Erythroid adhesion molecules in sickle cell disease: Effect of hydroxyurea

In sickle cell disease, the complex scenario of vaso-occlusive crisis (VOC) typical of this disease is clearly multifactorial and not fully understood. Cell-cell and cell-cell matrix interactions mediated by adhesive molecules present on blood cells and endothelial cells (ECs) are thought to play an important role. Early studies have shown that sickle red blood cells (RBCs) are abnormally adherent to ECs and some of the molecules involved in these interactions have been identified, such as the alpha4beta1 integrin and CD36, exclusively present on stress reticulocytes, and CD47 on mature RBCs. More recently, attention focused on Lu/BCAM, the unique RBC receptor for laminin, and on ICAM-4, a red cell-specific adhesion receptor, which is a ligand for a large repertoire of integrins (alphaLbeta2, alphaMbeta2, alphaxbeta2, alphaVbeta3). The counter-receptors on ECs and the role of plasma proteins forming bridges between blood cells and ECs have been clarified in part. It has also been shown that reticulocytes from SCD patients express higher levels of alpha4beta1 integrin and CD36, and that under hydroxyurea (HU) therapy, both cell adhesion to ECs or extracellular matrix proteins and the levels of these adhesion molecules are reduced. These findings are consistent with the view that enhanced adhesion of blood cells to ECs is largely determined by the membrane expression level of adhesion molecules and could be a crucial factor for triggering or aggravating vaso-occlusion. In SCD patients, membrane expression of Lu/BCAM (and perhaps ICAM-4) is enhanced on RBCs whose adherence to laminin or ECs is also increased. Interestingly, Lu/BCAM- and ICAM-4-mediated adhesion are enhanced by the stress mediator epinephrine through a PKA-dependent pathway initiated by a rise in intracellular cAMP and leading to receptor activation by phosphorylation according to the same signaling pathway. More recently, studies based on quantitative expression analysis of adhesion molecules on RBCs and during erythroid differentiation in patients undergoing HU therapy, surprisingly revealed that Lu/BCAM level was enhanced, although alpha4beta1, CD36 and ICAM-4 (to a lower extent) levels were indeed reduced. CD47 and CD147 expression were also enhanced in HU-treated patients. Based on these findings we suggest that the signalization cascade leading to receptor activation rather than the expression level only of adhesion molecules may be the critical factor regulating cell adhesion, although both mechanisms are not mutually exclusive.     

Role of Lu/BCAM in abnormal adhesion of sickle red blood cells to vascular endothelium

Lutheran (Lu) blood group and Basal Cell Adhesion Molecule (BCAM) antigens are both carried by two glycoprotein (gp) isoforms of the immunoglobulin superfamily representing receptors for laminin alpha5 chain. They are expressed in red blood cells, in endothelial cells of vascular capillaries and in epithelial cells of several tissues. Lu/BCAM gps are overexpressed in sickle red blood cells (SS RBCs). Stimulation of SS RBCs by epinephrine activates the PKA depending signaling pathway and induces reinforced Lu/BCAM-mediated adhesion to laminin10/11. We have analyzed the phosphorylation state of Lu/BCAM long isoform cytoplasmic tail and showed that it is phosphorylated by CKII, GSK3b and PKA. Phosphorylation of this isoform in transfected K562 cells is stimulated by effectors of the PKA pathway and induces cell adhesion to laminin10/11. Lu/BCAM gps are highly expressed in endothelial cells and exhibit potential integrin binding motifs. We showed that they interact with integrin alpha4beta1, the unique integrin expressed on the surface of young reticulocytes. Adhesion assays under flow conditions showed that SS RBCs adhere to primary human endothelial cells (HUVEC) after selective activation of intergin alpha4beta1 and that this adhesion is mediated by endothelial Lu/BCAM gps. Our studies show that Lu/BCAM gps expressed either on erythroid or on endothelial cells are involved in SS RBC-endothelium interactions and could play a role in the abnormal adhesion of SS RBCs to vascular endothelium contributing to the vaso-occlusive crises reported for sickle cell disease patients.     

Red cell transfusion in medicine: Future challenges

Many side-effects of red blood cell transfusion have been described. They include iron-overload, as well as allo- and autoantibody formation against red cells. During storage, erythrocytes undergo complex structural and biochemical changes. It has been suggested that accelerated and/or aberrant forms of the physiological erythrocyte aging process underlie the red cell storage lesion. This storage lesion may contribute to side-effects of transfusion as endothelial damage by release of internal erythrocyte constituents, (pro)inflammatory consequences, hampered microcirculation and oxygen delivery. Understanding the process that determines the fate of red blood cells after transfusion may contribute to the prevention of side-effects after red blood cell transfusion. This should be the focus of research on red blood cell transfusion in clinical transfusion medicine.     

Mouse models of sickle cell disease

In the absence of a natural animal model for sickle cell disease, transgenic mouse models have been generated to better understand the complex pathophysiology of the disease and to evaluate potential specific therapies. In the early nineties, the simple addition of human globin genes induced the expression of hemoglobin S (HbS) or HbS-related human hemoglobins in mice still expressing mouse hemoglobin. To increase the proportion of human hemoglobin and the severity of the mouse sickle cell syndrome, the proportion of mouse hemoglobin could be decreased by a combination of mouse alpha- and beta-thalassemic defects, leading to complex genotypes and mild disease. Following the discovery of gene targeting in the mouse embryonic stem cells (ES cells), it was made possible to knock out all mouse adult globin genes (2alpha and 2beta) and to add the human homologous genes elsewhere in the mouse genome. In addition, the human gamma gene of fetal hemoglobin was protecting the fetus from HbS polymer formation. Accordingly, the resulting adult mouse models obtained in 1997, expressing human HbS-only, had a very severe anemia (Hb=5-6 g/dL). In order to survive, these "HbS-only mice" had to reduce the HbS concentration within the red blood cells. The phenotype could be less severe by adding modified human gamma genes, still expressed in adult mice. In 2006, a last "S-only" model was obtained by homologous knock in, replacing the mouse globin genes by human genes. This array of models contributes to better understand the role of different interacting factors in the complexity of sickle cell events, such as red cell defects, changes in blood flow and vaso-occlusion, hyperhemolysis, vascular tone dysregulation, oxidations, inflammation, activation and adhesion of cells, ischemia, reperfusion... In addition, each model has an appropriate usefulness to evaluate experimental therapies in vivo and to perform preclinical studies.     

Interaction of macrophages with "old" red blood cells from syngeneic mice in vitro and the independence of the recognition process on macrophage Fc receptors

The interaction between peritoneal macrophages and "old" red blood cells (RBC) from syngeneic mice was studied in vitro. It seems that this interaction is not mediated directly via Fc receptors on the macrophage. (1) 2-deoxy-D-glucose did not inhibit the phagocytosis of "old" RBC, but did inhibit the phagocytosis of IgG-coated sheep RBC (SRBC). (2) Immobilization of Fc receptors by plating macrophages on coverslips coated with bovine serum albumin: anti-bovine serum albumin (BSA: anti-BSA) complexes had no effect on the phagocytosis of "old" RBC, but inhibited the phagocytosis of IgG-coated SRBC. The interaction between mouse macrophages and "old" RBC is temperature dependent. At 4 degrees C and 22 degrees C "old" RBC attach to macrophages but are not phagocytized; at 37 degrees C phagocytosis occurs. Fetal calf serum (FCS) is required for optimal phagocytosis. Sialic acid residues on the macrophage surface do not play a role in this recognition and phagocytosis process, as neuraminidase treatment of macrophages did not affect the interaction.     

小白鼠腹腔渗出细胞吞噬功能的研究

本实验比较糖元、鸡红细胞及两者复合使用对小鼠腹腔渗出细胞的影响,结果表明鸡红细胞和糖元一样能激活巨噬系和组织嗜碱系细胞,影响巨噬系细胞分化,鸡红细胞较糖元快;而影响组织嗜碱系细胞分化,糖元较鸡红细胞快。由于腹腔渗出细胞中存在原、幼巨噬细胞,按照血细胞发育是不可逆回的理论,推测腹腔巨噬细胞不是来源于血液循环中的单核细胞,而是来自血循环中的定向干细胞。本实验在腹腔渗出细胞中,首次发现组织嗜碱系细胞并描写其形态特征。     

A Novel Synthesis of Polyacrolein Microspheres and Their Application for Cell Labeling and Cell Separation

A novel method for the synthesis of polyacrolein microspheres with fluorescent or magnetic properties is described. These microspheres carry reactive aldehyde groups on their surface, which are used for covalent binding of various proteins at physiological pH. Polyacrolein microspheres may be used as a simple tool for cell labeling and cell separation. The feasibility of specific labeling of fresh human red blood cells and of the separation of human red blood cells from turkey red blood cells by means of a magnetic field is discussed.     

