Leukocyte integrin ?M?2 (Mac-1) plays a pivotal role in normal protective inflammatory response and pathological inflammation. It is also a potential therapeutic target in many diseases in which inflammation plays an essential role, including cardiovascular diseases. The diverse functions and activities ascribed to Mac-1 arise from its ability to bind a multitude of ligands. We have recently discovered a new function of Mac-1. In particular, we found that Mac-1 is involved in macrophage fusion, a salient feature of chronic inflammation in many diseases, including atherosclerosis and other vascular pathologies. Macrophage fusion requires a specific proinflammatory environment and leads to the formation of multinucleated giant cells (MGCs). Although MGCs have been first discovered in the middle of the 19th century in tuberculoid granuloma and since then were found in numerous inflammatory diseases, the molecular mechanisms of fusion bringing two membranes together remain unknown. In our preliminary studies we found that ICAM-1, a counter-receptor of Mac-1, also promotes macrophage fusion. However, deficiency of ICAM-1 resulted only in a partial decrease of fusion which could not be accounted for the strong impairment of fusion observed in Mac-1- deficient macrophages. These results suggested than other Mac-1 ligands are involved in fusion. We have found that MFR (macrophage fusion receptor/SIRP?), a receptor upregulated on the surface of fusing macrophage, interacts with Mac-1. Like ICAM-1, MFR is a member of the immunoglobulin superfamily of transmembrane receptors and one of the molecules implicated in macrophage fusion. Based on this discovery and some preliminary data, we hypothesize that Mac-1 is required for macrophage fusion during which it interacts with MFR.
Specific Aim 1 is to test this hypothesis. Recombinant techniques, studies of protein-protein interactions, RNA interference, bone marrow transplantations, and combinatorial peptide libraries will be used to elucidate how Mac-1 binds MFR and clarify the role of this interaction in fusion.
Specific Aim 2 will test the hypothesis that filopodia and microspikes are fusion intermediates in macrophage fusion and that Mac-1 is required for the formation of these structures. The hypothesis is based on our discovery of these hitherto unknown structures during macrophage fusion. Ultrastructural studies using high-resolution electron microscopy, confocal immunofluorescence microscopy, and TIRF will be used to characterize these fusion intermediates.
Specific Aim 3 is to test the hypothesis that MGCs, which secrete potent elastolytic enzymes, contribute to the progression of atherosclerosis. The formation of MGCs in atherosclerotic plaques and the role of Mac-1 will be examined in ApoE-/- and LDLR-/- mice and the mice crossbred with Mac-1. Overall, these studies will lead to an increased understanding of the molecular mechanisms of macrophage fusion, identify previously unconsidered structures involved in the fusion of two membranes, give new insights into the biology of Mac-1, and potentially reveal unsuspected therapeutic targets.
Inflammation is critically involved in the pathogenesis of many disorders, including cardiovascular disease. Integrin ?M?2 (Mac-1) is the most versatile receptor on leukocytes and mediates numerous responses of these cells during the inflammatory response. The proposed studies will establish the molecular basis for the newly discovered role of Mac-1 in macrophage fusion, a salient feature of chronic inflammation, leading to the formation of multinucleated giant cells. Elucidating this process will help us to understand how chronic inflammation is sustained and potentially develop new therapeutic strategies to treat atherosclerosis and other cardiovascular pathologies.
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