Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.     

Cerebral malaria: role of microparticles and platelets in alterations of the blood-brain barrier

Brain lesions of cerebral malaria (CM) are characterised by a sequestration of Plasmodium falciparum-parasitised red blood cells (PRBC), leucocytes and platelets within brain microvessels, by an excessive release of pro-inflammatory cytokines as well as by disruption of the blood-brain barrier (BBB). We evaluated the possibility that PRBC and platelets interact and induce functional alterations in brain endothelium. Using an in vitro model of endothelial lesion, we showed that platelets can act as bridges between PRBC and endothelial cells (EC) allowing the binding of PRBC to endothelium devoid of cytoadherence receptors. Furthermore, platelets potentiated the cytotoxicity of PRBC for brain EC by inducing an alteration of the integrity of their monolayer and increasing their apoptosis. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM. Another aspect of inflammatory and infectious diseases is that they often lead to activation of vascular and blood cells. Such activation results in an enhanced vesiculation, i.e. the release of circulating microparticles (MP). We thus explored plasma levels of endothelial MP in Malawian children with malaria. Plasma MP numbers were markedly increased on admission only in patients with severe malaria complicated with coma. Using the experimental mouse model of CM, we evaluated the pathogenic implications of MP using genetically deficient mice in which the capacity to vesiculate is impaired. Such mice, lacking the ABCA-1 gene, upon infection by Plasmodium berghei ANKA, showed complete resistance to CM. When purified from infected susceptible animals, MP were able to reduce normal plasma clotting time and to significantly enhance tumour necrosis factor release from na?ve macrophages. Altogether these data provide a novel insight into the pathogenic mechanisms leading to the neurological syndrome. The finding that ABCA-1 gene deletion confers complete protection against cerebral pathology, linked to an impaired MP production, provides new potential targets for therapeutic amelioration of severe malaria.     

Association of adhesion molecule PECAM-1/CD31 polymorphism with susceptibility to cerebral malaria in Thais

Adhesion molecules on endothelial cells are known to be important ligands for malaria infected red blood cells (PRBC) [Mol Biochem Parasitol, 76, (1996) 1], and may be involved in the pathogenic process of cerebral malaria (CM) which is the most serious complication of falciparum malaria, through enhancing micro embolism or sequestration in the capillaries of the brain. PECAM-1/CD31 is one of these candidate ligands and is coded by a polymorphic gene. Two hundred and ten Thai malaria patients (43 cerebral, 89 severe and 78 uncomplicated) were analyzed for their genetic polymorphism of CD31 to examine the clinical relationship between the disease and specific genotypes. Four alleles were defined 125 valine (V)-563 asparagine (N); 125V-563 serine (S); 125 leucine (L)-563N; and 125L-563S. We found that the frequency of the 125 V/V 563 N/N genotype was significantly high in CM patients as compared with severe cases without CM (P<0.01, OR=2.92), suggesting that this genotype is one of the risk factors for CM.     

Molecular mechanistic insights into the endothelial receptor mediated cytoadherence of Plasmodium falciparum-infected erythrocytes

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the 'holder' for providing stable binding.     

Membrane proteins involved in the adherence of Plasmodium falciparum-infected erythrocytes to the endothelium.

Plasmodium falciparum (human malaria) infections are characterized by the attachment of erythrocytes infected with mature stage parasites to endothelial cells lining the post-capillary venules, a phenomenon known as sequestration. In the human body, the microvessels of the heart, lungs, kidneys, small intestine, and liver are the principal sites of sequestration. Sequestered cells that clog the brain capillaries may reduce blood flow sufficiently so that there is confusion, lethargy, and unarousable coma--cerebral malaria. This review considers what is known about the molecular characteristics of the surface proteins, that is, the red cell receptors and the endothelial cell ligands, involved in sequestration. Recent work from our laboratory on the characterization of the adhesive proteins on the surface of the P falciparum-infected red cell, and the ligands to which they bind on human brain endothelial cells is also discussed. Finally, consideration is given to the multifactor processes involved in sequestration and cerebral malaria, as well as the possible role of 'anti-adhesion therapy' in the management of severe malaria.     

Inhibition of Plasmodium falciparum field isolates-mediated endothelial cell apoptosis by Fasudil: therapeutic implications for severe malaria

Plasmodium falciparum infection can abruptly progress to severe malaria, a life-threatening complication resulting from sequestration of parasitized red blood cells (PRBC) in the microvasculature of various organs such as the brain and lungs. PRBC adhesion can induce endothelial cell (EC) activation and apoptosis, thereby disrupting the blood-brain barrier. Moreover, hemozoin, the malarial pigment, induces the erythroid precursor apoptosis. Despite the current efficiency of antimalarial drugs in killing parasites, severe malaria still causes up to one million deaths every year. A new strategy targeting both parasite elimination and EC protection is urgently needed in the field. Recently, a rho-kinase inhibitor Fasudil, a drug already in clinical use in humans for cardio- and neuro-vascular diseases, was successfully tested on laboratory strains of P. falciparum to protect and to reverse damages of the endothelium. We therefore assessed herein whether Fasudil would have a similar efficiency on P. falciparum taken directly from malaria patients using contact and non-contact experiments. Seven (23.3%) of 30 PRBC preparations from different patients were apoptogenic, four (13.3%) acting by cytoadherence and three (10%) via soluble factors. None of the apoptogenic PRBC preparations used both mechanisms indicating a possible mutual exclusion of signal transduction ligand. Three PRBC preparations (42.9%) induced EC apoptosis by cytoadherence after 4 h of coculture ("rapid transducers"), and four (57.1%) after a minimum of 24 h ("slow transducers"). The intensity of apoptosis increased with time. Interestingly, Fasudil inhibited EC apoptosis mediated both by cell-cell contact and by soluble factors but did not affect PRBC cytoadherence. Fasudil was found to be able to prevent endothelium apoptosis from all the P. falciparum isolates tested. Our data provide evidence of the strong anti-apoptogenic effect of Fasudil and show that endothelial cell-P. falciparum interactions are more complicated than previously thought. These findings may warrant clinical trials of Fasudil in severe malaria management.     

A recombinant peptide based on Pf EMP-1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow

During falciparum malaria infection, severe complications ensue because parasitized red blood cells (PRBCs) adhere to endothelial cells and accumulate in the microvasculature. At the molecular level, adhesion is mediated by interaction of Plasmodium falciparum erythrocyte membrane protein 1 ( Pf  EMP-1) on the PRBC surface with receptors on the surface of endothelial cells, including CD36. We have shown that a recombinant 179-residue subfragment of Pf  EMP-1 (rC1-2[1–179]), which encompasses the CD36-binding region, inhibits and reverses adhesion of PRBCs to CD36 under physiologically relevant flow conditions. rC1-2[1–179] inhibited adhesion in a concentration-dependent manner over the range 100 pM to 2 μM, with up to 99% of adhesion blocked at the highest concentration tested. The antiadhesive activity of rC1-2[1–179] was not strain specific and almost totally ablated adhesion of four different parasite lines. Furthermore, rC1-2[1–179] showed remarkable ability to progressively reverse adhesion when flowed over adherent PRBCs for 2 h. The effect of rC1-2[1–179] was, however, specific for CD36-mediated adhesion and had no effect on adhesion mediated by CSA. Interference with binding of PRBCs to the vascular endothelium using rC1-2[1–179] or smaller organic mimetics may be a useful therapeutic approach to ameliorate severe complications of falciparum malaria.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Peroxisome proliferator-activated receptor gamma-retinoid X receptor agonists increase CD36-dependent phagocytosis of Plasmodium falciparum-parasitized erythrocytes and decrease malaria-induced TNF-alpha secretion by monocytes/macrophages

Severe and fatal malaria is associated with the failure of host defenses to control parasite replication, excessive secretion of proinflammatory cytokines such as TNF-alpha, and sequestration of parasitized erythrocytes (PEs) in vital organs. 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. This concept has been challenged by unexpected evidence that individuals deficient in CD36 are more susceptible to severe and cerebral malaria. In this study, we demonstrate that CD36 is the major receptor mediating nonopsonic phagocytosis of PEs by macrophages, a clearance mechanism of potential importance in nonimmune hosts at the greatest risk of severe malaria. CD36-mediated uptake of PEs occurs via a novel pathway that does not involve thrombospondin, the vitronectin receptor, or phosphatidylserine recognition. Furthermore, we show that proliferator-activated receptor gamma-retinoid X receptor agonists induce an increase in CD36-mediated phagocytosis and a decrease in parasite-induced TNF-alpha secretion. Specific up-regulation of monocyte/macrophage CD36 may represent a novel therapeutic strategy to prevent or treat severe malaria.     

Thrombin-Stimulated Calcium Mobilization Is Inhibited by Thrombospondin via CD36

Activation of the G-protein-linked thrombin receptor in endothelial cells normally leads to an increase in free intracellular calcium, [Ca²⁺]_i, which is the proximate stimulus for many important cell functions. We present evidence showing that signals from CD36, the thrombospondin (TSP) receptor, can inhibit this thrombin-mediated calcium response. Human endothelial cells preloaded with Indo-1 exhibited rapid calcium mobilization in response to thrombin. The presence of TSP inhibited the thrombin-stimulated calcium response in CD36-positive microvascular endothelial cells but not in CD36-negative umbilical vein endothelial cells. This TSP effect was mimicked by anti-CD36 antibodies and a TSP peptide (CSVTCG), but not by an alternative CD36 ligand (collagen IV) or an antibody to an alternative TSP receptor (α_vβ₃). TSP also inhibited the calcium response to the thrombin receptor-tethered ligand peptide, SFLLRN. In addition, TSP and anti-CD36 antibodies inhibited the calcium response of a closely related receptor, the trypsin/SLIGKVD-activated receptor PAR-2. TSP did not indiscriminately inhibit all calcium release pathways, since histamine- or VEGF-stimulated calcium responses were not inhibited by TSP. We conclude that cross-talk from the CD36 receptor influences the responsive state of the endothelial thrombin receptor family and/or its signaling pathway.     

CD44, a signal receptor for the inhibition of the cytoadhesion of CD36-binding Plasmodium falciparum-infected erythrocytes by CSA-binding infected erythrocytes

The cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) in organ microvessels is a key event in the pathogenesis of cerebral malaria and pulmonary edema. Identification of the molecules involved in the interaction between IEs and endothelial cells has been a major goal of research into severe forms of malaria. In contrast, the consequences of cytoadhesion for endothelial cells have been largely ignored. By combining phenotypic selection, cytoadhesion assays and flow cytometry, we demonstrated that the cytoadhesion of CSA-binding IEs inhibited the cytoadhesion of CD36-binding IEs. We identified CD44 as a signal receptor for CSA-binding IEs cytoadhesion, and demonstrated that the signal was transduced to CD36 through a pathway involving the Src-kinase family and MEK. CD36-mediated cytoadhesion was modulated independently of changes in CD36 expression. These results provide the first evidence that some IEs can downregulate the cytoadhesion of IEs of another phenotype, by modifying endothelial cells via a signaling pathway relating CD44 to CD36. Mimicking this phenomenon may constitute an interesting therapeutic strategy for inhibiting the adhesion of CD36-binding IEs -- the most abundant phenotype among field isolates -- and promoting their degradation in the spleen.     

Studies on the glycoprotein modification in erythrocyte membrane during experimental cerebral malaria

Plasmodium berghei ANKA (Pb ANKA) is a lethal strain of malaria that causes experimental cerebral malaria (ECM) in rodent models. Pathology of the disease is associated with the sequestration of the infected rbc (irbc) in the micro vessels of brain. In the present study, we analyzed the nature of the glycoprotein modification occurring in irbc membrane during erythrocytic stages of Pb ANKA infection. Titration of naturally occurring glycoproteins with concanavalin A (Con A) and wheat germ agglutinin (WGA) lectins revealed an enhanced lectin binding ability for the irbc membrane preparations. Partial characterization of the Con A specific determinants (alpha-d-methyl mannoside specificity) by lectin affinity chromatography followed by 2D electrophoresis and WGA specific determinants (sialic acid specificity) by Western analysis revealed the association of novel lectin specific determinants in irbc membrane. To correlate the biochemical changes with the morphological changes, SEM of irbc, and TEM of sequestered irbc were performed. These ultra structural studies revealed variable and irregular surface protrusions and deep surface indentations on irbc. These observations implicate that altered glycoprotein profiles may lead to cytoarchitectural changes in irbc membrane and such changes may be essential to establish contact with the host endothelial cells. These observations may be central to the microvascular sequestration and pathology of ECM.     

