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...
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