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.
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.
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