CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.
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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. (+info)
CD47 ligation selectively inhibits the development of human naive T cells into Th1 effectors.
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The CD47 Ag, also named integrin-associated protein, was recently reported to regulate the production of IL-12 by human monocytes and dendritic cells. The present study shows that CD47 ligation by CD47 mAb in primary cultures of cord blood mononuclear cells inhibits IL-12-driven Th1 cell development, as revealed by the cytokine secretion profile at restimulation and IFN-gamma production at the single-cell level. F(ab')(2) fragments of CD47 mAb or the synthetic peptide 4N1K, corresponding to the CD47 binding site of thrombospondin, display the same activity. CD47 engagement does not change the phenotype of IL-12-primed cells from Th1 to Th2 or affect IL-4-induced Th2 cell development. Moreover, CD47 mAb inhibits IL-12- but not IL-4-induced IL-2 production as well as IFN-gamma in primary cultures, which was correlated with a decrease of the IL-12Rbeta2 chain expression. Inclusion of exogenous IL-2 at priming corrects IL-12R expression as well as the inhibition of Th1 cell development. The data thus underline the role of IL-2 in Th1 cell development and further suggest that targeting IL-2 and IL-12 simultaneously may have some therapeutic advantage in Th1 autoimmune diseases. (+info)
Membrane raft association of CD47 is necessary for actin polymerization and protein kinase C theta translocation in its synergistic activation of T cells.
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CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Ctheta (PKCtheta) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCtheta cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCgamma and associated Ca(2+) signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca(2+)](i) is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCtheta translocation. (+info)
Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow.
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Integrin-associated protein (CD47/IAP) is a pentaspan molecule that regulates integrin functions. We prepared a CD47-deficient Jurkat T cell line to assess its role in the arrest of T cells on inflammatory endothelium. Under flow conditions, constitutive arrest of CD47-deficient cells is strongly decreased as compared to the original cell line, whereas reexpression of CD47 reestablishes their ability to stop. Moreover, cells transfected with a chimera made with the extracellular portion of CD47 and the transmembrane domain of CD7 or several truncated forms of CD47 show that the first transmembrane domain and a short cytoplasmic loop are sufficient for this process. CD47 effect is indirect and depends mainly on the alpha4beta1/VCAM-1 pathway, as shown by blocking antibodies. We detected on endothelium the two CD47 counter receptors known to date: thrombospondin and SIRP1alpha. Blocking experiments show that both are involved. Overall, CD47 participates in the constitutive arrest of T lymphocytes on inflamed vascular endothelium by up-regulating alpha 4beta1 integrins. (+info)
Thrombospondin-1 inhibits TCR-mediated T lymphocyte early activation.
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Biological activities of the matrix glycoprotein thrombospondin-1 (TSP1) are cell type specific and depend on the relative expression or activation of several TSP1 receptors. Although engaging individual TSP1 receptors in T lymphocytes can elicit costimulating signals, in this study we show that intact TSP1 inhibits TCR-mediated T cell activation, assessed globally using cDNA microarrays. TSP1 signaling suppressed expression of several genes induced in Jurkat T cells, including the T cell activation markers CD69, early growth response gene-1 (Egr-1), and phosphatase of activated cells (PAC-1). TCR-stimulated and CD47-costimulated IL-2 secretion and cell surface CD69 expression were also inhibited by TSP1. The specific inhibitory effect of TSP1 was verified in freshly isolated human PBMCs. TSP1 inhibited TCR-mediated but not protein kinase C-mediated T cell activation. Using CD69 expression as a marker, we demonstrated that the inhibitory activity of TSP1 depended on two TSP1 receptors, CD47 and integrin-associated protein heparan sulfate proteoglycans. Signals from these receptors inhibited TCR signaling downstream of ZAP70, but upstream of NF-AT. Therefore, the expression of TSP1 induced during wound repair and in tumor stroma may limit T cell activation at these sites. (+info)
Osteoclasts and giant cells: macrophage-macrophage fusion mechanism.
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Membrane fusion is a ubiquitous event that occurs in a wide range of biological processes. While intracellular membrane fusion mediating organelle trafficking is well understood, much less is known about cell-cell fusion mediating sperm cell-oocyte, myoblast-myoblast and macrophage-macrophage fusion. In the case of mononuclear phagocytes, their fusion is not only associated with the differentiation of osteoclasts, cells which play a key role in the pathogenesis of osteoporosis, but also of giant cells that are present in chronic inflammatory reactions and in tumours. Despite the biological and pathophysiological importance of intercellular fusion events, the actual molecular mechanism of macrophage fusion is still unclear. One of the main research themes in my laboratory has been to investigate the molecular mechanism of mononuclear phagocyte fusion. Our hypothesis has been that macrophage-macrophage fusion, similar to virus-cell fusion, is mediated by specific cell surface proteins. But, in contrast with myoblasts and sperm cells, macrophage fusion is a rare event that occurs in specific instances. To test our hypothesis, we established an in vitro cell-cell fusion assay as a model system which uses alveolar macrophages. Upon multinucleation, these macrophages acquire the osteoclast phenotype. This indicates that multinucleation of macrophages leads to a specific and novel functional phenotype in macrophages. To identify the components of the fusion machinery, we generated four monoclonal antibodies (mAbs) which block the fusion of alveolar macrophages and purified the unique antigen recognized by these mAbs. This led us to the cloning of MFR (Macrophage Fusion Receptor). MFR was cloned simultaneously as P84/SHPS-1/SIRPalpha/BIT by other laboratories. We subsequently showed that the recombinant extracellular domain of MFR blocks fusion. Most recently, we identified a lower molecular weight form of MFR that is missing two extracellular immunoglobulin (Ig) C domains. Shortly after we cloned MFR, CD47 was reported to be a ligand for P84/SIRPalpha. We have since generated preliminary results which suggest that CD47 interacts with MFR during adhesion/fusion and is a member of the fusion machinery. We also identified CD44 as a plasma membrane protein which, like MFR, is highly expressed at the onset of fusion. The recombinant soluble extracellular domain of CD44 blocks fusion by interacting with a cell-surface binding site. We now propose a model in which both forms of MFR, CD44, and CD47 mediate macrophage adhesion/fusion and therefore the differentiation of osteoclasts and giant cells. (+info)
A role for syndecan-1 in coupling fascin spike formation by thrombospondin-1.
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An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure. (+info)
Integrin-associated protein is an adhesion receptor on sickle red blood cells for immobilized thrombospondin.
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The adhesive protein thrombospondin (TSP) potentially mediates sickle (SS) red blood cell (RBC) adhesion to the blood vessel wall, thereby contributing to vaso-occlusive crises in sickle cell disease. We previously reported that SS RBCs bind to immobilized TSP under flow conditions, whereas normal (AA) red cells do not. However, the SS RBC receptors that mediate this interaction are largely unknown. Here it is reported that integrin-associated protein (IAP), or CD47, mediates the adhesion of these cells to immobilized TSP under both flow and static conditions. A peptide derived from the C-terminal IAP binding site of TSP also supports sickle cell adhesion; adhesion to this peptide or to TSP is inhibited specifically by the anti-IAP monoclonal antibody, 1F7. Furthermore, these data suggest that IAP on SS RBCs is structurally different from that expressed on AA RBCs but that IAP expression levels do not vary between AA and SS RBCs. This structural difference may contribute to the enhanced adhesion of SS RBCs to immobilized TSP. These results identify IAP as a TSP receptor on SS RBCs and suggest that this receptor and its binding site within TSP represent potential therapeutic targets to decrease vaso-occlusion. (Blood. 2001;97:2159-2164) (+info)