Induction of the CD23/nitric oxide pathway in endothelial cells downregulates ICAM-1 expression and decreases cytoadherence of Plasmodium falciparum-infected erythrocytes

Cytoadherence of parasitized red blood cells (PRBCs) to postcapillary venules and cytokine production are clearly involved in the pathogenesis of cerebral malaria. Nitric oxide and TNF-alpha have been proposed as major effector molecules both in protective and physiopathological processes during malaria infections. Nitric oxide production has been shown to be induced by engagement of CD23 antigen. This study aimed to investigate the potential role of the CD23/nitric oxide pathway in the control of the cytoadherence of PRBCs on human endothelial cells. We demonstrate that normal human lung endothelial cells (HLECs) are able to express the low affinity receptor for IgE (Fc in RII/CD23), following cell incubation with interleukin 4 or PRBCs. Ligation of the CD23 antigen by a specific anti-CD23 monoclonal antibody at the cell surface of HLECs was found to induce iNOS mRNA and protein expression, NO release and P. falciparum killing. In addition, the specific CD23-engagement on these cells also induced a significant decrease in ICAM-1 expression, an adhesion molecule implicated in PRBCs cytoadherence. These data not only described for the first time the expression of a CD23 antigen at the cell surface of endothelial cells but also suggest a possible new regulatory mechanisms via the CD23/NO pathway during malaria infection.     

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [¹²⁵I]-TSP1 and [¹²⁵I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,^18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd.     

Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhibiting myristic acid uptake

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.     

Peroxisome proliferator-activated receptor gamma ligands improve the antitumor efficacy of thrombospondin peptide ABT510

An expanding capillary network is critical for several pathologic conditions. In cancer, the decrease of antiangiogenic thrombospondin-1 (TSP1) often enables an angiogenic switch, which can be reversed with exogenous TSP1 or its peptide derivative ABT510. TSP1 acts by inducing endothelial cell apoptosis via signaling cascade initiated at CD36, a TSP1 antiangiogenic receptor. Here, we show that the ligands of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), 15-deoxy-delta(12,14)-prostaglandin J2, troglitazone, and rosiglitazone increased PPARgamma and CD36 expression in endothelial cells and improved the efficacy of TSP1 and ABT510 in a CD36-dependent manner. The ABT510 and PPARgamma ligands cooperatively blocked angiogenic endothelial functions in vitro and neovascularization in vivo. In tumor xenografts, 15-deoxy-delta(12,14)-prostaglandin J2 and troglitazone synergistically improved antiangiogenic and antitumor effects of ABT510. Our data provide one mechanism for the in vivo angioinhibitory effect of PPARgamma ligands and show fine-tuning of the antiangiogenic efficacy via targeted up-regulation of the endothelial receptor.     

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [¹²⁵I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28–40%, (ii) the RGD peptide or MoAbs against integrin α_vβ₃ or the calcium binding region of TSP inhibited binding by 18–28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.     

CD36 Contributes to Malaria Parasite-Induced Pro-Inflammatory Cytokine Production and NK and T Cell Activation by Dendritic Cells

The scavenger receptor CD36 plays important roles in malaria, including the sequestration of parasite-infected erythrocytes in microvascular capillaries, control of parasitemia through phagocytic clearance by macrophages, and immunity. Although the role of CD36 in the parasite sequestration and clearance has been extensively studied, how and to what extent CD36 contributes to malaria immunity remains poorly understood. In this study, to determine the role of CD36 in malaria immunity, we assessed the internalization of CD36-adherent and CD36-nonadherent Plasmodium falciparum-infected red blood cells (IRBCs) and production of pro-inflammatory cytokines by DCs, and the ability of DCs to activate NK, and T cells. Human DCs treated with anti-CD36 antibody and CD36 deficient murine DCs internalized lower levels of CD36-adherent IRBCs and produced significantly decreased levels of pro-inflammatory cytokines compared to untreated human DCs and wild type mouse DCs, respectively. Consistent with these results, wild type murine DCs internalized lower levels of CD36-nonadherent IRBCs and produced decreased levels of pro-inflammatory cytokines than wild type DCs treated with CD36-adherent IRBCs. Further, the cytokine production by NK and T cells activated by IRBC-internalized DCs was significantly dependent on CD36. Thus, our results demonstrate that CD36 contributes significantly to the uptake of IRBCs and pro-inflammatory cytokine responses by DCs, and the ability of DCs to activate NK and T cells to produce IFN-γ. Given that DCs respond to malaria parasites very early during infection and influence development of immunity, and that CD36 contributes substantially to the cytokine production by DCs, NK and T cells, our results suggest that CD36 plays an important role in immunity to malaria. Furthermore, since the contribution of CD36 is particularly evident at low doses of infected erythrocytes, the results imply that the effect of CD36 on malaria immunity is imprinted early during infection when parasite load is low.     

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

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.