A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

Background

Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and α_v-integrin. Phagocytosis via the phosphatidylserine-lactadherin-α_v-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes.

Design and Methods

Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity.

Results

Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape (‘rounding up’). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects.

Conclusions

Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.     

Preferential localisation of red cell vacuoles to pathologically shaped human red blood cells

The percentage of pitted erythrocytes and Howell-Jolly bodies in peripheral blood samples of 51 individuals following posttraumatic splenectomy and 20 patients splenectomized because of various haematological diseases differed significantly from each other (p less than 0.001) and from that of healthy controls (p less than 0.001). The percentage of pitted erythrocytes was significantly higher in pathologically shaped red blood cells (RBCs) (acanthocytes, schizocytes, elliptocytes) than in normal discoid shaped RBCs (p less than 0.001). As the number of pits per RBC showed great individual variations, a scoring system for the evaluation of pitted RBCs is proposed.     

Erythrophagocytosis in vivo in sickle cell anemia

Recent observations that the sickle RBC are excessively susceptible to phagocytosis by macrophages in vitro prompted me to look for evidence of in vivo erythrophagocytosis (Ep) in patients with sickle cell anemia (SS). Freshly prepared smears of unmanipulated blood of 27 patients with SS in steady state were examined for Ep by a 500-cell differential white blood cell (WBC) count performed in duplicate. Ten of 27 (37%) SS patients showed Ep (1-6/1,000 WBC or 1-10/100 monocytes). By contrast, no Ep was found in similarly prepared blood smears of 25 normal adult controls and nine splenectomized subjects. The mean hemotocrit value of the Ep(+) SS patients was significantly lower than that of the Ep(-) patients (21.0 +/- 1.7% vs 24.0 +/- 2.7% p less than 0.01). Considering the rarity of spontaneous Ep in unmanipulated blood from normal subjects and the relative insensitivity of the method used, the finding of Ep in over one third of SS patients indicates a significant membrane injury of the sickle RBC and serves to validate the in vitro observations. The possible role of the "senescence" mechanism in the induction of Ep is discussed.     

Ubiquitination of red blood cell alpha-spectrin does not affect heterodimer formation

Erythrocyte alpha-spectrin is ubiquitinated in repeats alpha20/alpha21, which also represents the nucleation site for contact with the beta subunit which leads to heterodimer formation by a zippering mechanism. In this study we have determined the second-order rate constant for association of ubiquitinated alpha'-spectrin, nonubiquitinated alpha-spectrin, and beta-spectrin into the alpha'beta or alphabeta heterodimer. The rate constant for incorporation of monomers into heterodimers at 37 degrees C were (5.181 +/- 0.001) x 10(5) M(-1) sec(-1) for total alpha-spectrin (alpha + alpha'), (5.121 +/- 0.001) x 10(5) M(-1) sec(-1) for alpha'-spectrin, and (5.178 +/- 0.003) x 10(5) M(-1) sec(-1) for beta-spectrin. We conclude that ubiquitination of alpha-spectrin does not regulate heterodimer formation.     

In vivo studies of sickle red blood cells

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.     

Some characteristics of human red blood cells separated according to their size: a comparison with density-fractionated red blood cells

During their in vivo ageing, red blood cells (RBC) increase in density and become smaller. Age-defined RBC subpopulations are usually collected by centrifugation. A fractionation according to RBC volume has been proposed as an improved alternative to such age separation. Because a few data reported in the literature indicate some discrepancies between the two methods, blood samples were separated either by centrifugation or by counterflow centrifugation, and some characteristics of the RBC thus fractionated were studied. The enzyme activities decrease either when the density rises or when the volume (MCV) decreases. However, the comparison of other RBC characteristics strongly suggests that these two procedures do not lead to the collection of the same RBC subpopulations: for instance, the hemoglobin content increases when the MCV rises, whereas it remains constant whatever the RBC density is. With radiolabelled cells, it is shown 1) that the most dense RBC are recovered in all the size-separated RBC subpopulations, even though they tend to concentrate in the fractions with the largest MCV, and 2) that the smallest RBC are almost fairly distributed in all the RBC subpopulations, whatever their density, whereas the largest RBC are mainly, but not exclusively, present in the high-density fractions. Thus, fractionation according to size does not match separation according to density. Taken together with results from in vivo experiments carried out in mice and with the fact that reticulocytes are present in all the size-separated fractions, these data suggest that counterflow centrifugation may be a very questionable procedure to achieve a RBC fractionation according to age and therefore that RBC volume might not be a reliable criterion of RBC age.     

显微操作法在获取孕妇外周血中胎儿有核红细胞的应用

目的：探讨显微操作法对获取孕妇外周血中胎儿有核红细胞（MRBCs）的可行性和准确性。方法：将妊娠6-38周41例孕妇的外周血进行不连续密度梯度离心，将分离后的细胞进行制片，在光学显微镜下识别MRBCs，然后用微操作仪获取NRBCs，并用聚合酶链反应（PCR）在基因水平进行验证。结果：41例孕妇有22例在外周血中检出NRBCs，检出率为53.7％，PCR结果显示22例中有10例呈现Y特异带，总符合率为95.4％。结论：显微操作法是获取孕妇外周血中NRBCs准确可靠的方法。     

Sickle cell disease: relation between procoagulant activity of red blood cells from different phenotypes and in vivo blood coagulation activation

In the present study we examined if, among other mechanisms, the abnormal exposure of phosphatidylserine at the surface of sickle red blood cells (RBCs) contributes to the hypercoagulability which characterizes homozygous sickle cell disease (SCD). The question was addressed by comparison of the procoagulant properties of RBCs from subjects with various phenotypes (SS, SC and AS) that differ in clinical presentation. As previously reported, SS-RBCs accelerated the prothrombin activation by factor Xa, by decreasing the K _m of the reaction compared to normal RBCs. SC-RBCs and AS-RBCs also promoted prothrombin activation although their procoagulant properties were milder compared to SS-RBCs. A significant increase of the thrombin–antithrombin complexes was observed in SS subjects. Prothrombin fragment 1+2 (F1+2) was elevated in half of the SS subjects, but the difference with controls did not reach significance. Elevated levels of thrombin–antithrombin complexes were observed in a number of SC (4/11) and AS (3/12) subjects, but the difference with controls was not significant. A significant correlation was observed between the plasma levels of thrombin–antithrombin complexes in the subjects with SS, AS and AA phenotypes, and the procoagulant properties of RBCs. Our results strongly suggest that the procoagulant properties which characterize SS-RBCs also affect SC-RBCs and AS-RBCs, and that exposure of phosphatidylserine by RBCs contributes to the hypercoagulable state observed in SCD.     

Aggregation of red blood cells in patients with Gaucher disease

Gaucher disease is associated with increased red blood cell (RBC) aggregation, but the pathophysiological significance of this phenomenon and its correlation with disease manifestations are unclear. RBC aggregation was evaluated in 43 patients with Gaucher disease and 53 healthy controls. Dynamic RBC aggregation was examined in a narrow-gap flow chamber at varying shear stress. Compared with the controls, RBC aggregation in Gaucher disease was increased by 25%. Comparison of RBC aggregation in autologous plasma and in dextran (500 kDa) showed an increase both in plasma-dependent (extrinsic) and -independent (intrinsic) RBC aggregation. Subgroup analysis revealed that increased RBC aggregation was limited to patients with an intact spleen. RBC aggregation in patients did not correlate with plasma fibrinogen concentration, disease severity, enzyme replacement therapy or genotype. We conclude that RBC aggregation is increased in patients with Gaucher disease and an intact spleen, possibly reflecting the accumulation of glucocerebroside and other substances in the plasma and RBC membranes of these patients. Our results do not support a role for RBC aggregation in the pathogenesis of vascular complications of Gaucher disease.     

Quantification of loss of haemoglobin components from the circulating red blood cell in vivo

Abstract: Previous studies have shown that a considerable amount of haemoglobin is lost from the intact red cell during its lifespan. The aim of this study was to determine the relative contribution of all the haemoglobin components to this process. Therefore, the relative amount of haemoglobins A0, A2, F and the glycated haemoglobins were determined in 24 fractions of different cell age. These fractions were obtained by the combination of counterflow and density centrifugation. When the absolute amount of all haemoglobin components were calculated using the MCH-values of each fraction, it appeared that the mean red cell loss of haemoglobins A0, A2, F, an unknown X and “rest” comprised, respectively, 440, 23, 1, 4 and 1 amol per cell, while the mean gain of the glycated haemoglobins was 84 amol per cell. This resulted in a net loss of 385 amol of haemoglobin per cell. One of the glycated haemoglobins (HbA1e2) turned out to be the product of further carbamylation. It was concluded that in the first half of the red cell lifespan HbA0 and HbA2 decreased by glycation and carbamylation and that in the second half some of the HbA0 and HbA2 but also some of the glycated and carbamylated haemoglobin components leave the red cell. The total loss amounted to about 20%.     

血吸虫病小鼠红细胞天然免疫的变化

昆明小鼠感染日本血吸虫尾蚴后第7周,红细胞C3b受体花环率显著下降,免疫复合物花环率升高,表明感染血吸虫后小鼠的红细胞免疫功能低下。     

Imaging red blood cells with the atomic force microscope

Summary. A novel technique for the reproduction of ultramorphological images and details of the surface of normal and pathological red blood cells (RBC) was investigated. The atomic force microscope (AFM) provided high-resolution images of cell surfaces. The RBC dimensions obtained by this technique revealed differences between native red cells in smears and glutaraldehyde-fixed red cells. It was shown that fixed red blood cells were best suited for the ultramorphological imaging of the cell surface.     

Increased susceptibility of microcytic red blood cells to in vitro oxidative stress

Abstract: Oxidative damage to erythrocytes in thalassaemia has been related to generation of free radicals by an excess of denaturated α- or β-globin chains, intracellular iron overload and low concentration of normal haemoglobin (HGB). Two good indicators of such oxidative damage are the high red blood cell (RBC) malonyldialdehyde (MDA) production detected following exogenous oxidant stress and the decrease of pyrimidine 5′-nucleotidase (P5N), the most sensitive enzyme to SH-group damage in vivo. Conflicting data, however, have so far accumulated in the literature concerning differences in oxidative damage between the different forms of thalassaemia and iron deficiency anaemia (IDA). In the present study, oxidative susceptibility, as defined by the production of MDA in vitro and antioxidant capacity, as measured by the activity of RBC glutathione peroxidase (GPx), superoxide dismutase (SOD) and by reduced glutathione (GSH), have been studied in microcytic RBCs from patients with β-thalassaemia trait, Spanish (δβ)°-thalassaemia heterozygotes (δβ-thalassaemia trait) and iron deficiency anaemia (IDA). The results are consistent with the existence of significant differences in the severity and pattern of oxidative stress susceptibility between β-thalassaemia trait (increased MDA production and higher SOD and GPx activities) and the other two forms of microcytosis (δβ thalassaemia trait and IDA). Furthermore, the finding of normal P5′N activity in δβ thalassaemia trait, gives further support to the less intense peroxidative environment of RBCs in this form of thalassaemia when compared to β-thalassaemia trait, characterized by acquired RBC P5′N deficiency due to oxidative damage.     

海藻糖联合葡萄糖负载红细胞后溶血效果

目的：探讨海藻糖和葡萄糖联合负载红细胞的效果,为冷冻干燥红细胞提供新的保存剂。方法：分别采用0、0.125、0.25、0.5和1 mol/L的海藻糖、葡萄糖以及海藻糖联合葡萄糖37℃负载红细胞6 h。然后采用邻甲苯胺法检测上清液中游离血红蛋白含量。结果：负载液浓度为1 mol/L时,3组的细胞溶血程度较重,即上清中游离血红蛋白浓度较高。在浓度低于1 mol/L时,海藻糖联合葡萄糖组与单独海藻糖负载组溶血程度没有明显差别,但是2者低于葡萄糖组。结论：在浓度小于1 mol/L时,海藻糖联合葡萄糖负载红细胞后对细胞的损伤较小,能够满足冻存的负载要求。     

The Rh complex exports ammonium from human red blood cells

The Rh blood group system represents a major immunodominant protein complex on red blood cells (RBC). Recently, the Rh homologues RhAG and RhCG were shown to promote ammonium ion transport in yeast. In this study, we showed that also in RBC the human Rh complex functions as an exporter of ammonium ions. We measured ammonium import during the incubation of RBC in a solution containing a radiolabelled analogue of NH4Cl (14C-methyl-NH3Cl). Rhnull cells of the regulator type (expressing no Rh complex proteins) accumulated significantly higher levels (P = 0.05) of radiolabelled methyl-ammonium ions than normal RBC, at room temperature. Rhnull cells of the amorph type (expressing limited amounts of Rh complex proteins) accumulated an intermediate amount of methyl-ammonium ions. To show that decreased ammonium export contributes to its accumulation, the release of intracellular methyl-ammonium from the cells was measured over time. In 30 s, normal RBC released 87% of the intracellular methyl-ammonium ions, whereas Rhnull cells of the regulator type released only 46%. We conclude that the Rh complex is involved in the export of ammonium from RBC.     

手工分少白细胞红细胞和悬浮红细胞输注疗效观察

目的:了解并比较手工分少白细胞红细胞和悬浮红细胞的治疗效果。方法:将2 582例患者分为输注悬浮红细胞组(1 450例)和输注手工分少白细胞红细胞组(1 132例),输注前测定Hb值,复查患者及献血者ABO和RH(D)血型并作凝聚胺法交叉配血试验,输血后给每例患者进行血液学分析,检查Hb值,然后再进行输血不良反应发生率统计。结果:输注手工分少白细胞红细胞效果好于普通悬浮红细胞,并且输注手工分少白细胞红细胞的输血不良反应发生率明显减少。结论:输注手工分少白细胞红细胞可以使输血后非溶血性输血反应发生率降低。     

Extended storage of whole blood before the preparation of blood components: in vitro effects on red blood cells in Erythro-Sol environment

Background and Objectives  Routine procedures for extended storage of whole blood (WB) before the preparation of blood components are of interest primarily for logistical reasons. We stored red cell units in either Erythro-Sol 2 (E-Sol 2, test units, 150 ml added) or in saline-adenine-glucose–mannitol (SAG-M) (reference units, 100 ml added) that were prepared after storage of WB at room temperature for 8, 12, 16 or 19 h after blood collection.
Study Design and Methods  Red blood cells were stored for 42 days. We measured pH, glucose, lactate, haemolysis, red blood cell adenosine triphosphate and 2,3-diphosphoglycerate on days 1, 7, 14, 21, 28, 35 and 42.
Results  Haematocrits were significantly lower in E-Sol 2 than in SAG-M due to the higher volume of E-Sol 2 added compared to SAG-M. Significantly reduced levels were found in E-Sol 2 of extracellular pH (throughout storage after 8-h hold and initially after 12-, 16- or 19-h hold), of lactate (initially after 8-h hold and throughout storage after 12-, 16- or 19-h hold), and of haemolysis from day 35 in the 8-h and on day 42 in the 12-h hold group. Sigificantly increased levels of adenosine triphosphate were seen in E-Sol 2 after 8-h hold (from day 14) and after 12-h hold (at days 21, 35 and 42) compared to SAG-M. Significantly higher concentrations of 2,3-diphosphoglycerate were noticed primarily after 8-h hold of WB.
Conclusion  The use of E-Sol 2 as a replacement for SAG-M does not significantly improve in vitro data after extended storage of WB at room temperature before preparation of blood components. However, after 8-h hold in vitro characteristics similar to or better than in fresh blood will be maintained for several weeks in E-Sol 2, a situation that makes E-Sol 2 superior to SAG-M when storage of WB is limited to 8 h. Some improvement was noted after 12-h hold as well.