A Flow Induced Autoimmune Response and Accelerated Senescence of Red Blood Cells in Cardiovascular Devices.

Red blood cells (RBCs) passing through heart pumps, prosthetic heart valves and other cardiovascular devices undergo early senescence attributed to non-physiologic forces. We hypothesized that mechanical trauma accelerates aging by deformation of membrane proteins to cause binding of naturally occurring IgG. RBCs isolated from blood of healthy volunteers were exposed to high shear stress in a viscometer or microfluidics channel to mimic mechanical trauma and then incubated with autologous plasma. Increased binding of IgG was observed indicating forces caused conformational changes in a membrane protein exposing an epitope(s), probably the senescent cell antigen of band 3. The binding of immunoglobulin suggests it plays a role in the premature sequestration and phagocytosis of RBCs in the spleen. Measurement of IgG holds promise as a marker foreshadowing complications in cardiovascular patients and as a means to improve the design of medical devices in which RBCs are susceptible to sublethal trauma.

Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70.

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

Separación de poblaciones de glóbulos rojos senescentes por gradientes preformados de Percoll / Separation of populations of senescent red blood cells by Percoll performed gradients

Los mecanismos que determinan la senescencia de los eritrocitos han sido extensamente estudiados, sin embargo, no se han logrado conclusiones definitivas debido a la ausencia de una técnica que permita el aislamiento de grupos etáreos bien definidos. Los métodos más comúnmente empleados se basan en el aumento de densidad de los eritrocitos durante el envejecimiento. En este trabajo desarrollamos una técnica para la separación de glóbulos rojos de distintas edades empleando gradientes preformados de Percoll, un polímero sintético con propiedades fisicoquímicas adecuadas para trabajar con células vivas. En las suspensiones eritrocitarias obtenidas se realizaron determinaciones hematológicas, actividades de enzimas antioxidantes y el ensayo de eritrofagocitosis. Los valores de los parámetros hematológicos evaluados fueron significativamente mayores en las suspensiones de glóbulos rojos jóvenes. Las actividades enzimáticas mostraron una disminución de la capacidad antioxidante en las poblaciones de eritrocitos senescentes. Este proceso favorecería la interacción de los hematíes envejecidos con las células fagocíticas, demostrada median­te el ensayo de eritrofagocitosis. Los resultados obtenidos indican que el método de gradientes de Percoll permite una adecuada separación de las suspensiones eritrocitarias de distintas edades, con una eficiencia comparable a la observada en la técnica de centrifugación diferencial considerada de referencia.

The mechanisms that determine the senescence of the erythrocytes have been extensively studied; however. definitive conclusions have not been achieved mainly because of the lack of a technique that allows the isolation of well-defined etarian groups. The methods most commonly used for separating erythrocytes from different ages are based on the increase in density that these cells present during their aging. In the present work we have developed a technique for obtaining red blood cells from different ages using Percoll preformed gradients, a synthetic polymer with adequate physic-chemic properties to work with lives cells. In the erythrocytes suspensions we have made hematological determinations. activities of antioxidants enzymes and the essay of erythrophagocytosis. The values of the hematological parameters were significantly higher in the suspensions of young red blood cells. In the measurements of the enzymatic activity we observed a decrease of the antioxidant capacity in the populations of senescent erythrocytes. This process would promote the interaction between the old erythrocytes and the phagocyte cells, demonstrated by the erythrophagocytosis essay. The results obtained indicate that the method Percoll density gradients allows an appropriate separation of the erythrocytes suspensions of different ages with a comparable efficiency to that observed in the technique differential centrifugation, considered as reference.

A fluorometric quantitative erythrophagocytosis assay using human THP-1 monocytic cells and PKH26-labelled red blood cells.

Removal of senescent, damaged or diseased red blood cells (RBCs) from the circulation in vivo occurs by a process known as erythrophagocytosis. The exact details of the signaling mechanisms that mark RBCs for recognition and influence erythrophagocytosis are still not completely understood. The aim of this study was to develop a quantitative, fluorometric erythrophagocytosis assay for human RBCs and phagocytes to aid elucidation of the biological mechanisms regulating erythrophagocytosis. RBCs were labelled with the lipophilic fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1 at 37 degrees C for 45 min. Non-phagocytosed RBCs were lysed with hypotonic saline. Phagocytosed PKH26-labelled RBCs within THP-1 cells were detected with a fluorescence plate-reader and quantitated using a standard curve of known numbers of PKH26-labelled RBCs. Assay conditions were optimised for the numbers of phagocytes and RBCs, incubation time and fluorescence excitation and emission wavelengths. Erythrophagocytosis was also assessed by flow cytometry to determine the proportion of THP-1 cells with ingested RBCs and showed good correlation (P=0.7) between the two methods. The quantitative, fluorometric plate assay is very sensitive and has good reproducibility, making it a useful tool to investigate the biological mechanisms that regulate erythrophagocytosis of normal and diseased RBCs.

Effect of membrane-bound IgG and desialysation in the interaction of monocytes with senescent erythrocytes.

Determination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDS-PAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked anti-immunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis.

Assessment of survival of aging erythrocyte in circulation and attendant changes in size and CD147 expression by a novel two step biotinylation method.

Three intravenous injections (1mg each) of biotin-X-NHS (BXN) given at 24h intervals labeled all circulating erythrocytes with biotin in C57Bl/6 mice. After 5 days, administration of another i.v. injection of BXN (0.6mg) resulted in the labeling of erythrocytes released in blood circulation after the first biotinylation step, with a lower intensity of biotin. The older erythrocyte population with high intensity of biotin (biotin(high) population) and the later population of newly formed erythrocytes with lower intensity of biotin (biotin(low) population) could be stained with streptavidin-APC (SAv) and identified by flow cytometry. Using the double biotinylation technique, we could examine the survival and age related changes in biotin(low) population of erythrocytes that was released in circulation during a defined time period (5 days). Our results indicate that the percentage of Biotin(low) erythrocytes in circulation remained static for 10 days after the second biotinylation step and than started to decline steadily with time. Mean fluorescence intensity of biotin label on surviving biotin(low) population of erythrocytes however remained stable. These results suggest that after 15 days of release in blood, erythrocytes may undergo random destruction. Furthermore, forward scatter as well as CD147 expression of Biotin(low) population also declined with age. Double biotinylation technique described in this communication offers an easy method for tracking age related changes in populations of erythrocytes released in circulation during a defined period of time.

SHPS-1 promotes the survival of circulating erythrocytes through inhibition of phagocytosis by splenic macrophages.

The lifespan of circulating red blood cells (RBCs) produced in bone marrow is determined by their elimination through phagocytosis by splenic macrophages. The mechanism by which RBC elimination is regulated has remained unclear, however. The surface glycoprotein SHPS-1, a member of the immunoglobulin superfamily, is abundant in macrophages. We have now examined the regulation of RBC turnover with the use of mice that express a mutant form of SHPS-1 lacking most of its cytoplasmic region. The mutant mice manifested mild anemia as well as splenomegaly characterized by expansion of the red pulp. The numbers of erythroid precursor cells in the spleen and of circulating reticulocytes were also increased in the mutant mice. In contrast, the half-life of circulating RBCs was reduced in these animals, and the rate of clearance of injected opsonized RBCs from the peripheral circulation was increased in association with their incorporation into splenic macrophages. Phagocytosis of opsonized RBCs by splenic macrophages from mutant mice in vitro was also increased compared with that observed with wild-type macrophages. These results suggest that SHPS-1 negatively regulates the phagocytosis of RBCs by splenic macrophages, thereby determining both the lifespan of individual RBCs and the number of circulating erythrocytes.

Determinación de IgG en eritrocitos senescentes por un ensayo enzimático con consumo de antiglobulina / Determination of senescent red blood cells bound IgG using an enzyme test based an the consuption of antiglobulin

Durante el envejecimiento de los GR se acumula IgG autóloga sobre la membrana eritrocitaria. Esta IgG esta dirigida a un neo antígeno de senescencia, ubicado en la proteína banda 3. El objetivo de este trabajo fue determinar pequeñas cantidades de IgG en la membrana del GR utilizando un inmunoensayo con antiglobulina conjugada con una enzima. Las suspensiones de GRSe y GRJ fueron incubadas con anti-IgG humana conjugada con fosfatasa alcalina. Se transfirieron alícuotas a microplacas sensibilizadas con IgG humana. Se agregó el sustrato de la enzima y se midió la IgG libre. Las concentraciones de IgG unida a la membrana eritrocitaria en GRSe (13.31 x 10-4(g/(L(1.57 x 10-4) fueron significativamente mayores (p(0.0001) que los valores observados en GR (3.35 x 10-4(g/(L(1.39 x 10-4). Los resultados obtenidos indican que esta metodología constituye una herramienta útil para determinar pequeñas contidades de IgG unida a la membrana eritrocitaria.

Band 3/complement-mediated recognition and removal of normally senescent and pathological human erythrocytes.

Band 3 modifications that normally occur during physiological red blood cell (RBC) senescence in humans, and occasionally in pathological conditions are described in the context of their role in enhancing RBC recognition and phagocytic removal. Band 3 modifications are mostly due to oxidative insults that gradually accumulate during the RBC lifespan or impact massively in a shorter time period in pathological conditions. The oxidative insults that impact on the RBC, the protective mechanisms that counteract those damages and the phenotypic modifications that accumulate during the RBC lifespan are described. It is shown how specific oxidative as well as non-oxidative band 3 modifications enhance RBC membrane affinity for normally circulating anti-band 3 antibodies, and how membrane-bound anti-band 3 antibodies bring about a limited complement activation and membrane deposition of complement C3 fragments. The partially covalent complexes between anti-band 3 antibodies and complement C3 fragments are very powerful opsonins readily recognized by the CR1 complement receptor on the phagocyte. Band 3 modifications typically encountered in old RBCs have crystallized to a number of band 3-centered models of RBC senescence. One of those band 3-centered models, the so-called 'band 3/complement RBC removal model' first put up by Lutz et al. is discussed in more detail. Finally, it is shown how the genetic deficiency of glucose-6-phosphate dehydrogenase (G6PD) plus fava bean consumption, and a widespread RBC parasitic disease, P. falciparum malaria, may lead to massive and rapid destruction of RBCs by a mechanism comparable to a dramatic, time-compressed enhancement of normal RBC senescence.

Selective increase of autoimmune epitope expression on aged erythrocytes in mice: implications in anti-erythrocyte autoimmune responses.

We investigated the impact of changes occurring during red blood cell (RBC) ageing on the RBC-binding activity of pathogenic anti-erythrocyte monoclonal antibodies derived from autoimmune-prone New Zealand black (NZB) mice. As assessed by flow cytometric analysis on in vivo biotinylated RBCs, all five NZB-derived anti-RBC mAb exhibited more efficient binding to aged RBCs than to young RBCs, and resulted in a selective elimination of more aged RBCs from the circulating blood. In addition, treatment of RBCs with proteases markedly enhanced the binding of all five anti-RBC mAb, raising the possibility that increased exposure of autoimmune epitopes on aged RBCs may be in part, a result of contacts with proteolytic enzymes during the lifetime of circulating RBCs. In marked contrast, the binding activity of mAb raised in non-autoimmune animals against antigens expressed on RBCs, such as CD44, CD47, CD147 and TER-119, was either decreased or unchanged with RBC ageing, and these epitopes, except for that recognized by anti-CD47 mAb, were highly sensitive to mild treatment with proteases. Our data unravel the unique molecular feature of RBC epitopes involved in autoimmune haemolytic anaemia, suggesting that membrane alterations in aged RBCs might play a significant role in the development of the autoantibody response to RBCs.

Binding of anti-spectrin antibodies to red blood cells and vesiculation in various in vivo and in vitro ageing conditions in the rat.

In this study, the binding of naturally occurring antibodies as well as of induced anti-spectrin antibodies to red blood cells (RBC), in relation with different ageing conditions, was investigated in the rat. RBC from aged animals, or from rats whose RBC were age-induced either by means of hypertransfusion (which blocks erythropoiesis) or by treatment with clodronate-containing liposomes (which reduces RBC removal from circulation), were used. Attainment of RBC ageing was demonstrated by MCV reduction and by an increase of both RBC density and 4.1a/4.1b RBC membrane protein ratio. The results demonstrate an augmented anti-spectrin antibody binding to RBC in relation with their ageing condition, especially when induced by hypertransfusion. The vesiculation process was also investigated and correlated with antibody binding: vesicles were found only in the plasma of clodronate-treated rats, whose RBC showed the lowest level of anti-spectrin antibody binding with respect to the other groups. In addition, RBC preserved in vitro in different media showed a binding of anti-spectrin antibody, which inversely correlated with the vesiculation process. On the whole, the latter results suggest a protective effect of vesicles towards IgG opsonization of aged RBC.

