Erythroblastic Island Formation Research Articles

Kindlin-3 deficiency leads to impaired erythropoiesis and erythrocyte cytoskeleton.

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4β1 and macrophage αVβ3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function.

CD169-CD43 interaction is involved in erythroblastic island formation and erythroid differentiation.

CD169, a specific marker for macrophages, is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family which acts as an adhesion molecule implicated in cell-cell interaction via sialylated glycoconjugates. Although CD169+ macrophages have been found to participate in erythroblastic island (EBI) formation and support erythropoiesis under homeostasis and stress, the exact role of CD169 and its counter receptor in EBI remains unknown. Herein, we generated CD169-CreERT knock-in mice and investigated the function of CD169 in EBI formation and erythropoiesis using CD169-null mice. EBI formation was impaired in vitro by both blockade of CD169 using anti-CD169 antibody and deletion of CD169 on macrophages. Furthermore, CD43 expressed by early erythroblasts (EB) was identified as the counter receptor for CD169 in mediating the EBI formation via surface plasmon resonance and imaging flow cytometry. Interestingly, CD43 was proven to be a novel indicator of erythroid differentiation due to the progressive decrease of CD43 expression as EB mature. Although CD169-null mice did not display defects in bone marrow (BM) EBI formation in vivo, CD169 deficiency impeded BM erythroid differentiation probably via CD43 under stress erythropoiesis, in concert with the role of CD169 recombinant protein in hemin-induced K562 erythroid differentiation. These findings have shed light on the role of CD169 in EBI under steady and stress erythropoiesis through binding with its counter receptor CD43, suggesting that CD169-CD43 interaction might be a promising therapeutic target for erythroid disorders.

GM-CSF impairs erythropoiesis by disrupting erythroblastic island formation via macrophages

Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.

Identification, Isolation and Transcriptome Analyses of Mouse, Rat and Man Erythroblastic Island Central Macrophages

Erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, is the first hematopoietic niche discovered for erythropoiesis. Yet, the identity of the central macrophage has so far remained elusive. Based on the previous findings that F4/80, VCAM1 and CD169 are potential mouse central macrophage markers, we first calculated the number of F4/80+VCAM1+CD169+ mouse macrophages in the mouse bone marrow and compared it to the number of Ter119+ erythroblasts. We found that the ratio of F4/80+VCAM1+CD169+ macrophage and erythroblasts is about 1:2. Given the fact that one central macrophage is surrounded by multiple erythroblasts, the above finding suggests that it is unlikely that all the F4/80+VCAM1+CD169+ macrophages are central macrophages.Erythropoietin (Epo) is essential for erythropoiesis. It has been reported that the Epo receptor (Epor) is expressed in peritoneal macrophages. These findings promoted us to speculate that EBI central macrophages may express Epor so that Epo acts on both erythroid cells and the central macrophages simultaneously in the niche to ensure efficient and optimal red cell production. To test this notion, we first examined whether mouse bone marrow and fetal liver macrophages express Epor using the Epor-GFPcre knockin mouse model. We found that ~5% of bone marrow F4/80+ macrophages and ~35% of fetal liver F4/80+ macrophages express Epor-GFP. As negative control, no Epor-GFP macrophages are noted in wild type F4/80+ macrophages. Importantly, ImageStream analyses revealed the native EBIs in bone marrow and fetal liver are formed by Epor+ but not Epor- macrophages. Bioinformatics analyses of RNA-seq data on the sorted Epor+ and Epor- macrophage populations revealed that molecules involved in central macrophage-erythroblast association such as VCAM1, CD169, and molecules known to be important for central macrophage function such as Dnase2a, ferroportin, are highly expressed in Epor+ macrophages. In marked contrast, highly expressed pathways in Epor- macrophages are associated with immune responses including antigen process and presentation. Intriguingly, the immune related pathways are dramatically downregulated in the Epor+ macrophages, suggesting that the Epor+ macrophages in bone marrow and fetal liver have evolved a specialized function in supporting erythropoiesis.To examine whether expression of Epor in EBI central macrophages is a conserved feature across species, we generated Epor-GFPcre knockin rat using the CRISP/Cas9 technology. Using CD163 as rat macrophage marker, we found that a subpopulation of rat bone marrow CD163+ macrophages expresses Epor-GFP. As a negative control, no Epor-GFP macrophages are noted in wild type CD163+ macrophages. To examine whether EPOR is expressed in human EBI central macrophages, antibody specificity for human EPOR is critical. To this end, we employed CRISP/Cas9 approach to knock out EPOR in K562 and Hela cell lines and validated the specificity of a commercially available anti-human EPOR antibody. Using CD163, CD169 as human macrophage markers, we found that EPOR is also expressed in a subpopulation of human macrophages. Moreover, in vitro EBI formation assay revealed that human EPOR+ but not EPOR- macrophages form EBIs with erythroid cells and that the EBI formation is enhanced by EPO.In summary, we for the first time, after discovery of the EBIs 60 years ago, have identified Epor+ macrophages in mouse bone marrow and fetal liver as EBI central macrophages. Our findings provide solid foundation for studying the mechanisms by which erythropoieis is supported EBI central macrophages. A better understanding of such mechanisms will provide extensive new knowledge on basic biology of erythropoiesis. It is also important to understand the pathology of erythropoietic disorders as well as to improve ex vivo erythrocyte production. DisclosuresNo relevant conflicts of interest to declare.

Analyzing the Formation, Morphology, and Integrity of Erythroblastic Islands.

The bone marrow is the primary site of erythropoiesis in healthy adult mammals. In the bone marrow, erythroid cells mature within specialized microenvironments termed erythroblastic islands (EBIs). EBIs are multi-cellular clusters comprised of a central macrophage surrounded by red blood cell (erythroid) progenitors. It has been proposed that the central macrophage functions as a "nurse-cell" providing iron, cytokines, and growth factors for the developing erythroid cells. The central macrophage also engulfs and destroys extruded erythroid nuclei. EBIs have recently been shown to play clinically important roles during human hematological disease. The molecular mechanisms regulating this hematopoietic niche are largely unknown. In this chapter, we detail protocols to study isolated EBIs using multiple microscopy platforms. Adhesion molecules regulate cell-cell interactions within the EBI and maintain the integrity of the niche. To improve our understanding of the molecular regulation of erythroid cells in EBIs, we have developed protocols for immuno-gold labeling of erythroid surface antigens to combine with scanning electron microscopy. These protocols have allowed imaging of EBIs at the nanometer scale, offering novel insights into the processes regulating red blood cell production.

From the Cradle to the Grave: The Role of Macrophages in Erythropoiesis and Erythrophagocytosis.

Erythropoiesis is a highly regulated process where sequential events ensure the proper differentiation of hematopoietic stem cells into, ultimately, red blood cells (RBCs). Macrophages in the bone marrow play an important role in hematopoiesis by providing signals that induce differentiation and proliferation of the earliest committed erythroid progenitors. Subsequent differentiation toward the erythroblast stage is accompanied by the formation of so-called erythroblastic islands where a central macrophage provides further cues to induce erythroblast differentiation, expansion, and hemoglobinization. Finally, erythroblasts extrude their nuclei that are phagocytosed by macrophages whereas the reticulocytes are released into the circulation. While in circulation, RBCs slowly accumulate damage that is repaired by macrophages of the spleen. Finally, after 120 days of circulation, senescent RBCs are removed from the circulation by splenic and liver macrophages. Macrophages are thus important for RBCs throughout their lifespan. Finally, in a range of diseases, the delicate interplay between macrophages and both developing and mature RBCs is disturbed. Here, we review the current knowledge on the contribution of macrophages to erythropoiesis and erythrophagocytosis in health and disease.

Cultured Human Macrophages As a Model for Central Macrophages in Erythroblastic Islands

Erythropoiesis occurs in erythroblastic islands, specific structures in the bone marrow comprising a central macrophage surrounded by erythroid precursors at different stages of terminal differentiation. The central macrophage of the erythroblastic island supports proliferation and differentiation of erythroblasts, as well as phagocytosis of the extruded erythroblast nuclei, the pyrenocytes. Its identity, however, has been poorly characterized. We previously showed that macrophages also enhance in vitro erythropoiesis because they support hematopoietic stem cell (HSC) survival [Heideveld et al. 2015]. Thus, bone marrow macrophages affect all stages of erythropoiesis. The aim of our study is to characterize the relevant human bone marrow macrophages and unravel the mechanism by which they support erythropoiesis with the ultimate goal (i) to optimize erythroblast culture systems that produce erythrocytes for transfusion purposes, and (ii) to target macrophages in vivo to improve erythropoiesis in anemic patients.Macrophages are a heterogeneous population, that can be divided into pro-inflammatory M1 and anti-inflammatory M2 macrophages. Macrophages that we showed to support stem cell survival, and subsequently enhance the yield of erythroid cell cultures, were characterized as a subclass: M2c-like macrophages. These macrophages were derived from CD14+ cells isolated from human peripheral blood mononuclear cells that were cultured in serum-free media supplemented with stem cell factor, erythropoietin and dexamethasone. Within three days these macrophages expressed CD163high, CD169, mannose receptor (MR), CXCR4 and HLA-DR and harbored characteristics of bone marrow resident macrophages. This differentiation process was dependent on glucocorticoid receptor activation. Mass spectrometry of monocytes cultured in presence and absence of dexamethasone showed that expression of CD163 and MR was strictly Dex-dependent, underscoring the role of glucocorticoids in the phenotype of M2c macrophages. Protein ontology analysis revealed dexamethasone-mediated enrichment of lysosome, endocytosis and endothelial development (e.g. STAB1, IL13RA1, CD81, SLC1A3 and FKBP5). We wondered whether these macrophages with increased endosomal and lysosomal capacity not only support stem cell survival and enable erythroid commitment, but also support erythroblastic islands. In mice, it has been shown that clearance of the pyrenocytes by central macrophages occurs presumably via TAM-receptors on the macrophages. Indeed, mRNA expression of cultured M2c-like macrophages showed increased levels of TAM family members MerTK and AXL. Functionally, these macrophages have the capacity to phagocytose zymosan and to bind nuclei. Furthermore, co-culture of the M2c-like macrophages with erythroblasts yielded GPA+(erythroid marker)/CD14+ cell aggregates that suggested the formation of erythroblastic islands. Interestingly, M2c-like macrophages expressing CD163high, MR and CD169 were also observed in human bone marrow aspirates and human fetal livers resembling macrophages induced in in vitro cultures in presence of dexamethasone.Currently, we investigate the mechanism by which glucocorticoids induce monocytes to differentiate into macrophages that may be used to model erythroblastic island-mediated erythropoiesis. Knowledge on the function of such a erythroblastic island is lacking by the absence of an in vitro model. Furthermore, targeting this mechanism in vivo may enhance the recovery of erythropoiesis following bone marrow transplantation.CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield. (2015) Heideveld E, Masiello F, Marra M, Esteghamat F, Yağcı N, von Lindern M, Migliaccio AR, van den Akker E. Haematologica. 100(11):1396-1406 DisclosuresNo relevant conflicts of interest to declare.

Identification of a novel agrin-dependent pathway in cell signaling and adhesion within the erythroid niche.

Establishment of cell–cell adhesion is crucial in embryonic development as well as within the stem cell niches of an adult. Adhesion between macrophages and erythroblasts is required for the formation of erythroblastic islands, specialized niches where erythroblasts proliferate and differentiate to produce red blood cells throughout life. The Eph family is the largest known family of receptor tyrosine kinases (RTKs) and controls cell adhesion, migration, invasion and morphology by modulating integrin and adhesion molecule activity and by modifying the actin cytoskeleton. Here, we identify the proteoglycan agrin as a novel regulator of Eph receptor signaling and characterize a novel mechanism controlling cell–cell adhesion and red cell development within the erythroid niche. We demonstrate that agrin induces clustering and activation of EphB1 receptors on developing erythroblasts, leading to the activation of α5β1 integrins. In agreement, agrin knockout mice display severe anemia owing to defective adhesion to macrophages and impaired maturation of erythroid cells. These results position agrin-EphB1 as a novel key signaling couple regulating cell adhesion and erythropoiesis.

Сравнительный анализ влияния препаратов рекомбинантного эритропоэтина эпокрин и рекормон на эритропоэз <i>in vivo</i> и <i>in vitro</i>

Исследование проведено на культурах эритробластических островков и на животных с посттрансфузионной полицитемией. Рекормон и Эпокрин стимулируют эритропоэз в эритробластических островках in vitro и in vivo. В культуре эритробластических островков рекормон в 1,3 раза активнее эпокрина (p < 0,05) воздействует на процесс образования эритробластических островков de novo и de repeto. В условиях in vivo у животных с экспериментальной полицитемией эпокрин и рекормон обеспечивают адекватный ответ эритроидного ростка кроветворения на эритропоэтический сигнал, в 3,4 раза (p < 0,05) увеличивая количество эритробластических островков в костном мозге и в 2,2 раза (p < 0,05) повышая число ретикулоцитов в крови.

Effect of Oxygen and 3D Microenvironment on Physiologic Erythropoiesis

Erythropoiesis is regulated by microenvironmental factors including bone marrow (BM) architecture, cell-cell and cell-matrix interactions, growth factors, oxygen, etc. Traditional 2D hematopoietic cell cultures require high concentrations of exogenous cytokines and neglect BM architecture which favours formation of niches, including erythroblastic islands, wherein paracrine and autocrine communication occurs. Consequently, these 2D systems do not facilitate the study of erythropoiesis within a biomimetic hematopoietic microenvironment, are costly due to high concentrations of cytokines required, and not scalable. We have previously created a 3D BM biomimicry using porous collagen-coated polyurethane scaffolds for culture of human cord blood mononuclear cells (MNCs) in cytokine-free and serum-free conditions for 28 days. In addition, low concentration (200 mU/mL) of EPO was shown to enhance erythroid differentiation. We now extend our studies to evaluate the role of oxygen and the microenvironmental niches in supporting erythropoiesis.MNCs were isolated from cord blood and seeded into the 3D system (4x106 cells/scaffold). Serum-free cultures were supplemented with near physiologic concentrations of cytokines: 10 ng/mL SCF (D0-D28), and 100 mU/mL EPO (D7-D28), with medium exchange every 3 days. Three conditions were investigated: normoxia (20%), hypoxia (5%) and 2-step oxygenation (hypoxia from D0-D7 and normoxia thereafter). Proliferation was evaluated in the scaffolds in situ, of the cells released into the supernatant, and of cells extracted from scaffolds at different time-points. Maximum cell expansion was detected in situ at D14 under normoxic (1.6±0.3-fold) and 2-step oxygenation (1.8±0.3-fold) conditions (p<0.05), whereas in hypoxia no significant expansion was detected (0.9±0.2-fold). Erythroid cell maturation was monitored weekly by flow cytometry (CD45, CD71 & CD235a). Hypoxia stimulated faster upregulation of CD235a (34% in hypoxia vs 27% in normoxia of CD45low CD71+ CD235alow/+ cells at D7, which evolve to 34% in hypoxia and 11% in normoxia of CD45- CD71+ CD235a+ supernatant cells at D10, p<0.05). However, normoxia was beneficial for further differentiation (19% in hypoxia vs 41% in normoxia of CD45- CD71low/+ CD235a+ cells at D21, p<0.05). Using the 2-step oxygenation, we combined the early effect of hypoxia with the later effect of normoxia and observed a continuous increase of CD45- CD71low CD235a+ cells from D14-D28. At D28, both normoxia and hypoxia showed a renewed population of early erythroid progenitor cells (90% in hypoxia and 74% in normoxia of CD45low CD71low CD235a- cells) but little terminal maturation (6% of CD45- CD71low/+ CD235a+ cells in hypoxia and 4% in normoxia), whereas 2-step oxygenation displayed enhanced erythroid maturation and an early erythroid progenitor population (25% of CD45- CD71low/+ CD235a+ and 57% of CD45low CD71low CD235a- cells, p<0.05, fig.1), which may enable the continuous self-renewal of the culture. Clonogenic capacity of the extracted cells from the scaffolds was assessed weekly. BFU-E and CFU-E were predominantly present in the first 14 days (127±30 BFU-E in normoxia and 200±42 in hypoxia at D7, which progress to 58±19 CFU-E in normoxia and 22±7 in hypoxia at D14), confirming propensity towards erythroid lineage commitment and the critical time-period for erythropoiesis within the first 14-21 days. Cells positive for STRO-1, osteopontin and vimentin were identified in situ by confocal immunofluorescence microscopy. CD68+ cells were also detected in proximity to CD71+ cells suggesting formation of erythroblastic islands. Niche-like structures were also observed in situ by scanning electron microscopy at D17 & D25, suggesting the creation of physiologic erythroid niches with the presence of stroma-like cells. SCF and EPO concentrations were measured using ELISA; EPO was detected at D4 in hypoxia (0.8±0.5 ng/mL) and D7 (2.7±0.7 ng/mL in normoxia; 3.0±0.5 ng/mL in hypoxia), prior to addition of EPO to the cultures suggesting endogenous production. From D4 to D7, consumption of SCF was 2.9±0.5 ng/mL in hypoxia; no consumption was detected in normoxia.The 3D BM biomimicry with near-physiologic oxygen and cytokine concentrations was suitable in reproducing physiologic erythropoiesis for further study. Early stage hypoxia with late stage normoxia enhanced and extended erythroid production. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiple functions of Maf in the regulation of cellular development and differentiation.

SummaryCellular muscular aponeurotic fibrosarcoma (c‐Maf) is a member of the large macrophage‐activating factor family. C‐Maf plays important roles in the morphogenetic processes and cellular differentiation of the lens, kidneys, liver, T cells and nervous system, and it is particularly important in pancreatic islet and erythroblastic island formation. However, the exact role of c‐Maf remains to be elucidated. In this review, we summarize the research to clarify the functions of c‐Maf in the cellular development and differentiation. The expression of c‐Maf is higher in pancreatic duct cells than in pancreatic islet cells. Therefore, we suggest that pancreatic duct cells may be converted to the functional insulin‐secreting cells by regulating c‐Maf. © 2015 National Natural Science Foundation of China. Diabetes/Metabolism Research and Reviews Published by John Wiley & Sons Ltd.

Phylogenetic and Ontogenetic View of Erythroblastic Islands.

Erythroblastic islands are a hallmark of mammalian erythropoiesis consisting of a central macrophage surrounded by and interacting closely with the maturing erythroblasts. The macrophages are thought to serve many functions such as supporting erythroblast proliferation, supplying iron for hemoglobin, promoting enucleation, and clearing the nuclear debris; moreover, inhibition of erythroblastic island formation is often detrimental to erythropoiesis. There is still much not understood about the role that macrophages and microenvironment play in erythropoiesis and insights may be gleaned from a comparative analysis with erythropoietic niches in nonmammalian vertebrates which, unlike mammals, have erythrocytes that retain their nucleus. The phylogenetic development of erythroblastic islands in mammals in which the erythrocytes are anucleate underlines the importance of the macrophage in erythroblast enucleation.

Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver.

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3(-/-) erythroblasts, and cell-cycle analysis reveals that there are more Ter119(hi) cells in S-phase in Tmod3(-/-) embryos. Notably, enucleating Tmod3(-/-) erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3(-/-) late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3(-/-) fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.

The secreted lymphangiogenic factor CCBE1 is essential for fetal liver erythropoiesis

AbstractThe secreted protein CCBE1 is required for lymphatic vessel growth in fish and mice, and mutations in the CCBE1 gene cause Hennekam syndrome, a primary human lymphedema. Here we show that loss of CCBE1 also confers severe anemia in midgestation mouse embryos due to defective definitive erythropoiesis. Fetal liver erythroid precursors of Ccbe1 null mice exhibit reduced proliferation and increased apoptosis. Colony-forming assays and hematopoietic reconstitution studies suggest that CCBE1 promotes fetal liver erythropoiesis cell nonautonomously. Consistent with these findings, Ccbe1lacZ reporter expression is not detected in hematopoietic cells and conditional deletion of Ccbe1 in hematopoietic cells does not confer anemia. The expression of the erythropoietic factors erythropoietin and stem cell factor is preserved in CCBE1 null embryos, but erythroblastic island (EBI) formation is reduced due to abnormal macrophage function. In contrast to the profound effects on fetal liver erythropoiesis, postnatal deletion of Ccbe1 does not confer anemia, even under conditions of erythropoietic stress, and EBI formation is normal in the bone marrow of adult CCBE1 knockout mice. Our findings reveal that CCBE1 plays an essential role in regulating the fetal liver erythropoietic environment and suggest that EBI formation is regulated differently in the fetal liver and bone marrow.

Evaluation of erythroblast macrophage protein related to erythroblastic islands in patients with hematopoietic stem cell transplantation

BackgroundHematopoietic evaluation of the patients after Hematopoietic stem cell transplantation (HSCT) is very important. Erythroblast macrophage protein (Emp) is a key protein with function in normal differentiation of erythroid cells and macrophages. Emp expression correlates with erythroblastic island formation, a process widely believed to be associated with hematopoiesis in bone marrow. We aimed to investigate the hematopoietic function of bone marrow from 46 HSCT patients and 16 inpatients with severe anemia applied to the treatment of EPO by measuring Emp expression level.MethodsEmp mRNA and protein expression levels in mononuclear cells of bone marrow and peripheral blood samples were detected by RT-PCR and Western blotting method respectively.ResultsWhile hematopoiesis occurs in bone marrow, Emp expression level was elevated and more erythroblastic islands were found , and Emp is upregulated in bone marrow in response to erythropoietin (EPO) treatment.Conclusions Emp expression correlates with erythroblastic island formation and has an important function for bone marrow hematopoiesis. Emp could be a potential biomarker for hematopoietic evaluation of HSCT patients.

Leukocyte Immunoglobulin-Like Receptor B Regulates Erythroblastic Island Size in Human in-Vitro Erythroblastic Islands

AbstractAbstract 3190Erythropoiesis occurs in a unique bone marrow (BM) microenvironment known as erythroblastic islands. Erythroblastic islands are composed of erythroid precursors in close contact with specialized stromal macrophages, also referred to as central or island macrophages. Human erythroblastic island macrophages have been difficult to discern from other macrophage subpopulations by cell surface markers, although they have reduced expression of C3b receptor (CD35) (Lee et al., 1988). The unique non-antigen presenting nature of erythroblastic island macrophages led us to hypothesize that it may share surface receptors of other tolerogenic macrophages subpopulations, including leukocyte immunoglobulin-like transcript B receptors (LILRB).We first identified a distinct subset of human bone marrow macrophages with flow cytometry, which express high amounts of LILRB1 and LILRB2 surface receptors, have low expression of C3b surface receptor (p<0.05), and have increased expression of VCAM-1 surface receptor. This macrophage subpopulation consisted of 1 in 500 (0.02%) of bone marrow mononuclear cells. Functionally, when we FACS sorted human BM enriched erythroid precursors (CD45−/CD71+) and combined them with FACS sorted LILRB1+/LILRB2+ (LILRB+) or LILRB1−/LILRB2- (LILRB−) BM macrophages, there were no significant differences in erythroblastic island reconstitution, suggesting that the adhesion of primary BM erythroblasts to island macrophages is not related to macrophage LILRB expression. Since our ex-vivo erythroid population was enriched for late precursors, we tested island reconstitution with CD34+ cell-derived cultured erythroblasts and FACS sorted LILRB+ or LILRB- BM macrophages, which resulted in a significantly higher number of large proliferative erythroblastic islands (>25 erythroblasts/island macrophage) in LILRB+ compared with LILRB- macrophages (p<0.05) (Figure 1). Incubation with neutralizing anti-LILRB1/LILRB2 antibodies did not prevent primary erythroblastic island reconstitution independent of expression of LILRB by macrophages. However, anti-LILRB1/LILRB2 did prevent the formation of large erythroblastic islands indicating a specific role of LILRB in optimal island reconstitution and erythropoiesis (p<0.05) (Figure 1). In conclusion, we demonstrate that LILRB+ BM macrophages increase the formation of large in-vitro erythroblastic islands and LILRB receptors are important candidate receptors for functional regulation of erythropoiesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

C-Maf plays a crucial role for the definitive erythropoiesis that accompanies erythroblastic island formation in the fetal liver

Abstractc-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.

Functional Analysis of Transcription Factor c-Maf in Embryonic Hematopoiesis.

Abstract 3646Poster Board III-582c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the AP1 super family of basic leucine zipper proteins. c-Maf has been shown to be important for the regulation of IL-4 and IL-21 gene expression in T-helper-2 cells and the formation of the lens structure. A recent report has demonstrated that the augmentation of c-Maf gene expression was observed in myeloma cell lines and bone marrow samples of multiple myeloma patients. It suggests that c-Maf plays important role in multiple myeloma pathogenesis. However, the physiological roles of c-Maf in normal hematopoiesis are largely unknown and have never been analyzed in vivo setting. In this study, we investigated that c-Maf is indispensable for hematopoesis via analyzing c-Maf deficient (c-Maf−/−) mice. In the C57BL/6J background, c-Maf−/− mice were embryonic lethal and it died between embryonic day (E) 15 and E18. c-Maf−/− embryo looked pale and had evidence of anemia. In c-Maf−/− peripheral blood at E14.5, the number of enucleated erythrocytes was decreased compared that in wild-type (WT) littermates. Flow cytometric analysis of fetal liver cells indicated that mature red blood cells were reduced in c-Maf−/− embryos. In addition, Annexin-V positive apoptotic cells were increased in c-Maf−/− fetal liver. Interestingly, there was no significant difference between WT and c-Maf−/− fetal liver cells in erythroid colony counts on the colony-forming unit assay. These results indicated that c-Maf−/− erythroid cells can develop to mature cells, and the impaired definitive erythropoiesis in the c-Maf−/− mice is due to a non-cell-autonomous effect. Immunohistochemical staining of WT fetal liver using anti-c-Maf antibody showed that c-Maf was highly expressed in macrophage but not in erythroid cells. For this reason, we hypothesized that c-Maf−/− macrophage was responsible for anemia of c-Maf−/− embryo. The erythropoietic microenvironment in fetal liver mainly consists of clusters of erythroblastic islands that made up of central macrophage and surrounding erythroblasts. The compromise of erythroblasitc islands through defect of macrophage causes anemia and embryonic lethality. To verify whether c-Maf−/− macrophage cause fail to form erythroblasitic island, we isolated the erythroblasitc island from WT and c-Maf−/− fetal liver. As a result, the number of erythroblast surrounding the macrophage in erythroblastic island was significantly reduced in c-Maf−/− mice. Moreover, in vitro reconstitution experiments showed that c-Maf−/− erythroblasts were able to form erythroblastic island with WT macrophages, while c-Maf−/− macrophages and WT erythroblasts could not form erythroblastic island. These results strongly suggest that the cause of anemia in c-Maf−/− mice is due to abnormal function of macrophages in forming erythroblastic island. Finally, to clarify the molecular mechanism in formation of erythroblastic island, we found the target gene of c-Maf in fetal liver macrophages. The DNA microarray and real time RT-PCR analyses showed that the decreased expression of vascular adhesion molecule 1 (VCAM-1), known as an adhesion molecule expressed on macrophage of erythroblastic island, was observed in c-Maf−/− fetal liver macrophage. We conclude that c-Maf is indispensable for definitive hematopoiesis and erythroblastic island formation through crucial roll in fetal liver macrophages. Disclosures:No relevant conflicts of interest to declare.

Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver

AbstractPalladin was originally found up-regulated with NB4 cell differentiation induced by all-trans retinoic acid. Disruption of palladin results in neural tube closure defects, liver herniation, and embryonic lethality. Here we further report that Palld−/− embryos exhibit a significant defect in erythropoiesis characterized by a dramatic reduction in definitive erythrocytes derived from fetal liver but not primitive erythrocytes from yolk sac. The reduction of erythrocytes is accompanied by increased apoptosis of erythroblasts and partial blockage of erythroid differentiation. However, colony-forming assay shows no differences between wild-type (wt) and mutant fetal liver or yolk sac in the number and size of colonies tested. In addition, Palld−/− fetal liver cells can reconstitute hematopoiesis in lethally irradiated mice. These data strongly suggest that deficient erythropoiesis in Palld−/− fetal liver is mainly due to a compromised erythropoietic microenvironment. As expected, erythroblastic island in Palld−/− fetal liver was found disorganized. Palld−/− fetal liver cells fail to form erythroblastic island in vitro. Interestingly, wt macrophages can form such units with either wt or mutant erythroblasts, while mutant macrophages lose their ability to bind wt or mutant erythroblasts. These data demonstrate that palladin is crucial for definitive erythropoiesis and erythroblastic island formation and, especially, required for normal function of macrophages in fetal liver.

Cellular Localization and Developmental Expression of Emp Correlates with Its Role in Erythroblastic Island Formation.

In mammals, erythropoiesis takes place in distinct anatomic units called erythroblastic islands that are consisted of a central macrophage surrounded by erythroblasts of varying maturity. The attachment of erythroblasts to macrophages within these islands promotes terminal maturation and enucleation of erythroid cells. Among a few other adhesion molecules known to date, Emp (erythroblast macrophage protein) is involved in this association. Our recent gene targeting studies showed that in the absence of Emp, erythroblastic islands are not formed and erythroid cells do not undergo enucleation. In addition, Emp null macrophages do not fully mature. Information on the changes in the expression level and cellular localization of Emp in differentiating erythroid and macrophage cells is essential for understanding the function of Emp both in the formation of erythroblastic islands and in the development of these two cell types. Previously we showed that Emp is expressed in immature erythroid precursors but not in mature erythrocytes, consistent with the fact that mature red blood cells do not adhere. However, no information is available on the expression of Emp in macrophages. To address this issue, we have used primary mouse fetal liver macrophages cultured for various time periods. Our studies showed that while Emp was expressed in all stages of maturation, the localization pattern changed dramatically during maturation: in immature macrophages, a substantial fraction of Emp was intracellular, whereas in more mature cells, Emp was expressed largely at the plasma membrane. To determine if the membrane-associated Emp is exposed on the cell surface, we employed biotin labeling of surface proteins, and compared the fraction of total Emp that is accessible to the biotinylation reagent with the fraction that is inaccessible. Our data showed that approximately 5–20% of total cellular Emp was present on the surface of immature macrophage precursors compared to 60–70% in fully matured macrophages. Cell surface expression of Emp was further confirmed by live cell staining, without prior fixation, of macrophages at different stages of maturation. Intracellular pool of Emp was present largely in the nucleus where it co-localized with the nuclear matrix marker, the spliceosome assembly factor SC35. To examine the trafficking of newly synthesized Emp, we performed pulse-chase experiments in macrophages of varying maturity. We found that nascent Emp migrated intracellularly from the nucleus to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture showed that erythroid cells attached to mature macrophages but not to immature macrophage precursors. Taken together, our data shows that the temporal and spatial expression of Emp in erythroid and myeloid cells correlates with its involvement in the attachment of these two cell types in mammalian erythropoiesis. Furthermore, localization of Emp at multiple sites within the cellular environment suggests functions not limited to cell attachment.