Novel Functions of Red Cell Proteins Lu and LW

描述（由申请人提供）：我们研究的长期目标是对细胞-细胞和细胞-细胞外基质粘附相互作用如何调节红细胞生成进行机理性理解。成红细胞在成红细胞岛中分化，成红细胞岛由包围巨噬细胞的成红细胞组成。然而，很少有人知道关于这些红系壁龛中的粘附附件的目的。本申请的目的是探索粘附分子LW（称为ICAM-4）和Lutheran（Lu）糖蛋白的功能。我们已经表明，红细胞系岛屿在ICAM-4空骨髓显着减少; ICAM-4/1V整合素粘附介导成红细胞-巨噬细胞附着;和正常的，但不是Lu空成红细胞结合基质层粘连蛋白在分化阶段的特定方式。我们建议1）探索ICAM-4是应激性红细胞生成中红细胞生成扩增所必需的假说。诱导红细胞生成应激后，我们将研究ICAM-4基因敲除小鼠中反应不足的潜在机制，包括：增殖潜能降低;早期成红细胞向红细胞岛的募集受损;岛完整性降低。我们将在骨髓和脾岛的情况下，采用活细胞红细胞系岛测定，以及集落测定，红细胞系增殖和分化的特点，以及红细胞系岛文化。2)通过与不同整合素的相互作用调节成红细胞-巨噬细胞和成红细胞-成红细胞粘附，检验ICAM-4在红系岛中具有双重功能的假设。基于成红细胞通过1421结合ICAM-4的初步证据，我们将鉴定参与1421结合的ICAM-4位点;使用荧光探针和生物成像确定阻断ICAM-4/1421结合对野生型和ICAM-4无效红细胞岛屿的影响;并分析抑制ICAM-4/1421和ICAM-4/1V结合是否是相加的。还将采用骨髓和脾巨噬细胞缺乏的小鼠模型来研究成红细胞-巨噬细胞相互作用对红细胞生成的体内重要性。3)探索Lu-层粘连蛋白粘附是否将红系岛定位于骨髓中的含层粘连蛋白的区域，以及它是否调节分化和/或增殖。我们将通过免疫荧光显微镜绘制15层粘连蛋白在骨髓切片中的分布，并确定其与岛屿的物理关系，比较野生型和Lu基因敲除小鼠;并通过表征骨髓岛屿和在存在和不存在层粘连蛋白的情况下进行集落测定和成红细胞培养来分析和对比Lu基因敲除小鼠和野生型小鼠的增殖和分化。所提出的目标与了解红细胞生成如何增加以应对多种遗传性和获得性贫血，包括镰状细胞病，遗传性膜疾病，自身免疫性红细胞破坏和失血有关。它们也与骨髓纤维化和异常红细胞生成相关的疾病有关。公共卫生相关性：这些研究目标是相关的，以获得更多的了解红细胞的生产是如何增加，以应对多种遗传性和获得性贫血，包括地中海贫血，镰状细胞病，遗传性膜疾病，自身免疫性红细胞破坏和失血。它们还与骨髓纤维化和异常红细胞生成相关的疾病有关。

DESCRIPTION (provided by applicant): The long-term objectives of our research are to develop a mechanistic understanding of how cell-cell and cell-extracellular matrix adhesive interactions regulate erythropoiesis. Erythroblasts differentiate in erythroblast islands, composed of erythroblasts surrounding a macrophage. However, little is known regarding the purpose of adhesive attachments in these erythroid niches. The goals of this application are to explore the function of adhesion molecules LW (termed ICAM-4) and Lutheran (Lu) glycoproteins. We have shown that erythroid islands are markedly decreased in ICAM-4 null bone marrow; ICAM-4/1V integrin adhesion mediates erythroblast-macrophage attachment; and normal but not Lu null erythroblasts bind matrix laminin in a differentiation stage-specific manner. We propose to 1) Explore the hypothesis that ICAM-4 is required for amplification of red blood cell production in stress erythropoiesis. After inducing erythropoietic stress, we will examine potential mechanisms for the deficient response in ICAM-4 knockout mice including: decreased proliferative potential; impaired recruitment of early stage erythroblasts to erythroid islands; and decreased island integrity. We will characterize erythroid proliferation and differentiation in the context of bone marrow and splenic islands employing live cell erythroid island assays, as well as colony assays, and erythroid island cultures. 2) Test the hypothesis that ICAM-4 has dual functions in erythroid islands by modulating both erythroblast-macrophage and erythroblast-erythroblast adhesion via interactions with different integrins. Building on preliminary evidence that erythroblasts bind ICAM-4 via 1421, we will identify the ICAM-4 site involved in 1421 binding; determine the effect on wild type and ICAM-4 null erythroid islands of blocking ICAM-4/1421 binding using fluorescent probes and bioimaging; and analyze whether inhibiting ICAM-4/1421 and ICAM-4/1V binding are additive. Mouse models for deficiency of marrow and splenic macrophages will also be employed to study the in vivo importance of erythroblast-macrophage interactions for erythropoiesis. 3) Explore whether Lu-laminin adhesion localizes erythroid islands to Laminin-containing regions in bone marrow and whether it regulates differentiation and/or proliferation. We will map the distribution of 15 laminin in bone marrow sections by immunofluorescence microscopy and determine its physical relationship to islands, comparing wild type and Lu knockout mice; and analyze and contrast proliferation and differentiation in Lu null and wild type mice by characterizing bone marrow islands and performing colony assays and erythroblast cultures in the presence and absence of laminin. The proposed objectives are relevant to obtaining an understanding of how red blood cell production is increased in response to multiple inherited and acquired anemias including sickle cell disease, hereditary membrane disorders, autoimmune red blood cell destruction and blood loss. They are also relevant to diseases associated with bone marrow fibrosis and abnormal red cell production. PUBLIC HEALTH RELEVANCE: These research objectives are relevant to obtaining increased understanding of how red blood cell production is increased in response to multiple inherited and acquired anemias including thalassemia, sickle cell disease, hereditary membrane disorders, autoimmune red blood cell destruction and blood loss. They are also relevant to diseases associated with bone marrow fibrosis and abnormal red blood cell production.