A Novel Synthesis of Polyacrolein Microspheres and Their Application for Cell Labeling and Cell Separation

A novel method for the synthesis of polyacrolein microspheres with fluorescent or magnetic properties is described. These microspheres carry reactive aldehyde groups on their surface, which are used for covalent binding of various proteins at physiological pH. Polyacrolein microspheres may be used as a simple tool for cell labeling and cell separation. The feasibility of specific labeling of fresh human red blood cells and of the separation of human red blood cells from turkey red blood cells by means of a magnetic field is discussed.     

Sickle red cell adhesion: Many issues and some answers

Among multiple pathologies associated with sickle cell disease, sickle red cell-endothelial interaction has been implicated as a potential initiating mechanism in vaso-occlusive events that characterize this disease. Vast literature exists on various aspects of sickle red cell adhesion, but many issues remain unresolved, especially pertaining to the role of sickle red cell heterogeneity, the relative role of multiple adhesion mechanisms and targets of antiadhesive therapy. This review briefly analyzes these issues.     

用血红蛋白酶释放法检测巨噬细胞的吞噬能力

以鸡红细胞作为靶细胞,小鼠腹腔巨噬细胞为效应细胞研究了巨噬细胞的吞噬能力。经低渗处理后,血红蛋白酶释放的多少以它氧化邻苯二胺所产生的颜色反应表示吞噬百分比。对红细胞数与OD值关系、不同粘附时间和不同吞噬时间与吞噬能力的关系进行了研究。方法灵敏、可靠、客观,有推广应用的价值。     

L'adhérence des érythrocytes à l'endothélium vasculaire

Blood cells are in continuous contact with the vascular endothelium. Endothelial cell culture, intravital videomicroscopy allowed the investigation of blood cell-endothelium interactions in dynamic conditions.In the various diseases, diabetes mellitus, sickle cell anemia and malaria, erythrocytes have an increased adhesion to endothelial cells. The presence of advanced glycation end products (AGE) on erythrocytes of diabetics is responsible for their binding to the receptor RAGE present on the endothelium. The AGE-RAGE binding provokes an oxidant stress and induces the expression of the adhesion molecule. Furthermore, erythrocyte AGE induce an increase in vascular permeability. In sickle cell anemia, the increased adhesiveness and the sickling of red blood cells are responsible for thrombosis. Plasmodium falciparum infestation of erythrocytes induces knob formation at the cell surface and the P. falciparum protein binding to CD36, ICAM-1 and thrombospondin present on the endothelium, and facilitates the parasite dissemination. © 2999 Éditions scientifiques et médicales Elsevier SAS     

Vascular biosynthesis of nitric oxide: effect on hemostasis and fibrinolysis

Nitric oxide (NO) is a multifunctional effector molecule that plays a central role in the regulation of vascular homeostasis. NO is synthesized from L-arginine by a family of enzymes called NO synthases. The principal source of NO in the vascular system of healthy mammals is the constitutively expressed NO synthase in endothelial cells. The basal endothelial formation of NO can be increased by receptor-dependent agonists (i.e., bradykinin) in a calcium-calmodulin-dependent manner, and also by physical forces (i.e., shear stress), predominantly without changes in the intracellular concentration of free calcium. Nitric oxide can diffuse toward the blood vessel wall where the major target is the smooth muscle cell. NO regulates vascular tone, and the free radical is also a potent inhibitor of smooth muscle cell proliferation, migration and synthesis of extracellular matrix proteins. NO can also diffuse toward the lumen of the blood vessel where it helps maintain blood fluidity. NO inhibits platelets' and leucocytes' adhesion to endothelial cells. In addition, NO inhibits platelet aggregation and facilitates the dissolution of small platelet aggregates. However, the regulatory action of NO on blood cells is most likely limited to the luminal surface of endothelial cells since NO is rapidly scavenged by hemoglobin in erythrocytes and inactivated by oxygen-derived radicals such as superoxide anions. NO can also affect the fibrinolytic activity by regulating the release of tissue-type plasminogen activator and plasminogen activator inhibitor-1. The crucial role of vascular NO in the control of blood fluidity has been demonstrated by the regulation of the bleeding time in humans.     

Modulation of Staphylococcus aureus adhesion by biofunctional copolymers derived from polystyrene

Biomaterials-centered infections are serious complications associated with the use of implants. The infection risk of biomaterials varies between different materials and is determined by the chemical composition of materials, the host proteins and the type of bacteria. In this study we measured the initial adhesion of Staphylococcus aureus onto polystyrene derivatives containing carboxylate and sulfonate groups. Five polymers were synthesized and characterized. We studied the role of the host protein fibronectin in promoting adhesion of Staphylococcus aureus. Fibronectin adsorption was comparable on all the tested polymers (pKd=7.2±0.2) whereas bacterial adhesion was dependent on surfaces chemical compositions. Polymers substituted with sulfonate groups showed the most important inhibition of initial bacterial adhesion.     

