Mechanisms tagging senescent red blood cells for clearance in healthy humans.

This review focuses on the analysis and evaluation of the diverse senescence markers suggested to prime red blood cells (RBC) for clearance in humans. These tags develop in the course of biochemical and structural alterations accompanying RBC aging, as the decrease of activities of multiple enzymes, the gradual accumulation of oxidative damage, the loss of membrane in form of microvesicles, the redistribution of ions and alterations in cell volume, density, and deformability. The actual tags represent the penultimate galactosyl residues, revealed by desialylation of glycophorins, or the aggregates of the anion exchanger (band 3 protein) to which anti-galactose antibodies bind in the first and anti-band 3 naturally occurring antibodies (NAbs) in the second case. While anti-band 3 NAbs bind to the carbohydrate-free portion of band 3 aggregates in healthy humans, induced anti-lactoferrin antibodies bind to the carbohydrate-containing portion of band 3 and along with anti-band 3 NAbs may accelerated clearance of senescent RBC in patients with anti-neutrophil cytoplasmic antibodies (ANCA). Exoplasmically accessible phosphatidylserine (PS) and the alterations in the interplay between CD47 on RBC and its receptor on macrophages, signal regulatory protein alpha (SIRPalpha protein), were also reported to induce erythrocyte clearance. We discuss the relevance of each mechanism and analyze the strength of the data.

Naturally occurring autoantibodies in mediating clearance of senescent red blood cells.

Germline-encoded naturally occurring autoantibodies (NAbs) developed about 400 to 450 million years ago to provide specificity for clearance ofbody waste in animals with 3 germ layers. Such NAbs became a necessity to selectively clear aged red blood cells (RBC) surviving 60 to 120 d in higher vertebrates. IgG NAbs to senescent RBC are directed to the most abundant integral membrane protein, the anion-transport protein or band 3 protein, but only bind firmly upon its oligomerization, which facilitates bivalent binding. The main constituent of RBC, the oxygen-carrying hemoglobin, is susceptible to oxidative damage. Oxidized hemoglobin forms hemichromes (a form of aggregates) that bind to the cytoplasmic portion of band 3 protein, induces their clustering on the cytoplasmic, as well as the exoplasmic side and thereby provides the prerequisites for the low affinity IgG anti-band 3 NAbs to bind bivalently. Bound anti-band 3 NAbs overcome their low numbers per RBC by stimulating complement amplification. An affinity for C3 outside the antigen binding region is responsible for a preferential formation of C3b(2)-IgG complexes from anti-band 3 NAbs. These complexes first bind oligomeric properdin, which enhances their affinity for factor B in assembling an alternative C3 convertase.

How immune complexes from certain IgG NAbs and any F(ab')2 can mediate excessive complement activation.

In sepsis death follows an excessive inflammatory response involving cytokines and complement that is activated primarily via the amplifying C3/C5 convertase. Excessive stimulation of complement amplification requires IgG-containing or F(ab')2-containing immune complexes (IC) that capture dimeric C3b on one of their heavy chains or heavy chain fragments. The ability of IgG-IC to capture dimeric C3b by the Fab portion is dependent on an affinity for C3 within the Fab portion, but outside the antigen-binding region. This property is rare among IgG NAbs. In contrast to this, the lack of the Fc portion renders the Fab regions of any F(ab')(2)-IC accessible to nascent C3b, but dimeric C3b deposits only if F(ab')2-IC form secondary IC with anti-hinge NAbs that rigidify the complex and thereby promote deposition of dimeric C3b. Both types of complexes, C3b2-IgG-IC and C3b2-F(ab')2-IC/anti-hinge NAbs, are potent precursors of alternative C3 convertases and stimulate complement amplification along with properdin up to 750 times more effectively than C3b and properdin. F(ab')2 fragments are not normally generated, but are formed from NAbs by enzymes from pathogens and neutrophils in sepsis. Unlike IgG-IC F(ab')2-IC are not cleared by Fc-receptor dependent processes and circulate long enough to form secondary IC with anti-hinge NAbs that rigidify the complexes such that they capture dimeric C3b and gain the potency to stimulate complement amplification.

Naturally occurring antibodies/autoantibodies in polyclonal immunoglobulin concentrates.

It was a long way from the use of hyperimmune animal sera for the treatment of toxin-producing infections to the production of polyclonal, polyspecific human immunoglobulin preparations and the use of NAbs as therapeutic tools for autoimmune and inflammatory diseases. Some highlights of the development of knowledge in blood fractionation techniques, basic science and clinical wisdom are reviewed in this chapter. Proudly we mention the outstanding contribution of Swiss scientists and clinicians in the development of IVIG as clinical tool for some otherwise untreatable diseases or taking advantage of its low adverse event profile in long-term treatment of other chronic autoimmune and inflammatory diseases. This chapter summarizes some of the characteristics and the effects in humans of NAbs which are present in IgG concentrates. We call attention to the fact that the human data remain, at least in part, incomplete, among others because even with the most efficient large-scale techniques available not more than approximately 50% of the total IgG in plasma can be fractionated into an immunoglobulin G concentrate.

Asymptomatic elevation of the hyperchromic red blood cell subpopulation is associated with decreased red cell deformability.

Hyperchromasia of the red blood cells (RBC), defined as an elevation of the hyperchromic subpopulation, has been described for various medical conditions. However, neither the association of hyperchromasia with an altered RBC membrane nor with other medical conditions has been investigated in a systematic way so far. Since the percentage of hyperchromic RBC is measured on a routine basis by many hematologic laboratories, we evaluated the predictive value of this parameter for the detection of RBC disorders. An extensive workup of all patients undergoing standard hematogram during a period of 6 months at our institution with a fraction of hyperchromic RBC larger than 10 % was collected by reviewing the medical history and performing osmotic gradient ektacytometry on RBC from a part of these patients. Thirty-two thousand two hundred twenty-six individuals were screened; of which, 162 (0.5 %) showed more than 10 % hyperchromic RBC. All of the patients examined by ektacytometry featured abnormal membrane deformability. Hereditary spherocytosis was found in 19 out of these 32 patients, in most cases unknown to the patient and currently asymptomatic. Another 17.9 % of the patients with an elevated subpopulation of hyperchromic RBC suffered from viral infection (human immunodeficiency virus, hepatitis). Our study shows that an elevated proportion of hyperchromic erythrocytes larger than 10 % is associated with both hereditary and acquired RBC membrane disorders and further follow-up should be considered.

A 4-h hyperglycaemic excursion induces rapid and slow changes in major electrolytes in blood in healthy human subjects.

Hyperglycaemia is well known to cause reductions in plasma Na(+) levels or even hyponatraemia due to an osmotically induced dilution of the interstitium and blood. It is, however, unclear whether this dilution is significantly counteracted by ion regulatory homeostatic mechanism(s) or not. Furthermore, the effects of moderate hyperglycaemia on other major ions are less well known. To further clarify these questions, we measured the changes in blood osmolarity and concentrations of Na(+), K(+), Cl(-), Mg(2+) and Ca(2+) during a 4-h-long experimental hyperglycaemia in healthy subjects rendered temporarily insulin deficient using the hyperglycaemic clamp. Hyperglycaemia, 16.8 mM, was rapidly imposed from a baseline of 4.4 mM by intravenous somatostatin and glucose infusions in 19 healthy subjects (10 m, 9 f; age 36 ± 5 years (mean ± SD); BMI 22.7 ± 2.9 kg/m(2)). Subsequently, glycaemia was returned to basal and measurements continued until all dynamic changes had stopped (at ~8 h). Osmolarity increased from 281.8 ± 0.7 to 287.9 ± 0.7, while Na(+) decreased from 143.9 ± 0.3 to 138.7 ± 0.2, Cl(-) from 101.7 ± 0.2 to 99.5 ± 0.1, Ca(2+) from 1.98 ± 0.04 to 1.89 ± 0.02 and Mg(2+) from 0.84 ± 0.01 to 0.80 ± 0.00 mM. All these changes were rapidly reaching stable levels. K(+) increased from 4.02 ± 0.02 to 4.59 ± 0.02 mM (P < 0.0001) also reaching stable levels but with some delay. Na(+), Cl(-), Mg(2+) and Ca(2+) are essentially determined by blood dilution, and their values will remain diminished as long as the hyperglycaemia lasts. Partial suppression of insulin-stimulated Na(+)/K(+) pumping lead to increased K(+) levels. The combination of elevated K(+) and decreased Mg(2+) and Ca(2+) levels may lead to an altered excitability, which is particularly relevant for diabetic patients with heart disease.

Naturally occurring anti-band 3 antibodies in clearance of senescent and oxidatively stressed human red blood cells.

SUMMARY: Naturally occurring anti-band 3 antibodies (anti-band 3 NAbs) are directed against the 55-kDa chymotryptic fragment of the anion transport protein (band 3) of red blood cells (RBCs). They bind to senescent and oxidatively stressed RBCs and induce their selective clearance. These IgG NAbs exist at low concentrations, and have a weak affinity that prevents them from actively recruiting second binding sites. Cellular senescence or oxidative damage induces a cascade of biochemical events that results in the detachment of band 3 from the cytoskeleton and in clustering of band 3 protein by bound hemichromes and Syk kinase. Clustered band 3 proteins allow bivalent binding of anti-band 3 NAbs. Bivalently bound anti-band 3 NAbs have the unique capacity to stimulate C3b deposition by preferentially generating C3b2-IgG complexes, which act as potent C3 convertase precursors of the alternative complement pathway. Antibody binding not only to clustered, but also to oligomerized band 3 protein further increases if the human plasma also contains induced anti-lactoferrin antibodies. These bind to the polylactosaminyl oligosaccharide, a carbohydrate that exists in lactoferrin and in the 38-kDa fragment of band 3 protein. Anti-lactoferrin antibodies are found primarily in plasma of patients with autoimmune diseases and who have anti-neutrophil cytoplasmic antibodies (ANCA).

Red blood cell (RBC) membrane proteomics--Part II: Comparative proteomics and RBC patho-physiology.

Membrane proteomics offers unprecedented possibilities to compare protein expression in health and disease leading potentially to the identification of markers, of targets for therapeutics and to a better understanding of disease mechanisms. From transfusion medicine to infectious diseases, from cardiovascular affections to diabetes, comparative proteomics has made a contribution to the identification of proteins unique to RBCs of patients with specific illnesses shedding light on possible RBC markers for systemic diseases. In this review we will provide a short overview of some of the main achievements obtained by comparative proteomics in the field of RBC-related local and systemic diseases and suggest some additional areas of RBCs research to which comparative proteomics approaches could be fruitfully applied or extended in combination with biochemical techniques.

Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.

Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases.

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation.

BACKGROUND: Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. DESIGN AND METHODS: Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient's erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient's erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. RESULTS: In the cold, the cryohydrocytosis patient's erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO(3)-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na(+)/K(+) exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K(+) uptake. Unidirectional K(+) influx measurements showed that the patient's cells have abnormally high activities of the cation-proton exchanger and the K(+),Cl(-) co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient's erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.

The role of GLUT1 in the sugar-induced dielectric response of human erythrocytes.

We propose a key role for the glucose transporter 1 (GLUT1) in mediating the observed changes in the dielectric properties of human erythrocyte membranes as determined by dielectric spectroscopy. Cytochalasin B, a GLUT1 transport inhibitor, abolished the membrane capacitance changes in glucose-exposed red cells. Surprisingly, D-fructose, known to be transported primarily by GLUT5, exerted similar membrane capacitance changes at increasing D-fructose concentrations. In order to evaluate whether the glucose-mediated membrane capacitance changes originated directly from intracellularly bound adenosine triphosphate (ATP) or other components of the glycolysis process, we studied the dielectric responses of swollen erythrocytes with a decreased ATP content and of nucleotide-filled ghosts. Resealed ghosts containing physiological concentrations of ATP yielded the same glucose-dependent capacitance changes as biconcave intact red blood cells, further supporting the finding that ATP is the effector of the glucose-mediated dielectric response where the ATP concentration is also the mediating factor in swollen red blood cells. The results suggest that ATP binding to GLUT1 elicits a membrane capacitance change that increases with the applied concentration gradient of D-glucose. A simplified model of the membrane capacitance alteration with glucose uptake is proposed.

Naturally occurring auto-antibodies in homeostasis and disease.

Antibodies with germline or close to germline configuration exist in vertebrates, and these so-called 'naturally occurring auto-antibodies' (NAb) are directed to self and altered self components. Such NAbs have been attracting increasing interest because several of them, including some in their recombinant forms, have therapeutic potential. Whereas a large number of IgM and IgG NAbs have tissue homeostatic roles, others modulate and regulate cellular and enzyme properties. This review describes some of these NAbs and emphasizes how these low-titer, low-affinity NAbs interact with self and altered self and show functional potency in homeostasis and regulation, in addition to in diseases such as infarction and systemic inflammatory response syndrome.

Stimulation of complement amplification by F(ab')(2)-containing immune complexes and naturally occurring anti-hinge antibodies, possible role in systemic inflammation.

A systemic inflammatory response syndrome follows excessive complement amplification, but how complement amplification is stimulated is unknown. Immune complexes containing IgG (IgG-IC) rarely stimulate complement amplification in human plasma. IgG molecules doing so may have an affinity for C3 within their framework and therefore preferentially generate C3b(2)-IgG complexes, potent C3 convertase precursors. However, immune complexes generated from F(ab')(2) fragments of any homologous and heterologous IgG antibody (F(ab')(2)-IC), which have been studied over 35 years, stimulate complement amplification in human plasma. Stimulation of complement amplification by F(ab')(2)-IC is not simply due to the lack of the Fc portion, but unexpectedly requires a naturally occurring antibody (NAb) directed to the hinge region, exposed on F(ab')(2)-IC. Anti-hinge NAbs and bound antigen stabilize F(ab')(2), allowing dimeric C3b molecules to deposit to one of its shortened heavy chains. Complement amplification can also be stimulated by this mechanism in vivo, because neutrophilic elastase can generate F(ab')(2) from IgG molecules. The concentrations of F(ab')(2) and of factor Bb correlated linearly with that of elastase in septic patients at the onset of SIRS and F(ab')(2) fragments migrated on gel filtration columns as immune complexes, also containing anti-hinge NAbs in septic patients at the onset of SIRS.

Human F(ab')2-containing immune complexes together with anti-hinge natural antibodies stimulate complement amplification in vitro and in vivo.

The systemic inflammatory response syndrome (SIRS) is triggered by C5a generation following an excessive complement amplification, but it has remained unclear how complement amplification is stimulated. It is known that neutrophilic elastase can cleave IgG to F(ab')(2) and that F(ab')(2)-containing immune complexes (F(ab')(2)-IC) stimulate complement amplification together with an unidentified plasma factor. We show that absorption of plasma on F(ab')(2) from human IgG removed this factor and prevented F(ab')(2)-IC from stimulating complement amplification. The required factor was purified from pooled whole human IgG (IVIG) as those naturally occurring antibodies (NAbs) that bind to F(ab')(2), but not to intact IgG. These "anti-hinge NAbs" restored complement amplification by F(ab')(2)-IC in absorbed plasma. Anti-hinge NAbs must have formed secondary, rigidified IC from F(ab')(2)-IC, because the F(ab')(2) fragments evidently captured dimeric C3b, known as a potent C3 convertase precursor. This process may also stimulate complement amplification in vivo, because plasma from septic patients at the onset of SIRS indeed contained F(ab')(2) fragments. The concentrations of F(ab')(2) and that of factor Bb, an unbiased measure of complement amplification, correlated linearly with that of released elastase. Moreover, the F(ab')(2) fragments migrated on gelfiltration columns together with anti-hinge NAbs as ICs with MW of up to approximately 750kDa, as verified on plasma of each of the nine patients studied. These findings provide for the first time a plausible mechanism of how F(ab')(2)-containing immune complexes stimulate complement amplification together with anti-hinge NAbs. The same mechanism may contribute to complement overreaction at the onset of SIRS.

Importance of molecular studies on major blood groups--intercellular adhesion molecule-4, a blood group antigen involved in multiple cellular interactions.

Several blood groups, including the LW-blood group were discovered in the first part of last century, but their biochemical characteristics and cellular functions have only more recently been elucidated. The LW-blood group, renamed ICAM-4 (CD242), is red cell specific and belongs to the intercellular adhesion molecule family. ICAM-4 binds to several integrin receptors on blood and endothelial cells and is thus able to form large cellular complexes containing red cells. Its physiological function(s) has remained incompletely understood, but recent work shows that macrophage integrins can bind red cells through this ligand. In this article we discuss molecular properties of major blood group antigens, describe ICAM-4 in more detail, and show that phagocytosis of senescent red cells is in part ICAM-4/beta(2)-integrin dependent.

Opinion paper: Stimulation of complement amplification or activation of the alternative pathway of complement?

In this opinion paper, we suggest that the scheme of the complement system should be redrawn in order to better illustrate its potencies. This can be achieved by putting the amplification loop of the alternative complement pathway at the center of the complement system. This arrangement emphasizes that C3b molecules, generated by any pathway, can stimulate complement amplification. Furthermore, it allows one to differentiate between this type of stimulation of amplification and that driven by those immune complexes that capture dimeric C3b molecules, which are more potent C3 convertase precursors than C3b. Schemes similar to the one drawn may help to better illustrate the interplay of the pathways and convey a clearer comprehension of the mechanics of the complement system.