RESUMEN
The macrophage fusion receptor (MFR), also called P84/BIT/SIRPalpha/SHPS-1, is a transmembrane glycoprotein that belongs to the superfamily of immunoglobulins. Previously, we showed that MFR expression is highly induced at the onset of fusion in macrophages, and that MFR appears to play a role in macrophage-macrophage adhesion/fusion leading to multinucleation. The recent finding that IAP/CD47 acts as a ligand for MFR led us to hypothesize that it interacts with CD47 at the onset of cell-cell fusion. CD47 is a transmembrane glycoprotein, which, like MFR, belongs to the superfamily of immunoglobulins. We show that macrophages express the hemopoietic form of CD47, the expression of which is induced at the onset of fusion, but to a lower level than MFR. A glutathione S-transferase CD47 fusion protein engineered to contain the extracellular domain of CD47, binds macrophages, associates with MFR, and prevents multinucleation. CD47 and MFR associate via their amino-terminal immunoglobulin variable domain. Of the nine monoclonal antibodies raised against the extracellular domain of CD47, three block fusion, as well as MFR-CD47 interaction, whereas the others have no effect. Together, these data suggest that CD47 is involved in macrophage multinucleation by virtue of interacting with MFR during adhesion/fusion.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación , Proteínas Portadoras/metabolismo , Núcleo Celular/ultraestructura , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptores Inmunológicos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Secuencia de Bases , Antígeno CD47 , Proteínas Portadoras/inmunología , Fusión Celular , Cartilla de ADN , Ligandos , Macrófagos/ultraestructura , Glicoproteínas de Membrana/ultraestructura , Moléculas de Adhesión de Célula Nerviosa/ultraestructura , RatasRESUMEN
Cells of the mononuclear phagocyte lineage have the capability to adhere to and fuse with each other and to differentiate into osteoclasts and giant cells. To investigate the macrophage adhesion/fusion mechanism, we focused our attention on CD44, a surface glycoprotein known to play a role in hematopoietic cell-cell adhesion. We report that CD44 expression by macrophages is highly and transiently induced by fusogenic conditions both in vitro and in vivo. We show that CD44 ligands, hyaluronic acid, chondroitin sulfates, and osteopontin prevent macrophage multinucleation. In addition, we report that the recombinant extracellular domain of CD44 binds fusing macrophages and prevents multinucleation in vitro. These data suggest that CD44 may control the mononucleated status of macrophages in tissues by virtue of mediating cell-cell interaction.
Asunto(s)
Células Gigantes/citología , Receptores de Hialuranos/fisiología , Macrófagos Alveolares/citología , Animales , Células COS , Adhesión Celular , Fusión Celular , Receptores de Hialuranos/biosíntesis , Ácido Hialurónico/farmacología , Ligandos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-DawleyRESUMEN
We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein is recognized by monoclonal antibodies that block macrophage fusion. We have now purified this protein and cloned its corresponding cDNA. This protein belongs to the superfamily of immunoglobulins and is similar to immune antigen receptors such as the T-cell receptor, B-cell receptor, and viral receptors such as CD4. We have therefore named this protein macrophage fusion receptor (MFR). We show that the extracellular domain of MFR prevents fusion of macrophages in vitro and therefore propose that MFR belongs to the fusion machinery of macrophages. MFR is identical to SHPS-1 and BIT and is a homologue of P84, SIRPalpha, and MyD-1, all of which have been recently cloned and implicated in cell signaling and cell-cell interaction events.
Asunto(s)
Antígenos de Diferenciación , Fusión Celular/fisiología , Macrófagos Alveolares/fisiología , Glicoproteínas de Membrana/química , Molécula L1 de Adhesión de Célula Nerviosa , Moléculas de Adhesión de Célula Nerviosa/química , Receptores de Superficie Celular/química , Receptores Inmunológicos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Células COS , Clonación Molecular , Regulación de la Expresión Génica/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/inmunología , Análisis de Secuencia de ADN , Transducción de Señal/fisiología , Transfección/genéticaRESUMEN
Multinucleated giant cells and osteoclasts arise through the fusion of mononuclear phagocyte precursors. To elucidate the mechanism by which cells of monocytic lineage fuse and differentiate into giant cells and osteoclasts, we hypothesized that, as with other cell fusion events, specific surface molecules mediate the adhesion/fusion process. It has been observed that macrophages can be induced to fuse with one another in response to specific stimuli or when placed in a specific microenvironment. The formation of giant cells is primarily associated with chronic inflammatory reactions and tumors, while osteoclasts differentiate on bone which they resorb. The fact that, under normal conditions, macrophages and monocytes fail to fuse in regions and tissues where they are present in large numbers suggests the regulated and transient expression of potential fusion molecules. To identify such a fusion-associated molecule, we established a macrophage fusion assay and generated monoclonal antibodies (mAbs) that alter the fusion of macrophages in vitro. We selected four mAbs that each had the ability to block the fusion but not the aggregation of macrophages in vitro. All four antibodies recognize surface proteins of 150 kDa. The expression of the antigens recognized by all four mAbs is restricted to macrophages that have been induced to fuse in vitro and in vivo and is inducible, transient, and regulated, as neither nonfusing macrophages nor macrophages fused in vitro express these antigens. These results support the hypothesis that macrophage fusion is mediated by specific fusion/adhesion molecules and also provide a means to study the molecular mechanisms of macrophage fusion.