Freshly isolated human red blood cells (RBC) bind to the signal regulatory protein alpha (SIRPα) on macrophages in a CD47-dependent manner (CD47 = integrin-associated protein). This interaction provides a ‘do not eat me signal’ such that these RBC are not phagocytosed and remain in circulation as exemplified for mouse RBC by Oldenborg and collaborators [1, 2]. Recently, Burger et al. have reported [3] and reviewed in this journal [4] that CD47 can function like a switch and then induces rather than inhibits phagocytosis of ‘experimentally aged human RBC’. In reality the studied RBC were oxidatively damaged by treating with CuSO4 and ascorbic acid. The ‘eat me signal’ could be induced by blocking CD47 on oxidized RBC with F(ab')2 anti-CD47. Data from others [5] suggested that thrombospondin-1, known to interact with CD47, may act similarly. Hence, the authors studied whether a particular peptide of thrombospondin-1 (4N1K) promoted the interaction of CD47 with SIRPα. Experiments with human red pulp macrophages and oxidized RBC showed that phagocytosis increased upon addition of the 4N1K decapeptide, but not when supplemented with an irrelevant peptide from thrombospondin-1. The 4N1K peptide was half as effective as blocking CD47 with F(ab’)2 anti-CD47. The F(ab’)2 fragment of the CD47-specific antibody prevented CD47 from interacting with SIRPα and this was sufficient to induce an ‘eat me signal’, because the inhibitory signal could not be induced by SIRPα. The authors think that binding of the 4N1K peptide to oxidized CD47 induces an ‘eat me signal’ not by blocking the interaction with SIRPα, but by conveying to oxidized CD47 the ability to interact in a new, so far unknown way with SIRPα which then induces an ‘eat me signal’. The authors suggest that thrombospondin-1 binding to CD47 can switch the role of CD47 to a promoter of erythrophagocytosis, which may even be responsible for in vivo clearance of aged RBC. This, however, is highly questionable, because the 4N1K peptide of thrombospondin-1 was applied at 3 × 10−5 mol/l, a concentration that exceeds the thrombospondin-1 concentration in plasma by a factor of 103 to 104 [6]. Correspondingly, Head et al. [7] found that the 4N1K peptide at the very same high concentration (50 µg/ml) binds to CD47 without the need to impose a conformational change by e.g. oxidation. Moreover, incubation of RBC with 50 µg/ml 4N1K peptide for 24 h induced phosphatidylserine exposure on these RBC, amounting to an annexin binding that was 5 times higher than in controls and resulted in 40% loss of viable RBC. The two sets of findings may explain the extra RBC destruction during vaso-occlusive crisis in sickle cell anemia, where local concentrations of thrombospondin may be considerably higher and the plasma concentration is 2–3 times higher than normal [6]. This type of induced RBC destruction is random and does not affect a particular RBC subpopulation. Otherwise the findings of Burger et al. [3, 4] can in no way provide mechanistic details on how aged RBC are selectively cleared in vivo at the end of their life span of 120 days. The CD47/thrombospondin/SIRPα interactions lack the subtleties required to signal a preferential clearance of senescent RBC at a controlled pace. One reason is that about 40% of CD47 are mobile within the plane of the membrane of RBC of any cell age [8]. Hence, oxidative damage, aggregation, and the altered conformation of CD47 are induced in an unrestricted manner. Furthermore, binding of thrombospondin-1 or its active peptide stabilizes the new CD47 conformation at any concentration above a minimal dose having sufficient affinity. Thus, the suggested recognition principle lacks means to selectively tag a particular RBC subpopulation. This becomes evident by comparing the properties of the CD47/thrombospondin/SIRPoα-induced RBC removal with those operating through a naturally occurring antibody (NAb) to band 3 protein [for review see 9]. In this system recognition of senescent or oxidatively stressed RBC depends on bivalent binding of anti-band 3 NAbs to band 3 oligomers, but not to preexisting band 3 dimers. Anti-band 3 NAbs [10] have a low affinity and require that their target is presented in form of oligomers. Cross-linkable band 3 oligomers represent a minute fraction of band 3 protein of 1.5 ± 0.3% on young and 1.9 ± 0.3% on senescent RBC (different at a confidence level of 0.06) despite a million copies of band 3 per cell [11]. Band 3 oligomers are formed upon detachment of band 3 protein from the cytoskeleton via selective phosphorylation [12] and binding of oxidatively generated hemichromes to the cytoplasmic portion of band 3 protein, promoting clusterization [13]. Finally, the few anti-band 3 NAbs associating with oligomerized band 3 protein represent an insufficient number to induce phagocytosis [14]. The low number of firmly bound anti-band 3 NAbs is, however, compensated by a massive deposition of C3b induced by bound anti-band 3 NAbs. The reason is that bound anti-band 3 NAbs have a unique affinity for C3 within their Fab arm [15] and therefore preferentially generate C3b2-IgG complexes in the presence of active complement [16]. C3b2-IgG complexes subsequently stimulate alternative complement pathway C3b deposition because these complexes first bind properdin that greatly enhances factor B binding [17]. This sequence of well controlled processes favors a selective opsonization of in vivo aged and oxidatively stressed RBC and at the same time prevents an excessive opsonization.

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 C3b(2)-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.

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.

This volume illustrates the functional properties of NAbs. Authors from pioneering groups report in their chapters on the tissue homeostatic, tissue regenerating and regulatory properties of NAbs and

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−, Mg2+ and Ca2+ 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/m²). 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, Ca2+ from 1.98 ± 0.04 to 1.89 ± 0.02 and Mg2+ 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−, Mg2+ and Ca2+ 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 Mg2+ and Ca2+ levels may lead to an altered excitability, which is particularly relevant for diabetic patients with heart disease.

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.

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.

Electrolyte disturbances are well-known consequences of the diabetic pathology. However, less is known about the cumulative effects of repeated changes in glycaemia, a characteristic of diabetes, on the electrolyte balance. We therefore investigated the ionic profiles of patients with type 1 diabetes during consecutive hyper- and/or hypoglycaemic events using the glucose clamp.

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 KNO3-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.

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.

Vox SanguinisVolume 99, Issue s1 p. 1-90 Free Access Oral abstracts First published: 25 June 2010 https://doi.org/10.1111/j.1423-0410.2010.01343_1.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume99, Issues1Special Issue: Abstracts of the XXXIst International Congress of the International Society of Blood Transfusion in joint cooperation with the 43rd Congress of the DGTI, Berlin, Germany, 26 June ‐ 1 July, 2010July 2010Pages 1-90 RelatedInformation

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.

Antibodies with germline or close to germline configuration exist in vertebrates, and these so-called 'naturally occurring auto-anti bodies' (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.

This Correspondence relates to "Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies" (Am J Pathol 2007, 170:52–64). This Correspondence relates to "Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies" (Am J Pathol 2007, 170:52–64). Last year J.C. Jennette and collaborators1Xiao H Schreiber A Heeringa P Falk RJ Jennette JC Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies.Am J Pathol. 2007; 170: 52-64Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar published an excellent paper in The American Journal of Pathology, demonstrating that anti-neutrophil cytoplasmic antibodies (ANCA) induce disease via complement amplification in a mouse model for ANCA-induced glomerulonephritis and vasculitis. The data imply that these autoantibodies do not generate a regular immune complex that would have induced classical pathway complement activation. Instead, by binding to neutrophils, these autoantibodies induce the release of factors capable of stimulating complement amplification. The authors could not address the nature of these factors but list a few components that might be involved, such as the release of properdin, which could bind apoptotic cells, attract C3b, and generate an amplifying C3 convertase, as shown recently.2Xu W Berger SP Trouw LA de Boer HC Schlagwein N Mutsaers C Daha MR van Kooten C Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation.J Immunol. 2008; 180: 7613-7621PubMed Google Scholar However, as indicated, neutrophils also release proteases, among which elastase,2Xu W Berger SP Trouw LA de Boer HC Schlagwein N Mutsaers C Daha MR van Kooten C Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation.J Immunol. 2008; 180: 7613-7621PubMed Google Scholar cathepsin G,3Baici A Knöpfel M Fehr K Cleavage of the four human IgG subclasses with cathepsin G.Scand J Immunol. 1982; 16: 487-498Crossref PubMed Scopus (20) Google Scholar and PR34Dolman KM Jager A Sonnenberg A von dem Borne AE Goldschmeding R Proteolysis of classic anti-neutrophil cytoplasmic autoantibodies (C-ANCA) by neutrophil proteinase 3.Clin Exp Immunol. 1995; 101: 8-12Crossref PubMed Scopus (18) Google Scholar are known to generate F(ab′)2-like fragments from IgG molecules. F(ab′)2-containing immune complexes have been known for many years to stimulate complement amplification together with a serum factor.5Reid KB Complement fixation by the F(ab′)2-fragment of pepsin-treated rabbit antibody.Immunology. 1971; 20: 649-658PubMed Google Scholar My group has recently identified this factor as anti-hinge natural antibodies, which together with F(ab′)2-containing immune complexes generate rigidified, secondary immune complexes that capture dimeric C3b, a potent precursor of the amplifying C3 convertase in human plasma.6Fumia S Goede JS Fischler M Luginbühl A Frick S Fodor P Lutz HU Human F(ab′)2-containing immune complexes together with anti-hinge natural antibodies stimulate complement amplification in vitro and in vivo.Mol Immunol. 2008; 45: 2951-2961Crossref PubMed Scopus (28) Google Scholar It is, however, unlikely that this very same mechanism may operate in mice, since mice lack IgG anti-hinge natural antibodies.7Yano S Kaku S Suzuki K Terazaki C Sakayori T Kawasaki T Kawamura K Sugita Y Hoshino K Masuho Y Natural antibodies against the immunoglobulin F(ab′)2 fragment cause elimination of antigens recognized by the F(ab′)2 from the circulation.Eur J Immunol. 1995; 25: 3128-3133Crossref PubMed Scopus (17) Google Scholar On the other hand, it is possible that certain anti-idiotypic antibodies exert a similar effect in mice as ANCA-induced glomerular damage is significantly higher when splenocytes rather than the IgG fraction from MPO-immunized Mpo−/− mice are injected.1Xiao H Schreiber A Heeringa P Falk RJ Jennette JC Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies.Am J Pathol. 2007; 170: 52-64Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 8Xiao H Heeringa P Hu P Liu Z Zhao M Aratani Y Maeda N Falk RJ Jennette JC Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (1019) Google Scholar I encourage researchers in this field to elucidate the actual mechanism of how ANCA antibodies mediate complement amplification in mice, so that we may gain better insight into how the mouse model differs from the events presumably operating in humans.

Evolved dependence is a process through which one species becomes 'dependent' on another following a long evolutionary history of interaction. This happens when adaptations selected in the first species for interacting lead to fitness costs when the second species is not encountered. Evolved dependence is frequent in host–parasite interactions, where hosts may achieve a higher fitness in the presence of the parasite than in its absence. Since oncogenic manifestations are (i) ubiquitous across multicellular life, (ii) involved in parasitic-like interactions with their hosts, and (iii) have effectively driven the selection of numerous adaptations, it is possible that multicellular organisms display evolved dependence in response to oncogenic processes. We provide a comprehensive overview of the topic, including the implications for cancer prevention and treatment.