Erythrocyte aging in sickle cell disease.

Physiological removal of old erythrocytes from the circulation by macrophages is initiated by binding of autologous IgG to senescent cell antigen (SCA). SCA is generated from the anion exchanger band 3. This process is accompanied by a number of alterations in the function and structure of band 3. We measured these aging-related parameters in erythrocytes from individuals with sickle cell anemia. Most sickle erythrocytes have characteristics that are also found in senescent normal erythrocytes, such as an increased density and considerable concentrations of cell-bound IgG. Together with the concomitant changes in structure and function of band 3, these data suggest that most sickle erythrocytes have undergone a process of accelerated aging. Preliminary results indicate that this process is reversed upon vitamin E supplementation. These data show that the erythrocyte aging paradigm may provide a useful conceptual framework for the study of the pathophysiology and the evalution of therapeutic intervention in sickle cell disease, and support the view that oxidation can generate neoantigens that are recognized by autoantibodies.     

Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes. Formation of senescent antigen on erythrocyte surface by an oxidative mechanism

Incubation of human erythrocytes oxidized by iron catalysts, ADP/Fe3+ or xanthine/xanthine oxidase/Fe3+, with autologous IgG resulted in IgG binding as detected by enzyme immunoassay using protein A-beta-galactosidase conjugate. The binding of autologous IgG to ADP/Fe3(+)-treated erythrocytes maximized when the cells were treated with 1.8:0.1 mM ADP/Fe3+, and declined when treated above this concentration, suggesting that autologous IgG binds to moderately but not to excessively oxidized erythrocytes. The antibody involved in the binding was anti-Band 3, the autoantibody known to bind to aged erythrocytes, because isolated anti-Band 3 bound to the oxidized cells, but anti-Band 3-depleted autologous IgG did not. In addition, purified Band 3 inhibited the autologous IgG binding. Anti-alpha-galactosyl IgG, another natural antibody which has been reported to bind to aged erythrocytes, did not bind to the oxidized cells. Oxidation of membrane lipids, SH-groups of membrane proteins, and Hb of these cells was slight, but the cells contained an increased amount of membrane-bound native Hb, indicating that the oxidized cell membrane has an altered property. alpha-Tocopherol prevented the lipid oxidation and the subsequent IgG binding. Reduction of the oxidized erythrocytes with dithiothreitol resulted in a loss of the IgG binding. These results suggest that anti-Band 3 binding sites (Band 3 senescent antigen) are formed on moderately oxidized erythrocytes as a result of oxidation of membrane protein SH-groups which can be mediated by the membrane lipid oxidation and that formation of the anti-Band 3 binding sites on the oxidized cells is an essentially reversible membrane event which is linked to oxidation and restoration of the protein SH-groups.     

Erythrocyte aging: a more than superficial resemblance to apoptosis?

In physiological circumstances, erythrocyte aging leads to binding of autologous IgG followed by recognition and removal through phagocytosis, mainly by Kupffer cells in the liver. This process is triggered by the appearance of a senescent erythrocyte-specific antigen. The functional and structural characteristics of senescent erythrocytes strongly suggest that this antigen originates on band 3, probably by calcium-induced proteolysis. Generation of vesicles enriched in denatured hemoglobin is an integral part of the erythrocyte aging process. These vesicles are also removed by Kupffer cells, with a major role for exposure of phosphatidylserine. Moreover, senescent erythrocyte-specific antigens are present on vesicles. Thus, vesicles and senescent erythrocytes may be recognized and removed through the same signals. These and other, recent data support the theory that erythrocyte aging is a form of apoptosis that is concentrated in the cell membrane, and provide the context for future studies on initiation and regulation of the erythrocyte aging process. Insight into the normal aging mechanism is essential for understanding the fate of erythrocytes in pathological circumstances and the survival of donor erythrocytes after transfusion.     

Comparative proteomics of erythrocyte aging in vivo and in vitro

During aging in vivo and in vitro, erythrocytes display removal signals. Phagocytosis is triggered by binding of autologous IgG to a senescent cell antigen originating on band 3. Erythrocytes generate vesicles as an integral part of the aging process in vivo and in vitro, i.e. during storage. These vesicles display senescent cell antigens as well as phosphatidylserine, that is recognized by scavenger receptors. Recent comparative proteomic analyses of erythrocytes and their vesicles support the hypothesis that aging is accompanied by increased binding of modified hemoglobins to band 3, disruption of the band 3-mediated anchorage of the cytoskeleton to the lipid bilayer, vesicle formation, and antigenic changes in band 3 conformation. Proteomic data also suggest an, until then unknown, involvement of chaperones, stress proteins, and proteasomes. Thus, the presently available comparative proteomic analyses not only confirm previous immunochemical and functional data, but also (1) provide new clues to the mechanisms that maintain erythrocyte homeostasis; (2) open new roads to elucidate the processes that regulate physiological erythrocyte aging and removal, and thereby; (3) provide the foundation for rational interventions to prevent untimely erythrocyte removal, and unwanted interactions between the erythrocyte and the immune system, especially after transfusion.     

