Showing papers on "Red blood cell published in 1991"

Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin.

Abstract: IL-8 (also known as neutrophil-activating peptide 1) is recognized as a potent effector of neutrophil functions. Several different cell types that contact blood, namely T lymphocytes, monocytes, and endothelial cells, secrete this polypeptide following stimulation by cytokines, or lipopolysaccharide. Here we show that when IL-8 is added to blood it rapidly partitions from the plasma fluid to the blood cells and that erythrocytes account for the vast majority of this binding. Analysis of 125I-IL-8 binding [( ala-IL-8]77 form) to human red cells indicates a single, 5 nM Kd affinity class of binding sites, present at approximately 2,000 per red cell representing approximately 15 nmol of red cell IL-8 binding sites per liter of blood. These sites are protease sensitive. Their binding of IL-8 is rapidly reversible and does not result in receptor internalization, although bound IL-8 is resistant to extraction by pH 3 buffer at 5 degrees C. 125I-IL-8 binding to red cells was not inhibited by epidermal growth factor or interleukin 1, but was inhibited by monocyte chemotactic peptide-1, which is not a neutrophil chemotaxin, but is a member of the same family of polypeptides as IL-8. FACS analysis of IL-8-mediated mobilization of Ca2+ in neutrophils indicates that the IL-8 bound to red cells is incapable of stimulating neutrophils. Thus, red cell absorption of IL-8 may function to limit stimulation of leukocytes by IL-8 released into blood.

Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins.

Abstract: We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.

Modification of host cell membrane lipid composition by the intra-erythrocytic human malaria parasite Plasmodium falciparum

Abstract: The phospholipid and fatty acid compositions of the host infected erythrocyte plasma membrane (IEPM) have been determined for erythrocytes infected with the human malaria parasite Plasmodium falciparum. IEPM were prepared by selective lysis of the host erythrocyte (but not of the parasite membranes) with 0.1% saponin, followed by differential centrifugation. The purity of the IEPM was determined by measuring the membrane-specific enzyme markers acetylcholinesterase, glutamate dehydrogenase and lactate dehydrogenase, and by immunoelectron microscopy using monoclonal antibodies specific for human erythrocyte glycophorin A (4E7) and for a 195 kDa parasite membrane glycoprotein (Pf6 3B10.1). Both approaches demonstrated that the host erythrocyte plasma membrane preparation was free from contamination by parasite membranes. During intra-erythrocytic development of the parasite, the phospholipid composition of the erythrocyte membrane was strikingly altered. IEPM contained more phosphatidylcholine (38.7% versus 31.7%) and phosphatidylinositol (2.1% versus 0.8%) and less sphingomyelin (14.6% versus 28.0%) than normal uninfected erythrocytes. Similar alterations in phospholipid composition were determined for erythrocyte membranes of parasitized cells isolated by an alternative method utilizing polycationic polyacrylamide microbeads (Affigel 731). The total fatty acid compositions of the major phospholipids in IEPM were determined by g.l.c. The percentage of polyunsaturated fatty acids in normal erythrocyte phospholipids (39.4%) was much higher than in phospholipids from purified parasites (23.3%) or IEPM (24.0%). The unsaturation index of phospholipids in IEPM was considerably lower than in uninfected erythrocytes (107.5 versus 161.0) and was very similar to that in purified parasites (107.5 versus 98.5). Large increases in palmitic acid (C16:0) (from 21.88% to 31.21%) and in oleic acid (C18:1) (from 14.64% to 24.60%), and major decreases in arachidonic acid (C20:4) (from 17.36% to 7.85%) and in docosahexaenoic acid (C22:6) (from 4.34% to 1.8%) occurred as a result of infection. The fatty acid profiles of individual phospholipid classes from IEPM resembled in many instances the fatty acid profiles of parasite phospholipids rather than those of uninfected erythrocytes. Analysis of IEPM from P. falciparum-infected erythrocytes (trophozoite stage) revealed that, during intra-erythrocytic maturation of the parasite, the host erythrocyte phospholipid composition was markedly refashioned. These alterations were not dependent on the method used to isolate the IEPM, with similar results obtained using either a saponin-lysis method or binding to Affigel beads. Since mature erythrocytes have negligible lipid synthesis and metabolism, these alterations must occur as a result of parasite-directed metabolism of erythrocyte lipids and/or trafficking of lipids between the parasite and erythrocyte membranes.

