Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1

The clinical complications associated with severe and cerebral malaria occur as a result of the intravascular mechanical obstruction of erythrocytes infected with the asexual stages of the parasite, Plasmodium falciparum. We now report that a primary P. falciparum-infected erythrocyte (parasitized red blood cell [PRBC]) isolate from a patient with severe complicated malaria binds to cytokine-induced human vascular endothelial cells, and that this adhesion is in part mediated by endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). PRBC binding to tumor necrosis factor alpha (TNF-alpha)-activated human vascular endothelial cells is partially inhibited by antibodies to ELAM-1 and ICAM-1 and the inhibitory effects of these antibodies is additive. PRBCs selected in vitro by sequential panning on purified adhesion molecules bind concurrently to recombinant soluble ELAM-1 and VCAM-1, and to two previously identified endothelial cell receptors for PRBCs, ICAM-1, and CD36. Post-mortem brain tissue from patients who died from cerebral malaria expressed multiple cell adhesion molecules including ELAM-1 and VCAM-1 on cerebral microvascular endothelium not expressed in brains of individuals who died from other causes. These results ascribe novel pathological functions for both ELAM-1 and VCAM-1 and may help delineate alternative adhesion pathways PRBCs use to modify malaria pathology.     

Intercellular adhesion molecule 1 is important for the development of severe experimental malaria but is not required for leukocyte adhesion in the brain.

Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit a systemic inflammatory response. Most investigators hypothesize that leukocytes bind to endothelial cells via intercellular adhesion molecule 1 (ICAM-1), which causes endothelial damage, increased microvascular permeability, and, ultimately, death. ICAM-1-deficient mice on an ECM-susceptible C57BL/6 background were significantly (p = .04) protected from P. berghei mortality compared with ICAM-1 intact controls. ICAM-1 expression assessed by the dual radiolabeled monoclonal antibody technique was increased in the brain and lung in C57BL/6 mice on day 6 of P. berghei infection compared with uninfected controls (5.3-fold, p = .0003 for brain and 1.8-fold, p = .04 for lung). The increase in ICAM-1 expression coincided with significant (p < .05) increases in microvascular permeability in the brain and lung. In contrast to the hypothesized role for ICAM-1, in vivo analysis by intravital microscopy of leukocyte rolling and adhesion in brain microvasculature of mice revealed markedly increased levels of leukocyte rolling and adhesion in ICAM-1-deficient mice on day 6 of P. berghei infection compared with uninfected controls. In addition, ICAM-1 expression and microvascular permeability were increased in infected ECM-resistant BALB/c mice compared with uninfected BALB/c controls. These results collectively indicate that although ICAM-1 contributes to the mortality of experimental malaria, it is not sufficient for the development of severe experimental malaria. In addition, ICAM-1 expressed on the endothelium or on leukocytes is not required for leukocyte rolling or adhesion to the brain microvasculature of mice during P. berghei malaria. Leukocyte rolling and adhesion in the brain vasculature during P. berghei malaria use different ligands than observed during inflammation in other vascular beds.     

Soluble intercellular adhesion molecule 1-immunoglobulin G1 immunoadhesin mediates phagocytosis of malaria-infected erythrocytes

We describe an immunoadhesin molecule containing intercellular adhesion molecule 1 (ICAM-1) molecularly fused to hinge and CH2 and CH3 domains of the human immunoglobulin G1 H chain that binds Plasmodium falciparum-infected erythrocytes. This receptor-based immunoadhesin is an effective and specific inhibitor of P. falciparum-infected erythrocyte adhesion to ICAM-1-bearing surfaces, but does not inhibit leukocyte function antigen 1 (LFA-1) interaction with ICAM-1. Furthermore, the immunoadhesin promotes phagocytosis and destruction of parasitized erythrocytes by human monocytes. Each of these modes of action has potential for the therapy of malaria.     

A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IE) specifically adhere to vascular endothelium in vivo and to human endothelial cells, some human melanoma cell lines, and human monocytes in vitro. The tissue cell receptor for a ligand on the surface of the infected erythrocytes is an Mr 88,000 glycoprotein (GP88) recognized by the MAb OKM5, which also blocks cytoadherence of IE. Isolated, affinity-purified GP88 (CD36) competitively blocks cytoadherence and when absorbed to plastic surfaces, specifically binds P. falciparum IE. Additionally, monoclonal and polyclonal antibodies to GP88 block cytoadherence to both target cells and immobilized GP88. Binding to GP88 by IE is unaffected by the absence of calcium or the absence of thrombospondin, a putative mediator for cytoadherence of P. falciparum IE. Thus, GP88 (CD36), which has been demonstrated to be the same as platelet glycoprotein IV, interacts directly with P. falciparum IE, presumably via a parasite-induced ligand exposed on the surface of the infected erythrocytes. CD36 is shown to be present on brain endothelium in both individuals without malaria and individuals with cerebral malaria. This would suggest that factors other than just cerebral sequestration of IE play an initiating role in the genesis of cerebral malaria.     

Plasmodium falciparum erythrocyte membrane protein 1 variants induce cell swelling and disrupt the blood–brain barrier in cerebral malaria

Cerebral malaria (CM) is caused by the binding of Plasmodium falciparum–infected erythrocytes (IEs) to the brain microvasculature, leading to inflammation, vessel occlusion, and cerebral swelling. We have previously linked dual intercellular adhesion molecule-1 (ICAM-1)– and endothelial protein C receptor (EPCR)–binding P. falciparum parasites to these symptoms, but the mechanism driving the pathogenesis has not been identified. Here, we used a 3D spheroid model of the blood–brain barrier (BBB) to determine unexpected new features of IEs expressing the dual-receptor binding PfEMP1 parasite proteins. Analysis of multiple parasite lines shows that IEs are taken up by brain endothelial cells in an ICAM-1–dependent manner, resulting in breakdown of the BBB and swelling of the endothelial cells. Via ex vivo analysis of postmortem tissue samples from CM patients, we confirmed the presence of parasites within brain endothelial cells. Importantly, this discovery points to parasite ingress into the brain endothelium as a contributing factor to the pathology of human CM.     

Visualization of Plasmodium falciparum-endothelium interactions in human microvasculature: mimicry of leukocyte recruitment

Plasmodium falciparum-infected erythrocytes roll on and/or adhere to CD36, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and P-selectin under shear conditions in vitro. However, the lack of an adequate animal model has made it difficult to determine whether infected erythrocytes do indeed interact in vivo in microvessels. Therefore, we made use of an established model of human skin grafted onto severe combined immunodeficient (SCID) mice to directly visualize the human microvasculature by epifluorescence intravital microscopy. In all grafts examined, infected erythrocytes were observed to roll and/or adhere in not just postcapillary venules but also in arterioles. In contrast, occlusion of capillaries by infected erythrocytes was noted only in approximately half of the experiments. Administration of an anti-CD36 antibody resulted in a rapid reduction of rolling and adhesion. More importantly, already adherent cells quickly detached. The residual rolling after anti-CD36 treatment was largely inhibited by an anti-ICAM-1 antibody. Anti-ICAM-1 alone reduced the ability of infected erythrocytes to sustain rolling and subsequent adhesion. These findings provide conclusive evidence that infected erythrocytes interact within the human microvasculature in vivo by a multistep adhesive cascade that mimics the process of leukocyte recruitment.     

Intercellular adhesion molecule-1 and CD36 synergize to mediate adherence of Plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells.

We have compared the adhesion of Plasmodium falciparum-infected erythrocytes to human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) and have assessed the relative roles of the receptors CD36 and intercellular adhesion molecule-1 (ICAM-1). HUVEC (a cell line that expresses high levels of ICAM-1 but no CD36) mediate low levels of adhesion, whereas HDMEC (which constitutively express CD36) mediate high levels of adhesion even before ICAM-1 induction ICAM-1 expression leads to yet greater levels of adhesion, which are inhibited both by anti-ICAM-1 and CD36 mAbs, despite no increase in the expression of CD36. The results indicate the presence of a substantial population of infected cells that require the presence of both receptors to establish adhesion. Synergy between these receptors could be demonstrated using a number of parasite lines, but it could not be predicted from the binding of these same parasite lines to purified ICAM-1 and CD36. This phenomenon could not be reproduced using either purified receptors presented on plastic, or formalin-fixed HDMEC, suggesting that receptor mobility is important. This is the first study to demonstrate receptor synergy in malaria cytoadherence to human endothelial cells, a phenomenon necessary for parasite survival and associated with disease severity.     