Band-3 polymers and aggregates, and hemoglobin precipitates in red cell aging

As part of our systematic ongoing studies of mechanisms of cellular and molecular aging, we developed a "biochemical profile" of senescent human red cells. This "red cell aging" panel allows us to assess functional red cell age independent of chronologic age. The panel used to obtain this profile includes IgG binding, phagocytosis, enzyme activity, anion transport, ankyrin binding, and immunoblotting with antibodies to band 3. We used this panel to compare the biochemical profile of glucose 6-phosphate dehydrogenase-deficient and hemoglobin K?ln cells containing high molecular weight protein polymers or hemoglobin precipitates with that of normal senescent cells. We found no evidence in support of the concept that aggregation of band 3 plays a role in the mechanism for generating senescent cell antigen. Observations such as these support the hypothesis that degradation of band 3, rather than aggregation is a critical event in IgG binding and normal erythrocyte aging.     

Binding of autologous IgG to human red blood cells before and after ATP-depletion. Selective exposure of binding sites (autoantigens) on spectrin-free vesicles

Binding of autologous IgG to fresh, ATP-depleted red blood cells as well as to spectrin-free vesicles was studied by a non-equilibrium binding assay using 125I-iodinated protein A from Staphylococcus aureus. IgG binding was 14-times higher to spectrin-free vesicles than to ATP-maintaining red blood cells and 4-times higher than to ATP-depleted erythrocytes from which these vesicles were released. Protein A binding to vesicles that were released from washed and nutrient-deprived erythrocytes, was dependent on added autologous IgG. However, spectrin-free vesicles that were spontaneously released from erythrocytes conserved in whole blood, bound similar amounts of protein A with or without added autologous IgG (0.45-0.55 ng/micrograms band 3 protein). These findings demonstrate that opsonization of spectrin-free vesicles by autologous IgG occurs not only in the test tube, but also under blood blank conditions. The binding characteristics of IgG to spectrin-free vesicles are indicative of a natural autoantibody rather than an unspecific binding of autologous IgG. The preferential binding of IgG to spectrin-free vesicles implies a selective exposure of corresponding autoantigens in membrane regions that have lost cytoskeletal anchorage and bud off.     

Hypothesis: synthetic aging antigen can be used to manipulate cellular lifespan

Physiologic removal of old and damaged erythrocytes, platelets, and other terminally differentiated cells is initiated by the appearance of an aging antigen that marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. We have developed a synthetic aging antigen peptide that blocks binding of IgG to senescent cells in vitro. We hypothesize that the synthetic antigen can be used to prevent cell destruction in diseases such as autoimmune hemolytic anemias and idiopathic thrombocytopenia purpura, and that the antigen itself can be used to manipulate cellular lifespan in vivo.     

Molecular mapping of human band 3 aging antigenic sites and active amino acids using synthetic peptides

An aging antigen, senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. This antigen is derived from the major anion transport protein, protein band 3, that is involved in respiration and acid base balance. We use synthetic peptides from the transmembrane, anion transport segment of band 3 to walk band 3 to identify potential aging antigenic sites. A competitive inhibition assay with affinity purified IgG autoantibody from senescent red cells was used. Results indicate that: aging antigenic sites reside on human band 3 residues 538–554, 593–601, and 812–830; and that the smallest residues which act as aging antigenic sites are 593–601 and 813–818. The contribution of lysine and/or arginine to antigenicity is examined by synthesizing peptide analogs in which glycines or arginines are substituted for lysines or arginines. Substitution of neutral glycine for the positively charged amino acids arginine or lysine or both arginine and lysine did not result in a significant difference in antigenicity between the analog and the native band 3 peptide. Substitution of the positively charged arginine for the positively charged lysine resulted in a significant reduction in antigenicity. The chicken sequence of band 3 peptides 538–554 and 812–827 differs from that of the human peptides at several sites. Antigenicity of these chicken analogs were tested and compared to the human peptides. The data suggest that the three-dimensional configuration of band 3 segments plays a dominant role in defining the antigenic determinants reactive with senescent cell IgG autoantibodies.     