Elevated lipid peroxidation levels in red blood cells of streptozotocin-treated diabetic rats.

Abstract: This study was performed to determine whether or not hyperglycemia in diabetes results in elevated levels of lipid peroxidation products in red blood cells (RBC). Diabetes was induced in rats by treatment with streptozotocin. The level of lipid peroxidation products was examined in fresh RBC by measuring their thiobarbituric acid (TBA) reactivity after 2 and 4 months of induction of diabetes. Hyperglycemia was assessed by measuring the level of glycosylated hemoglobin and blood glucose. Results show that lipid peroxidation levels were significantly higher (50% to 84%) in RBC of diabetic rats than in controls. The increase in the level of lipid peroxidation was blocked in diabetic rats in which hyperglycemia was controlled by insulin treatment. Among phospholipid classes, relative percentage of sphingomyelin (SM) was significantly reduced in RBC at both 2 and 4 months of diabetes; whereas phosphatidylethanolamine (PE) levels were higher in RBC at 4 months of diabetes only. The level of phosphatidylcholine (PC) did not differ significantly between RBC of control and diabetic rats. This study suggests a significantly altered lipid composition and an accumulation of lipid peroxidation products in RBC of streptozotocin-treated diabetic rats.

Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells.

Abstract: Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.

Intracellular free zinc and zinc buffering in human red blood cells.

Abstract: Zn2+ has been allowed to equilibrate across the red cell membrane using two agents that increase membrane permeability to this ion: the ionophore A23187 and the specific carrier ethylmaltol. Extracellular free Zn2+ was controlled with EGTA (1,2-di(2-aminoethoxy)ethane-NNN′N′tetra-acetic acid)) buffers, except in the case of ethylmaltol, which itself acts as a buffer. Measurement of cellular zinc content at different levels of free Zn2+ facilitated the study of intracellular Zn2+ binding. It was also possible to estimate intracellular free Zn2+ concentration in untreated cells using a “null-point” technique. Intracellular zinc was found to consist of an inexchangeable component of about 129 μmol/1013 cells and an exchangeable component of 6.7±1.5 μmol/1013 cells, with a free concentration of about 2.4×10−11 m. The main component of Zn2+ buffering is hemoglobin, with a dissociation constant of about 2×10−8 m.

Laboratory hemostatic abnormalities in massively transfused patients given red blood cells and crystalloid.

Abstract: Most of the literature on massive transfusion concerns whole blood replacement, whereas clinically, packed red blood cells are commonly given. To determine when hemostatic abnormalities occur in patients resuscitated primarily with packed red blood cells and crystalloid, the cases of 39 consecutive patients who were transfused with 10 or more red blood cell units of any kind within 24 hours were reviewed. After transfusion with 20 or more units of red blood cell products of any kind (packed red blood cells, cell-saver units, or whole blood), 75% (3 of 4) of patients had platelet counts less than 50 x 10(9)/L, compared to 0 of 29 patients given less than 20 units (P less than 0.001). After transfusion of 12 units of relatively plasma-free red blood cell products (packed red blood cells or cell-saver units), 100% (8 of 8) of patients had prothrombin time prolonged by more than 1.5 times mid-range of normal, compared to 36% (5 of 14) of patients given less than 12 units (P = 0.012). These data confirm that patients massively transfused with red blood cells of any kind develop significant thrombocytopenia after 20 units. Importantly, probably clinically significant prothrombin time and partial thromboplastin time prolongations occurred consistently after transfusion of 12 units of relatively plasma-free red blood cells in unselected patients at an urban trauma hospital. These data suggest that coagulation factor replacement is necessary in patients who receive 12 or more units of packed red blood cells or cell-saver blood, and platelet replacement is necessary in patients who receive 20 or more units of any red blood cell product. A prospective study is needed to determine whether the expected abnormal clinical bleeding indeed occurs in patients with such laboratory coagulation abnormalities and to determine when plasma transfusion is indicated in patients massively transfused with red blood cells.

Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia.

Abstract: In an attempt to determine the mechanism whereby thalassemia in its milder forms may protect against malaria, we have examined the expression of neoantigen at the surface of Plasmodium falciparum-parasitized thalassemic red cells. Neoantigen expression was estimated by measurement of antibody bound after incubation in serum from adults living in a malaria-endemic area, using a quantitative radiometric antiglobulin assay. We found that P. falciparum-parasitized alpha- and beta-thalassemic red cells bind greater levels of antibody from endemic serum than controls: mean binding ratios (+/- SE), respectively, for alpha- and beta-thalassemia compared with controls were 1.69 +/- 0.12 and 1.23 +/- 0.06 on a cell for cell basis, and 1.97 +/- 0.11 and 1.47 +/- 0.08 after a correction for surface area differences. Binding of antibody increased exponentially during parasite maturation. In addition, we found a small but significant degree of binding of naturally occurring antibody to parasitized red cells, the extent of which was also greater in thalassemia. The apparent protective effect of thalassemia against malaria may be related to enhanced immune recognition and hence clearance of parasitized erythrocytes.

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

Abstract: In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

Kinetic abnormalities of the red blood cell sodium-proton exchange in hypertensive patients.

Abstract: The present study was designed to examine the kinetics of Na(+)-H+ exchange in red blood cells of normotensive and hypertensive subjects and its relation to the previously reported abnormalities in Na(+)-Li+ exchange. The Na(+)-H+ antiporter activation kinetics were studied by varying cell pH and measuring net Na+ influx (mmol/l cell x hr = units) driven by an outward H+ gradient. The Na(+)-Li+ exchange was determined at pH 7.4 as sodium-stimulated Li+ efflux. Untreated hypertensive patients (n = 30) had a higher maximal rate of Na(+)-Li+ exchange (0.43 +/- 0.05 versus 0.26 +/- 0.02 units, p less than 0.0003), a higher maximal rate of Na(+)-H+ exchange (62.3 +/- 6.2 versus 47 +/- 4 units; p less than 0.02), but a similar affinity for cell pH compared with normotensive subjects (n = 46). The cell pH activation of the Na(+)-H+ antiporter exhibited a lower Hill coefficient than that of normotensive subjects (1.61 +/- 0.12 versus 2.56 +/- 0.14; p less than 0.0001). This index of occupancy of internal H+ regulatory sites was found reduced in most of the hypertensive patients (73%) whether their hypertension was untreated or treated. Hypertensive patients with Na(+)-Li+ exchange above 0.35 units (0.68 +/- 0.057 units, n = 16) did not exhibit elevated maximal rates of Na(+)-H+ exchange (57.3 +/- 10 units, NS) in comparison with those with Na(+)-Li+ exchange below 0.35 units (66.4 +/- 7.6 units, n = 26), but both groups exhibited reduced Hill coefficients. Hypertensive patients with enhanced Na(+)-H+ exchange activity (more than 90 units) had normal maximal rates of Na(+)-Li+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Aging: Physical and Chemical Membrane Changes

Abstract: The mammalian erythrocyte is an interesting model for studies of membrane aging. Experimental approaches to this problem involve, first of all, comparison of properties of erythrocytes separated by density because red blood cell age correlates with density in principle. Other approaches to study red cell membrane aging, such as hypertransfusion, are also discussed. A number of physical and chemical changes occur in erythrocytes with aging. Crucial to the elucidation of aging mechanisms is to determine which are primary and which are secondary. Immunoglobulin G binding triggers cellular removal and seems necessary for the recognition of senescent erythrocytes. Cellular deformability decreases while fragility to hemolytic factors generally increases and surface charge density does not alter. Perhaps more important are reactions of reactive oxygen species with membrane constituents and subsequent proteolysis. These are implicated as causative factors in red cell aging, immunoglobulin binding and recognition of senescent erythrocytes.

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

Abstract: The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical mw to that isolated from the same cells using murine mAb CR1 antisera Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag

Endothelial cells create a hematopoietic inductive microenvironment preferential to erythropoiesis in the mouse spleen.

Abstract: The spleen is an erythropoietic organ in mouse. To reconstruct a microenvironment essential for erythropoiesis in vitro, the stroma (MSS31) cell line had been established from newborn mouse spleens. MSS31 cells exhibited properties of endothelial cells: (a) the cells showed the activity to uptake acetylated low-density lipoprotein (Ac-LDL) and (b) the cells can form a capillarylike structure by a phenotypic modulation in collagen matrices. MSS31 cells selectively supported the proliferation and differentiation of the erythroid progenitor cells by direct cell-to-cell contact in a semisolid medium in the presence of erythropoietin. These layers also supported erythrocyte maturation and enucleation of erythroblasts. This suggests that spleen endothelial cells are a new type of stromal cell with erythropoietic stimulation activity and may have a critical function in the hemopoietic inductive microenvironment of the mouse spleen.

Influence of prolonged physical exercise on the erythropoietin concentration in blood.

Abstract: Erythropoietin (EPO) and red blood cells were studied in 15 well-trained men before and several times after a marathon run. Changes in red blood cells reflected changes of plasma volume. Immediately after the run, red blood cells were increased due to haemoconcentration, whereas 31 h later the values were decreased due to haemodilution. The EPO concentration was increased 3 h, and more impressive 31 h, after the run. This long-lasting increase in EPO concentration after the marathon run would seem to be responsible for the increased red blood cell mass in long distance runners.

Isolation and partial characterization of antibody- and globin-enriched complexes from membranes of dense human erythrocytes

Abstract: In previous studies we have described a process whereby an erythrocyte in biochemical distress can initiate its own removal by macrophages of the reticuloendothelial system. This process involves the clustering of the integral membrane protein band 3 by denatured haemoglobin and the subsequent recognition of the exofacial poles of clustered band 3 and associated proteins by autologous antibodies. To determine whether this clearance pathway might mediate normal cell turnover, the fraction of normal erythrocytes containing the 0.5% densest cells, which are known to be destined for immediate removal, was isolated and characterized biochemically. This densest fraction was found to contain 6 times more membrane-bound globin (haemichromes) and 10 times more surface-bound autologous IgG than the other fractions containing cells of lower density. To determine whether the autologous IgG was physically associated with the haemichrome-stabilized membrane protein clusters, a procedure was developed for isolation and characterization of the microscopic aggregates. The isolated aggregates were found to contain a disulphide-cross-linked mixture of several membrane proteins, predominantly haemichromes, spectrin and band 3. Although the aggregates constituted only 0.09% of the total membrane protein, they still contained approximately 55% of the total cell-surface IgG. Since in control studies anti-(blood group A) antibodies, which are distributed randomly over the surface of type A cells, could not be recovered in the aggregate, we conclude that the autologous cell-surface IgGs were physically associated with the membrane protein clusters when they were co-isolated with them in our procedure. Thus the 640-fold enrichment of autologous IgG in the aggregates compared with regions of the membrane devoid of tightly clustered protein suggests that sites of integral protein clustering either are non-specifically sticky to IgG or are viewed as foreign or 'non-self' by the immune system and aggressively opsonized with IgG.

Alterations in erythrocyte membrane phospholipid organization due to the intracellular growth of the human malaria parasite, Plasmodium falciparum.

Abstract: The asymmetric distribution of phospholipids in the erythrocyte membrane during the intracellular development of the human malaria parasite Plasmodium falciparum was studied. Infected cells of high parasitaemia were treated with phospholipase A2 or sphingomyelinase C, followed by isolation of the host red cell membrane using the Affigel (731) bead method. Additionally, phosphatidylserine on the surface of infected cells was probed using a phosphatidylserine-sensitive prothrombinase assay. Trophozoite-infected cells showed an increase in phosphatidylethanolamine and phosphatidylserine and a decrease in phosphatidylcholine in the outer leaflet. In addition to the changes already present in trophozoite-infected cells, schizont-infected cells showed a decrease in sphingomyelin as well as a further increase in phosphatidylserine in the outer leaflet. The results are discussed with respect to possible mechanisms and consequences of these changes.

Lovastatin therapy in hypercholesterolemia : effect on fibrinogen, hemorrheologic parameters, platelet activity, and red blood cell morphology

Abstract: The effect of lovastatin therapy on blood rheology was investigated in 26 hypercholesterolemia patients. Treatment with lovastatin was associated with a significant improvement in whole blood filtration time and a tendency toward normalization in red blood cell morphology. A significant increase was observed in fibrinogen level, in ADP-induced platelet aggregation, in the percentage of “big” platelets, and in platelet count. The viscosity of whole blood and plasma and the percentage of aggregated platelets did not change significantly. The cause for these hemorrheologic changes and their role in influencing the coronary risk of lovastatin-treated hypercholesterolemia patients should be further investigated.

Full-length CD4 electroinserted in the erythrocyte membrane as a long-lived inhibitor of infection by human immunodeficiency virus.

Abstract: Recombinant full-length CD4 expressed in Spodoptera frugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was "correctly" oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments (125I-CD4 and 51Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, we showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance of HIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cells. RBC-CD4, but not RBCs, aggregated with chronically HIV-1-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.

Red blood cell mechanics and capillary blood rheology

Abstract: Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessels walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic "tank-treading" motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow.