Clearance Of Red Blood Cells Research Articles

In Vivo Splenic Clearance Correlates with In Vitro Deformability of Red Blood Cells from Plasmodium yoelii-Infected Mice

Recent experimental and clinical studies suggest a crucial role of mechanical splenic filtration in the host's defense against malaria parasites. Subtle changes in red blood cell (RBC) deformability, caused by infection or drug treatment, could influence the pathophysiological outcome. However, in vitro deformability measurements have not been directly linked in vivo with the splenic clearance of RBCs. In this study, mice infected with malaria-inducing Plasmodium yoelii revealed that chloroquine treatment could lead to significant alterations to RBC deformability and increase clearance of both infected and uninfected RBCs in vivo. These results have clear implications for the mechanism of human malarial anemia, a severe pathological condition affecting malaria patients.

Developmental plasticity of red blood cell homeostasis

Most human physiologic set points like body temperature are tightly regulated and show little variation between healthy individuals. Red blood cell (RBC) characteristics such as hematocrit and mean cell volume are stable within individuals but can vary by 20% from one healthy person to the next. The mechanisms for the majority of this inter-individual variation are unknown and do not appear to involve common genetic variation. Here, we show that environmental conditions present during development, namely in utero iron availability, can exert long-term influence on a set point related to the RBC life cycle. In a controlled study of rhesus monkeys and a retrospective study of humans, we use a mathematical model of in vivo RBC population dynamics to show that in utero iron deficiency is associated with a lowered threshold for RBC clearance and turnover. This in utero effect is plastic, persisting at least 2 years after birth and after the cessation of iron deficiency. Our study reports a rare instance of developmental plasticity in the human hematologic system and also shows how mathematical modeling can be used to identify cellular mechanisms involved in the adaptive control of homeostatic set points.

Of macrophages and red blood cells; a complex love story

Macrophages tightly control the production and clearance of red blood cells (RBC). During steady state hematopoiesis, approximately 1010 RBC are produced per hour within erythroblastic islands in humans. In these erythroblastic islands, resident bone marrow macrophages provide erythroblasts with interactions that are essential for erythroid development. New evidence suggests that not only under homeostasis but also under stress conditions, macrophages play an important role in promoting erythropoiesis. Once RBC have matured, these cells remain in circulation for about 120 days. At the end of their life span, RBC are cleared by macrophages residing in the spleen and the liver. Current theories about the removal of senescent RBC and the essential role of macrophages will be discussed as well as the role of macrophages in facilitating the removal of damaged cellular content from the RBC. In this review we will provide an overview on the role of macrophages in the regulation of RBC production, maintenance and clearance. In addition, we will discuss the interactions between these two cell types during transfer of immune complexes and pathogens from RBC to macrophages.

Storage-Induced Clearance Of RBCs Following Transfusion Occurs Independent Of RBC Age

BackgroundDuring red blood cell (RBC) storage, changes occur that increase the clearance of RBCs following transfusion. RBCs also display unique age related changes that target them for removal in vivo, a process commonly termed RBC senescence. However, whether storage related changes simply reflect acceleration of the normal senescent programs that occur in vivo, or whether all RBCs display equal sensitivity to storage induced alterations that enhance clearance following transfusion remains unknown. As a result, we sought to determine whether enhanced RBC removal following storage simply reflects preferential clearance of older RBCs. MethodsB6 mice expressing green fluorescent protein (GFP) under a H-2Kb promoter were injected with N-hydroxysulfosuccinimide biotin followed by evaluation of biotinylated RBCs at weekly time points post transfusion by staining RBCs with strepavidin and flow cytometric analysis. At approximately 1 or 35 days post-biotinylation, blood was harvested into citrate phosphate dextrose adenine (CPDA) and RBCs were immediately transfused or stored in CPDA for 21 days prior to transfusion. Prior to transfusion, the percent of RBCs that remained biotinylated was enumerated. Following transfusion, mice were bleed at 10 minutes, 1 hour or 2 hours followed by daily bleeds for 7 days and weekly thereafter and the ratio of biotin and GFP double positive to GFP single positive RBCs was examined by staining RBCs with strepavidin followed by flow cytometric analysis. ResultsImmediately after whole mouse biotinylation, nearly 100% of donor RBCs became strepavidin positive. The percent strepavidin positivity gradually decreased until approximately 20% of the total RBCs remained strepavidin positive 35 days post-biotinylation. RBCs harvested 35 days post biotinylation into CPDA and transfused immediately exhibited gradual clearance over time, such that very few transfused RBCs were detectable 40 days post transfusion. In contrast, the percent of transfused RBCs that remained biotin positive rapidly declined to undetectable levels within 7 days following transfusion. Transfused RBCs harvested only 1 day following biotinylation failed to display enhanced clearance during the same 7 day interval. In contrast, the percent of biotin positive RBCs remained unchanged during storage. In addition, transfusion of stored RBCs failed to result in selective clearance of biotin positive RBCs during the initial clearance phase, although biotin positive RBCs appeared to retain enhanced rates of removal following the initial phase of RBC removal. ConclusionOlder RBCs appear to retain signals that result in preferential removal compared to younger RBCs following harvesting, processing and transfusion into a new recipient. Preferential clearance of older RBCs does not appear to reflect a biotinylation artifact, as newly biotinylated RBCs failed to display similar increases in RBC clearance. However, storage induced changes do not appear to result in selective removal of older RBCs, as biotinylated and non-biotinylated RBCs displayed significant removal during the initial phase of clearance following transfusion. Taken together, these results suggest that older RBCs retain senescent markers that results in enhanced clearance, but that RBC storage induces unique RBC changes that marks them for rapid clearance largely independent of RBC age. Disclosures:Zimring:Immucor Inc.: Research Funding; Terumo: Research Funding; Haemonetics: Consultancy; Cerus: Honoraria.

DMT1-Mutant Erythrocytes Have Shortened Life Span, Accelerated Glycolysis and Increased Oxidative Stress

Divalent metal transporter 1 (DMT1, also known as NRAMP2 and SLC11A2) is a transmembrane protein important for intestinal iron (Fe2+) absorption and erythroid iron utilization. Homozygous or compound heterozygous mutations in DMT1 are associated with moderate to severe hypochromic microcytic anemia in human patients and a mouse model - mk/mk mice. We have previously reported that DMT1 deficiency leads to an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast which also showed increased rate of apoptosis. For human samples we observed suppression of colony-forming capacity of erythroid progenitors that can be corrected by the addition of iron saturated chelate Fe-SIH. Later we proved this result also for mk/mk progenitors and showed reduced number of mk/mk CFU-E (164±25 vs. 283±50) and BFU-E (9±4 vs. 22±5) colonies in comparison to the colonies of wild-type (wt) mice and improvement of the colony growth with Fe-SIH.In our following studies we focused on mature erythrocytes, the last stage of erythroid differentiation that has not been analyzed yet. We first determined the in vivo half-life of red blood cells (RBC). Isolated RBCs from mk/mk mice and wt controls were in vitro labeled with CFSE fluorescent dye and injected into the wt mice. The intensity of RBCs fluorescence was measured on the 1st, 7th, 10th, 14th, 19th, 26th and 30th day following the injection. We observed an accelerated clearance of CFSE-labeled mk/mk RBCs from circulating blood when compared to wt RBCs, which indicates increased destruction of DMT1-mutant erythrocytes in vivo. It is known, that mature RBCs retain the ability to undergo stress-induced death (eryptosis), characterized by their shrinkage, membrane blebbing and phosphatidylserine surface exposure. This process may be triggered by iron deficiency. To determine the involvement of eryptosis in mk/mk RBCs clearance, RBCs were exposed to different stress conditions in vitro. A significantly increased number of Annexin V-positive RBCs was detected for mk/mk RBCs when compared to wt RBCs after 5 and 7 hour exposure to hyperosmotic shock (400mM sucrose) and glucose depletion, respectively. These results indicate shortened life span of DMT1-mutant erythrocytes and their reduced ability to cope with stress.To unravel the possible underlying mechanisms we focus on two processes important for RBC survival; anti-oxidative defense and anaerobic glycolysis. We observed 1.5 to 2-fold higher activity of glutathione peroxidase, catalase and methemoglobin reductase and elevated levels of methemoglobin in mk/mk RBCs in comparison to wt RBCs, indicating increased oxidative stress in mk/mk RBCs. Increased activity of hexokinase (2.5 times) and pyruvatkinase (2.4 times) together with reduced ratio of ATP/ADP in mk/mk mice compared with wt mice (from 2.89±0.56 μmol/L to 1.71±0.49 μmol/L) shows enhanced demand for glycolytically derived ATP to maintain the stability of RBC membrane in mk/mk mice.Our analyzes suggest that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis contributing to the pathophysiology of the disease. Grant supportCzech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic Grant No. NT11208 and Internal Grant of Palacky University Olomouc (LF_2013_010). Disclosures:No relevant conflicts of interest to declare.