Mapping of senescent cell antigen on brain anion exchanger protein (AE) isoforms using HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS)

Molecular recognition of senescent cells involves oxidation of a crucial membrane protein leading to generation of a neoantigen, called 'senescent cell antigen' (SCA), and binding of physiologic autoantibodies. These IgG autoantibodies trigger macrophage removal of the cell prior to its lysis at a time when anion transport has decreased but the membrane is still grossly intact. The neoantigen SCA is generated by oxidation of a major anion transport protein called band 3 or anion exchange protein. In this study, we use IgG physiologic autoantibodies from senescent red cells to isolate SCA from brain, and HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS) to compare brain SCA to band 3. HPLC peptide maps of band 3 and SCA showed substantial homology, suggesting that SCA is a subset of band 3, and includes an estimated >/=45% of the band 3 molecule. FAB-MS results indicate that residues matching all three band 3 isoforms (AE1, AE2 and AE3) are detected in SCA fractions. These findings suggest that other isoforms of band 3 may undergo the same aging changes that AE1 on red blood cells undergoes to generate SCA. This provides confirmation that SCA is on non-erythroid cell types. Implications of these studies to the generation of neoantigens by oxidation and their recognition by autoantibodies to them are discussed.     

Band 3 and its alterations in health and disease.

Band 3 proteins, members of the anion exchange family of proteins (AE 0-3), are involved in a number of physiological activities such as cell volume and osmotic homeostasis, HCO3-/Cl- exchange, red cell aging, IgG binding and cellular removal, and the maintenance of the structural integrity of cells. They are present in the membranes of all cells and cellular organelles examined including Golgi, mitochondria and nuclei. The first polymorphisms of band 3 discovered were the asymptomatic band 3 Memphis variants carrying the Lys --> Gly substitution at position 56 in the cytoplasmic tail, and band 3 Texas (high transport band 3 Texas) with a mutation in the critical transmembrane, anion transport domain (Pro --> Leu substitution at position 868). The rate at which band 3 mutations were discovered accelerated in the mid 1990s and there are now over 50 known. The most common polymorphisms of band 3 are the Diego blood group antigens which reside on extracellular loops of the protein. Southeast Asia ovalocytosis (SAO; a nine amino acid deletion of residues 400-408) is a band 3 mutation known only in the heterozygous state in which it does not cause disease. It is thought to confer resistance to malaria by altering red cell deformability. Band 3 mutations are responsible for a subset of the heterogeneous group of disorders known as hereditary spherocytosis (HS). HS is a relatively common congenital or inherited group of anemias characterized by chronic hemolysis and abnormal red cell morphology. Red cells in the subset of HS with band 3 mutations behave like they are band 3 deficient either because the mutant protein is not incorporated into the membrane or because it is not functional. HS can be caused by mutations in any of at least 5 proteins involved in membrane stability. Band 3 mutations are associated with diseases in cells besides erythrocytes. For example, 2 types of distal renal tubular acidosis are the result of band 3 mutations either alone or combined with SAO. Band 3 alterations are implicated in neurological diseases such as familial paroxysmal dyskinesia, idiopathic generalized epilepsies, and neuro- or choreoacanthocytosis although they have not been demonstrated to be causative. Mutations in other genes can cause changes in band 3. An example is sickle cell anemia where the increased oxidation causes accelerated aging of band 3 and increased IgG binding and cellular removal.     

Immunocytochemical determination of membrane-bound IgG using physiologically aged and experimentally aged erythrocytes

The expression of IgG receptor sites at the band 3 protein is important for the recognition and elimination of aged and experimentally altered erythrocytes. Membrane bound IgG was detected in different erythrocyte preparations and microvesicles by means of electron microscopic procedures (protein A-gold-, protein A-gold-silver- and anti-ferritin-sandwich-technique) and light microscopic procedures (immunofluorescence). Physiologically "old", pronase and neuraminidase as well as diamide treated erythrocytes and microvesicles demonstrated significant IgG loading. An increased IgG binding of erythrocytes treated with phenylhydrazine was only evident when higher phenylhydrazine concentrations were used. Both, the alteration of the glycocalyx (conformational changes of the external segment of the glycophorins) and the alteration of the membrane skeleton lead to an unmasking of the IgG receptor site at band 3 proteins (transmembrane effect). The result is an overcritical loading of cells with IgG molecules which initiate the elimination of the erythrocytes by macrophages of the Reticulo-Histiocytic-System.     

Phagocytosis of senescent erythrocytes by autologous monocytes: requirement of membrane-specific autologous IgG for immune elimination of aging red blood cells

The role of membrane-bound IgG present on the membrane of senescent erythrocytes in immune eliminations of aging red cells was investigated. Phagocytosis of populations of red blood cells (RBC) of different ages by autologous monocytes was assessed both by direct phagocytosis and by induction of microsomal heme oxygenase. Removal of IgG from older RBCs inhibited their phagocytosis; in contrast, preincubation of neuraminidase-treated young or in vitro aged RBCs with IgG eluted from old cells led to phagocytosis of RBCs treated by autologous monocytes. It was also found that the Fc portion of membrane-bound IgG is essential for the elimination of senescent cells; less than 15% of old heat-inactivated RBCs coated with F(ab)2 fragment of membrane-bound IgG were phagocytosed. In contrast, more than 50% of old heat-inactivated RBCs coated with heat-eluted IgG were phagocytosed by autologous monocytes. A possible mechanism of elimination of aged cells is discussed.     

Chronic myelogenous leukemia: alterations in red cell membrane band 3 and increased IgG binding

Chronic myelogenous leukemia (CML) is a hematologic malignancy arising from an abnormal hemopoietic stem cell. Our earlier studies have identified defects in spectrin tetramer formation and organization of cytoskeletal proteins (Basu et al., Biochim. Biophys. Acta 1988, 121-126); and decreased ankyrin binding to ankyrin-depleted vesicles in CML patients. These may lead to clustering of band 3 and increased binding of autologous IgG. This has now been explored by studying the binding of 125I-protein A to normal and CML erythrocytes. There is increased binding of 125I-protein A in CML erythrocytes compared to normal erythrocytes. Since binding of autologous IgG is responsible for removal of erythrocytes from the circulation, the above findings suggest that CML erythrocytes are likely to be prematurely removed from the circulation, accounting for anemia.     

Oxidative stress and autologous immunoglobulin G binding to band 3 dimers in newborn erythrocytes

Since birth-induced oxidative stress (OS) results in the removal of erythrocytes from the blood stream, we studied the binding of autologous IgG to erythrocyte band 3 dimers (the 170-kDa band, which marks the erythrocytes for removal) in preterm and term newborns and in adults. The 170-kDa band was present in as much as 74% of preterm, in 21% of term newborns, and in 10% of adults. During erythrocyte ageing "in vitro" (0, 24, and 48 h aerobic incubation), the appearance of the band occurred much faster with erythrocytes from newborns (particularly preterm) than with those from adults. When the blots for the 170-kDa band were quantified by scanning densitometry, it was seen that the 0 time values were significantly higher in preterm compared to term and adult values. After aerobic incubation a progressive increase in the optical density was observed in each group and the densities were higher in preterm than in the other groups. The course of iron release during the various incubations was analogous to that of the 170-kDa band blots, and significant correlations were found at 0 and 48 h. Methemoglobin formation roughly paralleled iron release. Esterified F(2)-isoprostanes (markers of OS) and O(2)(-) production in the nonincubated (0 time) erythrocytes were much higher in newborn (preterm and term) than in adult erythrocytes. Plasma free F(2)-isoprostanes were significantly higher in preterms than in terms and in terms than in adults. Plasma non-protein-bound iron (NPBI) was higher in preterm than in term newborns and not detectable in adults. In conclusion dimers of band 3 with autologous IgG are found under conditions in which OS can be detected in erythrocytes or in plasma: namely in newborns or in aged erythrocytes.     

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.     

Sickling-induced binding of autologous IgG to erythrocytes heterozygous for sickle hemoglobin

This study reports that sickling-induced increased autoantibody binding can be demonstrated in varying degrees for deoxygenated S/beta-thalassemic (2-fold) and hemoglobin-SC (1.2-fold) erythrocytes as compared with oxygenated paired samples. In contrast, HbAS erythrocytes deoxygenated in autologous plasma exhibited less than 2% morphologic sickling and no increased IgG binding as compared with control samples. Sickling in the presence or absence of plasma increased the IgG binding capacity of S/beta-thalassemic erythrocytes, comparable to previous findings for HbSS erythrocytes, while increased IgG binding to HbSC erythrocytes was detected only after deoxygenation in plasma. It is concluded that specific IgG binding to deoxygenated S/beta-thalassemic RBCs results from subtle permanent sickling-induced alterations of the membrane surface, while IgG binding to HbSC erythrocytes sickled in plasma results from transitory membrane changes. These findings suggest that sickling in vivo will produce cumulative autoantibody binding to S/beta-thalassemic erythrocytes, a process which could lead to immune-mediated erythrocyte destruction. In contrast, comparatively small fractions of the autoantibody bound to HbSC erythrocytes in vivo would result from sickling-induced membrane alterations. These studies indicate that sickling-associated autoantibody binding in vivo will not occur for sickle cell trait (HbAS) erythrocytes protected by plasma.     

Erythrocyte aging in neurodegenerative disorders.

In the present paper, we have reviewed the principal studies on red cell membrane abnormalities associated with neurodegenerative disorders. In the literature, two lines of investigation may be recognized: one based on the hypothesis of the presence of an oxidative environment responsible for red cell oxidative damage in Alzheimer's disease (AD), Alzheimer's dementia type (DAT) and Parkinson' disease (PD); the other one based on the identification of structural and/or functional abnormalities in red cell membrane band 3 and/or in red cell membrane lipid composition in "neuroacanthocytosis". In AD, DAT and PD patients, an increased red cell membrane lipid peroxidation suggests an increase red cell oxidative damages and precocious red cell aging. In "neuroacanthocytosis", grouping chorea-acanthocytosis, Mcleod syndrome and abetalipoproteinemia, the red cells are characterized by thorn or spur-like protrusions, known as "acanthocytes". The presence of circulating acanthocytes, characterized by abnormalities in red cell band 3 structure and/or function, is associated with increase levels of anti-band 3 antibodies which are physiologically produced against aged red cells and are known to mediate red cell removal from the peripheral circulation by macrophages. We have reviewed the mechanism(s) of the loss of red cell membrane stability and of the precocious red cell aging in neurodegenerative disorders.     

Antibody formation to autologous erythrocytes following the immunization with rat erythrocytes of normal animals and mice tolerant to the immunizing antigen

(CBA X C57B1/6)F1 mice immunized three times with rat erythrocytes produced antibodies both to this antigen and to autologous erythrocytes. Most of the antibodies to rat erythrocytes belonged to IgM isotype while antibodies to autologous red cells were of IgG isotype. Combined injection of thymectomized (CBA X C57B1/6)F1 mice with a massive dose of rat spleen cells and cyclophosphamide induced in animals stable tolerance to rat cells. Inducibility of antibodies to autologous red cells in tolerant mice injected 3-5 times with rat erythrocytes was drastically reduced. Nonspecific suppression (thymectomy and cyclophosphamide) did not prevent production of autoantibodies.     

Red blood cell aging--membrane skeleton alteration and IgG receptor expression

Investigations were performed on aging of erythrocytes. It has been assumed that structural changes of the membrane result after exposer of the cells to certain environmental influences in vivo or in vitro. Cell aging can be connected with varying combinations of membrane structure disturbances. It is postulated that the messenger which signals membrane structure lesion is involved in a mechanism given by the expression of immunoglobulin G (IgG) receptor sites which bind autologous IgG1 and IgG3. This antibodies are cytophilic for macrophages. The performed studies demonstrated that an intact molecular arrangement of the membrane skeleton is not only a supposition for stabilization of the membrane asymmetry but also for IgG receptor masking to prevent an early elimination of the red blood cells from the organism.     

Autologous immunoglobulin-G binding to erythrocytes subjected to cyclical deoxygenation in vitro

This study demonstrates that low-density metabolically replete HbSS erythrocytes suspended in heat-inactivated autologous plasma and subjected to 15 hr of cyclical deoxygenation (under nitrogen) bind significantly increased quantities of autologous IgG as compared with oxygenated paired samples. IgG binding to the erythrocyte surface was quantified by a nonequilibrium 125-iodinated protein A binding assay and by flow cytometry. Sickle cells deoxygenated 15 hr (37 degrees C) in the presence of 2 mM calcium bound 2.2 +/- 0.2 (mean +/- SD)-fold more IgG (p less than 0.01) than oxygenated paired samples. Sickle erythrocytes deoxygenated in 0.4 mM EDTA bound 1.7 +/- 0.3 (mean +/- SD)-fold more autologous IgG than oxygenated controls (p less than 0.05). Indirect immunofluorescence assays also demonstrated that the relative levels of autologous IgG bound to sickle cells after 15 hr cyclical deoxygenation in the presence or absence of calcium was increased as compared with IgG binding by oxygenated paired samples. After 3 hr of cyclical deoxygenation in the presence of 2 mM calcium sickle erythrocytes exhibited a 40-60% increase in IgG binding, as compared with 10-20% increased IgG binding by paired samples treated in EDTA. These findings demonstrate that repeated morphologic sickling will increase the IgG binding capacity of low-density sickle cells, and suggest that sickling-associated alterations of the cell surface will produce new binding sites recognized by autologous IgG. These studies also show that the sickling-induced increase in IgG binding may be slightly enhanced by the presence of extracellular calcium.