Disturbed Red Blood Cell Structure and Function: An Exploration of the Role of Red Blood Cells in Neurodegeneration.

The structure of red blood cells is affected by many inborn and acquired factors, but in most cases this does not seem to affect their function or survival in physiological conditions. Often, functional deficits become apparent only when they are subjected to biochemical or mechanical stress in vitro, or to pathological conditions in vivo. Our data on the misshapen red blood cells of patients with neuroacanthocytosis illustrate this general mechanism: an abnormal morphology is associated with an increase in the susceptibility of red blood cells to osmotic and mechanical stress, and alters their rheological properties. The underlying mutations may not only affect red cell function, but also render neurons in specific brain areas more susceptible to a concomitant reduction in oxygen supply. Through this mechanism, an increased susceptibility of already compromised red blood cells to physiological stress conditions may constitute an additional risk factor in vulnerable individuals. Also, susceptibility may be induced or enhanced by systemic pathological conditions such as inflammation. An exploration of the literature suggests that disturbed red blood cell function may play a role in the pathophysiology of various neurodegenerative diseases. Therefore, interventions that reduce the susceptibility of red blood cells to physiological and pathological stress may reduce the extent or progress of neurodegeneration.

Red Blood Cell Homeostasis: Mechanisms and Effects of Microvesicle Generation in Health and Disease.

Red blood cells (RBCs) generate microvesicles to remove damaged cell constituents such as oxidized hemoglobin and damaged membrane constituents, and thereby prolong their lifespan. Damage to hemoglobin, in combination with altered phosphorylation of membrane proteins such as band 3, lead to a weakening of the binding between the lipid bilayer and the cytoskeleton, and thereby to membrane budding and microparticle shedding. Microvesicle generation is disturbed in patients with RBC-centered diseases, such as sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, spherocytosis or malaria. A disturbance of the membrane-cytoskeleton interaction is likely to be the main underlying mechanism, as is supported by data obtained from RBCs stored in blood bank conditions. A detailed proteomic, lipidomic and immunogenic comparison of microvesicles derived from different sources is essential in the identification of the processes that trigger vesicle generation. The contribution of RBC-derived microvesicles to inflammation, thrombosis and autoimmune reactions emphasizes the need for a better understanding of the mechanisms and consequences of microvesicle generation.

Acetylcholinesterase provides new insights into red blood cell ageing in vivo and in vitro.

BACKGROUND: During its 120 days sojourn in the circulation, the red blood cell (RBC) remodels its membrane. Acetylcholinesterase (AChE) is a glycosylphosphatidylinositol (GPI)-linked enzyme that may serve as a marker for membrane processes occurring this ageing-associated remodelling process. MATERIALS AND METHODS: Expression and enzymatic activity of AChE were determined on RBCs of various ages, as obtained by separation based on volume and density (ageing in vivo), and on RBCs of various times of storage in blood bank conditions (ageing in vitro), as well as on RBC-derived vesicles. RESULTS: During ageing in vivo, the enzymatic activity of AChE decreases, but not the AChE protein concentration. In contrast, neither AChE activity nor concentration show a consistent, significant decrease during ageing in vitro. CD59, another GPI-linked protein that protects against complement-induced removal, also remains constant during storage. The cellular content of the integral membrane protein glycophorin A, however, decreases with storage time in the more dense RBC fractions. The latter are enriched in echinocytes and other misshapen cells during storage. DISCUSSION: Our findings suggest that, during RBC ageing, GPI-linked proteins and integral membrane proteins are differentially sorted. Also, the vesicles that are generated in vitro show a fast and extensive loss of AChE activity, but not of AChE expression. Thus, AChE characteristics may constitute sensitive biomarkers of RBC ageing in vivo, and a source of information on the structural and functional changes that GPI-linked proteins undergo during ageing in vivo and in vitro. This information may help to understand RBC homeostasis and the effects of transfusion, especially in immunologically compromised patients.

Inflammation-associated changes in lipid composition and the organization of the erythrocyte membrane.

BACKGROUND: Reduced erythrocyte survival and deformability may contribute to the so-called anemia of inflammation observed in septic patients. Erythrocyte structure and function are affected by both the membrane lipid composition and the organization. We therefore aimed to determine whether these parameters are affected during systemic inflammation. METHODS: A sensitive matrix-assisted laser desorption and ionization time-of-flight mass spectrometric method was used to investigate the effect of plasma components of 10 patients with septic shock and of 10 healthy volunteers subjected to experimental endotoxemia on erythrocyte membrane lipid composition. RESULTS: Incubation of erythrocytes from healthy control donors with plasma from patients with septic shock resulted in membrane phosphatidylcholine hydrolysis into lysophosphatidylcholine (LPC). Plasma from volunteers undergoing experimental human endotoxemia did not induce LPC formation. The secretory phospholipase A2 IIA concentration was enhanced up to 200-fold in plasma of septic patients and plasma from endotoxin-treated subjects, but did not correlate with the ability of these plasmas to generate LPC. Erythrocyte phosphatidylserine exposure increased up to two-fold during experimental endotoxemia. CONCLUSIONS: Erythrocyte membrane lipid remodeling as reflected by LPC formation and/or PS exposure occurs during systemic inflammation in a secretory phospholipase A2 IIA-independent manner. GENERAL SIGNIFICANCE: Sepsis-associated inflammation induces a lipid remodeling of the erythrocyte membrane that is likely to affect erythrocyte function and survival, and that is not fully mimicked by experimental endotoxemia.

The involvement of erythrocyte metabolism in organismal homeostasis in health and disease.

Historically, study of erythrocyte homeostasis has focussed on the survival of erythrocytes in the blood bank and, especially in pathological circumstances, on the mechanisms leading to accelerated aging and removal from the circulation. Recent proteomic and metabolomic data suggest that erythrocyte metabolism involves more than ATP production and transport of oxygen and carbondioxide; is subject to regulation; and is likely to reflect organismal metabolism. Also, it has become clear that systemic diseases affect erythrocyte homeostasis. The perspectives emerging from these data include new possibilities to manipulate erythrocyte function and survival in vivo, and thereby organismal homeostasis.

Platelet microparticles inhibit IL-17 production by regulatory T cells through P-selectin.

Self-tolerance and immune homeostasis are orchestrated by FOXP3(+)regulatory T cells (Tregs). Recent data have revealed that upon stimulation, Tregs may exhibit plasticity toward a proinflammatory phenotype, producing interleukin 17 (IL-17) and/or interferon γ (IFN-γ). Such deregulation of Tregs may contribute to the perpetuation of inflammatory processes, including graft-versus-host disease. Thus, it is important to identify immunomodulatory factors influencing Treg stability. Platelet-derived microparticles (PMPs) are involved in hemostasis and vascular health and have recently been shown to be intimately involved in (pathogenic) immune responses. Therefore, we investigated whether PMPs have the ability to affect Treg plasticity. PMPs were cocultured with healthy donor peripheral blood-derived Tregs that were stimulated with anti-CD3/CD28 monoclonal antibodies in the presence of IL-2, IL-15, and IL-1ß. PMPs prevented the differentiation of peripheral blood-derived Tregs into IL-17- and IFN-γ-producing cells, even in the presence of the IL-17-driving proinflammatory cytokine IL-1ß. The mechanism of action by which PMPs prevent Treg plasticity consisted of rapid and selective P-selectin-dependent binding of PMPs to a CCR6(+)HLA-DR(+)memory-like Treg subset and their ability to inhibit Treg proliferation, in part through CXCR3 engagement. The findings that ~8% of Tregs in the circulation of healthy individuals are CD41(+)P-selectin(+)and that distinct binding of patient plasma PMPs to Tregs was observed support in vivo relevance. These findings open the exciting possibility that PMPs actively regulate the immune response at sites of (vascular) inflammation, where they are known to accumulate and interact with leukocytes, consolidating the (vascular) healing process.

The Proteome of the Red Blood Cell: An Auspicious Source of New Insights into Membrane-Centered Regulation of Homeostasis.

During the past decade, the hand-in-hand development of biotechnology and bioinformatics has enabled a view of the function of the red blood cell that surpasses the supply of oxygen and removal of carbon dioxide. Comparative proteomic inventories have yielded new clues to the processes that regulate membrane-cytoskeleton interactions in health and disease, and to the ways by which red blood cells communicate with their environment. In addition, proteomic data have revealed the possibility that many, hitherto unsuspected, metabolic processes are active in the red blood cell cytoplasm. Recent metabolomic studies have confirmed and expanded this notion. Taken together, the presently available data point towards the red blood cell membrane as the hub at which all regulatory processes come together. Thus, alterations in the association of regulatory proteins with the cell membrane may be a sine qua non for the functional relevance of any postulated molecular mechanism. From this perspective, comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red blood cell homeostasis. Additionally, this perspective provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood.

Neuroacanthocytosis: Observations, Theories and Perspectives on the Origin and Significance of Acanthocytes.

The presence of acanthocytes in the blood is characteristic of patients suffering from neuroacanthocytosis (NA). Recent studies have described abnormal phosphorylation of the proteins involved in connecting the membrane and cytoskeleton in patient-derived erythrocytes. The involvement of lipids in the underlying signaling pathways and recent reports on in vitro disease-associated lipid alterations support renewed research into lipid composition, signal transduction, and metabolism in patient erythrocytes. In addition to morphology, changes in membrane organization affect erythrocyte function and survival. Patient erythrocytes may have a decreased ability to deform, and this may contribute to accelerated erythrocyte removal and a decreased oxygen supply, especially in vulnerable brain regions. The presently available data indicate that acanthocytes are likely to originate in the bone marrow, making erythropoiesis an obvious new focus in NA research. Moreover, new, detailed morphological observations indicate that acanthocytes may be the tip of the iceberg with regard to misshapen erythrocytes in the circulation of patients with NA. A systematic assessment of patient erythrocyte morphology, deformability, oxygen delivery, and metabolism will be instrumental in determining the putative contribution of erythrocyte function to NA clinical symptoms.

Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

Alterations in red blood cell deformability during storage: a microfluidic approach.

Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability.

Abnormal red cell features associated with hereditary neurodegenerative disorders: the neuroacanthocytosis syndromes.

PURPOSE OF REVIEW: This review discusses the mechanisms involved in the generation of thorny red blood cells (RBCs), known as acanthocytes, in patients with neuroacanthocytosis, a heterogenous group of neurodegenerative hereditary disorders that include chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS). RECENT FINDINGS: Although molecular defects associated with neuroacanthocytosis have been identified recently, their pathophysiology and the related RBC abnormalities are largely unknown. Studies in ChAc RBCs have shown an altered association between the cytoskeleton and the integral membrane protein compartment in the absence of major changes in RBC membrane composition. In ChAc RBCs, abnormal Lyn kinase activation in a Syk-independent fashion has been reported recently, resulting in increased band 3 tyrosine phosphorylation and perturbation of the stability of the multiprotein band 3-based complexes bridging the membrane to the spectrin-based membrane skeleton. Similarly, in MLS, the absence of XK-protein, which is associated with the spectrin-actin-4.1 junctional complex, is associated with an abnormal membrane protein phosphorylation state, with destabilization of the membrane skeletal network resulting in generation of acanthocytes. SUMMARY: A novel mechanism in generation of acanthocytes involving abnormal Lyn activation, identified in ChAc, expands the acanthocytosis phenomenon toward protein-protein interactions, controlled by phosphorylation-related abnormal signaling.

Phosphatidylserine exposure on stored red blood cells as a parameter for donor-dependent variation in product quality.

BACKGROUND: Exposure of phosphatidylserine on the outside of red blood cells contributes to recognition and removal of old and damaged cells. The fraction of phosphatidylserine-exposing red blood cells varies between donors, and increases in red blood cell concentrates during storage. The susceptibility of red blood cells to stress-induced phosphatidylserine exposure increases with storage. Phosphatidylserine exposure may, therefore, constitute a link between donor variation and the quality of red blood cell concentrates. MATERIALS AND METHODS: In order to examine the relationship between storage parameters and donor characteristics, the percentage of phosphatidylserine-exposing red blood cells was measured in red blood cell concentrates during storage and in fresh red blood cells from blood bank donors. The percentage of phosphatidylserine-exposing red blood cells was compared with red blood cell susceptibility to osmotic stress-induced phosphatidylserine exposure in vitro, with the regular red blood cell concentrate quality parameters, and with the donor characteristics age, body mass index, haemoglobin level, gender and blood group. RESULTS: Phosphatidylserine exposure varies between donors, both on red blood cells freshly isolated from the blood, and on red blood cells in red blood cell concentrates. Phosphatidylserine exposure increases with storage time, and is correlated with stress-induced phosphatidylserine exposure. Increased phosphatidylserine exposure during storage was found to be associated with haemolysis and vesicle concentration in red blood cell concentrates. The percentage of phosphatidylserine-exposing red blood cells showed a positive correlation with the plasma haemoglobin concentration of the donor. DISCUSSION: The fraction of phosphatidylserine-exposing red blood cells is a parameter of red blood cell integrity in red blood cell concentrates and may be an indicator of red blood cell survival after transfusion. Measurement of phosphatidylserine exposure may be useful in the selection of donors and red blood cell concentrates for specific groups of patients.

Gateway to understanding microparticles: standardized isolation and identification of plasma membrane-derived vesicles.

Microparticles (MPs) are small plasma membrane-derived vesicles that can expose molecules originating from their parental cells. As vectors of biological information they are likely to play an active role in both homeostasis and pathogenesis, making them promising biomarkers and nanomedicine tools. Therefore, there is an urgent need for standardization of MP isolation and analysis protocols to propel our understanding of MP biology to the next level. Based on current methodology and recent insights, this review proposes an optimized protocol for the isolation and biochemical characterization of MPs.

Survival of red blood cells after transfusion: processes and consequences.

THE CURRENTLY AVAILABLE DATA SUGGEST THAT EFFORTS TOWARD IMPROVING THE QUALITY OF RED BLOOD CELL (RBC) BLOOD BANK PRODUCTS SHOULD CONCENTRATE ON: (1) preventing the removal of a considerable fraction of the transfused RBCs that takes place within the first hours after transfusion; (2) minimizing the interaction of the transfused RBCs with the patient's immune system. These issues are important in reducing the number and extent of the damaging side effects of transfusions, such as generation of alloantibodies and autoantibodies and iron accumulation, especially in transfusion-dependent patients. Thus, it becomes important for blood bank research not only to assess the classical RBC parameters for quality control during storage, but even more so to identify the parameters that predict RBC survival, function and behavior in the patient after transfusion. These parameters are likely to result from elucidation of the mechanisms that underly physiological RBC aging in vivo, and that lead to the generation of senescent cell antigens and the accumulation of damaged molecules in vesicles. Also, study of RBC pathology-related mechanisms, such as encountered in various hemoglobinopathies and membranopathies, may help to elucidate the mechanisms underlying a storage-associated increase in susceptibility to physiological stress conditions. Recent data indicate that a combination of new approaches in vitro to mimick RBC behavior in vivo, the growing knowledge of the signaling networks that regulate RBC structure and function, and the rapidly expanding set of proteomic and metabolomic data, will be instrumental to identify the storage-associated processes that control RBC survival after transfusion.

Storage-induced changes in erythrocyte membrane proteins promote recognition by autoantibodies.

Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation.

The proteome of erythrocyte-derived microparticles from plasma: new clues for erythrocyte aging and vesiculation.

Vesicle formation is an integral part of the physiological erythrocyte aging process. Recent biophysical and immunochemical data have suggested that vesicles originate by the extrusion of membrane patches that, during aging, have become damaged and simultaneously enriched in removal signals. Thereby, vesiculation may serve to postpone the untimely removal of functional cells. As a first step toward the identification of the underlying mechanisms, we isolated erythrocyte-derived vesicles from plasma by fluorescence-activated cell sorting, analyzed their proteome by mass spectrometry, and compared this with the membrane proteomes of erythrocytes that were separated according to cell age. The presence of band 3 and actin in the vesicles together with the absence of almost all other integral membrane and cytoskeletal proteins, and the specific, aging-associated alterations in band 3 aggregation and degradation shown by proteomics as well as immunochemistry, all suggest that the erythrocyte aging process harbors a specific, band 3-centered mechanism for vesicle generation. The age-related recruitment of plasma proteins, proteins of the ubiquitin-proteasome system, and small G proteins to the erythrocyte membrane supports the hypothesis that modification of band 3 and/or degradation initiate vesiculation, and the subsequent recognition and fast removal of vesicles by the immune system. This article is part of a Special Issue entitled: Integrated omics.

The impact of erythrocyte age on eryptosis.

Mature, circulating erythrocytes undergo senescence, which limits their life span to approximately 120 d. Upon injury, erythrocytes may undergo suicidal erythrocyte death or eryptosis, which may accelerate senescence and shorten their survival. Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine at the cell surface. Triggers of eryptosis include oxidative stress. The present study addresses the impact of erythrocyte age on the relative susceptibility to eryptosis. Erythrocytes were separated into five fractions, based on age-associated differences in density and volume. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, the cell volume from forward scatter, and the Ca(2+) level from Fluo-3-dependent fluorescence. In addition, glutathione (GSH) concentrations were measured by an enzymatic/colourimetric method. After 48 h incubation in Ringer solution, Annexin V binding increased significantly with erythrocyte age. The differences were not accompanied by altered GSH concentrations, but were reversed by addition of the antioxidant N-acetyl-L-cysteine in vitro. Also, N-acetyl-L-cysteine significantly prolonged the half-life of circulating mouse erythrocytes in vivo. Thus, the susceptibility to eryptosis increases with the age of the erythrocytes, and this effect is at least partially due to enhanced sensitivity to oxidative stress.

Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity.

Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)-phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, ß-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of ß-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium.

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for (15)N and (13)C,(15)N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.