MOLECULAR MECHANISMS OF MACROPHAGE MULTINUCLEATION

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Osteoclasts and multinucleated giant cells originate from the fusion of mononuclear precursors of the monocyte-macrophage lineage. Multinucleation is one of the main phenotypic characteristics of these cells. Although fusion is an essential step in formation of these cells, the molecular mechanisms by which it occurs, and the functional consequences of multinucleation are poorly understood. Osteoclast and giant cells share some similarities but are clearly distinct cell types and reside in different microenvironments. As far as their similarities are concerned, we have shown that multinucleated alveolar macrophages express, like osteoclasts, a high and polarized concentration of plasma membrane Na,K-ATPases as well as a high copy number of receptors for calcitonin. Most importantly, it has been recently demonstrated that alveolar macrophages, cultured under appropriate conditions, are capable of resorbing bone in vitro. In this application we propose to use macrophages as a model system to study the mechanism of fusion of cells of the monocyte- macrophage lineage. To investigate the mechanism of cell fusion, we hypothesize that the formation of polykaryons must depend upon cell surface molecules specific for monocyte-macrophages. During the previous funding period, we have identified macrophage- specific surface molecules which potentially mediate fusion. We have generated monoclonal antibodies selected for their ability to prevent fusion of macrophages in vitro. Preliminary biochemical character- ization of one of the antigens has revealed a surface glycoprotein which is specifically detected in macrophages at the time of their fusion. A glycoprotein of similar molecular weight is detected by three additional monoclonal antibodies which we also selected for their ability to prevent fusion. The long-term goal of this project is to elucidate the fusion mechanism of macrophages which leads to the formation of polykaryons. The SPECIFIC AIMS of the present application are: 1. To characterize at the molecular level the structure of the antigens recognized by our monoclonal antibodies 12D6, 10B11, 10C4 and 10C5 which block macrophage fusion in vitro. This will be accomplished by cloning cDNAs coding for the corresponding antigens. 2. To establish a suitable cell expression system for the functional analysis of these antigens. 3. To determine the role of 12D6 antigen in osteoclast and giant cell formation in vivo, and the regulation of its expression by agents that affect osteoclast differentiation.