The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34 ؉ cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis.Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and ␤1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin Adecorated vesicles of autophagic origin. IntroductionThe human erythrocyte is one of the best characterized mammalian cells, yet the process through which the enucleated erythroblast (reticulocyte) is converted to an erythrocyte remains poorly understood. Two distinct stages in reticulocyte maturation are evident from microscopic studies in animals. 1 Reticulocytes formed immediately after enucleation (denoted R1) are motile, multilobular, and normally confined to the BM. Mature (R2) reticulocytes are nonmotile and much more mechanically stable than their multilobular predecessors and are released from the BM into the peripheral circulation. 1,2 From the moment of enucleation until formation of the erythrocyte, the reticulocyte must lose approximately 20% of its surface area, reduce its volume, and degrade or eliminate residual cytosolic organelles. Current opinion is that the loss of surface area and degradation or elimination of residual organelles is achieved through 2 separate mechanisms. Plasma membrane loss is assumed to occur through the multivesicular endosome-exosome pathway in which small plasma membrane vesicles are endocytosed and incorporated into multivesicular endosomal bodies that subsequently fuse with the plasma membrane, releasing unwanted material as exosomes. 3,4 Degradation and elimination of organelles is effected by autophagy, a process whereby unwanted materials are enclosed in a double membrane to form autophagosomes that are delivered to lysosomes and expelled from the cell. 4 Substantial evidence suggests the endocytic and autophagic pathways converge. 5 In addition, fusion of multivesicular bodies (which are derived from endosomes) and autophagosomes has been described in the erythroleukemic cell line K562. 6 Early (R1) and late (R2) reticulocytes have different morphological and mechanical properties, but few studies have considered that different processes might be operative in the 2 cell types.A clearer understanding of this final step in the maturation of human erythroid cells would facilitate the study of ...

Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified ␣ V integrins as ICAM-4 counterreceptors. Because macrophages express ␣ V , ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/␣ V adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage ␣ V functions in island integrity. Importantly, we documented that ␣ V integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/␣ V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis. IntroductionErythroid progenitors proliferate, differentiate, and enucleate within specialized bone marrow niches, termed erythroblastic islands. [1][2][3][4] These structural units are composed of developing erythroblasts surrounding a central macrophage. It is apparent from ultrastructural studies that extensive cell-cell interactions, both erythroblastmacrophage, as well as erythroblast-erythroblast, occur within these 3-dimensional structures. However, little is known regarding either the molecular nature or functional role of the specific adhesive interactions. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. ICAM-4 expression is limited to erythroid and placental tissue 5 but, to date, there is no information on its role in erythropoiesis. We earlier identified ␣ 4 ␤ 1 and ␣ V family integrins as ICAM-4-binding partners. 6 Because macrophages express ␣ V and erythroblasts exhibit ␣ 4 ␤ 1 , ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast interactions via ICAM-4/␣ 4 ␤ 1 binding and regulating adhesion of erythroblasts to central macrophages via ICAM-4/␣ V binding.ICAM-4, which carries the Lansteiner Wiener (LW) blood group antigen system, has strong sequence homology with other members of the ICAM protein superfamily. 7,8 It is composed of 2 extracellular immunoglobulin-like domains, an N-terminal I set and a membrane proximal I2 set, and a single membr...

Intercellular adhesion molecule-4 (ICAM-4, syn. LW glycoprotein) interacts with the integrins alpha(L)beta(2), alpha(M)beta(2), A(4)beta(1), the alpha(V) family, and alpha(IIb)beta(3). Systematic mutagenesis of surface-exposed residues conserved between human and murine ICAM-4 defined 12 single amino-acid changes that affect the interaction of ICAM-4 with alpha(V) integrins. Mutation of 10 of these residues, 8 of which are spatially close on the surface of the molecule, led to a reduction in adhesion. Moreover, peptides corresponding to regions of ICAM-4 involved in its interaction with alpha(V) integrins inhibited these interactions. The other 2 mutations increased the extent of interaction of ICAM-4 with alpha(V) integrins. These mutations appear to prevent glycosylation of N160, suggesting that changes in glycosylation may modulate ICAM-4-alpha(V) integrin interactions. The region of ICAM-4 identified as the binding site for alpha(V) integrins is adjacent to the binding sites for alpha(L)beta(2) and alpha(M)beta(2). Selective binding of ICAM-4 to different integrins may be important for a variety of normal red cell functions and also relevant to the pathology of thrombotic disorders and vasoocclusive events in sickle cell disease. Our findings suggest the feasibility of developing selective inhibitors of ICAM-4-integrin adhesion of therapeutic value in these diseases.

The erythrocyte membrane has been extensively studied, both as a model membrane system and to investigate its role in gas exchange and transport. Much is now known about the protein components of the membrane, how they are organised into large multi-protein complexes and how they interact with each other within these complexes. Many links between the membrane and the cytoskeleton have also been delineated and have been demonstrated to be crucial for maintaining the deformability and integrity of the erythrocyte. In this study we have refined previous, highly speculative molecular models of these complexes by including the available data pertaining to known protein-protein interactions. While the refined models remain highly speculative, they provide an evolving framework for visualisation of these important cellular structures at the atomic level.

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the ␣5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease. IntroductionThe Lutheran glycoprotein (Lu gp) cellular adhesion molecule is widely expressed in human tissues 1 and is known for carrying antigens of the Lutheran blood group system. On erythrocytes, Lu gp is expressed as 2 isoforms of 78 and 85 kDa. 2 Both share a common extracellular portion that has previously been predicted to comprise 5 immunoglobulin superfamily (IgSF) domains. 1 The 78-kDa isoform (also known as BCAM 3 or Lu[v13] 4 ) results from alternative splicing and lacks 40 C-terminal amino acids within the cytoplasmic domain that contain an SH3-binding motif, a dileucine motif responsible for basolateral targeting, 5 and 5 potential phosphorylation sites. 1 Lu gp binds specifically and with high affinity to the extracellular matrix (ECM) protein Laminin (Ln) containing the ␣5 subunit 6-9 (Laminin 511 and Laminin 521 (Ln511/521) (numbering as in Aumailley et al 10 ). This interaction plays a direct role in the pathophysiology of sickle cell disease by mediating adhesion of sickle cells, via Lu gp, to exposed Ln511/521 of inflamed or damaged vascular endothelium. 9,11,12 Recent studies have shown that higher than normal intracellular levels of cAMP in sickle erythrocytes influence a protein kinase A-mediated or Rap1-mediated signaling pathway resulting in increased adhesion of sickle cells to the basement membrane glycoproteins Ln511/ 521. 13,14 Furthermore, phosphorylation of the 85-kDa Lu gp at S621 in epinephrine-stimulated K562 cells alters adhesion to Ln511/521. 15 The Ln-binding activity of Lu gp is a property of the amino terminal region of Lu gp, and has been localized to a region predicted to form the first 3 IgSF domains (D1D2D3). 6,16 The complementary binding site on Ln is located within a large carboxyl-terminal globular domain of the ␣5 subunit, known to comprise 5 ...

The SARS-CoV-2 virus causes COVID-19, an infection capable of causing severe disease and death but which can also be asymptomatic or oligosymptomatic. We investigated whether ABO blood group or secretor status was associated with COVID-19 severity. We investigated secretor status because expression of ABO glycans on secreted proteins and non-erythroid cells are controlled by a fucosyltransferase (FUT2), and inactivating FUT2 mutations result in a non-secretor phenotype which protects against some viral infections. Data combined from healthcare records and our own laboratory tests (n = 275) of hospitalized SARS-CoV-2 polymerase chain reaction positive patients confirmed higher than expected numbers of blood group A individuals compared to O (RR = 1.24, CI 95% [1.05, 1.47], p = 0.0111). There was also a significant association between group A and COVID-19-related cardiovascular complications (RR = 2.56, CI 95% [1.43, 4.55], p = 0.0011) which is independent of gender.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Key Points• Reticulocyte maturation involves the release of intact, inside-out autophagic vesicles with PS exposed on their surface.• Elevated levels of autophagic vesicles on circulating reticulocytes cause PS exposure in patients with SCD.During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 mm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia. (Blood. 2015;126(15):1831-1834) IntroductionThe erythrocyte is one of the most abundant, accessible, and best characterized of human cells but until the recent development of in vitro erythroid culture systems 1,2 obtaining large numbers of its precursor cell, the human reticulocyte has been problematic. Reticulocytes are broadly grouped into R1, motile multilobular, and normally confined to bone marrow, and R2, which are nonmotile, much more mechanically stable, and released into peripheral circulation where they comprise ;2% of red blood cells.3,4 During maturation to an erythrocyte, the reticulocyte must lose ;20% of its surface area, reduce its volume, and degrade or eliminate residual cytosolic organelles. Current dogma considers that loss of plasma membrane is through the release of endocytosed plasma membrane as exosomes, whereas purging of cellular organelles is executed by autophagy. 5Surface phosphatidylserine (PS) exposure is a well characterized signal for initiating phagocytosis of unwanted cells or cellular material. 6PS is normally located on the intracellular surface of plasma membranes. Relocation to the extracellular surface may occur by activation of a scramblase 7 or a bidirectional trafficking process involving cytosolic vesicles.8 PS-exposed red cells are found in the peripheral blood of patients who have undergone splenectomy, or have sickle cell disease (SCD) or thalassemia. 9-13 Study designFor details of anonymized patient samples, antibodies used, and full methods, see supplemental Methods, available on the Blood Web site. Briefly, reticulocytes were derived from erythroid cultures and confocal microscopy was performed as described.2 PS was detected using Annexin V fluore...

Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds alphaV-integrins, including alphaVbeta3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on alphaV-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and alphaV-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to alphaVbeta3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via alphaVbeta3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential.