Novel Functions of Red Cell Proteins Lu and LW

描述(由申请者提供)：我们研究的长期目标是发展对细胞-细胞和细胞-细胞外基质黏附相互作用如何调节红细胞生成的机械性理解。红母细胞在红细胞岛上分化，由围绕巨噬细胞的红细胞团组成。然而，关于粘附物在这些红细胞性壁龛中的作用人们知之甚少。这项应用的目的是探索黏附分子LW(称为ICAM-4)和路德(Lu)糖蛋白的功能。我们发现，在ICAM-4缺失的骨髓中，红细胞岛明显减少；ICAM-4/1V整合素的黏附介导了红细胞-巨噬细胞的黏附；正常而不是Lu缺失的红细胞以分化阶段特异性的方式与基质层粘连蛋白结合。我们建议1)探索在应激性红细胞生成中ICAM-4是红细胞产生放大所必需的假说。在诱导红细胞生成应激后，我们将研究ICAM-4基因敲除小鼠反应不足的潜在机制，包括：增殖潜力降低；早期红细胞向红系岛屿的招募受损；以及岛屿完整性降低。我们将在骨髓和脾岛的背景下，使用活细胞红细胞岛分析、集落分析和红细胞岛培养来表征红系的增殖和分化。2)通过与不同的整合素相互作用，调节红细胞-巨噬细胞和红细胞-红细胞的黏附，验证ICAM-4在红系细胞岛上具有双重功能的假设。基于红细胞通过1421与ICAM-4结合的初步证据，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来确定阻断ICAM-4/1421结合的野生型和ICAM-4缺失红细胞岛的作用；并分析抑制ICAM-4/1421和ICAM-4/1V结合是否具有相加性。骨髓和脾巨噬细胞缺乏的小鼠模型也将被用来研究红细胞-巨噬细胞相互作用在体内对红细胞生成的重要性。3)探讨Lu-laminin黏附是否将红细胞岛定位于骨髓中含有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个层粘连蛋白在骨髓切片中的分布图，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；并通过表征骨髓岛以及在存在和不存在层粘连蛋白的情况下进行集落分析和红细胞培养，分析和对比Lu缺失和野生型小鼠的增殖和分化。拟议的目标与了解如何增加红细胞产量以应对包括镰状细胞病、遗传性膜疾病、自身免疫性红细胞破坏和失血在内的多种遗传性和获得性贫血有关。它们也与与骨髓纤维化和异常红细胞产生有关的疾病有关。公共卫生相关性：这些研究目标与进一步了解如何增加红细胞产量以应对包括地中海贫血、镰状细胞病、遗传性膜疾病、自身免疫性红细胞破坏和失血在内的多发性遗传性和获得性贫血有关。它们也与与骨髓纤维化和异常红细胞产生有关的疾病有关。

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.