Senescent erythrocytes: modification of rheologic properties, antigenic expression and interaction with monocytes.

Human erythrocytes have a well-defined lifespan of 120 days. Their eventual removal from circulation is a complex process affected by many cellular parameters, making them susceptible to sequestration in the spleen and other organs. The purpose of this study was to investigate putative changes in rheologic properties, antigenic expression and interaction with monocytes of senescent erythrocytes (SE). SE and young erythrocyte (YE) fractions were obtained by differential centrifugation from 20 healthy donor blood samples. Membrane rheomechanic properties (by diffractometric method), ABO and MN antigens reactivity and erythrophagocytosis by peripheral monocytes were investigated in each fractions. SE showed a little decrease in the deformability index and an increase of both membrane elastic modulus and surface viscosity. The studies performed indicate a decreased expression in the antigens of both blood group systems studied (p < 0.01) and an increased rate of erythrophagocytosis by monocytes in SE compared to YE (p < 0.01). The significant modifications in the biomechanic properties of senescent red blood cell membrane and the loss of antigenic expression could lead to physiological phagocytosis.

Senescent erythrocytes: modification of rheologic properties, antigenic expression and interaction with monocytes

Human erythrocytes have a well-defined lifespan of 120 days. Their eventual removal from circulation is a complex process affected by many cellular parameters, making them susceptible to sequestration in the spleen and other organs. The purpose of this study was to investigate putative changes in rheologic properties, antigenic expression and interaction with monocytes of senescent erythrocytes (SE). SE and young erythrocyte (YE) fractions were obtained by differential centrifugation from 20 healthy donor blood samples. Membrane rheomechanic properties (by difractometric method), ABO and MN antigens reactivity and erythrophagocytosis by peripheral monocytes were investigated in each fractions. SE showed a little decrease in the deformability index and an increase of both membrane elastic modulus and surface viscosity. The studies performed indicate a decreased expression in the antigens of both blood group systems studied (p < 0.01) and an increased rate of erythrophagocytosis by monocytes in SE compared to YE (p < 0.01). The significant modifications in the biomechanic properties of senescent red blood cell membrane and the loss antigenic expression could lead to physiological phagocytosis.

Senescent erythrocytes: modification of rheologic properties, antigenic expression and interaction with monocytes

Human erythrocytes have a well-defined lifespan of 120 days. Their eventual removal from circulation is a complex process affected by many cellular parameters, making them susceptible to sequestration in the spleen and other organs. The purpose of this study was to investigate putative changes in rheologic properties, antigenic expression and interaction with monocytes of senescent erythrocytes (SE). SE and young erythrocyte (YE) fractions were obtained by differential centrifugation from 20 healthy donor blood samples. Membrane rheomechanic properties (by difractometric method), ABO and MN antigens reactivity and erythrophagocytosis by peripheral monocytes were investigated in each fractions. SE showed a little decrease in the deformability index and an increase of both membrane elastic modulus and surface viscosity. The studies performed indicate a decreased expression in the antigens of both blood group systems studied (p < 0.01) and an increased rate of erythrophagocytosis by monocytes in SE compared to YE (p < 0.01). The significant modifications in the biomechanic properties of senescent red blood cell membrane and the loss antigenic expression could lead to physiological phagocytosis. (AU)

Autoimmune hemolytic anemia.

Immune hemolytic anemia can be either isoimmune or autoimmune. Autoimmune hemolytic anemias (AIHA) consist of group of disorders whose common characteristics are the presence of an antibody which in turn causes short red blood cell (RBC) life. The rate and site of hemolysis and hence the clinical manifestations depends on the type of antibody attached and its propensity to fix complement. Antibodies of the IgG class are most commonly responsible for AIHA in children. Rh erythrocyte antigen is involved in more than 70% of cases. Since the antibody has its maximal activity at 37 degrees C, the resultant hemolysis is called warm antibody induced hemolytic anemia. This is a severe life threatening condition, the clinical features are: sudden onset of pallor, jaundice and dark urine. The cornerstone of diagnosis is a positive Coomb's antiglobulin test in the presence of hemolysis. Coomb's test has false negative and false positive rates in about 2-4% and 8% of all cases respectively. The modalities for treatment of warm AIHA include blood transfusion, steroid therapy, intravenous gammaglobulin, plasma-pheresis and splenectomy. The choice depends on the severity of the disease and child's response to therapy.

The functional assessment of autotransplanted splenic tissue by its capacity to remove oxidatively modified erythrocytes.

Free radicals and reactive oxygen species have been implicated in the pathogenesis of a variety of hematologic diseases and erythrocyte aging. Aged erythrocytes are removed from the circulation primarily by the spleen. In this study, we aimed to determine the functional effectiveness of autotransplanted splenic tissue by its capacity to remove oxidatively modified erythrocytes from the circulation. Our experimental model in rats includes splenectomy with autotransplantation of 80% of the excised splenic tissue into the omental pouch. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as carbonyl content and thiobarbituric acid reactive substances (TBARS), together with Heinz body formation. Our results have shown that splenic autotransplantation was effective in removing oxidatively modified, aged erythrocytes from the circulation.

At(a-) phenotype: description of a family and reduced survival of At(a+) red cells in a proposita with anti-Ata.

BACKGROUND: There is a paucity of data on the August (At) blood group antigen and clinical significance of anti-Ata. STUDY DESIGN AND METHODS: A proposita with the At(a-) phenotype was identified by the finding of anti-Ata in the cord blood eluate of her fifth live infant. Family members were studied, and a small aliquot of 51Cr-labeled At(a+) red cells was transfused to determine survival. RESULTS: There was no evidence of hemolytic disease of the newborn, as determined by the normal hemoglobin and bilirubin and normal clinical conditions. Six of seven siblings were tested, and two At(a-) female siblings were identified. In contrast to the proposita, neither sister had detectable anti-Ata in her serum, although each has had only one pregnancy. A monocyte monolayer assay performed on serum from the proposita gave a result of 20-percent hemolysis (normal, <3%), which is consistent with a clinically significant antibody. Transfusion of a small volume of allogeneic red cells that were phenotypically matched with the proposita, except for Ata, resulted in a 1-hour survival of 95 percent, but a 24-hour survival of only 18 percent, of the transfused cells. The survival pattern was exponential, which is characteristic of a non-complement-binding IgG antibody. CONCLUSION: Despite the absence of hemolytic disease of the newborn, this example of anti-Ata would appear to be a clinically significant antibody for the purposes of transfusion practice. Therefore, approaches to the management of clinical situations in which transfusion is required or likely should focus on the availability of autologous cells or frozen allogeneic At(a-) red cells.

Circulatory clearance of transfused antibody-sensitized red cells in an entirely allogenic rabbit model.

BACKGROUND: Antibodies against histocompatibility antigens on phagocytes abrogate their Fc gamma-receptor-mediated functions in vitro. Studies were carried out to determine whether this phenomenon also exists in vivo. METHODS: An allogenic blood group antibody was generated in rabbits. Red cells sensitized with this antibody were internalized in vitro by rabbit phagocytes. Another allogenic antibody specific for major histocompatibility complex (MHC) antigens of rabbits was produced. Plasma containing this antibody was infused into rabbits with phagocytes expressing the corresponding MHC antigens. Thereafter, the sensitized rabbit red blood cells were transfused into the rabbits. RESULTS: The following 3 phases of the circulatory clearance of transfused sensitized red cells could be observed when MHC antibodies were not given prior to the transfusion: 1. initial rapid clearance of the cells (t/2 = 1.7-3.3 min), 2. release of the cells back into the circulation after 0.5-24 h and 3. terminal slow clearance which was, however, faster than that with unsensitized cells. Two independent experiments carried out on the same recipient out of the 3 recipients analysed showed that the prior treatment of the recipient with MHC-alloantibodies extended the circulatory half-life of the sensitized red cells during the terminal phase from t/2 = 1 day to t/2 = 3 and 4 days, respectively. CONCLUSION: Antibodies against MHC antigens on phagocytes can reduce the Fc gamma-receptor-mediated immune elimination of sensitized red cells. This corresponds to the observation that many cases of morbus haemolyticus neonatorum show unexpectedly reduced levels of foetal red cell destruction and a good clinical outcome if mothers not only produce antibodies to Rh (D) but also to foetal MHC antigens.