1 This work was supported by a grant from World Health Organization Global Program for Vaccines and Immunization (VRD.11/3 V25/18/193; to M.A.), a grant from ISS (PNR-AIDS 50B.1; to M.A.), Fondazione Piemontese Studi e Ricerche sulle Ustioni (to M.A.), MURST (Programmi di Ricerca di Rilevante Interesse Nazionale) and Compagnia di San Paolo, Torino (to P.A.), F ISS (PNR-AIDS 40B.19 and 30B.5, Program on Tumor Therapy; to F.B.), MURST (60% and Programmi di Ricerca di Rilevante Interesse Nazionale, 1998 and 1999; to F.B.), and Regione Piemonte (to F.B.). Back

2 Address correspondence and reprint requests to Dr. Massimo Alessio, DIBIT, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy. E-mail address: m.alessio{at}hsr.it Back

3 Abbreviations used in this paper: IRBC, infected RBC; CD36-EC, CD36-transfected human endothelial cells; DIG, detergent-insoluble glycocomplex; EC, endothelial cells; Nac, N-acetylcysteine; NEM, N-ethylmaleimide; {beta}-OGTP, {beta}-octylglucothiopyranoside; oxLDL, oxidized low density lipoprotein; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; TX100, Triton X-100. Back

Received for publication June 8, 2001. Accepted for publication October 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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