Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion

The effect of flow on the adhesion of polymorphonuclear leukocytes (PMNL) to vascular endothelium was investigated using a parallel plate chamber with a well-defined flow field. Washed PMNL were perfused over a monolayer of primary human umbilical vein endothelial cells (HUVEC) pretreated with formyl-methionyl-leucyl-phenylalanine (FMLP, 1 X 10(-7) mol/L) for five minutes. In other experiments HUVEC were pretreated with interleukin 1 (IL1,2 U/mL) for four hours. PMNL adhesion to stimulated and control HUVEC was measured over a physiologic range of wall shear stresses. PMNL adhesion to nylon-coated surface was also studied. At a wall shear stress of 0.98 dynes/cm2,283 +/- 37.3 PMNL/mm2 (mean +/- SEM) adhered to FMLP-treated HUVEC while 195 +/- 20.3 PMNL/mm2 adhered to control HUVEC. At 1.96 dynes/cm2, 68 +/- 14.1 PMNL/mm2 adhered to FMLP-treated HUVEC and 42 +/- 6.0 PMNL/mm2 adhered to control HUVEC. At 3.92 dynes/cm2, virtually no PMNL adherence was noted on either control or FMLP-treated HUVEC. On IL 1-treated HUVEC at 1.96 dynes/cm2, 371 +/- 25.8 PMNL/mm2 adhered while 28 +/- 2.9 PMNL/mm2 adhered to control HUVEC. PMNL adhesion to IL 1-treated and control HUVEC dropped to 10.2 +/- 3.8 and 6.8 +/- 3.5 PMNL/mm2, respectively, at 3.01 dynes/cm2. The effect of flow on PMNL adhesion appears to be an important factor in determining the outcome of the PMNL/HUVEC adhesive interaction under these experimental conditions.     

Inflammatory mediators promote strong sickle cell adherence to endothelium under venular flow conditions

Adherence of sickle erythrocytes to endothelium in venules is thought to initiate or propagate vaso-occlusive episodes. Because of blood shear forces with normal microvascular flow, adherence in post-capillary venules requires binding via high-affinity receptor-mediated pathways. Microvascular flow in sickle patients is episodic, even in asymptomatic patients, so adherence may also occur at low shear not requiring high-affinity binding. Sickle cell binding to endothelium was quantified under flow or static incubation with unusually large vWF, thrombospondin, alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/fibronectin (FN). Adherence under flow at 0.5 dyne/cm(2) shear stress leads to the greatest number of adherent sickle cells. Adherence under flow at 1.0 dyne/cm(2) leads to the strongest adherence. Static incubation conditions promote weak adherence of low numbers of sickle cells to endothelium. Following attachment at 1.0 dyne/cm(2), adherence strength was 2.5 +/- 0.1 or 2.6 +/- 0.2 dynes/cm(2) for alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/FN pathways, a level 50% greater than adherence strength mediated by thrombospondin or ULvWF (1.7 +/- 0.08 or 1.6 +/- 0.07 dynes/cm(2), respectively). Sickle cell adhesion promoted by simultaneous activation of alpha(4)beta(1)/VCAM-1 and alpha(4)beta(1)/FN pathways is the strongest at 6.2 +/- 0.2 dynes/cm(2) and adherent red cells resist detachment shear stresses up to 10 dynes/cm(2). These data demonstrate that sickle cell adhesion to endothelium is regulated both by receptor/ligand affinity and flow conditions. Thus, both microvascular flow conditions and receptor-ligand interactions may regulate sickle cell adherence in vivo.     

Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium

The CD11/CD18 family of glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to inflammatory responses. Using a monoclonal antibody (MoAb) against CD18 (TS1/18), the role of these molecules in polymorphonuclear neutrophil (PMNL) adhesion to cultured primary human umbilical vein endothelial cells (HUVEC) was examined under venous flow conditions. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to interleukin-1 (IL-1)-treated HUVEC at 2.0 dynes/cm2 (TS1/18-treated 305 +/- 58 PMNL/mm2 v 334 +/- 63 PMNL/mm2 on control). Furthermore, incubation of HUVEC with R6.5.D6, an MoAb against intercellular adhesion molecule-1 (ICAM-1) did not significantly inhibit PMNL adhesion to IL-1-treated HUVEC at 2.0 dynes/cm2 (P greater than .3). In contrast to the lack of inhibition of adhesion under conditions of flow, incubation of PMNL with TS1/18 reduced PMNL adherence in static adhesion assays. PMNL migration beneath HUVEC monolayers has been shown to be stimulated by 4-hour IL-1 treatment. TS1/18 and R6.5.D6 significantly inhibited migration of PMNL beneath IL-1-treated HUVEC monolayers under flow conditions by slightly more than 80% (P less than .005). In flow experiments with CD18-deficient PMNL, virtually no transendothelial migration was observed. The effect of FMLP (10(-8) mol/L) on PMNL adhesion to untreated HUVEC at wall shear stresses ranging from 0.25 to 2.0 dynes/cm2 was also investigated. FMLP had little effect on PMNL adherence at shear stresses above 0.5 dynes/cm2 (P greater than .45). In response to FMLP exposure at lower wall shear stresses, PMNL adherence to untreated HUVEC increased 6.9-fold at 0.5 dynes/cm2 (P less than .001). At 0.25 dynes/cm2, FMLP stimulation increased PMNL adherence to untreated HUVEC 6.5-fold compared with controls (P less than .005), and FMLP failed to make CD18-deficient PMNL more adherent. In experiments with PMNL pretreated with TS1/18 (anti-CD18), there was a 67% inhibition of FMLP-stimulated adhesion at 0.5 dynes/cm2 (P less than .025). The upper threshold of CD18-mediated PMNL adhesion appears to be between 0.5 and 1.0 dyne/cm2. Above these wall shear stresses, the initial attachment of PMNL to cultured endothelium was mediated almost exclusively by CD18-independent mechanisms. By simulating some of the flow parameters in the microcirculation with well-characterized shear forces, PMNL adhesion by CD18-independent and dependent mechanisms can be differentiated. These data also indicate that CD18 is an important mediator of transendothelial migration by PMNL, which have attached to the endothelium by a CD18-independent mechanism.     

Thrombospondin from activated platelets promotes sickle erythrocyte adherence to human microvascular endothelium under physiologic flow: a potential role for platelet activation in sickle cell vaso-occlusion

Adherence of erythrocytes to vascular endothelium likely contributes to the pathophysiology of episodic vascular occlusion in patients with sickle cell disease (SCD). In addition, coagulation activation has been reported in sickle patients during complications such as pain episodes. To test the hypothesis that platelet activation contributes to sickle erythrocyte binding, we investigated whether factors released from activated sickle platelets promote adherence of sickle erythrocytes to human microvascular endothelial cells (MEC) under flow conditions. Activated sickle platelet supernatant (ASPS) promoted high levels of sickle erythrocyte adherence to MEC (55.4 +/- 3.9 erythrocytes/mm2) but only moderate adherence of normal erythrocytes to MEC (14.1 +/- 0.7 erythrocytes/mm2). When MEC were incubated with an antibody (OKM5) against CD36 (a thrombospondin [TSP] receptor), platelet supernatant mediated sickle erythrocyte adherence was inhibited 86%, suggesting that TSP participated in the adherence. To further define the role of TSP in adherence, additional studies using purified TSP were performed. At a concentration of 0.2 micrograms/mL TSP in serum-free media (SFM), sickle erythrocyte adherence to MEC was 33.9 +/- 2.7 erythrocytes/mm2 and sixfold greater than either sickle erythrocyte adherence in the absence of TSP or normal erythrocyte adherence in the presence of TSP. Doubling the concentration of TSP to 0.4 micrograms/mL proportionally increased adherence of sickle erythrocytes. Incubation of MEC with OKM5 or anti-alpha v monoclonal antibodies inhibited TSP-mediated sickle erythrocyte adherence more than 95%. These data suggest that activated platelet release factors, including alpha-granule TSP, which promote receptor-mediated sickle erythrocyte adherence to microvascular endothelium. Such factors released during in vivo platelet activation could contribute to vaso-occlusive complications by promoting erythrocyte adherence and microvascular occlusion.     

Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells

We investigated the effect of hemodynamic shear forces on the expression of adhesive molecules, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC) exposed to laminar (8 dynes/cm2) or turbulent shear stress (8.6 dynes/cm2 average), or to a static condition. Laminar flow induced a significant time-dependent increase in the surface expression of ICAM- 1, as documented by flow cytometry studies. Endothelial cell surface expression of ICAM-1 in supernatants of HUVEC exposed to laminar flow was not modified, excluding the possibility that HUVEC exposed to laminar flow synthetize factors that upregulate ICAM-1. The effect of laminar flow was specific for ICAM-1, while E-selectin expression was not modulated by the flow condition. Turbulent flow did not affect surface expression of either E-selectin or ICAM-1. To evaluate the functional significance of the laminar-flow-induced increase in ICAM-1 expression, we studied the dynamic interaction of total leukocyte suspension with HUVEC exposed to laminar flow (8 dynes/cm2 for 6 hours) in a parallel-plate flow chamber or to static condition. Leukocyte adhesion to HUVEC pre-exposed to flow was significantly enhanced, compared with HUVEC maintained in static condition (233 +/- 67 v 43 +/- 16 leukocytes/mm2, respectively), and comparable with that of interleukin-1 beta treated HUVEC. Mouse monoclonal antibody anti-ICAM-1 completely blocked flow-induced upregulation of leukocyte adhesion. Interleukin-1 beta, which upregulated E-selectin expression, caused leukocyte rolling on HUVEC that was significantly lower on flow- conditioned HUVEC and almost absent on untreated static endothelial cells. Thus, laminar flow directly and selectively upregulates ICAM-1 expression on the surface of endothelial cells and promotes leukocyte adhesion. These data are relevant to the current understanding of basic mechanisms that govern local inflammatory reactions and tissue injury.     

Multiple ligands for cytoadherence can be present simultaneously on the surface of Plasmodium falciparum-infected erythrocytes.

A major virulence factor of Plasmodium falciparum is the adherence of parasitized erythrocytes to the wall of postcapillary venules via a specific interaction between parasite-derived erythrocyte surface ligands and receptors on endothelial cells. To study this phenomenon in vitro, we selected a parasite population that expressed at least two different ligands and demonstrated that parasitized cells may coexpress ligands with specificity for multiple receptors. This selected parasite line had several antigenic and cytoadherence characteristics that were different from those of the parent line. Single parasitized erythrocytes were able to adhere to three distinct receptors via at least two separate ligands; a trypsin-sensitive molecule mediated cytoadherence to CD36 and intercellular adhesion molecule 1 and a trypsin-insensitive molecule(s) was responsible for adherence to a third receptor on the surface of melanoma cells. We present evidence that this newly discovered receptor for cytoadherence is an N-linked glycosaminoglycan, as treatment of melanoma cells with endoglycosidase H abolished cytoadherence. These observations emphasize the adaptability of P. falciparum and the complexity of the cytoadherence phenomenon.     

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes.

Cerebral involvement in Plasmodium falciparum malaria is associated with sequestration of infected red blood cells and occlusion of cerebral vessels. Adhesion of infected erythrocytes along the vascular endothelium as well as binding of uninfected erythrocytes to cells infected with late-stage asexual parasites (rosetting) may be important in erythrocyte sequestration. We report that the recently discovered rosetting phenomenon shares characteristics with other human cell-cell interactions (heparin sensitivity, temperature independence, Ca2+/Mg2+ and pH dependence). Mono- and polyclonal antibodies specific for PfHRP1, a histidine-rich protein present in the membrane of P. falciparum-infected erythrocytes, disrupt rosettes but do not affect attachment of infected erythrocytes to endothelial cells. The inhibitory anti-PfHRP1 antibodies reacted with rosetting parasites in indirect immunofluorescence and with P. falciparum polypeptides of Mr 28,000 and Mr 90,000 in immunoprecipitation and immunoblotting, respectively. No inhibitory effects on erythrocyte rosetting were obtained with antibodies to related histidine-rich or other antigens of P. lophurae or P. falciparum. Whether the epitope that mediates rosetting, and is recognized by the anti-PfHRP1 antibodies, is located on PfHRP1 or on a crossreactive antigen remains to be established. The results suggest that endothelial cytoadherence and erythrocyte rosetting involve different molecular mechanisms.     

Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.     

Abnormal membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes was analysed by employing phospholipase A2 and Merocyanine 540 as external membrane probes. Both bee venom and pancreatic phospholipases A2 failed to hydrolyse phosphatidylserine in uninfected human red cells isolated from in vitro P. falciparum cultures. However, these enzymes under identical conditions readily degraded this aminophospholipid in P. falciparum-infected erythrocytes. Phosphatidylethanolamine hydrolysis also increased in parasitized cells. The degree to which these aminophospholipids were cleaved by the enzymes in intact infected cells depended on the developmental stage of the intracellular parasite, and was maximum at the schizont stage. This was consistent with the finding that the 'fluid-sensing' fluorescent dye, Merocyanine 540, readily labelled both the schizont and trophozoite-infected cells but not the fresh, uninfected or ring-infected erythrocytes. These results demonstrate that P. falciparum produces stage-dependent changes in the membrane phospholipid organization of its host erythrocyte.     

约氏疟原虫感染对小鼠红细胞膜蛋白组分及其与内皮细胞黏附力的影响

目的分析约氏疟原虫感染后小鼠红细胞膜蛋白及受染红细胞与内皮细胞黏附能力的变化,为进一步探索脑型疟的发生机制提供线索。方法提取约氏疟原虫感染的BALB/c小鼠红细胞膜蛋白,进行SDS-PAGE电泳,检测红细胞膜蛋白的变化。用内皮细胞株与小鼠红细胞共同培养,观察受染红细胞与内皮细胞黏附能力的改变。结果 SDS-PAGE显示约氏疟原虫感染后的小鼠红细胞膜蛋白中含有分子质量约137ku组分,53/54ku固有膜蛋白减少。与未感染对照组比较,受染红细胞对内皮细胞的黏附能力增强约3倍,差异有统计学意义(P<0.05)。结论疟原虫感染可改变宿主红细胞膜蛋白组成,增强受染红细胞与内皮细胞的黏附力。这些变化可能与脑型疟的发生有关。     

Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and receptors on sickle erythrocytes. It has previously been reported that sickle reticulocytes express the alpha 4 beta 1 integrin receptor and bind to cytokine-activated endothelium via an alpha 4 beta 1/vascular- cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other roles for alpha 4 beta 1 in sickle-cell adherence, the ability of activated alpha 4 beta 1 to promote adhesion to endothelium via a ligand different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm2. Preincubation of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increased adherence of sickle cells eightfold as compared with untreated sickle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta 1 antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blood cell (RBC) adhesion to endothelium. Anti-alpha 4 antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle-cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha 4 beta 1, was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PDBu-treated sickle cells was completely inhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endothelium using an antifibronectin antibody in enzyme-linked immunosorbent assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/- 17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adherence 86% +/- 8%. Taken together, these data suggest that endothelial-bound fibronectin mediates adherence of PDBu- treated sickle cells. Interleukin-8 (IL-8), a chemokine released in response to bacterial infection, viral infection, or other injurious agents, and known to activate integrins, also increased adherence of sickle erythrocytes to endothelial cells via fibronectin. This novel adherence pathway involving sickle-cell alpha 4 beta 1 activated by PDBu or IL-8 may therefore be relevant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observations describe ways in which inflammation and immune responses cause vasoocclusive complications in sickle-cell disease.     

DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes/cm(2) for 6 or 24 h (1 dyne = 10 microN), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF/GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine/spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells.     

Abnormal erythrocyte fragmentation and membrane deformability in paroxysmal nocturnal hemoglobinuria

Hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) is considered to be a result of an intrinsic membrane defect. This defect may result in abnormal material properties of PNH erythrocytes. To examine this hypothesis, fragmentation failure, and membrane deformability were assessed in the absence of complement by micropipette techniques. Membrane viscosity was determined by observing relaxation of deformed cells. Results show a bimodal distribution of force for membrane failure, membrane viscoelasticity, and elastic shear modulus. One population requires significantly less force for fragmentation, mean 0.56 X 10(-6) dyne; has increased membrane viscosity, mean 0.205 X 10(-2) dyne sec/cm; and has decreased elastic shear modulus, mean 0.56 X 10(-2) dyne/cm. A second population resembles control with fragmentation force, mean 1.19 X 10(-6) dyne, control 1.05 X 10(-6) dyne; membrane viscosity, mean 0.112 X 10(-2) dyne/cm, control 0.102 X 10(-2) dyne sec/cm; elastic shear modulus, mean 0.70 X 10(-2) dyne/cm, control 0.78 X 10(-2) dyne/cm. The percent of cells with abnormal material properties corresponds to the percent of PNH III cells determined by complement lysis. Thus, the hemolysis attributed to an abnormal clone of erythrocytes in PNH is associated with an intrinsic membrane abnormality which predisposes to lysis.     

Effect of anti TNFalpha therapy on arterial diameter and wall shear stress and HDL cholesterol

It has been recently hypothesized that both TNFalpha and anti TNFalpha treatment have a stimulating effect on nitric oxide synthesis and release. Moreover, an in vitro experiment has demonstrated that HDL-cholesterol binds TNFalpha. Aims of our study were to investigate wall shear stress of peripheral arteries and endothelial function of brachial artery in subjects with Rheumatoid Arthritis (RA) at baseline and after infliximab. Moreover, we evaluated the effect of anti TNFalpha therapy on lipid profile. Ten patients with RA received infliximab therapy at weeks 0, 2 and 6. Lipids and vascular parameters were measured before and the day after each infusion. After the first treatment, FMD increased (3.7 +/- 1.9% versus 17.5 +/- 2.9%, P <0.01) and common carotid and brachial artery diameters decreased (5.9 +/- 0.2 mm versus 5.5 +/- 0.2 mm; 3.5 +/- 0.4 mm versus 3.1 +/- 0.4 mm, respectively, P <0.005). Common carotid and brachial artery wall shear stress increased (21.1 +/- 1.1 dynes/cm2 versus 23.9 +/- 1.4 dynes/cm2; 42.0 +/- 4.7 dynes/cm2 versus 51.6 +/- 5.7 dynes/cm2, P <0.01). Similar results were observed after the second and third infusion. All these parameters returned to pre-treatment level at the following infusion. HDL-cholesterol and apolipoprotein AI significantly decreased after each treatment (1st treatment: 1.4 +/- 0.05 mmol/L versus 1.2 +/- 0.06 mmol/L, P <0.01; 1.73 +/- 0.05 g/L versus 1.57 +/- 0.02 g/L, P <0.03). The present data show vasoconstriction and an increase of wall shear stress in studied arteries after infliximab. HDL cholesterol is reduced by treatment and does not seem to influence FMD.     

Sickle erythrocyte adherence to endothelium at low shear: role of shear stress in propagation of vaso-occlusion

Under venular flow conditions, sickle cell adherence to endothelium is mediated by cell adhesion molecules and adhesive proteins associated with inflammation, coagulation, and endothelial perturbation. Periodic and reduced blood flow are observed in sickle microcirculation during hematologic steady state, suggesting that blood flow is compromised in sickle microcirculation. We tested the hypothesis that low blood flow enhances adherence by quantifying sickle cell adhesion to endothelium under venular flow (1.0 dyne/cm(2) shear stress) and low flow (0.1 dyne/cm(2) shear stress), with and without addition of adhesion promoting agonists. Under low flow, sickle cell adherence to endothelium increases with contact time in the absence of endothelial activation or adhesive protein addition. In contrast, at venular shear stress, sickle cell adherence only occurs following endothelial activation with TNF-alpha or addition of thrombospondin. Analysis of these data with a mathematical model reveals that at low flow adherence is "transport-controlled," meaning that contact time between sickle cells and endothelium is a more important determinant of adherence than high-affinity receptor-ligand interactions. Low-affinity interactions are sufficient for adhesion at low flow. In contrast, at venular flow (1 dyne/cm(2) shear stress) adherence is "affinity-controlled," meaning that adherence requires induction of specific high-affinity receptor-ligand interactions. These findings demonstrate that in addition to activating factors and adherence proteins, microvascular shear stress is an important determinant of sickle cell adhesion to endothelium. This suggests that in vivo, erythrostasis is an important determinant of adhesion that can act either independently or concurrently with ongoing acute events to induce adhesive interactions and vaso-occlusion.     

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.     

Double-stranded RNA induces sickle erythrocyte adherence to endothelium: a potential role for viral infection in vaso-occlusive pain episodes in sickle cell anemia

Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double- stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1- -dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double- stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes.     

Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions

The occurrence of rosetting of Plasmodium falciparum-infected human red blood cells (IRBC) with uninfected red blood cells (RBC) and its potential pathophysiologic consequences were investigated under flow conditions using the perfused rat mesocecum vasculature. Perfusion experiments were performed using two knobby (K+) lines of P falciparum, ie, rosetting positive (K+R+) and rosetting negative (K+R-). The infusion of K+R+ IRBC resulted in higher peripheral resistance (PRU) than K+R- IRBC (P less than .0012). Video microscopy showed that under conditions of flow, in addition to cytoadherence of K+R+ IRBC to the venular endothelium, rosette formation was also restricted to venules, especially in the areas of slow flow. Rosettes were absent in arterioles and were presumably dissociated by higher wall shear rates. The presence of rosettes in the venules must therefore reflect their rapid reformation after disruption. Cytoadherence of K+R+ IRBC was characterized by formation of focal clusters along the venular wall. In addition, large aggregates of RBC were frequently observed at venular junctions, probably as a result of interaction between flowing rosettes, free IRBC, and uninfected RBC. In contrast, the infusion of K+R+ IRBC resulted in diffuse cytoadherence of these cells exclusively to the venular endothelium but not in rosetting or large aggregate formation. The cytoadherence of K+R+ IRBC showed strong inverse correlation with the venular diameter (r = -.856, P less than .00001). Incubation of K+R+ IRBC with heparin and with monoclonal antibodies to glycoprotein IV/CD36 abolished the rosette formation and resulted in decreased PRU and microvascular blockage. These findings demonstrate that rosetting of K+R+ IRBC with uninfected RBC enhances vasocclusion, suggesting an important in vivo role for rosetting in the microvascular sequestration of P falciparum-infected RBC.     

Heparan sulfate on endothelial cells mediates the binding of Plasmodium falciparum-infected erythrocytes via the DBL1alpha domain of PfEMP1

Plasmodium falciparum may cause severe forms of malaria when excessive sequestration of infected and uninfected erythrocytes occurs in vital organs. The capacity of wild-type isolates of P falciparum-infected erythrocytes (parasitized red blood cells [pRBCs]) to bind glycosaminoglycans (GAGs) such as heparin has been identified as a marker for severe disease. Here we report that pRBCs of the parasite FCR3S1.2 and wild-type clinical isolates from Uganda adhere to heparan sulfate (HS) on endothelial cells. Binding to human umbilical vein endothelial cells (HUVECs) and to human lung endothelial cells (HLECs) was found to be inhibited by HS/heparin or enzymes that remove HS from cell surfaces. (35)S-labeled HS extracted from HUVECs bound directly to the pRBCs' membrane. Using recombinant proteins corresponding to the different domains of P falciparum erythrocyte membrane protein 1 (PfEMP1), we identified Duffy-binding-like domain-1alpha (DBL1alpha) as the ligand for HS. DBL1alpha bound in an HS-dependent way to endothelial cells and blocked the adherence of pRBCs in a dose-dependent manner. (35)S-labeled HS bound to DBL1alpha-columns and eluted as a distinct peak at 0.4 mM NaCl. (35)S-labeled chondroitin sulfate (CS) of HUVECs did not bind to PfEMP1 or to the pRBCs' membrane. Adhesion of pRBCs of FCR3S1.2 to platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, mediated by the cysteine-rich interdomain region 1alpha (CIDR1alpha), was found be operative with, but independent of, the binding to HS. HS and the previously identified HS-like GAG on uninfected erythrocytes may act as coreceptors in endothelial and erythrocyte binding of rosetting parasites, causing excessive sequestration of both pRBCs and RBCs.