Phagocytosis of Colloidal Carbon and Heterologous Red Blood Cells in the Bone Marrow of Rats and Rabbits

Phagocytosis in the bone marrow of rats and rabbits was studied following intravenous injections of colloidal carbon and chicken red blood cells. In both animal species the marrow response to these different foreign particles was quite similar. There was an initial aggregation and degranulation of platelets around the injected particulate matter within the marrow sinuses. Then pseudopods of marrow macrophages appeared in the sinus lumen, forming a web-like structure which trapped and phagocytosed the injected foreign material as well as the platelets. Within the phagosomes of these macrophages, the injected material and platelets were withdrawn into the parenchyma, where degradation or storage occurred. This sequence of events suggests that platelets may play an important role in marrow phagocytosis. The most active cells in marrow phagocytosis are the macrophages. The endothelial cells participated in the phagocytosis of colloidal carbon. The amount of carbon within these cells, however, was small in comparison with that trapped by macrophages. Further, the endothelial cells did not phagocytose chicken red blood cells. These results, being similar to those obtained in lymph, spleen and liver, challenge the concept of the reticuloendothelial system. The term of macrophage system is proposed as a replacement.     

Adhesion of sickle red cells to endothelium: Myths and future directions

Sickle RBC are abnormally adherent to vascular endothelial cells. We briefly review the mechanisms that underlie this type of cell/cell adhesion, expose a number of extant myths about RBC adhesion, and discuss some aspects that need consideration via future experimentation. The relationship of this phenomenon of RBC-endothelial adhesion to the cytoadherence of parasitized sickle RBC is not yet clear.     

Phagocytosis of immunoglobulin-coated emulsion droplets

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. Herein we designed monodisperse, IgG-coated emulsion droplets that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. We show that, contrary to solid polymeric beads, droplet uptake is efficient even for low IgG densities, and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake.     

Erythrophagocytosis and recycling of heme iron in normal and pathological conditions; regulation by hepcidin]

Most of the iron required for erythropoiesis is provided by heme iron recycling following degradation of senescent erythrocytes by tissue macrophages. Accumulation of biochemical modifications at the red blood cell membrane during ageing (externalisation of phosphatidyl-serine, peroxydation of membrane-bound lipoproteins, loss of sialic acid residues and formation of senescence neoantigens) constitute a series of signals that will allow the macrophage to identify the red blood cells to be eliminated, through interaction with specific receptors. After this initial recognition step, the red blood cell is internalised by phagocytosis, and phagosome maturation, which can comprise recruitment of the endoplasmic reticulum, will favour degradation of red blood cell constituents. Heme is catabolised by heme oxygenase 1, anchored in the endoplasmic reticulum membrane. A fraction of the released iron will be recycled back to the plasma through ferroportin, a membrane-bound Fe (II) export molecule, and a fraction will retained within the ferritin molecules, to be released at later stages. Multiple evidence coming from human diseases (type 4 hemochromatosis) and animal models indicate that ferroportin is essential for heme iron recycling by macrophages. Furthermore, ferroportin seems to be the molecular target of hepcidin, this circulating peptide synthesized by the liver and acting as a negative regulator of intestinal iron absorption and iron recycling by macrophages. Perturbations in erythrophagocytosis play a physiopathological role in several diseases, including hemochromatosis, anemia of chronic disorders and thalassemia.     

Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and non-lymphoid organs

Macrophage scavenger receptors are trimeric integral membrane glycoproteins which have been implicated in various macrophage functions including uptake of oxidized lipoprotein and the serum-dependent, divalent cation-independent adhesion of macrophages to tissue culture-treated plastic. In this study we have used a recently defined monoclonal antibody (2F8) which recognizes murine macrophage scavenger receptor, to explore its expression in lymphoid and non-lymphoid organs of the normal adult. Scavenger receptor was detected in the red pulp and marginal zone of normal adult mouse spleen, medulla of the thymus and subcapsular region of lymph nodes. Kupffer cells in the liver, alveolar macrophages in the lung and lamina propria macrophages in the gut all reacted with 2F8 monoclonal antibody. The antigen was not detected on any nonmacrophage cells, with the exception of sinusoidal endothelial cells in the liver. In the spleen, lymph node and liver, scavenger receptor antigen expression was associated specifically with phagocytic cells which had taken up colloidal carbon. To examine macrophage adhesion in a context relevant to the interactions occurring within lymphoid and non-lymphoid organs, and the contribution of macrophage scavenger receptor to this adhesion, we designed an assay of macrophage adhesion to frozen tissue sections. Adhesion to most tissues was high and uniform in the absence of any chelating agents. The chelation of Ca²⁺ and Mg²⁺ revealed specific patterns of macrophage adhesion in lymphoid and non-lymphoid organs which was completely inhibited by 2F8. The ability of this antibody to block the EDTA-resistant adhesion correlated with tissue expression of the antigen in some tissues. Unlike adhesion to tissue culture-treated plastic, macrophage scavenger receptor-dependent adhesion of macrophages to frozen tissue sections did not exhibit an absolute requirement for exogenous fetal bovine serum indicating the presence of an endogenous ligand for scavenger receptor within the tissues. We propose that macrophage scavenger receptor is a candidate homing or retention molecule for macrophage localization within ligand-rich tissues.     

Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas' disease, interferes with the host immune response to establish a persistent infection. In this report, we demonstrate that macrophages infected with T. cruzi are unable to effectively present antigens to CD4 T cells. The interference is due to defective antigen-presenting cell (APC) function, as antigen-independent stimulation of the T cell in the presence of infected macrophages is not affected. The defect is distal to antigen processing and is not due to decreased major histocompatibility complex (MHC) class II expression, decreased viability, defective peptide loading in the infected macrophages, nor absence of CD28 co-stimulation. There was a role for gp39:CD40 co-stimulation during antigen presentation to the T cells we studied, but the expression of CD40 on T. cruzi-infected macrophages was not decreased. Antigen-specific adhesion between macrophages and T cells was reduced by infection. Equivalent levels of the adhesion molecules lymphocyte function-associated antigen-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or very late antigen-4 are found on infected and uninfected APC, suggesting that reduced expression of these adhesion molecules was not responsible for the defect in antigen-specific adhesion. The defective T cell:macrophage adhesion may be due to the reduced expression of other adhesion molecules or other changes in the cell induced by infection. Interfering with MHC class II antigen presentation in infected macrophages may help T. cruzi to blunt the immune response by the host.     

Clinical effects of different types of red cell concentrates in patients with thalassemia and sickle cell disease

The treatment of thalassemia is still essentially based on continuous transfusion supporting using red cell concentrates (RCC) prepared in different ways. For patients with sickle-cell disorders, either urgent or chronic red blood cell transfusion therapy, is widely used in the management of sickle cell disease (SCD) because it reduces HbS level and generally prevents recurrent vaso-occlusive disease (VOD). Recently, the introduction of pre-storage filtration to remove leukocytes and the use of techniques for multicomponent donation have increased the types of blood components available for transfusion purposes. The clinical effects of different types of blood components in thalassaemic and sickle-cell patients have not been extensively studied so far. We evaluated the impact of the various different blood components currently available on transfusion needs, transfusion intervals and adverse reactions in order to determine which is the most advantageous for transfusion-dependent thalassaemic and sickle-cell patients followed in our centre. We believe that the optimal characteristics of the RCC are aged less than 10 days from time of collection; Hb content greater than 56 g per unit; Hct: 55-60%; volume (including additive) 300 mL+/-20%; leucodepleted to less than 200,000 leukocytes per unit; low cytokine content (achievable by pre-storage filtration carried out between two and 24 hours after the collection); lack of microaggregates (achievable by pre-storage filtration or filtration in the laboratory) and protein content less than 0.5 g per unit for patients allergic to plasma proteins (achievable with manual or automated washing). It is still recommended that the blood transfused should be as fresh as possible, compatible with the centre's product availability and the centre's organisation should be continuously adapted to this aim. We always transfuse blood within 10 days of its collection, respecting Rh and Kell system phenotypes. Pre-storage filtration is strongly recommended, both in order to prevent adverse reactions through the marked leucodepletion (less than 200,000 leukocytes per unit) and for a better standardisation of the final product, including the certainty that the product does not contain clots, an assurance that bed-side filtration cannot give. The RCC should be produced using a method causing as little as possible stress to the red cell membrane. The use of RCC with a high content of Hb (less than 56 g per unit) is strongly recommended, because our study clearly shows that this reduces the number of exposures to donors and the number of accesses to hospital, thus improving the patient's quality of life.