Erythroblastic Islands: Specialized Mircoenvironmental Niches forErythropoiesis

This review focuses on current understanding of molecular mechanisms operating within erythroblastic islands including cell-cell adhesion, regulatory feedback, and central macrophage function. RECENT FINDINGS: Erythroblasts express a variety of adhesion molecules and recently two interactions have been identified that appear to be critical for island integrity. Erythroblast macrophage protein, expressed on erythroblasts and macrophages, mediates cell-cell attachments via homophilic binding. Erythroblast intercellular adhesion molecule-4 links erythroblasts to macrophages through interaction with macrophage alphav integrin. In intercellular adhesion molecule-4 knockout mice, erythroblastic islands are markedly reduced, whereas the erythroblast macrophage protein null phenotype is severely anemic and embryonic lethal. Retinoblastoma tumor suppressor (Rb) protein stimulates macrophage differentiation by counteracting inhibition of Id2 on PU.1, a transcription factor that is a crucial regulator of macrophage differentiation. Rb-deficient macrophages do not bind Rb null erythroblasts and the Rb null phenotype is anemic and embryonic lethal. Lastly, extruded nuclei rapidly expose phosphatidylserine on their surface, providing a recognition signal similar to apoptotic cells. SUMMARY: Although understanding of molecular mechanisms operating within islands is at an early stage, tantalizing evidence suggests that erythroblastic islands are specialized niches where intercellular interactions in concert with cytokines play critical roles in regulating erythropoiesis

The effects of spinal anesthesia on the circulation in normal unoperated man with reference to the autonomy of the arterioles and especially those of the renal circulation

The present study, comprising observations on 21 subjects given high spinal anesthesia, is an investigation of the effects of anesthetic denervation in normal man, uncomplicated by surgical intervention, on the circulation with particular reference to the vascular bed of the kidney. It is concluded that the renal arterioles are distinctly autonomous, in the sense that under basal conditions the renal vascular tone is not affected by anesthetic denervation. Our observations further suggest that the arteriolar bed generally (apart from the skin) possesses considerably more autonomy than is usually attributed to it-sufficient, in fact, in the normal supine individual at rest, to maintain an essentially normal arterial pressure. We find no evidence of significant arteriolar dilatation during high spinal anesthesia, such reduction in blood pressure as occurs being attributable, we believe, to diminished circulating blood volume in consequence of dilatation of the capillaries, venules, and veins. Part I deals with the following topics: (1) Methods; (2) the effects of spinal anesthesia on renal blood flow; (3) on the arterial pressure, and (4) on the reflex responses to posture, hypercapnia, and anoxemia. Part II consists of a review of the evidence on (5) the existence of tonic vasoconstrictor activity and on (6) the existence of autonomy in the peripheral arterioles generally, and (7) in the kidney in particular, and (8) the peripheral effects of hypercapnia and anoxemia. In (5) it is brought out that the notion of tonic vasoconstrictor activity in the sympathetic nervQus system, apart from the skin, is based largely upon animal experiments which are seriously complicated by general anesthesia, venous dilatation, etc., and that the information so obtained cannot be transferred with confidence to normal animals, and certainly not to man. PART I Methods The subjects were male convalescent patients ranging in age from 18 to 50 years who, with a single exception, presented no abnormal signs contraindicating selection for this study. They were examined in the morning in the basal, fasting condition, and were prepared for the measurement of renal blood flow and filtration rate by the clearance method, as described by Smith, Goldring, and Chasis (93). In the earlier observations the phenol red and inulin (35) clearances were followed, but after the introduction of the diodrast clearance (93), all three clearances were used. The phenol red clearance serves as a check on the diodrast clearance, the constancy of the phenol red/diodrast clearance ratio before and after anesthesia demonstrating that procaine per se has no effect upon the tubular excretory mechanism. The infusions corresponded to the typical infusion cited by Chasis, Ranges, Goldring, and Smith (18). Zero time was taken as the beginning of the priming infusion, which occupied about 10 minutes; the infusion was then changed to the sustaining infusion, the first urine collection period being started at about 30 minutes. The sustaining infusion was usually interrupted momentarily to permit injection of the anesthetic, and a short washout period was allowed to reestablish blood levels of diodrast, etc. before the next urine collection period was started. The urine collection periods (10 to 15 minutes in length) were timed to 15 seconds, and all urine samples were collected by catheter with sterile precautions, the bladder being washed out with 20 cc. of saline. (In our opinion, single urine collection periods obtained by voluntary voiding, or even by catheterization without rinsing the bladder, may be highly inaccurate; we have made nearly 3000 catheterized and rinsed collections, and we recognize that it is impossible even by this method to empty the bladder completely every time.) All our observations have been made on the descending limb of water diuresis, and to prevent an excessive reduction of urine flow we have 31

Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis

ABSTRACTTerminal erythroid differentiation is the process during which proerythroblasts differentiate to produceenucleated reticulocytes. While it is well established that during murine erythropoiesis in vivo, oneproerythroblast undergoes three mitosis to generate sequentially 2 basophilic, 4 polychromatic and 8orthochromatic erythroblasts, currently there is no method to quantitatively monitor this highly regulatedprocess. Here we outline a method that distinguishes each distinct stage of erythroid differentiation in cellsfrom mouse bone marrow and spleen based on expression levels of TER119, CD44 and cell size.Quantitative analysis revealed that the ratio of proerythroblasts:basophilic:polychromatic:orthromaticerythroblasts follows the expected 1:2:4:8 ratio, reflecting the physiological progression of terminalerythroid differentiation in normal mice. Moreover, in two stress erythropoiesis mouse models,phlebotomy-induced acute anemia and chronic hemolytic anemia due to 4.1R deficiency, the ratio of theseerythroblast populations remains the same as that of wild type bone marrow. In contrast, in anemic\u3b2-thalassemia intermedia mice, there is altered progression which is restored to normal by transferrintreatment which has been previously shown to ameliorate the anemic phenotype. The means to quantitate invivo murine erythropoiesis using our approach is likely to have broad application in the study of alterederythropoiesis in various red cell disorders

Macrophages: supportive cells for tissue repair and regeneration.

International audienceMacrophages, and more broadly inflammation, have been considered for a long time as bad markers of tissue homeostasis. However, if it is indisputable that macrophages are associated with many diseases in a deleterious way, new roles have emerged, showing beneficial properties of macrophages during tissue repair and regeneration. This discrepancy is likely due to the high plasticity of macrophages, which may exhibit a wide range of phenotypes and functions depending on their environment. Therefore, regardless of their role in immunity, macrophages play a myriad of roles in the maintenance and recovery of tissue homeostasis. They take a major part in the resolution of inflammation. They also exert various effects of parenchymal cells, including stem and progenitor cell, of which they regulate the fate. In the present review, few examples from various tissues are presented to illustrate that, beyond their specific properties in a given tissue, common features have been described that sustain a role of macrophages in the recovery and maintenance of tissue homeostasis

Carbon Monoxide Induced Erythroid Differentiation of K562 Cells Mimics the Central Macrophage Milieu in Erythroblastic Islands

Growing evidence supports the role of erythroblastic islands (EI) as microenvironmental niches within bone marrow (BM), where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb) is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule - CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms

Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

Erythroid Promoter Confines FGF2 Expression to the Marrow after Hematopoietic Stem Cell Gene Therapy and Leads to Enhanced Endosteal Bone Formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1+ hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1+ cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation