Plasmodium falciparum is the most lethal form of malaria and is increasing both in incidence and in its resistance to antimalarial agents. An improved understanding of the mechanisms of malarial clearance may facilitate the development of new therapeutic interventions. We postulated that the scavenger receptor CD36, an important factor in cytoadherence of P falciparum–parasitized erythrocytes (PEs), might also play a role in monocyte- and macrophage-mediated malarial clearance. Exposure of nonopsonized PEs to Fc receptor–blocked monocytes resulted in significant PE phagocytosis, accompanied by intense clustering of CD36 around the PEs. Phagocytosis was blocked 60% to 70% by monocyte pretreatment with monoclonal anti-CD36 antibodies but not by antibodies to αvβ3, thrombospondin, intercellular adhesion molecule-1, or platelet/endothelial cell adhesion molecule-1. Antibody-induced CD36 cross-linking did result in the early increase of surface CD11b expression, but there was no increase in, or priming for, tumor necrosis factor (TNF)-α secretion following either CD36 cross-linking or PE phagocytosis. CD36 clustering does support intracellular signaling: Antibody-induced cross-linking initiated intracellular tyrosine phosphorylation as well as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Both broad-spectrum tyrosine kinase inhibition (genistein) and selective ERK and p38 MAPK inhibition (PD98059 and SB203580, respectively) reduced PE uptake to almost the same extent as CD36 blockade. Thus, CD36-dependent binding and signaling appears to be crucial for the nonopsonic clearance of PEs and does not appear to contribute to the increase in TNF-α that is prognostic of poor outcome in clinical malaria.

Plasmodium falciparum malaria is the world's most important parasitic disease, responsible for an estimated 300 million to 500 million cases and 1.5 million to 2.7 million deaths annually.1-3 Deaths occur primarily in young children and other nonimmune individuals who are at greatest risk for developing severe and cerebral malaria.1-3 The central pathophysiologic events in falciparum malaria are the sequestration ofP falciparum–parasitized erythrocytes (PEs) in the microvasculature of vital organs and the release of proinflammatory cytokines from cells of the monocyte/macrophage (mφ) lineage.1,4-6 A number of receptors have been implicated in the cytoadherence of PEs to endothelial cells, including intercellular adhesion molecule (ICAM)-1,7,8 vascular cell adhesion molecule (VCAM)-1,9,10 E-selectin,10platelet/endothelial cell adhesion molecule (PECAM)-1/CD31,11 chondroitin 4-sulfate,12thrombospondin (TSP),13αvβ3,14 and CD36.15-19 CD36, an 88-kd integral protein found on endothelial cells, platelets, monocytes, and mφs, has been shown to be a major sequestration receptor recognized by almost all natural isolates of P falciparum.7,20 However, its role in cerebral malaria is unclear because little CD36 is expressed on cerebral microvasculature endothelial cells.6,21 Other adhesion receptors such as ICAM-1 are expressed in cerebral endothelial cells and may be up-regulated by inflammatory cytokines such as tumor necrosis factor (TNF)-α.1 Elevated levels of proinflammatory cytokines such as TNF-α have been associated with severe malaria and a poor prognosis,22-25 and it has been reported that a genetic predisposition to overproduce TNF-α may be associated with the development of cerebral malaria.1,26-28 Taken together, these data suggest that the sequestration of PEs observed in cerebral malaria may result from up-regulated ICAM-1 and other adhesion molecules on the cerebral microvasculature because of excessive or unchecked proinflammatory responses.1,24,26 28 

Phagocytic cells are an essential first line of defense against malaria. Circulating monocytes and tissue resident mφs in the liver, spleen, and elsewhere facilitate the control and resolution of infection by clearing PEs.29,30 However, the molecular mechanisms by which these cells recognize PEs is not well understood. To date most studies have concentrated on the phagocytosis of opsonized PEs31-33; however, it is unclear what role this type of clearance plays in the nonimmune individuals most at risk for severe and cerebral malaria. Monocytes can bind PEs in the absence of antibody,34 an event mediated at least in part by CD36,33 and engulf PEs even when complement and Fc receptor pathways are blocked.32 Recent studies in scavenger receptor knockout mice have suggested that this class of monocyte/mφ receptors may be involved in protection against murine malaria.35 

We undertook a study of the molecular mechanisms of P falciparum phagocytosis based on the hypothesis that monocyte/mφ scavenger receptor CD36 may play a role in the nonopsonic clearance of PEs. Because a recent study demonstrated that CD36 engagement by TSP activates the p38 mitogen-activated protein kinase (MAPK),36 we further postulated that clustering of CD36 would occur during PE phagocytosis and activate the p38 and the related extracellular signal-regulated kinase (ERK) MAPKs and that this activation would contribute to the ingestion process. We found that nonopsonic phagocytosis of P falciparum PEs occurs in a CD36-dependent fashion that is associated with pronounced clustering of CD36. Both the ERK and p38 MAPKs are phosphorylated upon cross-linking of CD36, and selective pharmacologic inhibition of either MAPK decreases the nonopsonic uptake of PEs by half. Our results suggest that monocytic CD36 uses both the ERK and p38 MAPK signaling cascades to actively participate in the nonopsonic phagocytosis of PEs.

Media and reagents

Endotoxin-free RPMI media were purchased from Life Technologies, Inc (Burlington, ON, Canada). Fetal calf serum (FCS) was from Hyclone (Logan, UT) and was heat-inactivated at 55°C for 30 minutes. Genistein (Calbiochem, La Jolla, CA), piceatannol (Sigma, Oakville, ON), and PD98059 (RBI, Natich, MA) were prepared in dimethyl sulfoxide. SB203580 was the kind gift of Dr J. C. Lee (SmithKline Beecham) and was suspended in dimethyl sulfoxide. The following mouse immunoglobulin (Ig) G1 monoclonal antibodies were used in phagocytosis or cross-linking studies: anti-CD36 OKM5 (Ortho Diagnostic Systems, Raritan, NJ), FA6-152 (Immunotech, Marseille, France), anti-TSP clone C6.7 (Medicorp, Montreal, QC, Canada), anti-αvβ3 clone 23C6 (Serotec, Raleigh, NC), anti–ICAM-1 clone 15.2 (Santa Cruz Biotech, CA), anti-CD49d hp2.1 (Immunotech), anti-CD45 monoclonal antibody 1214 (PDI Bioscience/Coulter, Montreal, QC, Canada), anti-FcγRIII 3G8 (Immunotech), and anti-CD18 7E4 (Immunotech). IgG2banti–HLA-DR BL2 was from Immunotech, as was goat F(ab′)2antimouse IgG. IgG2a anti-CD31 hec7 (PECAM-1) was from Endogen (Woburn, MA). Escherichia coli O111:B4 endotoxin (LPS) was purchased from Sigma and made up in sterile water. Human IgG Fc fragments were purchased from Calbiochem and trypsin-ethylenediaminetetraacetic acid (EDTA) from Sigma.

Monocyte and PE preparation

Human monocytes were isolated from the blood of healthy volunteers as previously described.37 Briefly, the buffy coat fraction from heparinized whole blood was slowly brought to an osmolarity of 360 mOsm by the addition of sterile 9% NaCl. After centrifugation over a 40/55/58 Percoll gradient at 600g for 30 minutes, the monocyte-rich layer was collected and washed 3 times in cold RPMI. This procedure consistently yields a platelet-free population of purified monocytes (> 96% neutral red granule positive; > 80% CD14+ by flow cytometry), which are not activated (minimal baseline procoagulant activity and TNF-α secretion) and have more than 98% viability by trypan blue exclusion. In some experiments, monocyte-derived mφs were prepared by in vitro incubation of purified monocytes on tissue culture wells for 5 days and maintained in RPMI–10% FCS at 37°C, 5% CO2. P falciparumcultures of the laboratory clone ITG38 were grown and synchronized by sorbitol lysis followed by 24 hours of culture as described previously.39 40 Synchronized trophozoite-stage infected erythrocytes were carefully washed 3 times in RPMI prior to phagocytosis and TNF-α assays. In experiments using trypsinised PEs, PEs were suspended in a 0.05% trypsin-EDTA solution, incubated at 37°C for 30 minutes, and washed twice in RPMI prior to phagocytosis assays.

Phagocytosis assay

About 2.5 × 105 monocytes were adhered to autoclaved glass coverslips placed in 12-well polystyrene culture plates. For studies of nonopsonic phagocytosis, Fc receptors were first blocked by incubating cells with human IgG Fc fragments (Calbiochem, San Diego, CA) at 20 μg/mL for 25 minutes at room temperature. Following incubation with Fc receptors, monocytes or culture-derived mφs were incubated with 10 μg/mL of anti-CD36, CD49d, PECAM-1, ICAM-1, TSP, or αvβ3 antibodies for an additional 25 minutes and washed with 2 changes of RPMI. PEs were suspended in 500 μL of RPMI–10% FCS–L-glutamine (L-G) and added to the monocytes/mφs at a PE:cell ratio of 20:1. In experiments to examine opsonic phagocytosis, PEs were opsonized by exposure to 50% patient serum (heat-inactivated for 30 minutes at 55°C) for 1 hour at 37°C. Control monocytes/mφs were exposed to equivalent numbers of uninfected erythrocytes (UEs). Plates were rotated gently for 4 hours at 37°C, 5% CO2. At the end of this time, nonadherent erythrocytes were washed away with 3 changes of RPMI, and adherent but nonphagocytosed erythrocytes were lysed in ice-cold distilled water for 30 seconds. Cell preparations were fixed and stained with Giemsa. Phagocytosis was assessed by light microscopy. From 500 to 1000 monocytes/mφs were counted for each coverslip and scored for the presence or absence of phagocytosed PEs. Criteria for phagocytosis required the PEs to be contained completely within the monocyte/mφ cell outline. The phagocytic index was calculated as the percentage of monocytes/mφs with clear evidence of phagocytosis.

Immunofluorescence

After lysis of nonphagocytosed PEs and UEs, monocytes were fixed and permeabilized in ice-cold 100% methanol for 60 seconds. Cells were stained for 1 hour with the monoclonal, fluorescein isothiocynate (FITC)-labeled anti-CD36 OKM5 or with murine monoclonal IC4 antibody specific to antigens expressed on the surface of P falciparum–infected erythrocytes.41 Following 5 washes in phosphate-buffered saline (PBS), Texas red–labeled goat antimouse antibody was added to the IC4 preparations and incubated for an additional hour, after which coverslips were washed in PBS and mounted. In colocalization studies, fixed cell preparations were stained first with IC4/antimouse IgG and then with FITC-labeled OKM5. Microscopy was performed with a Bio-Rad MRC 1024ES confocal microscope and analyzed with Lasersharp software.

Surface antigen cross-linking

Surface antigens were cross-linked on purified human monocytes using monoclonal antibodies as previously described.42About 5 × 105 monocytes were suspended in 100-μL RPMI–2% FCS–L-G at 4°C. Fc receptors were blocked by incubating the cells with 20-μg/mL IgG Fc fragments (Calbiochem) at 4°C for 20 minutes followed by washing in RPMI. Surface antigens were then ligated with 10 μg/mL of monoclonal antibody directed against CD36 (OKM5, FA6-152), very late antigen 4 (CD49d, hp2.1), CD18 (7E4), HLA-DR (BL2), or Fc-γRIII (3G8). After a 20-minute incubation at 4°C, cells were washed twice in RPMI, and 5-μg/mL goat antimouse F(ab′)2was added for an additional 20 minutes. After washing in cold RPMI, cells were resuspended in 500-μL RPMI–2% FCS–L-G and placed in the 37°C, 5% CO2 incubator for times ranging from 5 minutes to 4 hours.

Assessment of protein, ERK, and p38 MAPK phosphorylation

Following antigen cross-linking, monocytes were placed on ice, sedimented, and lysed in ice-cold lysis buffer containing 1% Triton X-100, 150-mmol/L NaCl, 10-mmol/L Tris-HCl (pH 7.4), 2-mmol/L sodium orthovanadate, 100-μg/mL leupeptin, 50-mmol/L NaF, 5-mmol/L EDTA, 1-mmol/L EGTA, and 1-mmol/L phenylmethylsulfonyl fluoride. Postnuclear supernatants were collected after centrifugation at 10 000gfor 5 minutes, diluted with 2 × Laemmli buffer, 0.1-mol/L dithiothreitol, and boiled for 4 minutes. Lysates prepared from 100 000 cells were separated on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore). Blots were then probed with polyclonal rabbit antiphosphotyrosine (Transduction Laboratories, Mississauga, ON, Canada) or antibody specific to the dually phosphorylated, activated forms of the ERK and p38 MAPKs (New England Biolabs, Burlington, ON, Canada). Following incubation with horseradish peroxidase–conjugated secondary antibody (Amersham, Montreal, QC, Canada), blots were developed using an ECL-based system (Amersham).

Measurement of monocyte TNF-α secretion and surface CD11b expression

Four hours after surface antigen cross-linking or exposure to PEs, cell-free supernatants were collected and assayed for soluble TNF-α using a sandwich enzyme-linked immunosorbent assay as described previously.43 Surface CD11b was measured 1 hour after cross-linking of cell surface antigens or exposure of the cells to 1-μg/mL LPS by flow cytometry. Suspended monocytes were washed in cold RPMI and resuspended in RPMI–10% FCS–L-G. Phycoerythrin-labeled anti-CD11b monoclonal antibody (Becton Dickinson) was incubated with the cells for 20 minutes at 4°C. Cells were washed in cold RPMI and evaluated for staining on a Coulter EPICS XL Cytofluorometer.

Statistical analysis

The data are represented as the mean and standard error of the indicated number of experiments. Where representative studies are shown, they are indicative of at least 3 equivalent and independent studies. Statistical comparisons were made for continuous data using one-way ANOVA with post hoc Tukey.

Nonopsonic phagocytosis of P falciparum–infected erythrocytes

Monocytes are able to phagocytose P falciparum–infected erythrocytes in the absence of opsonization and in the relatively stringent conditions created by a low PE:monocyte ratio. As shown in Figure 1A (light microscopy) and Figure 1B (immunofluorescence with the PE-specific IC4 monoclonal antibody), the incubation of monocytes with PEs in a complement-free environment results in PE uptake despite Fc receptor blockade of the monocytes and no prior PE opsonization. To avoid quantitating PEs attached to the monocyte but not internalized, we employed both a strict lysis procedure to remove adherent erythrocytes and morphologic criteria to ensure that only phagocytosed PEs were counted. As well, PE phagocytosis was routinely confirmed by confocal microscopy. UEs are not taken up by the monocytes. Monocytes did not have to be adherent for phagocytosis to occur: leukocytes in suspension demonstrated an equivalent ability to ingest PEs (data not shown). In time course studies, we found that nonopsonic PE phagocytosis increased in a linear fashion over the 4-hour course of the assay, with 1 to 4 PEs being ingested by monocytes (data not shown).

Fig. 1.

Nonopsonic phagocytosis of

P falciparum PEs. Synchronized, nonopsonizedP falciparum trophozoite PE cultures were incubated with adherent, Fc receptor–blocked human monocytes in 10% heat-inactivated fetal calf serum for 4 hours as described in “Materials and methods.” Nonadherent erythrocytes were washed away and adherent erythrocytes removed by hypotonic lysis. After washing, monocytes were fixed and prepared for light microscopic examination (A) or for IC4 monoclonal antibody immunofluoresence (B). (A) Three panels of representative monocytes are shown under light microscopy. Each panel contains 1 or 2 monocytes, with several ingested PEs. (B) Three panels of representative monocytes analyzed by IC4 monoclonal antibody immunofluorescence following PE phagocytosis. The typical speckled pattern produced by IC4 binding to the PE surface can be readily appreciated in the ingested PEs. All results shown are indicative of results obtained on at least 3 independent occasions. Original magnification, × 1000.

Fig. 1.

Nonopsonic phagocytosis of

P falciparum PEs. Synchronized, nonopsonizedP falciparum trophozoite PE cultures were incubated with adherent, Fc receptor–blocked human monocytes in 10% heat-inactivated fetal calf serum for 4 hours as described in “Materials and methods.” Nonadherent erythrocytes were washed away and adherent erythrocytes removed by hypotonic lysis. After washing, monocytes were fixed and prepared for light microscopic examination (A) or for IC4 monoclonal antibody immunofluoresence (B). (A) Three panels of representative monocytes are shown under light microscopy. Each panel contains 1 or 2 monocytes, with several ingested PEs. (B) Three panels of representative monocytes analyzed by IC4 monoclonal antibody immunofluorescence following PE phagocytosis. The typical speckled pattern produced by IC4 binding to the PE surface can be readily appreciated in the ingested PEs. All results shown are indicative of results obtained on at least 3 independent occasions. Original magnification, × 1000.

Close modal

Noncomplement-mediated phagocytosis of PEs has been noted previously, albeit in previously opsonized PEs with a higher PE:monocyte ratio (200-300:1 vs 20:1).32 A direct comparison of opsonic versus nonopsonic PE phagocytosis was undertaken to determine the relative efficiencies of both processes over a 4-hour time span. As seen in Figure 2, opsonic phagocytosis leads to more PE uptake than the nonopsonic process. Moreover, opsonized PEs are consumed more avidly, with the typical monocyte ingesting 4 to 6, as compared with 1 to 4 for nonopsonized PEs. On the other hand, our results with freshly explanted monocytes likely underestimate the potential for nonopsonic PE uptake in vivo. Tissue mφs would be expected to behave more like culture-matured mφs and have been reported to express increased levels of surface CD36.44 To address this issue, we examined the ability of culture-derived mφs to phagocytose PEs. Of note, monocytes induced to differentiate into mφs by a 5-day culture on culture plates expressed significantly higher levels of CD36 and ingested significantly more nonopsonized PEs than did freshly isolated monocytes (Table1). CD36 expression in culture-derived mφs increased a mean of 83.9%, and phagocytosis increased a mean of 124.6%. Anti-CD36 antibodies significantly inhibited phagocytosis by both fresh monocytes and monocyte-derived mφs. Taken together, these results demonstrate that nonopsonic PE phagocytosis occurs and, although less efficient than opsonic uptake, nonetheless could account for considerable ingestion of PEs. In addition, the observation of increased uptake of nonopsonized PEs coincident with increased surface expression of CD36 supports a role for CD36 in this process.

Fig. 2.

A comparison of nonopsonic and opsonic PE phagocytosis.

PEs were prepared as before or were opsonized by the addition of malarial patient antiserum for 60 minutes. Phagocytosis was allowed to proceed for 4 hours, at which time nonadherent cells were washed away and adherent erythrocytes lysed. The phagocytic index for the 2 groups was calculated as described in “Materials and methods.” Data = mean ± SEM; n ≥ 8 per group. δ, P < .001 vs control UEs; ***, P < .001 vs nonopsonized PEs (ANOVA with post hoc Tukey).

Fig. 2.

A comparison of nonopsonic and opsonic PE phagocytosis.

PEs were prepared as before or were opsonized by the addition of malarial patient antiserum for 60 minutes. Phagocytosis was allowed to proceed for 4 hours, at which time nonadherent cells were washed away and adherent erythrocytes lysed. The phagocytic index for the 2 groups was calculated as described in “Materials and methods.” Data = mean ± SEM; n ≥ 8 per group. δ, P < .001 vs control UEs; ***, P < .001 vs nonopsonized PEs (ANOVA with post hoc Tukey).

Close modal

CD36 is clustered during phagocytosis of PEs

To begin to investigate the molecular mechanisms underlying nonopsonic PE phagocytosis, monocytes that had ingested PEs were stained with the OKM5 antibody specific to CD36. Cells that have not ingested PEs are outlined by antibodies specific for CD36 but do not show evidence of CD36 clustering (Figure3A). By contrast, the process of PE uptake by monocytes is accompanied by intense clustering of CD36 in the region of the phagocytosed PEs (Figure 3B). Consistent with a selective role for CD36 in the ingestion of PEs, CD36 clustering is not observed upon ingestion of latex beads coated with Fc fragments (data not shown). Confirmation of the effect is seen in Figure 3C, which demonstrates colocalization of CD36 clustering with ingested PEs as determined by confocal microscopy. As evidenced by these colocalization studies, CD36 clustering was observed in virtually all cells that had ingested PEs. This type of clustering is similar to the clustering of Fc receptors observed during Fc-dependent phagocytosis45and suggests that CD36 is actively involved in the process of PE phagocytosis.

Fig. 3.

CD36 is clustered during PE phagocytosis.

Purified human monocytes were incubated with PE preparations for 4 hours, at which time the cells were extensively washed, adherent erythrocytes lysed, and monocytes fixed in 100% methanol. (A) A typical monocyte that has not ingested any PEs is stained with FITC-labeled anti-CD36 OKM5 and analyzed by immunofluorescent microscopy. The cell outline can be readily appreciated. (B) A monocyte that has ingested 2 PEs. The position of the PEs as determined by light microscopy is indicated by arrows. The cell is stained with FITC-labeled OKM5 antibody; immunofluorescent microscopy demonstrates the dense clustering of CD36 at the site of PE ingestion. (C) After a 4-hour nonopsonic phagocytosis assay, monocytes were fixed in 100% methanol, stained first with the PE-specific IC4 monoclonal antibody, then with Texas red–labeled antimouse secondary, and finally with FITC-labeled anti-CD36 OKM5 antibody. Each panel shows single monocytes that have ingested 1 (right panel) or 2 (left panel) PEs. Note the dense clustering of CD36 (green) surrounding the PEs (red) and colocalizing with them (as evidenced by yellow staining). Results shown are representative of 4 independent experiments.

Fig. 3.

CD36 is clustered during PE phagocytosis.

Purified human monocytes were incubated with PE preparations for 4 hours, at which time the cells were extensively washed, adherent erythrocytes lysed, and monocytes fixed in 100% methanol. (A) A typical monocyte that has not ingested any PEs is stained with FITC-labeled anti-CD36 OKM5 and analyzed by immunofluorescent microscopy. The cell outline can be readily appreciated. (B) A monocyte that has ingested 2 PEs. The position of the PEs as determined by light microscopy is indicated by arrows. The cell is stained with FITC-labeled OKM5 antibody; immunofluorescent microscopy demonstrates the dense clustering of CD36 at the site of PE ingestion. (C) After a 4-hour nonopsonic phagocytosis assay, monocytes were fixed in 100% methanol, stained first with the PE-specific IC4 monoclonal antibody, then with Texas red–labeled antimouse secondary, and finally with FITC-labeled anti-CD36 OKM5 antibody. Each panel shows single monocytes that have ingested 1 (right panel) or 2 (left panel) PEs. Note the dense clustering of CD36 (green) surrounding the PEs (red) and colocalizing with them (as evidenced by yellow staining). Results shown are representative of 4 independent experiments.

Close modal

Nonopsonic phagocytosis of PEs is CD36 dependent but does not use the αvβ3-TSP-CD36 phagocytic mechanism

CD36 is involved in PE binding to monocytic cells.16 19 To assess the role of CD36 in nonopsonic PE phagocytosis, CD36 adhesive interactions were blocked by monoclonal antibodies. As seen in Figure 4, pretreatment of monocytes with OKM5 or FA6-152 results in a dramatic decrease in PE phagocytosis, whereas no inhibition was observed with the IgG isotype–matched hp2.1 (CD49d), anti–PECAM-1, or anti–ICAM-1 monoclonal antibodies. All antibodies bound to the monocytes as judged by flow cytometry (data not shown).

Fig. 4.

Nonopsonic PE phagocytosis is inhibited with anti-CD36 antibodies.

(A) Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti-CD36 (OKM5, FA6-152), anti-CD49d (HP2.1), or anti–PECAM-1 (HEC7) antibodies for 20 minutes at room temperature and then washed. Phagocytosis assays with synchronized PE cultures were performed as before. (B) Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti-TSP (C6.7), anti-αvβ3 (23C6), or anti-CD36 antibodies for 30 minutes and washed. Phagocytosis assays were performed as before. (C) Synchronized PEs were suspended in a 0.05% trypsin-EDTA solution and incubated at 37°C for 30 minutes. PEs were then washed twice, and phagocytosis assays were performed as before. Alternatively, Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti-CD36, and phagocytosis assays were performed as before. (D) Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti–ICAM-1 or anti-CD36 antibody for 30 minutes and washed. Phagocytosis assays were performed as before. In all cases, cumulative data from at least 3 independent experiments are presented as mean ± SEM; n ≥ 3 per group. **, P < .01; ***,P < .001 vs PEs (ANOVA with post hoc Tukey).

Fig. 4.

Nonopsonic PE phagocytosis is inhibited with anti-CD36 antibodies.

(A) Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti-CD36 (OKM5, FA6-152), anti-CD49d (HP2.1), or anti–PECAM-1 (HEC7) antibodies for 20 minutes at room temperature and then washed. Phagocytosis assays with synchronized PE cultures were performed as before. (B) Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti-TSP (C6.7), anti-αvβ3 (23C6), or anti-CD36 antibodies for 30 minutes and washed. Phagocytosis assays were performed as before. (C) Synchronized PEs were suspended in a 0.05% trypsin-EDTA solution and incubated at 37°C for 30 minutes. PEs were then washed twice, and phagocytosis assays were performed as before. Alternatively, Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti-CD36, and phagocytosis assays were performed as before. (D) Fc receptor–blocked monocytes were pretreated with 10-μg/mL monoclonal anti–ICAM-1 or anti-CD36 antibody for 30 minutes and washed. Phagocytosis assays were performed as before. In all cases, cumulative data from at least 3 independent experiments are presented as mean ± SEM; n ≥ 3 per group. **, P < .01; ***,P < .001 vs PEs (ANOVA with post hoc Tukey).

Close modal

Previous studies examining the phagocytosis of apoptotic cells have shown that monocyte/mφ CD36 cooperates with the vitronectin receptor (αvβ3) and TSP to clear apoptotic cells.46-48 To determine whether CD36 was cooperating in a similar manner in the phagocytosis of nonopsonized PEs, CD36, αvβ3, and TSP were blocked by preincubation of monocytes with the monoclonal antibodies FA6-152, 23C6 (anti-αvβ3), and C6.7 (anti-TSP) alone and in combination. The anti-αvβ3 and anti-TSP antibodies had no effect on phagocytosis of nonopsonised PEs alone or in combination (Figure 4B, and data not shown).

PE adhesion to CD36 is mediated by the variant malarial antigen,P falciparum erythrocyte membrane protein (PfEMP)-1. PfEMP-1 is removed from the surface of the PEs following mild protease treatment.49 We investigated whether the phagocytosis of PEs is dependent upon this CD36 ligand on the infected erythrocyte surface. Proteolytic cleavage of PfEMP-1 from PEs prior to incubation with monocytes reduced their phagocytic clearance to that observed after CD36 receptor blockade with the monoclonal antibodies (Figure4C). These results suggest that CD36-mediated adhesion is central to the nonopsonic phagocytosis of PEs, although our data do not exclude the possible involvement of other receptors.

Induction of tyrosine phosphorylation, ERK, and p38 MAPK phosphorylation by CD36 cross-linking: effect of genistein, PD98059, and SB203580

A cell surface receptor actively involved in phagocytosis would be expected to generate intracellular signaling. We mimicked the intense clustering of CD36 observed during PE phagocytosis by first binding the surface antigen with specific monoclonal antibodies and then adding F(ab′)2 fragments directed against the antibody. The method allowed us to study signaling effects specific to CD36 without the confounding effect of other potential PE-monocyte interactions. Cross-linking of CD36 with primary anti-CD36 monoclonals followed by antimouse F(ab′)2 fragments results in intense clustering of CD36, similar to that observed with PE phagocytosis, and an increase in intracellular tyrosine phosphorylation (Figure5A-B), peaking 10 to 20 minutes after cross-linking and then fading (Figure 5B). Importantly, although simple antibody ligation of CD36 did generate a mild increase in tyrosine phosphorylation, cross-linking of the receptor with the addition of F(ab′)2 fragments results in considerably more signaling (Figure 5A). The F(ab′)2 fragments alone did not stimulate an intracellular signal (Figure 5A); nor did they bind to the monocytes (flow cytometric analysis, data not shown). Together these data are consistent with a signaling role for CD36 clustering.

Fig. 5.

CD36 clustering induces protein tyrosine and MAPK phosphorylation.

Monocyte surface antigen was ligated with primary murine anti-CD36 FA6-152 antibody and cross-linked with the addition of goat antimouse F(ab′)2 fragments. Ligation and cross-linking were performed at 4°C, and cellular reactions were initiated by bringing the cells to 37°C in a 5% CO2incubator. Western blot analysis was performed after lysis of the cells at the times indicated. (A) Western blot staining for tyrosine-phosphorylated proteins after simple antibody ligation of surface CD36 or clustering of CD36 by antibody cross-linking. Note the more intense pattern of tyrosine phosphorylation that follows cross-linking; the secondary F(ab′)2 alone does not induce intracellular tyrosine phosphorylation. (B) Western blot staining for tyrosine-phosphorylated proteins after cross-linking of CD36 in the presence or absence of genistein (10 μg/mL). Note the time course of phosphotyrosine accumulation, which peaks 10 to 20 minutes after cross-linking. (C) Accumulation of dually phosphorylated forms of the ERK and p38 MAPKs after cross-linking of surface CD36. In both cases, phosphorylated forms can be appreciated 60 to 120 minutes after cross-linking. Pretreatment of monocytes with genistein abolishes the induction of ERK phosphorylation and inhibits p38 MAPK phosphorylation. (D) Effect of monocyte pretreatment with PD98059 (50 μmol/L) or SB203580 (30 μmol/L) on the induction of tyrosine phosphorylation following cross-linking of CD36. Cells were lysed after 10 minutes of incubation at 37°C, 5% CO2. (E) Effect of monocyte pretreatment with PD98059 (50 μmol/L) or SB203580 (30 μmol/L) on CD36-dependent induction of ERK and p38 MAPK phosphorylation. Cells were lysed after a 20-minute incubation at 37°C, 5% CO2. All studies are representative of results obtained on at least 3 separate occasions. Although only data with the FA6-152 clone are presented, similar results were obtained with the OKM5 antibody.

Fig. 5.

CD36 clustering induces protein tyrosine and MAPK phosphorylation.

Monocyte surface antigen was ligated with primary murine anti-CD36 FA6-152 antibody and cross-linked with the addition of goat antimouse F(ab′)2 fragments. Ligation and cross-linking were performed at 4°C, and cellular reactions were initiated by bringing the cells to 37°C in a 5% CO2incubator. Western blot analysis was performed after lysis of the cells at the times indicated. (A) Western blot staining for tyrosine-phosphorylated proteins after simple antibody ligation of surface CD36 or clustering of CD36 by antibody cross-linking. Note the more intense pattern of tyrosine phosphorylation that follows cross-linking; the secondary F(ab′)2 alone does not induce intracellular tyrosine phosphorylation. (B) Western blot staining for tyrosine-phosphorylated proteins after cross-linking of CD36 in the presence or absence of genistein (10 μg/mL). Note the time course of phosphotyrosine accumulation, which peaks 10 to 20 minutes after cross-linking. (C) Accumulation of dually phosphorylated forms of the ERK and p38 MAPKs after cross-linking of surface CD36. In both cases, phosphorylated forms can be appreciated 60 to 120 minutes after cross-linking. Pretreatment of monocytes with genistein abolishes the induction of ERK phosphorylation and inhibits p38 MAPK phosphorylation. (D) Effect of monocyte pretreatment with PD98059 (50 μmol/L) or SB203580 (30 μmol/L) on the induction of tyrosine phosphorylation following cross-linking of CD36. Cells were lysed after 10 minutes of incubation at 37°C, 5% CO2. (E) Effect of monocyte pretreatment with PD98059 (50 μmol/L) or SB203580 (30 μmol/L) on CD36-dependent induction of ERK and p38 MAPK phosphorylation. Cells were lysed after a 20-minute incubation at 37°C, 5% CO2. All studies are representative of results obtained on at least 3 separate occasions. Although only data with the FA6-152 clone are presented, similar results were obtained with the OKM5 antibody.

Close modal

Antibody-induced clustering of CD36 also leads to accumulation of the dually phosphorylated, active forms of the p42 ERK2, p44 ERK1, and p38 MAPK as judged by Western blot analysis (Figure 5C). Phosphorylation of both the ERK and p38 MAPK proteins peaks within 10 to 20 minutes of CD36 cross-linking but persists through 60 to 120 minutes (Figure 5C). Pretreatment of monocytes with the broad-spectrum tyrosine kinase inhibitor genistein greatly attenuates the accumulation of tyrosine phosphoproteins after CD36 cross-linking, abrogates the induction of phosphorylated ERK, and attenuates the phosphorylation of the p38 MAPK (Figure 5B-C). By contrast, CD36 cross-linking continues to induce phosphorylation of cellular proteins on tyrosine residues after monocyte pretreatment with the MEK-1 selective inhibitor, PD98059, or the p38 MAPK-selective inhibitor, SB203580 (Figure 5D). Consistent with their mechanisms of action, PD98059 abolishes CD36-dependent phosphorylation of the ERK MAPK but has no effect on p38 MAPK phosphorylation, and SB203580, which directly inhibits p38 MAPK activity, does not inhibit either ERK or p38 MAPK phosphorylation (Figure 5E). Taken together, these studies demonstrate that cross-linking of CD36 results in the prolonged phosphorylation of both the ERK and p38 MAPKs.

In PE phagocytosis studies, we also observed a similar increase in phosphorylation of the ERK and p38 MAPKs associated with the ingestion of PEs by human monocytes (data not shown). However, because it is difficult to exclude other possible PE-monocyte–induced signaling events associated with these complex cell-cell interactions, these studies are less specific than the antibody cross-linking studies described above.

PE phagocytosis is dependent upon the signaling cascades induced by CD36 clustering

To determine whether the intracellular signals induced by CD36 cross-linking contribute to the process of PE phagocytosis, monocytes were pretreated with the broad-spectrum tyrosine kinase inhibitor genistein or with selective ERK and p38 pathway inhibitors (PD98059 and SB203580, respectively) or with the Syk kinase–specific inhibitor piceatannol. Genistein, PD98059, and SB203580 diminished phagocytosis by 60%, 40%, and 50%, respectively, and piceatannol had no inhibitory effect (Figure 6). These data suggest that the ERK and p38 MAPK signals are important to nonopsonic PE phagocytosis.

Fig. 6.

Tyrosine phosphorylation, ERK, and p38 MAPK play roles in nonopsonic PE phagocytosis.

Monocytes were pretreated with genistein (10 μg/mL), PD98059 (50 μmol/L), SB203580 (30 μmol/L), or piceatannol (30 μg/mL) for 1 hour at room temperature, and PE phagocytosis was evaluated as before. Cumulative data from at least 4 independent experiments are presented as mean ± SEM; n ≥ 6 per group. **, P < .01; ***, P < .001 vs PEs (ANOVA with post hoc Tukey).

Fig. 6.

Tyrosine phosphorylation, ERK, and p38 MAPK play roles in nonopsonic PE phagocytosis.

Monocytes were pretreated with genistein (10 μg/mL), PD98059 (50 μmol/L), SB203580 (30 μmol/L), or piceatannol (30 μg/mL) for 1 hour at room temperature, and PE phagocytosis was evaluated as before. Cumulative data from at least 4 independent experiments are presented as mean ± SEM; n ≥ 6 per group. **, P < .01; ***, P < .001 vs PEs (ANOVA with post hoc Tukey).

Close modal

Several lines of evidence suggest that our results are specific and not due to nonspecific pharmacologic effects of PD98059 or SB203580. First, we found no evidence of a nonspecific toxic effect. None of the inhibitors employed exerted monocyte toxicity, as evidenced by more than 96% cell viability (trypan blue exclusion) after 6-hour incubations in their presence. Importantly, the pharmacologic agents did not affect baseline surface CD36 expression over a 6-hour incubation period (flow cytometric data, not shown) and did not affect the number of PEs adhering to the monocyte surface (data not shown). Secondly, both PD98059 and SB203580 may inhibit the cyclooxygenase enzyme.50 However, pretreatment of monocytes with a potent cyclooxygenase inhibitor, indomethacin (100 μmol/L), has no effect on the ingestion of nonopsonized PEs (data not shown). Third, in dose-response experiments we found that 1-, 10-, and 30-μmol/L doses of SB203580 were equally effective at inhibiting nonopsonic PE ingestion (data not shown). Finally, the inhibition of nonopsonized PE uptake by PD203580 and SB203580 is quantifiably different from the effects of these inhibitors on opsonized PE ingestion. In contrast to the 40% inhibition observed with nonopsonic uptake, we found that pretreatment of monocytes with PD98059 (50 μmol/L) inhibited phagocytosis of opsonized PEs by only 9.7% ± 3.5% (mean ± SEM; n = 6). Similarly, SB203580 (1 μmol/L) inhibited nonopsonic ingestion of PEs by 50%, but the same dose had little to no effect on opsonic phagocytosis (inhibition 3.0 ± 3.6%; n = 3). In addition, piceatannol,51 a known inhibitor of Fc-mediated phagocytosis,52 had no inhibitory effect on the phagocytosis of nonopsonized PEs (Figure 6). Thus, the inhibition of nonopsonized PE uptake by PD98059 and SB203580 appears to reflect selective effects of the ERK and p38 MAPK inhibitors and to differ from opsonic phagocytosis.

CD36 cross-linking induces increased CD11b expression but not increased TNF-α secretion: implications for PE phagocytosis

Previous work has established that CD36 engagement by monoclonal antibody or binding of PEs can result in an early increase in monocyte oxidative burst.53 Consistent with the finding that CD36 engagement triggers early monocyte responses, we found that within 1 hour of CD36 cross-linking there is increased surface expression of CD11b (Figure 7). The increase was similar to that observed with a 1-μg/mL dose of LPS; cross-linking of a variety of other monocyte surface antigens, including CD49d, CD18, HLA-DR, and FcγRIII, results in no or less marked CD11b up-regulation.

Fig. 7.

Up-regulation of surface monocyte CD11b by cross-linking of CD36.

One hour after treatment with a 1-μg/mL dose of LPS or cross-linking of surface CD36 (FA6-152), HLA-DR (BL2), FcγRIII (3G8), CD18 (7E4), or CD49d (hp2.1), monocytes were washed extensively and evaluated for surface expression of CD11b as described in “Materials and methods.” Points are taken in triplicate and are representative of results obtained in 4 separate experiments. MCF indicates median channel fluoresence; **, P < .01; ***,P < .001 vs mock–cross-linked cells (ANOVA with post hoc Tukey).

Fig. 7.

Up-regulation of surface monocyte CD11b by cross-linking of CD36.

One hour after treatment with a 1-μg/mL dose of LPS or cross-linking of surface CD36 (FA6-152), HLA-DR (BL2), FcγRIII (3G8), CD18 (7E4), or CD49d (hp2.1), monocytes were washed extensively and evaluated for surface expression of CD11b as described in “Materials and methods.” Points are taken in triplicate and are representative of results obtained in 4 separate experiments. MCF indicates median channel fluoresence; **, P < .01; ***,P < .001 vs mock–cross-linked cells (ANOVA with post hoc Tukey).

Close modal

Human monocytes/mφs produce TNF-α in response to falciparum malaria, and Nagao et al5 have reported that fresh human monocytes produce significantly more TNF-α than culture-derived mφs in response to infection. To determine if CD36 binding and activation was involved in TNF-α production, we examined human monocytes for TNF-α secretion following CD36 cross-linking and CD36-mediated PE ingestion. In contrast to the observed up-regulation of CD11b, CD36 cross-linking does not result in increased secretion of TNF-α after a 4-hour incubation; a 100-ng/mL dose of LPS leads to a pronounced up-regulation of soluble TNF-α over the same time span (Figure8A). Although cross-linking of CD45 primes the monocyte for increased TNF-α secretion in response to low-dose LPS (0.1 ng/mL), CD36 cross-linking does not. Similarly, we did not find an increase in 4-hour monocyte procoagulant activity with CD36 cross-linking (data not shown). These results suggest that, despite the up-regulation of earlier monocyte responses, CD36 cross-linking does not lead to activation of monocyte cytokine secretion or to the induction of monocyte procoagulant inflammatory mediators. Similarly, the phagocytosis of intact PEs by monocytes does not lead to a significant increase in TNF-α release (Figure 8B). However, consistent with the observation that a glycosyl-phosphatidyl inositol (GPI) toxin released at schizont rupture induces TNF-α secretion,54 55 the incubation of lysed PE preparations did induce a marked increase in soluble TNF-α release (14.99 ± 1.33 ng/mL in a typical experiment; n = 3). These data suggest that CD36 clustering, either by antibody or during PE phagocytosis, leads to an increase in early markers of monocyte activation but that the secretion of TNF-α is not increased by CD36 engagement and signaling.

Fig. 8.

CD36 cross-linking and PE phagocytosis do not induce TNF-α secretion.

(A) 5 × 105 monocytes were treated with F(ab′)2 fragments alone or cross-linking with 10-μg/mL CD36 (FA6-152, OKM5) or CD45 (monoclonal antibody 1214) followed by 10-μg/mL F(ab′)2 fragments, suspended in 500-μL RPMI–10% FCS–L-G, and exposed to the presence or absence of low-dose LPS (0.1 ng/mL). High-dose (100 ng/mL) LPS stimulation was used as a positive control. Cells were incubated for 4 hours at 37°C, 5% CO2; pelleted; and soluble TNF-α determined from the supernatants by enzyme-linked immunosorbent assay. CD36 cross-linking neither increased baseline TNF-α secretion nor primed for increased secretion in response to low-dose LPS, but CD45 cross-linking did prime for increased TNF-α following low-dose LPS. (B) 2.5 × 105 monocytes were allowed to adhere to plastic culture wells and incubated with medium alone, UEs, PEs (carefully synchronized to the trophozoite stage), or 1-μg/mL LPS for 4 hours at 37°C, 5% CO2. During this time, there was minimal PE rupture. At the end of 4 hours, the medium was collected, nonadherent cells pelleted, and soluble TNF-α determined. In panels A and B, n ≥ 3 per group and data are representative of results obtained in 3 separate experiments. Data = mean ± SEM. ***,P < .001 vs mock–cross-linked or unstimulated control cells (ANOVA with post hoc Tukey).

Fig. 8.

CD36 cross-linking and PE phagocytosis do not induce TNF-α secretion.

(A) 5 × 105 monocytes were treated with F(ab′)2 fragments alone or cross-linking with 10-μg/mL CD36 (FA6-152, OKM5) or CD45 (monoclonal antibody 1214) followed by 10-μg/mL F(ab′)2 fragments, suspended in 500-μL RPMI–10% FCS–L-G, and exposed to the presence or absence of low-dose LPS (0.1 ng/mL). High-dose (100 ng/mL) LPS stimulation was used as a positive control. Cells were incubated for 4 hours at 37°C, 5% CO2; pelleted; and soluble TNF-α determined from the supernatants by enzyme-linked immunosorbent assay. CD36 cross-linking neither increased baseline TNF-α secretion nor primed for increased secretion in response to low-dose LPS, but CD45 cross-linking did prime for increased TNF-α following low-dose LPS. (B) 2.5 × 105 monocytes were allowed to adhere to plastic culture wells and incubated with medium alone, UEs, PEs (carefully synchronized to the trophozoite stage), or 1-μg/mL LPS for 4 hours at 37°C, 5% CO2. During this time, there was minimal PE rupture. At the end of 4 hours, the medium was collected, nonadherent cells pelleted, and soluble TNF-α determined. In panels A and B, n ≥ 3 per group and data are representative of results obtained in 3 separate experiments. Data = mean ± SEM. ***,P < .001 vs mock–cross-linked or unstimulated control cells (ANOVA with post hoc Tukey).

Close modal

In these studies we describe a novel role for the monocyte/mφ scavenger receptor CD36 in a P falciparum clearance mechanism that is likely to be relevant to the nonimmune patient population. Highly purified human monocytes and culture-derived mφs ingested large numbers of nonopsonized PEs over 4 hours when presented with PEs at a relatively low target:effector ratio. PE uptake was strongly inhibited by CD36 blockade or by cleavage of the CD36 ligand from the surface of PEs. PE uptake was accompanied by intense CD36 clustering around the phagocytosed PEs. Although CD36 clustering leads to increased surface CD11b within 1 hour, neither CD36 cross-linking nor PE phagocytosis increases the 4-hour secretion of TNF-α. Consistent with an active role of CD36 engagement in PE phagocytosis, cross-linking of CD36 initiates an intracellular signal characterized by protein tyrosine phosphorylation and MAPK family member recruitment. Just as the majority of nonopsonic phagocytosis is inhibited by CD36 blockade, so is PE uptake attenuated by pharmacologic inhibition of CD36-dependent signaling. These results suggest that monocytic CD36 contributes to the nonopsonic phagocytosis of P falciparum–infected erythrocytes.

In this study the majority of nonopsonic P falciparum–infected erythrocyte phagocytosis is dependent on surface CD36 and not on other described malarial receptors such as ICAM-1, TSP, αvβ3, or PECAM-1. These data are consistent with a large body of evidence demonstrating that CD36 plays an important role in PE binding to monocytes, melanoma C32 cells, and endothelial cells.15,16,18,19 Interestingly, CD36 transfection to Bowes melanoma cells has been described to directly confer the ability to phagocytose apoptotic neutrophils, lymphocytes, and fibroblasts,56 and several studies have implicated CD36 in the ingestion of apoptotic cells by primary phagocytes.46-48 These findings provide theoretical support for the participation of CD36 in phagocytic processes. It is possible that the nonopsonic phagocytosis of PEs is part of a general mechanism of clearance of senescent cells. Earlier work suggested that PE uptake was partially dependent on phosphatidyl serine,32 a phospholipid expressed in increased amounts in both senescent erythrocytes and PEs.57-60 Recent studies found that PE uptake was increased in oxidation-sensitive glucose-6-phosphate dehydrogenase–deficient erythrocytes, suggesting that the additional oxidative stress of P falciparum infection might promote red blood cell senescence and, therefore, phagocytosis of parasitized cells.31 However, increased phagocytosis was observed primarily in ring-stage PEs, not in the trophozoite-stage PEs used in our study. Furthermore, the uptake of senescent cells is clearly a function of multiple mφ surface antigens,47,48 and it has been postulated that the principal mφ receptor for senescent erythrocytes is distinct from CD36.57,61 Given that we found a large inhibition of PE uptake either by blockade of CD36 alone or by the proteolytic removal of the CD36 ligand on PEs, it seems more likely that monocyte/mφ phagocytosis of PEs is dependent on the PfEMP-1 family of variable surface proteins that are expressed in PEs and mediate adherence to CD36.49,62,63 Furthermore, the inability to block phagocytosis with anti-αvβ3 and anti-TSP antibodies suggests that the mechanism of CD36-mediated phagocytosis of PEs is distinct from the cooperative αvβ3-TSP-CD36 mechanism previously described for the clearance of apoptotic cells.46 48 

It is unknown why only a small proportion of P falciparum–infected individuals develop severe or cerebral malaria. Elevated levels of TNF-α are associated with severe malaria and a poor prognosis, leading to the hypothesis that excessive secretion of proinflammatory cytokines such as TNF-α may promote cerebral malaria. The finding that monocyte CD36 engagement does not lead to increased TNF-α secretion is consistent with a protective effect in malarial pathogenesis. We found that neither CD36 cross-linking nor nonopsonic PE ingestion led to monocyte TNF-α release (Figure 8). In contrast, previous studies have noted an association between PE uptake and TNF-α secretion64; however, these were performed with less tightly synchronized P falciparum cultures than are used in the studies presented here and may reflect the effects of GPI toxins released during schizont rupture.54 Alternatively, TNF-α release may have been secondary to Fc receptor–mediated phagocytosis of opsonized PEs, because Fc receptor cross-linking has been noted to induce monocyte TNF-α secretion.65 66 These stimuli for TNF-α secretion were not present in our studies because we used only washed, tightly synchronized P falciparum cultures, and Fc receptors were blocked prior to PE phagocytosis.

Previous studies have described a role for CD36 in the induction of an early monocyte respiratory burst.53 Although we did find an early, CD36-dependent increase in surface CD11b (Figure 7), the significance of this up-regulation is unclear. CD11b functions in part to promote leukocyte adhesion, but human malaria—unlike rodent malaria—is not generally characterized by leukocyte adhesion to the vascular bed.6 Because CD11b can function in complement-mediated phagocytosis, it may be that the increased CD11b is related to an increased potential for opsonic PE phagocytosis.

It has been reported that mφ ingestion of opsonized PEs or large amounts of hemozoin results in impaired phagocytic function and decreased expression of major histocompatibility complex class II antigen.32,67,68 These observations have led to the suggestion that hemozoin loading of mφs may impair both nonspecific and specific immune responses by inhibiting phagocytosis and antigen presentation, respectively. Despite these considerations, most falciparum-infected individuals do not progress to severe or cerebral malaria and control their infections due, at least in part, to the activity of circulating and tissue-resident monocytes/mφs. Whether CD36-mediated PE phagocytosis will lead to similar mφ impairment is currently under investigation. However, it should be stressed that even if the phagocytic function of the circulating pool of monocytes is permanently decreased, these cells are replaced during the course of clinical infection by fresh phagocytes. In fact, although the early phases of falciparum infection are characterized by loss of phagocytic activity in monocyte-derived mφs, the recovery phase is accompanied by the return of highly phagocytic cells.69 In other words, the recovery phase of human malaria is coincident with an increased capacity for its clearance.

We present evidence that protein tyrosine phosphorylation and, specifically, recruitment of the ERK and p38 MAPKs follows CD36 clustering. CD36 has been associated with Src family kinases and recently in the activation of p38 MAPK,36,70,71 providing a possible link between CD36 clustering, increased tyrosine phosphorylation, and accumulation of active ERK and p38 MAPK moieties. Src family kinases have been demonstrated to be upstream of the low molecular weight GTPases involved in activation of MAPK members.72,73 Importantly, we found that clustering of surface CD36 produced far more signaling than simple divalent ligation of the receptor (Figure 5A). This mechanism of signaling is similar to that seen with integrins42 74 and argues strongly that the clustering observed during PE uptake is sufficient to induce intracellular signaling. Pharmacologic inhibition of the intracellular signals induced by CD36 clustering reduces PE phagocytosis, suggesting that CD36 is an active participant in the phagocytic process. The mechanism linking CD36 clustering to PE phagocytosis involves the ERK and p38 MAPKs (Figure 6); further studies are required to elucidate the precise link between CD36 clustering and these pathways.

Similarly, it will be of interest to define how the MAPKs contribute to monocyte PE phagocytosis. Recent studies indicate that the ERK and p38 MAPKs may be directly or indirectly associated with cytoskeletal elements such as microtubules and actin and phosphorylate regulatory proteins, providing a possible link between their activation and phagocytosis.75-77 However, the roles of the ERK and p38 MAPKs in phagocytosis are likely to be cell- and stimulus-specific.78-80 

The identification of CD36 as a major sequestration receptor has led to the assumption that it contributes to the pathophysiology of severe malaria and has prompted the development of antiadherence therapies to disrupt the CD36-PE interaction.7,16,62,81 However, unlike ICAM-1, little if any CD36 is expressed on cerebral endothelial cells or renal glomeruli.6,21,82 CD36 is known to be well expressed in microvascular endothelial cells from skin, muscle, and sites rich in resident mφs, such as liver, spleen, and the reticuloendothelial system.6 Almost all natural P falciparum isolates bind CD36, but only a small proportion of infected individuals develop severe or cerebral malaria. Furthermore, recent studies have reported that significantly higher binding of PEs to CD36 occurs in cases of nonsevere malaria.83 84Collectively, these observations suggest that the CD36-PE interaction might represent a parasite-host adaptation, evolved for improved survival of the parasite with limited pathogenicity for the host (parasite sequestration in nonvital vascular beds with parasite replication balanced by host clearance). In support of this hypothesis, our data are most consistent with a protective role for monocyte/mφ CD36 by mediating clearance of P falciparum PEs and by not contributing to TNF-α release. If confirmed in vivo, these results have important therapeutic implications. First, strategies to disrupt CD36-PE interactions may be deleterious if they inhibit CD36-mediated PE phagocytosis and displace PEs from CD36 on endothelial cells in nonvital sites to receptors on the cerebral vasculature such as ICAM-1. Secondly, clinical malaria might benefit from selective up-regulation of monocyte/mφ CD36, particularly in the nonimmune host where opsonic phagocytosis would be expected to be less.

The authors thank Ziyue Lu (Toronto Hospital) for technical assistance and are indebted to Dr M. J. Phillips (Department of Pathology, University of Toronto, Toronto Hospital) for his help with light microscopic analysis, Dr I. Crandall (University of Toronto) for the kind gift of antibody IC4, and Drs. A. Gotlieb and A. Rosenthal (Department of Pathology, University of Toronto, Toronto Hospital) for their kind assistance with confocal microscopy.

Supported by the Medical Research Council of Canada operating grants MT-13721 (K.C.K.) and GR-13298 (O.R.), World Health Organization TDR Programme (TDR 920223; KC.K.), the Heart and Stroke Foundation of Canada (NA-3391; K.C.K.), and a Career Scientist Award from the Ontario Ministry of Health (K.C.K.).

I.D.M. is the recipient of a Medical Research Council of Canada fellowship. L.S. is the recipient of a Medical Research of Canada studentship.

I.D.M. and L.S. contributed equally to this work.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Miller
LH
Good
MF
Milon
G
Malaria pathogenesis.
Science.
264
1994
1878
1883
2
Kain
KC
Keystone
JS
Malaria in travelers: epidemiology, disease and prevention.
Infect Dis Clin North Am.
12
1998
267
284
3
Anonymous
Malaria, 1982-1997.
WHO Weekly Epidemiological Record.
74
1999
265
270
4
MacPherson
GG
Warrell
MJ
White
NJ
Looareesuwan
S
Warrell
DA
Human cerebral malaria: a quantitative ultrastructural analysis of parasitized erythrocyte sequestration.
Am J Pathol.
119
1985
385
401
5
Nagao
TH
Uemura
T
Yanagi
K
Oishi
T
Nagatake
Kanbara
H
Loss of tumor necrosis factor production by human monocytes in falciparum malaria after their maturation in vitro.
Am J Trop Med Hyg.
55
1996
562
566
6
Turner
GD
Morrison
H
Jones
M
et al
An immunohistochemical study of the pathology of fatal malaria.
Am J Pathol.
145
1994
1057
1069
7
Ockenhouse
CF
Ho
M
Tandon
NN
et al
Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1.
J Infect Dis.
164
1991
163
169
8
Berendt
AR
Simmons
D
Tansey
J
Newbold
CK
Marsh
K
Intercellular adhesion molecule 1 (ICAM-1) is an endothelial cytoadherence receptor for Plasmodium falciparum.
Nature.
341
1989
57
59
9
Udomsangpetch
R
Reinhardt
PH
Schollaardt
T
Elliott
JF
Kubes
P
Ho
M
Promiscuity of clinical Plasmodium falciparum isolates for multiple adhesion molecules under flow conditions.
J Immunol.
158
1997
4358
4364
10
Ockenhouse
CF
Tegoshi
T
Maeno
Y
et al
Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1.
J Exp Med.
176
1992
1183
1189
11
Treutiger
CJ
Heddini
A
Fernandez
V
Muller
WA
Wahlgren
M
PECAM-1/CD31 and endothelial receptor for binding Plasmodium falciparum-infected erythrocytes.
Nat Med.
3
1997
1405
1408
12
Chaiyaroj
SC
Angkasekwinai
P
Buranakiti
A
Looareesuwan
S
Rogerson
SJ
Brown
GV
Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate as a cytoadherence receptor.
Am J Trop Med Hyg.
55
1996
76
80
13
Roberts
DD
Sherwood
JA
Spitalnik
SL
et al
Thrombospondin binds falciparum parasitized erythrocytes and may mediate cytoadherence.
Nature.
318
1985
64
66
14
Siano
JP
Grady
KK
Millet
P
Wick
TM
Short report: Plasmodium falciparum: cytoadherence to αvβ3 on human microvascular endothelial cells.
Am J Trop Med Hyg.
59
1998
77
79
15
Oquendo
P
Hundt
E
Lawler
J
Seed
B
CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes.
Cell.
58
1989
95
101
16
Ockenhouse
CF
Chulay
JD
Plasmodium falciparum sequestration: OKM5 antigen (CD36) mediates cytoadherence of parasitized erythrocytes to a myelo-monocytic cell line.
J Infect Dis.
157
1988
584
588
17
Ockenhouse
CF
Tandon
NN
Magowan
C
Jamieson
GA
Chulay
JD
Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor.
Science.
243
1989
1469
1471
18
Panton
LJ
Leech
JH
Miller
LH
Howard
RJ
Cytoadherence of Plasmodium falciparum-infected erythrocytes to human melanoma cells lines correlates with surface OKM5 antigen.
Infect Immun.
55
1987
2754
2758
19
Barnwell
JW
Ockenhouse
CF
Knowles
DM
Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells.
J Immunol.
135
1985
3494
3497
20
Newbold
C
Warn
P
Black
G
et al
Receptor-specific adhesion and clinical disease in Plasmodium falciparum.
Am J Trop Med Hyg.
57
1997
389
398
21
Aikawa
M
Iseki
M
Barnwell
JW
Taylor
D
Oo
MM
Howard
RJ
The pathology of human cerebral malaria.
Am J Trop Med Hyg.
43
2 pt 2
1990
30
37
22
Grau
GE
Taylor
TE
Molyneux
ME
et al
Tumour necrosis factor and disease severity in children with falciparum malaria.
N Engl J Med.
320
1989
1586
1591
23
Molyneux
ME
Taylor
TE
Wirima
JJ
Borgstein
A
Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children.
Q J Med.
71
1989
441
459
24
Day
NP
Hien
TT
Schollaardt
T
et al
The prognostic and pathophysiologic role of pro- and antiinflammatory cytokines in severe malaria.
J Infect Dis.
180
1999
1288
1297
25
Brown
H
Turner
G
Rogerson
S
et al
Cytokine expression in the brain in human cerebral malaria.
J Infect Dis.
180
1999
1742
1746
26
McGuire
W
Hill
AVS
Allsopp
CEM
Greenwood
BM
Kwiatkowski
D
Variation in the TNF-α promoter region associated with susceptibility to cerebral malaria.
Nature.
371
1994
508
511
27
Knight
JC
Udalova
I
Hill
AV
et al
A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria.
Nat Genet.
22
1999
145
150
28
Wahlgren
M
Creating deaths from malaria.
Nat Genet.
22
1999
120
121
29
Shear
HL
Srinivasan
R
Nolan
T
Ng
C
Role of IFN-gamma in lethal and nonlethal malaria in susceptible and resistant murine hosts.
J Immunol.
143
1989
2038
2044
30
Urquhart
AD
Putative pathophysiological interactions of cytokines and phagocytic cells in severe human falciparum malaria.
Clin Infect Dis.
19
1994
117
131
31
Cappadoro
M
Giribaldi
G
O'Brien
E
et al
Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency.
Blood.
92
1998
2527
2534
32
Turrini
F
Ginsburg
H
Bussolino
F
Pescarmona
GP
Serra
MV
Arese
P
Phagocytosis of Plasmodium falciparum-infected human red blood cells by human monocytes: involvment of immune and non-immune determinants and dependence on parasite developmental stage.
Blood.
80
1992
801
808
33
Staunton
DE
Ockenhouse
CF
Springer
TA
Soluble intercellular adhesion molecule 1-immunoglobulin G1 immunoadhesin mediates phagocytosis of malaria-infected erythrocytes.
J Exp Med.
176
1992
1471
1476
34
Ruangjirachuporn
W
Afzelius
BA
Helmby
H
et al
Ultrastructural analysis of fresh Plasmodium falciparum-infected erythrocytes and their cytoadherence to human leukocytes.
Am J Trop Med Hyg.
46
1992
511
519
35
Nogami
S
Watanabe
J
Nakagaki
K
et al
Involvement of macrophage scavenger receptors in protection against murine malaria.
Am J Trop Med Hyg.
59
1998
843
845
36
Jimenez
B
Volpert
OV
Crawford
SE
Febbraio
M
Silverstein
RL
Bouck
N
Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.
Nat Med.
6
2000
41
48
37
Chuluyan
HE
Issekutz
AC
VLA-4 can mediate CD11/CD18-independent transendothelial migration of human monocytes.
J Clin Invest.
92
1993
2768
2777
38
Robson
KJH
Walliker
D
Creasey
A
McBride
J
Beale
G
Wilson
RJM
Cross-contamination of Plasmodium cultures.
Parasitol Today.
8
1992
38
39
39
Tragger
W
Jensen
JB
Human malaria parasites in continuous culture.
Science.
193
1976
673
675
40
Serghides
L
Crandall
I
Hull
E
Kain
KC
The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36.
Blood.
92
1998
1814
1819
41
Crandall
I
Guthrie
N
Sherman
IN
Plasmodium falciparum: sera of individuals living in a malaria-endemic region recognize peptide motifs of the human erythrocyte anion transport protein.
Am J Trop Med Hyg.
52
1995
450
455
42
McGilvray
ID
Lu
Z
Bitar
R
Dackiw
APB
Davreux
CJ
Rotstein
OD
VLA-4 crosslinking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase.
J Biol Chem.
272
1997
10287
10294
43
Brisseau
GF
Dackiw
APB
Cheung
PYC
Christie
N
Rotstein
OD
Posttranscriptional regulation of macrophage tissue factor expression by antioxidants.
Blood.
85
1995
1025
1035
44
Huh
HY
Pearce
SF
Yesner
LM
Schindler
JL
Silverstein
RL
Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation.
Blood.
87
1996
2020
2028
45
Hackam
DJ
Rotstein
OD
Schreiber
A
Zhang
WJ
Grinstein
S
Rho is required for the initiation of calcium signaling and phagocytosis by Fc gamma receptors in macrophages.
J Exp Med.
186
1997
955
966
46
Fadok
VA
Warner
ML
Bratton
DL
Henson
PM
CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (αvβ3).
J Immunol.
161
1998
6250
6257
47
Savill
J
Dransfield
L
Hogg
N
Haslett
C
Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis.
Nature.
343
1990
170
173
48
Savill
J
Hogg
N
Ren
Y
Haslett
C
Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis.
J Clin Invest.
90
1989
1513
1522
49
Baruch
DI
Gormley
JA
Ma
C
Howard
RJ
Pasloske
BL
Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.
Proc Natl Acad Sci U S A.
93
1996
3497
3502
50
Borsch-Haubold
AG
Pasquet
S
Watson
SP
Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase.
J Biol Chem.
273
1998
28766
28772
51
Oliver
JM
Burg
DL
Wilson
BS
McLaughlin
JL
Geahlen
RL
Inhibition of mast cell Fc epsilon R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol.
J Biol Chem.
269
1994
29697
29703
52
Matsuda
M
Park
JG
Wang
DC
Hunter
S
Chien
P
Schreiber
AD
Abrogation of the Fc gamma receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides.
Mol Biol Cell.
7
1996
1095
1106
53
Ockenhouse
CF
Magowan
C
Chulay
JD
Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro.
J Clin Invest.
84
1989
468
475
54
Kwiatkowski
D
Cannon
JG
Manogue
KR
Cerami
A
Dinarello
CA
Greenwood
BM
Tumor necrosis factor production in falciparum malaria and its association with schizont rupture.
Clin Exp Immunol.
77
1989
361
366
55
Schofield
L
Hackett
F
Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites.
J Exp Med.
177
1993
145
153
56
Ren
Y
Silverstein
RL
Allen
J
Savill
J
CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis.
J Exp Med.
181
1995
1857
1862
57
Sambrano
GR
Steinberg
D
Recognition of oxidatively damaged and apoptotic cells by an oxidized low density lipoprotein receptor on mouse peritoneal macrophages: role of membrane phosphatidylserine.
Proc Natl Acad Sci U S A.
92
1995
1396
1400
58
Sambrano
GR
Parthasarathy
S
Steinberg
D
Recognition of oxidatively damaged erythrocytes by a macrophage receptor with specificity for oxidized low density lipoprotein.
Proc Natl Acad Sci U S A.
91
1994
3265
3269
59
Schwartz
RS
Olson
JA
Raventos-Suarez
C
et al
Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes.
Blood.
69
1987
401
407
60
Facer
CA
Agiostratidou
G
High levels of anti-phospholipid antibodies in uncomplicated and severe Plasmodium falciparum and in P. vivax malaria.
Clin Exp Immunol.
95
1994
304
309
61
Ottnad
E
Parthasarathy
S
Sambrano
GR
et al
A macrophage receptor for oxidized low density lipoprotein distinct from the receptor for acetyl low density lipoprotein: partial purification and role in recognition of oxidatively damaged cells.
Proc Natl Acad Sci U S A.
92
1995
1391
1395
62
Baruch
DI
Ma
XC
Singh
HB
Bi
X
Pasloske
BL
Howard
RJ
Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: conserved function with variant sequence.
Blood.
90
1997
3766
3775
63
Baruch
DI
Pasloske
BL
Singh
HB
et al
Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized erythrocytes.
Cell.
82
1995
77
87
64
Picot
S
Peyron
F
Vuillez
J-P
Barbe
G
Marsh
K
Ambroise-Thomas
P
Tumor necrosis factor production by human macrophages stimulated in vitro by Plasmodium falciparum.
Infect Immun.
58
1990
214
216
65
Debets
JMH
Van Der Linden
CJ
Dieteren
IEM
Leewenberg
JFM
Buurman
WA
Fc-receptor crosslinking induces rapid secretion of tumor necrosis factor (cachectin) by human peripheral blood monocytes.
J Immunol.
141
1988
1197
1201
66
Debets
JMH
Van de Winkel
JGJ
Ceuppens
JL
Dieteren
IEM
Buurman
WA
Cross-linking of both FcγR I and FcγR II induces secretion of tumor necrosis factor by human monocytes, requiring high affinity Fc-FcγR interactions: functional activation of FcgRII by treatment with proteases or neuraminidase.
J Immunol.
144
1990
1304
1310
67
Schwarzer
E
Turrini
F
Ulliers
D
Giribaldi
G
Ginsburg
H
Arese
P
Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment.
J Exp Med.
177
1993
1033
1041
68
Schwarzer
E
Alessio
M
Ulliers
D
Arese
P
Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes.
Infect Immun.
66
1998
1601
1606
69
Leitner
WW
Krzych
U
Plasmodium falciparum malaria blood stage parasites preferentially inhibit macrophages with high phagocytic activity.
Parasite Immunol.
19
1997
103
110
70
Bull
HA
Brickell
PM
Dowd
PM
Src-related protein tyrosine kinases are physically associated with the surface antigen CD36 in human dermal microvascular endothelial cells.
FEBS Lett
351
1994
41
44
71
Huang
MM
Bolen
JB
Barnwell
JW
Shattil
SJ
Brugge
JS
Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn and Yes protein tyrosine kinases in human platelets.
Proc Natl Acad Sci U S A.
88
1991
7844
7848
72
Stokoe
D
McCormick
F
Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vitro and in vivo.
EMBO J.
16
1997
2384
2396
73
Erpel
T
Courtneidge
SA
Src family protein tyrosine kinases and cellular signal transduction pathways.
Curr Opin Cell Biol.
7
1995
176
182
74
Miyamoto
S
Teramoto
H
Coso
O
et al
Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.
J Cell Biol.
131
1995
791
805
75
Morishima-Kawashima
M
Kosik
KS
The pool of map kinase associated with microtubules is small but constitutively active.
Mol Biol Cell.
7
1996
893
905
76
Earnest
S
Khokhlatchev
A
Albanesi
JP
Barylko
B
Phosphorylation of dynamin by ERK2 inhibits the dynamin-microtubule interaction.
FEBS Lett.
396
1996
62
66
77
Huang
CK
Zhan
L
Ai
Y
Jongstra
J
LSP1 is the major substrate for mitogen-activated protein kinase-activated protein kinase 2 in human neutrophils.
J Biol Chem.
272
1997
17
19
78
Suchard
SJ
Mansfield
PJ
Boxer
LA
Shayman
JA
Mitogen-activated protein kinase activation during IgG-dependent phagocytosis in human neutrophils: inhibition by ceramide.
J Immunol.
158
1997
4961
4967
79
Downey
GP
Butler
JR
Tapper
H
et al
Importance of MEK in neutrophil microbicidal responsiveness.
J Immunol.
160
1998
434
443
80
McLeish
KR
Klein
JB
Coxon
PY
Head
KZ
Ward
RA
Bacterial phagocytosis activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in human neutrophils.
J Leukoc Biol.
64
1998
835
844
81
Cooke
BM
Nicoll
CL
Baruch
DI
Coppel
RL
A recombinant peptide based on PfEMP1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow.
Mol Microbiol.
30
1998
83
90
82
Silamut
K
Phu
NH
Whitty
C
et al
A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain.
Am J Pathol.
155
1999
395
410
83
Newbold
C
Craig
A
Kyes
S
Rowe
A
Fernandez-Reyes
D
Fagan
T
Cytoadherence, pathogenesis and the infected red cell surface in Plasmodium falciparum.
Int J Parasitol.
29
1999
927
937
84
Traore
B
Muanza
K
Looareesuwan
S
et al
Cytoadherence characteristics of Plasmodium falciparum isolates in Thailand using an in vitro human lung endothelial cells model.
Am J Trop Med Hyg.
62
2000
38
44

Author notes

Kevin Kain, Tropical Disease Unit, EN G-224, Toronto General Hospital, 200 Elizabeth St, Toronto, Ontario, Canada M5G 2C4; e-mail: kevin.kain@uhn.on.ca.

Sign in via your Institution