Why it might be bad for brain cells to eat malaria parasites

In this issue, Adams et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20201266) show that red blood cells infected with strains of Plasmodium falciparum, which are commonly found in cerebral malaria patients, are specifically internalized by brain endothelial cells, perhaps contributing to the symptoms of the disease.

Malaria becomes very dangerous when it affects the brain. Cerebral malaria is caused when red blood cells, infected by the parasite Plasmodium falciparum, accumulate within tiny brain blood vessels, blocking blood flow (White et al., 2013). This can increase blood pressure and induce brain swelling, crushing precious parts of the brain against the skull (Newton et al., 1991; Seydel et al., 2015). As a result, patients may suffer from seizures and coma and, if they survive, experience permanent brain damage. But why do some malaria episodes cause cerebral symptoms while the majority do not?The surfaces of Plasmodium falciparum–infected erythrocytes display a small number of parasite-derived molecules, organized into families (Wahlgren et al., 2017). One of these, the PfEMP1 proteins, mediates binding of infected erythrocytes to tissue and blood vessel surfaces, preventing their destruction by the spleen (Jensen et al., 2020). Each parasite genome contains many PfEMP1 proteins that form a highly diverse protein family. Parasites switch which of these they produce to avoid antibody-mediated detection. But this also varies the binding properties of infected erythrocytes as different PfEMP1 bind to different endothelial receptors.Insights from Matthew K. Higgins.Two of the major human receptors recognized by PfEMP1 are endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1; Jensen et al., 2020). PfEMP1 that bind to these receptors are associated with parasites that cause different malaria symptoms. Parasites expressing PfEMP1 that bind EPCR are more commonly found in children suffering from severe malaria episodes (Turner et al., 2013), while those that can simultaneously bind to both EPCR and ICAM-1 are more commonly associated with episodes of cerebral malaria (Lennartz et al., 2017). But why is this? How does binding to particular receptors lead to the symptoms of cerebral disease? The study in this issue by Adams et al. presents a novel discovery that leads the authors to propose a novel hypothesis.They started by taking a panel of parasite strains that produce different PfEMP1, selecting some that bind to just ICAM-1, some that bind to just EPCR, and some of the dual EPCR and ICAM-1 binders. They incubated human brain endothelial cells with erythrocytes infected with these parasites and observed what happened to the ICAM-1. When, and only when, the erythrocytes used in this study expressed dual binding PfEMP1, they observed an interesting phenomenon. ICAM-1 became enriched on endothelial cell surfaces, forming unusual rings and protrusions, changing in both surface density and in distribution.Looking more closely, they spotted something very unexpected. As well as observing infected erythrocytes attached to the surfaces of brain endothelial cells, they found infected erythrocytes inside these cells. This occurred in three independent experiments using three different brain endothelial cell lines. In each case, only infected erythrocytes that can bind both ICAM-1 and EPCR were internalized. The same effect was also seen using a spheroid model. These spheroids are clusters of cells that assemble to mimic the blood–brain barrier. Once again, only dual ICAM-1– and EPCR-binding infected erythrocytes were internalized by the endothelial cells of the spheroids. What was going on? Brain endothelial cells have previously been shown to internalize damaged erythrocytes, perhaps to clear them from the blood. Could they be doing the same with parasite-infected erythrocytes?The next set of experiments suggested that internalization of infected erythrocytes is not a good move. Two distinct and potentially deleterious effects were observed. First, Adams et al. (2021) studied the uptake of a large fluorescent dye, which cannot enter healthy cells. Spheroids containing internalized dual ICAM-1– and EPCR-binding infected erythrocytes took up more of this dye. Could this indicate that internalization of infected erythrocytes causes damage to brain endothelial cells, increasing their permeability? Second, the authors noticed that the spheroids increased in volume when incubated with dual ICAM-1– and EPCR-binding infected erythrocytes. Could this swelling of endothelial cells play a role in brain swelling in cerebral disease? In both cases, internalization of infected erythrocytes seems likely to spell trouble for the brain endothelium.Human erythrocytes infected with the malaria parasite, Plasmodium falciparum, produce adhesive PfEMP1 proteins that allow them to interact with human endothelial receptors. Some of the these PfEMP1 bind to ICAM-1 and some bind to EPCR, while some can simultaneously bind to both ICAM-1 and EPCR. Adams et al. (2021) show that brain endothelial cells can specifically internalize infected erythrocytes that bind to both ICAM-1 and EPCR. This leads to endothelial cell swelling and increased permeability, perhaps contributing to the severe symptoms of cerebral malaria.This fascinating discovery raises a number of questions. The first relates to the specificity of internalization. It appears as though only infected erythrocytes producing dual ICAM-1– and EPCR-binding PfEMP1 are internalized. As these are the same PfEMP1 whose expression in patients is associated with development of cerebral symptoms (Lennartz et al., 2017), this is tantalizing. But how does this selectivity arise, and what is the mechanism of internalization?Adams et al. (2021) show clearly that the interaction between PfEMP1 and ICAM-1 is critical for internalization, with antibodies targeting either ICAM-1 or the ICAM-1–binding domain of the PfEMP1, preventing the effect. However, infected erythrocytes that express a PfEMP1 protein that binds to ICAM-1 alone are not internalized. While it is true that PfEMP1 that bind to ICAM-1, but not EPCR, bind ICAM-1 with a subtly different binding mode than those that bind both ICAM-1 and EPCR (Lennartz et al., 2019), it seems unlikely that this is the cause of a major difference in internalization efficiency. It is much more likely that EPCR binding is also involved in uptake into the brain endothelium. In the highlighted study, the authors added an antibody that targets EPCR and found that this led to a nonsignificant reduction in internalization (Adams et al., 2021). It will be interesting to probe the role of the EPCR–binding domain of these PfEMP1 with further experiments—for example, assessing internalization in the presence of soluble EPCR or antibodies against the EPCR-binding domain—to see if this reduction is real.If both ICAM-1 and EPCR binding prove to be involved in internalization, how do they do it? Previous studies have shown that dual EPCR- and ICAM-1–binding PfEMP1 are able to simultaneously bind to both receptors, with dual binding tethering infected erythrocytes more tightly to endothelial cells than when either receptor is used for adhesion alone (Avril et al., 2016; Lennartz et al., 2017). Could this tighter binding provide more time for internalization? Alternatively, and perhaps more likely, EPCR-binding PfEMP1 have been shown to block the natural ligand of EPCR, protein C, from mediating signaling (Lau et al., 2015; Turner et al., 2013). One consequence of this could be up-regulation of ICAM-1 expression on the endothelium (Moxon et al., 2013; Turner et al., 2013). Could EPCR-mediated signaling lead to the up-regulation of ICAM-1 seen in the brain endothelial cells and, together with tighter binding due to simultaneous dual receptor attachment, trigger internalization? More studies are needed to dissect these mechanisms and to understand their specificity.The second question is the degree to which internalization of infected erythrocytes contributes to cerebral disease and, if it does, how. The authors speculate that swelling of spheroids on infected erythrocyte internalization could contribute to brain swelling seen in cerebral disease (Seydel et al., 2015). Indeed, upon observing a brain section taken postmortem from a victim of cerebral malaria, they observe internalized infected erythrocytes, showing that internalization does happen during natural infection as well as in culture conditions. But, do infected erythrocytes need to enter into endothelial cells in order to cause brain swelling? Will infected erythrocytes that tightly adhere to the surface of the endothelium not have just the same effect in increasing the volume of cellular material in the brain? Estimates suggest that as much as 50 ml of parasite biomass might be sequestered in the brain of a cerebral malaria sufferer (White et al., 2013). It will be fascinating to see how much of this parasite material is inside cells and how much outside, and whether it matters for the brain swelling.Another way in which internalization of infected erythrocytes could cause harm is through damage to the brain endothelium. The increased permeability of spheroids after infected erythrocyte internalization could contribute to disruption of the integrity of the endothelium. This could combine with an effect already observed for EPCR-binding PfEMP1. Signaling through EPCR-mediated pathways has been shown to protect the endothelium from thrombin-mediated disruption, and PfEMP1 binding to EPCR interferes with this protective effect (Bernabeu et al., 2016; Kessler et al., 2017). Could the increased permeability of the endothelium resulting from infected erythrocyte internalization combine with this effect from infected erythrocytes attached to cell surfaces, spelling trouble for endothelial integrity?Whatever the answers to these questions, the discovery that brain endothelium can internalize infected erythrocytes, and that this internalization is specific to parasite variants found more commonly in sufferers of cerebral malaria, is fascinating and tantalizing. It opens up a series of questions, the answers to which will require complex and challenging in vivo experiments. But one thing appears clear. Those brain cells need to be careful what they eat. Gobbling up parasite-infected erythrocytes might not be good for them.     

Rational design of anticytoadherence inhibitors for Plasmodium falciparum based on the crystal structure of human intercellular adhesion molecule 1

Adhesion of Plasmodium falciparum-infected erythrocytes (IE) to host endothelium has been associated with pathology in malaria. Although the interaction with endothelial cells can be complex due to the relatively large number of host receptors available for binding, specific proteins have been identified that are more commonly used than others. For example, binding to intercellular adhesion molecule 1 (ICAM 1) is found frequently in parasites from pediatric cases of malaria. The binding site for P. falciparum-infected erythrocytes on ICAM 1 has been mapped in some detail and is distinct from the site for lymphocyte function-associated antigen 1 (LFA-1). Part of the ICAM 1 binding site for P. falciparum-infected erythrocytes (the DE loop) was used to screen a library of compounds based on its structure (derived from the crystal structure of human ICAM 1). This resulted in the identification of 36 structural mimeotopes as potential competitive inhibitors of binding. One of these compounds, (+)-epigalloyl-catechin-gallate [(+)-EGCG], was found to inhibit IE adhesion to ICAM 1 in a dose-dependent manner with two variant ICAM 1-binding parasite lines, providing the first example of a potential mimeotope-based anticytoadherence inhibitor for Plasmodium falciparum.     

Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at 10 mg/ml and by 72.5 +/- 3.8% at 1 mg/ml, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell- associated or immobilized CS.     

Cytoadherence between endothelial cells and P. falciparum infected and noninfected normal and thalassemic red blood cells

BACKGROUND: Cytoadhesion of P. falciparum infected red blood cells (RBCs) to endothelial cells (ECs) is an important phenomenon that causes cerebral malaria in man. Reduced adhesion especially in thalassemia and hemoglobinopathies may be related to a protective mechanism against malaria in such people. METHODS: The cytoadherence assay was performed using both conventional and floating conditions between ECs (ECV 304) and P. falciparum infected and noninfected RBCs from both normal and thalassemia subjects. In floating condition, RBC was fluorescently labeled with anti-glycophorin A antibody, whereas EC was identified by surface expression of PECAM-1, CD-36, ICAM-1, and E-selectin. The condition of floating EC was similar to the condition for subcultivation as they can adhere or bind to any surface. The phosphatidylserine (PS) exposure was also determined by using flow cytometer. RESULTS: The adhesion of noninfected heterozygous thalassemic RBCs (all genotypes) to ECs was significantly increased as compared with normal RBCs (P < 0.02). Interestingly, after P. falciparum infection, the number of normal RBCs bound to ECs was significantly increased as compared with noninfected RBCs (P < 0.01), whereas heterozygous thalassemic RBCs infected by P. falciparum showed no significant difference compared with noninfected RBCs. In addition, we found a similar level of PS exposure in normal and thalassemic infected RBCs, which was related to the cytoadherence phenomenon. CONCLUSION: The reduced adhesion between heterozygous thalassemic RBCs infected by P. falciparum to ECs provides an explanation for their protective mechanism against malaria, as increased adhesion is a high risk for cerebral malaria and nonbinding infected RBCs can be removed by the reticuloendothelial system and other mechanism(s) in vivo.     

Plasmodium falciparum- infected erythrocytes induce tissue factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes

BACKGROUND: Plasmodium falciparum malaria infects 300-500 million people every year, causing 1-2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. OBJECTIVES: We have asked whether interaction of parasitized red blood cells (pRBC) with EC induces tissue factor (TF) expression in vitro and in vivo. The role of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated. RESULTS: We demonstrate that mature forms of pRBC induce functional expression of TF by EC in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late-stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, post-mortem brain sections obtained from P. falciparum-infected children who died from cerebral malaria and other causes display a consistent staining for TF in the EC. CONCLUSIONS: These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications.     

Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36

The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.     

Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke.

Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.     

Plasmodium Falciparum Malaria: AN AMELANOTIC MELANOMA CELL LINE BEARS RECEPTORS FOR THE KNOB LIGAND ON INFECTED ERYTHROCYTES

Erythrocytes infected with Plasmodium falciparum trophozoites and schizonts are not seen in the peripheral circulation because they attach to venular endothelium via knoblike structures on the infected erythrocyte membrane. We have recently shown that erythrocytes containing P. falciparum trophozoites and schizonts likewise attach to cultured human venous endothelial cells via knobs. In search of a more practical target cell for large scale binding studies designed to characterize and isolate the knob ligand, we tested various normal cells and continuous cell lines for their ability to bind P. falciparum-infected erythrocytes. Of the 18 cell types tested, binding of infected erythrocytes was observed to a human amelanotic melanoma cell line and amnion epithelial cells as well as to human aortic and umbilical vein endothelial cells. 96-100% of amelanotic melanoma cells bound 17±4 (±1 SEM) infected erythrocytes per positive cell, whereas fewer endothelial cells (4-59%) and amnion epithelial cells (8-19%) were capable of binding 12±5 and 4±1 infected erythrocytes per positive cell, respectively. Further studies designed to compare the mechanism of binding to the amelanotic melanoma cell line and endothelial cells showed the following results. First, that adhesion of infected erythrocytes to these two cell types was parasite stage-specific in that only erythrocytes containing late ring forms, trophozoites, and schizonts bound. Erythrocytes containing early ring forms, which do not attach to venular endothelium in vivo, did not bind to either cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. We conclude that cultured human endothelial cells and an amelanotic melanoma cell line share common determinants on their surface and that the mechanism of binding to these two different cell types is similar. The amelanotic melanoma cell line offers a useful substitute for endothelial cells in binding studies requiring large numbers of target cells.     

Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue

To determine the role of vascular cell adhesion molecule 1 (VCAM- 1)/very late activation antigen 4 (VLA-4) and intercellular adhesion molecule 1 (ICAM-1)/lymphocyte function-associated antigen 1 (LFA-1) interactions in causing antigen-induced eosinophil and T cell recruitment into the tissue, we studied the effect of the in vivo blocking of VCAM-1, ICAM-1, VLA-4, and LFA-1 by pretreatment with monoclonal antibodies (mAb) to these four adhesion molecules on the eosinophil and T cell infiltration of the trachea induced by antigen inhalation in mice. The in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, prevented antigen-induced eosinophil infiltration into the mouse trachea. On the contrary, the in vivo blocking of VCAM-1 and VLA-4, but not of ICAM-1 and LFA-1, increased blood eosinophil counts after antigen challenge, but did not affect blood eosinophil counts without antigen challenge in sensitized mice. Furthermore, the expression of VCAM-1 but not ICAM-1 was strongly induced on the endothelium of the trachea after antigen challenge. In addition, pretreatment with anti-IL-4 mAb decreased the antigen-induced VCAM-1 expression only by 27% and had no significant effect on antigen-induced eosinophil infiltration into the trachea. The in vivo blocking of VCAM- 1 and VLA-4 inhibited antigen-induced CD4+ and CD8+ T cell infiltration into the trachea more potently than that of ICAM-1 and LFA-1. In contrast, regardless of antigen challenge, the in vivo blocking of LFA- 1, but not of ICAM-1, increased blood lymphocyte counts more than that of VCAM-1 and VLA-4. These results indicate that VCAM-1/VLA-4 interaction plays a predominant role in controlling antigen-induced eosinophil and T cell recruitment into the tissue and that the induction of VCAM-1 expression on the endothelium at the site of allergic inflammation regulates this eosinophil and T cell recruitment.     

大鼠局灶性脑缺血再灌注后ICAM-1表达及甲基强的松龙干预实验研究

目的:探讨细胞间粘附分子 -1(ICAM -1)在脑缺血再灌注损伤中的作用及甲基强的松龙(MP)干预后的影响。方法 :采用栓线法制备大鼠大脑中动脉(MCA)缺血再灌模型 ,运用免疫组化方法检测ICAM -1的表达 ,并观察脑组织病理学改变。结果 :ICAM -1明确表达于脑缺血再灌组手术侧半球的微血管内皮 ,MP干预后阳性微血管数减少 ,多形核白细胞浸润减少 ,组织损伤程度相对较轻。结论 :局灶性脑缺血再灌注可诱导局部脑微血管内皮细胞ICAM -1的表达 ,其表达与脑组织坏死关系密切。MP可减少ICAM -1的表达及多形核白细胞浸润。     

Targets of antibodies against Plasmodium falciparum-infected erythrocytes in malaria immunity

Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum-infected erythrocytes (P. falciparum-IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development.     

大鼠脑出血后细胞黏附分子1 mRNA的表达与血脑屏障通透性的关系

目的探讨大鼠脑出血后细胞间黏附分子1(ICAM-1)mRNA的表达与血脑屏障(BBB)通透性的关系。方法采用自体动脉血注入大鼠尾状核建立脑出血模型,将50只大鼠分成对照组、脑出血组,两组分6小时、1天、3天、5天、7天5个时点,每个时点各5只大鼠。分别采用干湿重法、伊文思兰染色法、逆转录聚合酶链法(RT-PCR)测定不同时间点的脑水含量、血脑屏障通透性(EB含量)和ICAM-1mRNA表达量的变化。结果大鼠出血造模后6小时BBB通透性和ICAM-1表达开始升高,3天达高峰,7天开始下降,出血组和对照组EB含量6小时(0.592±0.106)OD/mg vs(0.309±0.044)OD/mg,3天(0.791±0.520)OD/mg vs(0.315±0.342)OD/mg,7天(0.455±0.082)OD/mg vs(0.308±0.032)OD/mg(均P<0.01)。ICAM-1 6小时1.220±0.044vs 0.884±0.336,3天1.638±0.07vs 0.860±0.057,7天1.282±0.144vs 0.872±0.048(均P<0.01)。BBB通透性与ICAM-1mRNA表达呈正相关(r=0.433,P<0.01),而且与脑水含量变化趋势相一致。结论脑出血后血肿周围组织ICAM-1mRNA表达增高,可能增加血脑屏障通透性,并参与脑水肿形成。     

Invasion of mouse erythrocytes by the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum malaria merozoites require erythrocyte sialic acid for optimal invasion of human erythrocytes. Since mouse erythrocytes have the form of sialic acid found on human erythrocytes (N-acetyl neuraminic acid), mouse erythrocytes were tested for invasion in vitro. The Camp and 7G8 strains of P. falciparum invaded mouse erythrocytes at 17-45% of the invasion rate of human erythrocytes. Newly invaded mouse erythrocytes morphologically resembled parasitized human erythrocytes as shown on Giemsa-stained blood films and by electron microscopy. The rim of parasitized mouse erythrocytes contained the P. falciparum 155-kD protein, which is on the rim of ring-infected human erythrocytes. Camp but not 7G8 invaded rat erythrocytes, indicating receptor heterogeneity. These data suggest that it may be possible to adapt the asexual erythrocytic stage of P. falciparum to rodents. The development of a rodent model of P. falciparum malaria could facilitate vaccine development.