A novel role for C3 in antibody-induced red blood cell clearance and antigen modulation

AbstractHemolytic transfusion reactions (HTRs) due to incompatible red blood cell (RBC) transfusions are a leading cause of transfusion associated death. Although many transfused incompatible RBCs are cleared, some remain in circulation despite the presence of RBC-specific antibodies, potentially due to “antigen modulation.” With a goal of better understanding incompatible RBC clearance, we generated a murine model with RBC-specific expression of a clinically significant human antigen (KEL2) known to be involved in antigen modulation as well as in HTRs. Wild-type (WT) recipients transfused with transgenic KEL2 RBCs generated anti-KEL glycoprotein alloantibodies, which fixed complement, led to intravascular hemolysis, and resulted in decreased levels of KEL2 antigen detectable on cells remaining in circulation. Antigen modulation did not appear to solely reflect removal of RBCs with higher antigen expression, because cells continued to display antigen modulation in the absence of significant clearance. Recipients genetically lacking complement exhibited lesser degrees of incompatible RBC clearance and antigen modulation in comparison with WT or FcγR knock-out (KO) animals, suggesting a role for complement in RBC clearance. In summary, this HTR model may serve as a platform to test strategies to downmodulate antigen and inhibit incompatible RBC clearance, thus potentially mitigating transfusion dangers.

Alloantibodies to a paternally derived RBC KEL antigen lead to hemolytic disease of the fetus/newborn in a murine model

AbstractExposure to nonself red blood cell (RBC) antigens, either from transfusion or pregnancy, may result in alloimmunization and incompatible RBC clearance. First described as a pregnancy complication 80 years ago, hemolytic disease of the fetus and newborn (HDFN) is caused by alloimmunization to paternally derived RBC antigens. Despite the morbidity/mortality of HDFN, women at risk for RBC alloimmunization have few therapeutic options. Given that alloantibodies to antigens in the KEL family are among the most clinically significant, we developed a murine model with RBC-specific expression of the human KEL antigen to evaluate the impact of maternal/fetal KEL incompatibility. After exposure to fetal KEL RBCs during successive pregnancies with KEL-positive males, 21 of 21 wild-type female mice developed anti-KEL alloantibodies; intrauterine fetal anemia and/or demise occurred in a subset of KEL-positive pups born to wild type, but not agammaglobulinemic mothers. Similar to previous observations in humans, pregnancy-associated alloantibodies were detrimental in a transfusion setting, and transfusion-associated alloantibodies were detrimental in a pregnancy setting. This is the first pregnancy-associated HDFN model described to date, which will serve as a platform to develop targeted therapies to prevent and/or mitigate the dangers of RBC alloantibodies to fetuses and newborns.

Therapeutic Cells via Functional Modification: Influence of Molecular Properties of Polymer Grafts on In Vivo Circulation, Clearance, Immunogenicity, and Antigen Protection

Modulation of cell surface properties via functional modification is of great interest in cell-based therapies, drug delivery, and in transfusion. We study the in vivo circulation, immuogenicity, and mechanism of clearance of hyperbranched polyglycerol (HPG)-modified red blood cells (RBCs) as a function of molecular properties of HPGs. The circulation half-life of modified cells can be modulated by controlling the polymer graft concentration on RBCs; low graft concentrations (0.25 and 0.5 mM) showed normal circulation as that of control RBCs. Molecular weight of HPG did not affect the circulation of modified RBCs. HPG grafting on RBCs reduced CD47 self-protein accessibility in a graft concentration-dependent fashion. HPG-grafted RBCs are not immunogenic, as is evident from their similar circulation profile upon repeated administration in mice and monitoring over 100 days. Histological examination of the spleen, liver, and kidneys of the mice injected with modified RBCs revealed distinct differences, such as elevated iron deposits and an increase in the number of CD45 expressing cells at high graft concentration of HPGs (1.5 mM); no changes were seen at low graft concentration. The absence of iron deposits in the white pulp region of the spleen and its presence in the red pulp region indicates that the clearance of functional RBCs occurs in the venous sinuses mechanical filtering system, similar to the clearance of unmodified senescent RBCs. HPG modification at grafting concentrations that yield long circulation in mice produced camouflage of a large number of minor blood group antigens on human RBCs, demonstrating its utility in chronic transfusion. The normal circulation, nonimmunogenic nature, and the potential to modulate the circulation time of modified cells without toxicity make this HPG-based cell surface modification approach attractive for drug delivery and other cell-based therapies.

Calcium in red blood cells-a perilous balance.

Ca2+ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.

Relationship of Hemoglobin, Packed Cell Volume and Total Count of RBC with the Severity of Chronic Renal Failure

Background: Anemia is one of the most consistent and severe hematological complication in chronic renal failure (CRF) patients. Lowered Hb concentration, packed cell volume (PCV) and total count (TC) of red blood cell (RBC) in such patients and the relation of changes of these values with the severity of renal failure is important to detect anemia earlier.. Subjects and Methods: The present study has been designed to observe hemoglobin concentration, PCV and TC of RBC in different stages of CRF patients suffering from anemia. For this purpose, 65 male CRF patients with anemia and 25 healthy male (control) subjects, age ranged from 30–50 years were selected randomly from BSMMU outpatient department. Hb concentration, PCV, TC of RBC and creatinine clearance were estimated by usual laboratory technique. Data were analyzed statistically by ANOVA and Pearson’s correlation coefficient test. Among 65 CRF patients, 15 were mild, 25 were moderate and 25 were severe with renal failure associated with anemia. Results: In this study, the mean Hb concentration, PCV and total count of RBCs were significantly lower in three stages of CRF patients with anemia compared to those of healthy subjects. Again, these hematological values were significantly ( P < 0.001) lower in moderate and severe CRF patients compared to mild cases and also in severe cases than those of moderate CRF cases with anemia. All the hematological parameters showed positive correlation with creatinine clearance in three stages of CRF and it was statistically significant in moderate and severe group. Conclusion: From the present study it may be concluded that CRF patients with anemia had lower Hb concentration, PCV and TC of RBC and the degree of changes depend on severity of renal failure. DOI: http://dx.doi.org/10.11566/cmoshmcj.v12i2.36 Chatt Maa Shi Hosp Med Coll J 2013; 12(2): 31-34

CD169+ macrophages provide a niche promoting erythropoiesis under homeostasis and stress

The role of macrophages in erythropoiesis was suggested several decades ago with the description of “erythroblastic islands” in the bone marrow (BM) composed of a central macrophage surrounded by developing erythroblasts. However, the in vivo role of macrophages in erythropoiesis under homeostasis or disease remains unclear. Specific depletion of CD169+ macrophages markedly reduced erythroblasts in the BM but did not result in overt anemia under homeostasis likely due to concomitant alterations in RBC clearance. However, CD169+ macrophage depletion significantly impaired erythropoietic recovery from hemolytic anemia, acute blood loss and myeloablation. Furthermore, macrophage depletion normalized the erythroid compartment in a JAK2V617F-driven murine model of polycythemia vera (PV), suggesting that erythropoiesis in PV, unexpectedly, remains under the control of macrophages in the BM and splenic microenvironments. These data indicate that CD169+ macrophages promote late erythroid maturation and that modulation of the macrophage compartment represents a novel strategy to treat erythropoietic disorders.

GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance

Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize ‘eat me’ signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80+ macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.

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.

The Role of Iron in Toxicity of Stored Red Blood Cell Units

AbstractAbstract SCI-46Accumulating evidence suggests that transfusion of red blood cells (RBCs) stored for longer durations is associated with increased rates of infection, morbidity, and mortality in hospitalized patients. During refrigerated storage in vitro, RBCs undergo cumulative biochemical and biomechanical changes that reduce their survival in vivo. After transfusion, storage-damaged RBCs are rapidly cleared from the circulation by reticuloendothelial macrophages, usually within the first hour. The associated hemoglobin iron is then rapidly catabolized and returned to the plasma at a pace that can exceed the rate of uptake by the physiologic iron transporter, transferrin, thereby producing plasma non-transferrin-bound iron. Our overarching hypothesis is that the rapid clearance of storage-damaged RBCs is responsible for some of the adverse effects of transfusion, specifically by delivering a large bolus of toxic hemoglobin-derived iron to macrophages. In particular, plasma non-transferrin-bound iron can produce oxidative damage to lipids, proteins, and DNA, thereby activating endothelial cells and platelets to increase thrombotic risk. Furthermore, increased macrophage iron and plasma non-transferrin-bound iron can enhance infectious risk by providing sources of iron to microbial pathogens both within macrophages and within the systemic circulation. Our studies in healthy human volunteers demonstrate that transfusions of “older” RBCs (stored for 40–42 days), but not “fresh” RBCs (stored for less than seven days), significantly increase circulating non-transferrin-bound iron levels in vivo (1) and enhance bacterial growth in vitro in posttransfusion serum samples. Animal studies (2) confirm these findings and provide additional evidence that transfusion exacerbates infection in vivo with Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus. In addition, murine studies suggest that rapid transfusion of the equivalent of two units of older, stored RBCs produces a proinflammatory cytokine response, which was not seen after slow transfusion of one unit of older, stored RBCs to healthy human volunteers. Studies in dogs are currently under way to test whether the RBC transfusion rate and dose affects the proinflammatory response. In conclusion, transfusion of older, stored RBCs results in delivery of a potentially toxic dose of iron to macrophages that both increases macrophage intracellular iron and produces plasma non-transferrin-bound iron. This iron toxicity may increase infectious and thrombotic risks and potentially exacerbate an existing proinflammatory response. Disclosures:No relevant conflicts of interest to declare.

Marginal Zone B Cells Mediate Alloantibody Formation to a Clinically Significant Human RBC Antigen in a Murine Model

Abstract 843 Introduction:Alloantibodies generated against red blood cell (RBC) antigens can lead to severe and potentially fatal outcomes such as hemolytic transfusion reactions (HTRs) or hemolytic disease of the fetus and newborn (HDFN). Several mouse models have been generated in efforts to study the alloimmune response to RBC antigens; however, few express clinically significant human RBC antigens. As a result, we developed a novel transgenic mouse model with RBC specific expression of the human KEL antigen, one of the most common non-ABO(H) antigens implicated in HTRs and HDFN. Using this model, we examined the rate, magnitude and consequence of alloimmunization following transfusion of RBCs expressing the human KEL2 antigen in moderate copy number (approximately 1200/RBC). In addition, we evaluated the potential involvement of marginal zone B (MZ B) cells, a B cell population involved in antibody-mediated immunity toward blood borne antigens, in the RBC alloimmunization process. Materials and Methods:C57BL/6 or KEL2 transgenic control recipient mice were transfused with KEL2 and C57BL/6 RBCs labeled with distinct lipophilic dyes to facilitate detection of cells following transfusion. To determine the role of MZ B cells, an additional group of C57BL/6 mice was treated with a-LFA-1 and a- CD49d blocking antibodies, four days prior to transfusion, in order to selectively deplete B cells from the marginal zone; additional animals were treated with an isotype matched control antibody or were surgically splenectomized. Post-transfusion survival and RBC bound antibody levels were determined by flow cytometry at specific timepoints after transfusion utilizing the fluorescent lypophilic dyes, anti-IgM, and anti-IgG. Serum antibodies were also analyzed by indirect immunofluorescence using flow cytometry with KEL2 and control C57BL/6 RBCs as targets. All experiments were completed at least three times with 3–5 recipients per group per experiment. Results:In C57BL/6 recipient mice, anti-KEL glycoprotein IgM was detectable within 2 days after KEL2 RBC transfusion, and peaked at day 5 post transfusion (compilation data of 3 experiments: adjusted MFI of IgM bound to KEL2 RBCs was 24.5 +/− 8.2 (mean +/− SD) on D5). Anti-KEL glycoprotein IgG was detectable on the transfused KEL2 RBCS and in the serum by day 5 and peaked between 14 and 21 days post transfusion (compilation data of 3 experiments: adjusted MFI of IgG bound to KEL2 RBCs was 19.7 +/− 16.7 on D14). Clearance of KEL2 RBCs began as early as 2 days following transfusion and continued to largely parallel the formation of anti-KEL alloantibodies. In contrast, KEL2 recipients failed to make anti-KEL alloantibodies (adjusted MFI of IgM bound to KEL2 RBCs was 2.1 +/− 2.7 on D5; adjusted MFI of IgG bound to KEL2 RBCs was 1.2 +/− 0.6 on D14, p<0.001 (IgM) and <0.05 (IgG) compared to KEL2 RBCs into C57BL/6 recipients). Furthermore, KEL2 recipients failed to clear transfused cells, strongly suggesting that KEL2 RBC clearance in C57BL/6 recipient mice was an antibody-mediated process. Splenectomy abrogated the alloantibody response to KEL2 RBCs, and selective depletion of MZ B cells greatly reduced alloimmunization (adjusted MFI of IgM bound to KEL2 RBCs was 9.3 +/− 6.1 on D5, adjusted MFI of IgG bound to KEL2 RBCs was 1.7 +/− 3.2 on D14, p<0.001 (IgM) and p<0.01 (IgG) compared to KEL2 RBCs into C57BL/6 recipients); furthermore, MZ B cell depletion also prevented KEL2 RBC clearance. Discussion:Transfusion of RBCs expressing the KEL2 antigen elicits a robust alloimmune response in recipient mice lacking expression of the human KEL glycoprotein. Given the complete lack of the human KEL glycoprotein antigen on recipient RBCs, the described system more closely models Rh(D) than KEL1/KEL2 (which involves a single amino acid polymorphism). The anti-KEL alloantibody response observed during primary exposure in the described model system leads to clearance of the transfused KEL2 RBCs, and suggests that initial alloantibody formation may reduce therapeutic efficacy when antigenic differences exist on transfused RBCs. In addition, these results suggest a key role for splenocytes in RBC alloimmunization, specifically MZ B-cells. Ongoing studies are further examining alloimmune responses and clearance patterns within this model system, with a focus on manipulation of MZ B cells as a potential method to prevent RBC alloimmunization. Disclosures:No relevant conflicts of interest to declare.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is defined as the increased destruction of red blood cells (RBCs) in the presence of anti-RBC autoantibodies with or without complement activation. However, the underlying mechanism for the development of AIHA remains largely unclear. In this study, we carefully evaluated the potential role of Th17 cells in the development of AIHA. We found an elevated frequency of Th17 cells in patients with AIHA, which were closely correlated with their disease activity, including the level of anti-RBC IgG antibodies, hemoglobin, serum C3, and lactate dehydrogenase activity. Furthermore, we observed that interleukin (IL)-17 was also closely correlated with the disease activity in AIHA patients. To further elucidate the potential role of Th17 cells in induction of AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA. Notably, we found that Th17 cells affected development of AIHA by enhancing the adaptive humoral responses. Specifically, we found that adoptive transfer of Th17 cells heightened the initial anti-rat RBC antibody responses and concomitantly increased the onset of AIHA. In addition, invivo neutralization of IL-17 abrogated the development of AIHA, while initiation of anti-rat RBC IgG responses and induction of AIHA in IL-17(-/-) mice were impaired. Our findings suggest that Th17 cells contribute to the development of AIHA, which could facilitate our better understanding of AIHA pathogenesis and provide clues to developing novel forms of immunotherapy against AIHA.

Biphasic clearance of incompatible red blood cells through a novel mechanism requiring neither complement nor Fcγ receptors in a murine model

Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA. C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy. This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice. These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs.

A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and α(v)-integrin. Phagocytosis via the phosphatidylserine-lactadherin-α(v)-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes. Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity. Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape ('rounding up'). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects. Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.

Apolipoprotein J/clusterin in human erythrocytes is involved in the molecular process of defected material disposal during vesiculation.

BackgroundWe have showed that secretory Apolipoprotein J/Clusterin (sCLU) is down-regulated in senescent, stressed or diseased red blood cells (RBCs). It was hypothesized that sCLU loss relates to RBCs vesiculation, a mechanism that removes erythrocyte membrane patches containing defective or potentially harmful components.Methodology/Principal FindingsTo investigate this issue we employed a combination of biochemical and microscopical approaches in freshly prepared RBCs or RBCs stored under standard blood bank conditions, an in vitro model system of cellular aging. We found that sCLU is effectively exocytosed in vivo during membrane vesiculation of freshly prepared RBCs. In support, the RBCs' sCLU content was progressively reduced during RBCs ex vivo maturation and senescence under cold storage due to its selective exocytosis in membrane vesicles. A range of typical vesicular components, also involved in RBCs senescence, like Band 3, CD59, hemoglobin and carbonylated membrane proteins were found to physically interact with sCLU.Conclusions/SignificanceThe maturation of RBCs is associated with a progressive loss of sCLU. We propose that sCLU is functionally involved in the disposal of oxidized/defected material through RBCs vesiculation. This process most probably takes place through sCLU interaction with RBCs membrane proteins that are implicit vesicular components. Therefore, sCLU represents a pro-survival factor acting for the postponement of the untimely clearance of RBCs.

Rapid clearance of transfused murine red blood cells is associated with recipient cytokine storm and enhanced alloimmunogenicity

Fourteen-day stored red blood cells (RBCs) containing an RBC-specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes. HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti-HOD alloimmunization response were measured after transfusion. Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine- or heat-treated RBCs had elevated circulating levels of keratinocyte-derived chemokine/CXCL-1, monocyte chemoattractant protein-1, and interleukin-6 after transfusion. Furthermore, phenylhydrazine- or heat-treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1- and 10.3-fold enhancement, respectively, of anti-HOD alloimmunization magnitude by flow cytometric crossmatch. Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic.