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发展对细胞-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的机制理解。红母细胞在由巨噬细胞周围的红母细胞组成的红母细胞岛中分化。然而，关于这些红系壁龛中黏附的目的，我们所知甚少。本应用程序的目的是探索粘附分子LW（称为ICAM-4）和路德（Lu）糖蛋白的功能。我们发现ICAM-4阴性骨髓中红系岛明显减少；ICAM-4/1V整合素粘附介导红细胞-巨噬细胞粘附；而正常但非Lu null的红母细胞以分化阶段特异性的方式结合基质层粘连蛋白。我们建议：1)探索在应激性红细胞生成过程中，ICAM-4是扩增红细胞生成所必需的假设。在诱导红细胞生成应激后，我们将研究ICAM-4敲除小鼠的缺陷反应的潜在机制，包括：增殖潜力降低；早期红母细胞向红岛的募集受损；岛屿完整性下降。我们将利用活细胞红细胞岛测定、集落测定和红细胞岛培养来描述骨髓和脾岛中红细胞的增殖和分化。2)通过ICAM-4与不同整合素的相互作用，调节红母细胞-巨噬细胞和红母细胞-红母细胞的粘附，验证ICAM-4在红系岛屿中具有双重功能的假说。基于红细胞通过1421结合ICAM-4的初步证据，我们将确定参与1421结合的ICAM-4位点；利用荧光探针和生物成像技术检测阻断ICAM-4/1421结合对野生型和ICAM-4无红系岛的影响；并分析抑制ICAM-4/1421和ICAM-4/1V结合是否具有可加性。小鼠骨髓和脾巨噬细胞缺乏模型也将用于研究红细胞-巨噬细胞相互作用对红细胞生成的体内重要性。3)探讨Lu-laminin粘附是否将红细胞岛定位到骨髓中含有laminin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种层粘连蛋白在骨髓切片中的分布，并确定其与孤岛的物理关系，比较野生型和Lu敲除小鼠；并通过表征骨髓岛，进行集落测定和红母细胞培养在层粘连蛋白存在和不存在的情况下分析和对比Lu null和野生型小鼠的增殖和分化。提出的目标与了解红细胞生成如何在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）的反应中增加有关。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：这些研究目标与进一步了解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）如何增加红细胞生成有关。它们也与骨髓纤维化和异常红细胞生成相关的疾病有关。

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.