Key Points• In steady state, where no IgGs against RBCs are present, macrophages are the primary phagocytes of RBCs.• In conditions where RBCs are IgGopsonized, neutrophils can have a major effect on RBC clearance.Red blood cell (RBC) clearance is known to occur primarily in the spleen, and is presumed to be executed by red pulp macrophages. Erythrophagocytosis in the spleen takes place as part of the homeostatic turnover of RBCs to remove old RBCs. It can be strongly promoted by immunoglobulin G (IgG) opsonization of RBCs, a condition that can occur as a consequence of autoantibody or alloantibody formation. The purpose of our study was to investigate which phagocytes are involved in IgG-mediated RBC clearance in the human spleen. We developed a highly specific in vitro assay to monitor RBC phagocytosis in total human splenocytes. Surprisingly, we have found that whereas homeostatic clearance of RBCs is primarily a task for splenic macrophages, neutrophils and, to a lesser extent, also monocytes can be a major factor in clearance of IgG-opsonized RBCs. Erythrophagocytosis by neutrophils is strongly dependent on the degree of opsonization of the RBCs. Additionally, the process is enhanced after blocking the "do not eat me" signal CD47 on the opsonized RBCs, which binds signal regulatory protein a, a myeloid inhibitory receptor that restricts phagocytosis. Moreover, RBCs isolated from autoimmune hemolytic anemia patients, opsonized by auto-IgGs, were shown to be readily phagocytosed by neutrophils. Finally, priming of neutrophils by inflammatory mediators such as tumor necrosis factor a and lipopolysaccharide further increases the magnitude of erythrophagocytosis. Collectively, our data suggest that neutrophils contribute significantly to the phagocytosis of antibody-opsonized RBCs, especially under inflammatory conditions. This indicates a hereto unanticipated contribution of neutrophils in RBC phagocytosis, especially under pathological conditions such as alloimmunization or autoimmunization.

Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4+ T-cell response. Lastly, although the “don’t eat me” signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.

Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

Key Points• The Lu/BCAM adhesion molecule is gradually activated during erythrocyte aging due to loss of sialic acid on glycophorin-C.• Upon activation, Lu/ BCAM engages a sialic acid-dependent interaction with the extracellular matrix protein laminin-a5.Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-a5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises.On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-a5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-a5.

Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV-and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant noncoding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EVassociated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases.

An easy, rapid (<2 hr), and robust method was developed to manufacture biotin-labeled RCCs for clinical research compliant to GPG.

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)–containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel–mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

Background Clearance of transfused red blood cells (RBCs) is thought to be related to both storage time of the transfusion product and to inflammatory status of the recipient. We investigated these effects in a randomized “two hit” healthy volunteer transfusion model, comparing autologous RBCs stored for 35 days with RBCs stored for 2 days. Study Design and Methods Healthy male volunteers donated one unit of autologous RBCs either 2 (2D) or 35 days (35D) before the study day. The experiment was started by infusion of 2 ng/kg LPS (“first hit”). Two hours later the stored RBCs (“second hit”) were reinfused followed by RBCs labeled with biotin. Clearance of biotin labeled RBCs (BioRBCs) was measured during the 5 h post-transfusion endotoxemia period, along with measurement of PS-exposure, lactadherin binding and CD47 expression. Results In the 2D stored RBCs group, 1.5±3.4% of infused BioRBCs were cleared from the circulation 5 h post-transfusion versus 4.8±4.0% in the 35D stored group (p=0.1). There were no differences in PS-exposure, lactadherin binding, and CD47 expression between fresh and stored RBCs or between pre- and post-transfusion. Conclusion Our study shows a low clearance of RBCs even during endotoxemia. Furthermore, short-term clearance of BioRBCs during endotoxemia was not related to storage duration. Consistent with these observations, PS-exposure, lactadherin binding and CD47 expression did not differ between 2D and 35D stored cells before or after transfusion. We conclude that in the presence of endotoxemia, clearance of 35D stored autologous RBCs is not increased compared to 2D fresh RBCs.