Macrophage Multinucleation Research Articles

Investigation of multinucleation and apoptosis of macrophages of BCG-infected mice and their production of cathepsins and matrix metalloproteinases

The relevance of the study of the role of macrophages and their multinucleated forms in the pathogenesis of tuberculous granulomatosis is determined by its wide prevalence, the presence of severe socio-economic consequences of its morbidity and necrotic complications, which are based on the high destructive potential of macrophages associated with the role of hydrolases in the degradation of extracellular matrix components. Aim of the study was to investigate the features of the multinucleation, apoptosis and expression of a number of hydrolases in macrophages of BCG-infected mice. Material and methods. The intensity of macrophage multinucleation and apoptosis, the peculiarities of their expression of matrix metalloproteinases (MMP-1, MMP-9), catepsins (CatB, CatD), caspase-3, and p53 protein were studied in peritoneal cells cultures of intact and BCG-infected BALB/c mice. Results. The number of multinucleated macrophages increased according to the terms of the experiment, having a maximum value for 3 months of observation, but after 2 months almost reaching this level. The realization of apoptosis, multinucleation of macrophages had a complex character, determining the composition of their subpopulations. The dynamics of the expression of the studied hydrolases by macrophages indicated their unequal role in tissue necrosis at various stages of granulomogenesis. The high functional ability of multinucleated macrophages to produce hydrolases of certain types is shown. Intense expression of MMP-1 in the early stages of granulomogenesis and its maximum value, as well as CatD expression for 3 months, and strong expression of MMP-9 for 6 months were noted. Conclusions. Stimulation of plastic processes in macrophages under conditions of BCG-granulomatosis determines the formation of multinucleated macrophages with high functional potential and intensive expression of hydrolases by macrophages for 2 and 3 months of granulomogenesis. These are periods of high risk of necrotic complications of tuberculous granulomatosis, which should be taken into account when developing methods for their prevention and therapeutic correction.

Leishmania infection-induced multinucleated giant cell formation via upregulation of ATP6V0D2 expression.

Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. In both clinical and experimental visceral leishmaniasis, macrophage multinucleation is observed in parasitized tissues. However, the feature and the mechanism of macrophage multinucleation remained unclear. Here, we report that infection of Leishmania donovani, a causative agent of visceral leishmaniasis, induces multinucleation of bone marrow-derived macrophages (BMDMs) in vitro. When these infection-induced multinucleated macrophages were compared with cytokine-induced multinucleated giant cells, the former had higher phagocytic activity on red blood cells but no apparent changes on phagocytosis of latex beads. BMDMs infected with L. donovani had increased expression of ATP6V0D2, one of the components of V-ATPase, which was also upregulated in the spleen of infected mice. Infection-induced ATP6V0D2 localized in a cytoplasmic compartment, which did not overlap with the mitochondria, endoplasmic reticulum, or lysosomes. When ATP6V0D2 expression was recombinantly induced in BMDMs, the formation of multinucleated macrophages was induced as seen in the infected macrophages. Taken together, L. donovani infection induces multinucleation of macrophages via ATP6V0D2 upregulation leading to a unique metamorphosis of the macrophages toward hemophagocytes.

Macrophage-specific responses to human- and animal-adaptedtubercle bacilli reveal pathogen and host factors drivingmultinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Re-establishment of macrophage homeostasis by titanium surface modification in type II diabetes promotes osseous healing

Titanium surface mediated immunomodulation may address compromised post-implantation bone healing in diabetes mellitus. To assess in vitro phenotypic changes, M1 and M2 polarised Type 2 diabetic rat (Goto Kakizaki, GK) macrophages were cultured on micro-rough (SLA) or hydrophilic nanostructured SLA (modSLA) titanium. The in vivo effects of the SLA and modSLA surfaces on macrophage phenotype, wound-associated protein expression and bone formation were investigated using a critical-sized calvarial defect model. Compared to healthy macrophages, GK M2 macrophage function was compromised, secreting significantly lower levels of the anti-inflammatory cytokine IL-10. The modSLA surface attenuated the pro-inflammatory cellular environment, reducing pro-inflammatory cytokine production and promoting M2 macrophage phenotype differentiation. ModSLA also suppressed gene expression associated with macrophage multinucleation and giant cell formation and stimulated pro-osteogenic genes in co-cultured osteoblasts. In vivo, modSLA enhanced osteogenesis compared to SLA in GK rats. During early healing, proteomic analysis of both surface adherent and wound exudate material showed that modSLA promoted an immunomodulatory pro-reparative environment. The modSLA surface therefore successfully compensated for the compromised M2 macrophage function in Type 2 diabetes by attenuating the pro-inflammatory response and promoting M2 macrophage activity, thus restoring macrophage homeostasis and resulting in a cellular environment favourable for enhanced osseous healing.

Heterogeneity and Actin Cytoskeleton in Osteoclast and Macrophage Multinucleation.

Osteoclast signatures are determined by two transcriptional programs, the lineage-determining transcription pathway and the receptor activator of nuclear factor kappa-B ligand (RANKL)-dependent differentiation pathways. During differentiation, mononuclear precursors become multinucleated by cell fusion. Recently, live-cell imaging has revealed a high level of heterogeneity in osteoclast multinucleation. This heterogeneity includes the difference in the differentiation states and the mobility of the fusion precursors, as well as the mode of fusion among the fusion precursors with different numbers of nuclei. In particular, fusion partners often form morphologically distinct actin-based linkages that allow two cells to exchange lipids and proteins before membrane fusion. However, the origin of this heterogeneity remains elusive. On the other hand, osteoclast multinucleation is sensitive to the environmental cues. Such cues promote the reorganization of the actin cytoskeleton, especially the formation and transformation of the podosome, an actin-rich punctate adhesion. This review covers the heterogeneity of osteoclast multinucleation at the pre-fusion stage with reference to the environment-dependent signaling pathway responsible for reorganizing the actin cytoskeleton. Furthermore, we compare osteoclast multinucleation with macrophage fusion, which results in multinucleated giant macrophages.

A trans-eQTL network regulates osteoclast multinucleation and bone mass.

Functional characterisation of cell-type-specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans-regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (Pik3cb-/-, Atp8b2+/-, Igsf8-/-, Eml1-/-, Appl2-/-, Deptor-/-) and myeloid-specific Slc40a1 knockout mice displayed abnormal bone phenotypes. We report opposing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast multinucleation and bone mass.

Integrating Phosphoproteome and Transcriptome Reveals New Determinants of Macrophage Multinucleation

Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.

Kcnn4 Is a Regulator of Macrophage Multinucleation in Bone Homeostasis and Inflammatory Disease

SummaryMacrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca2+ signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.

THU0511 Macrophage Growth by Polyploidization in Chronic Inflammation

BackgroundHow macrophages grow at sites of chronic inflammation is not well understood. In rheumatoid arthritis and several granulomatous autoimmune diseases, such as sarcoidosis, giant cell arteriitis and granulomatosis with polyangiitis,...

Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

Signal regulatory protein-α (SIRPA/SIRPα) is a transmembrane protein that is expressed in various tissues, including the heart. Previous studies have demonstrated that SIRPA is involved in multiple biological processes, including macrophage multinucleation, skeletal muscle differentiation, neuronal survival, protection against diabetes mellitus, and negative regulation of immune cells. However, the role of SIRPA in cardiac hypertrophy remains unknown. To examine the role of SIRPA in pathological cardiac hypertrophy, we used SIRPA knockout mice and transgenic mice that overexpressed mouse SIRPA in the heart. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. We observed downregulation of SIRPA expression in dilated cardiomyopathy human hearts and in animal hearts after aortic banding surgery. Accordingly, SIRPA(-/-) mice displayed augmented cardiac hypertrophy, which was accompanied by increased cardiac fibrosis and reduced contractile function, as compared with SIRPA(+/+) mice 4 weeks after aortic banding. In contrast, transgenic mice with the cardiac-specific SIRPA overexpression exhibited the opposite phenotype in response to pressure overload. Likewise, SIRPA protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we revealed that SIRPA-mediated protection during cardiac hypertrophy involved inhibition of the Toll-like receptor 4/nuclear factor-κB signaling axis. Furthermore, we demonstrated that the disruption of Toll-like receptor 4 rescued the adverse effects of SIRPA deficiency on pressure overload-triggered cardiac remodeling. Thus, our results identify that SIRPA plays a protective role in cardiac hypertrophy through negative regulation of the Toll-like receptor 4/nuclear factor-κB pathway.

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2.p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF-I-stimulated signal transduction and biological actions.

The Tec Family Tyrosine Kinase Btk Regulates RANKL-induced Osteoclast Maturation

A spontaneous mutation in Bruton's tyrosine kinase (Btk) induces a defect in B-cell development that results in the immunodeficiency diseases X-linked agammaglobulinemia in humans and X-linked immunodeficiency (Xid) in mice. Here we show an unexpected role of Btk in osteoclast formation. When bone marrow cells derived from Xid mice were stimulated with receptor activator of NF-kappaB ligand, an osteoclast differentiation factor, they did not completely differentiate into mature multinucleated osteoclasts. Moreover, we found that the defects appeared to occur at the stage in which mononuclear preosteoclasts fuse to generate multinucleated cells. Supporting this notion, macrophages from Xid mice also failed to form multinucleated foreign body giant cells. The fusion defect of the Xid mutant osteoclasts was caused by decreased expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, as well as reduced expression of various osteoclast fusion-related molecules, such as the d2 isoform of vacuolar H(+)-ATPase V0 domain and the dendritic cell-specific transmembrane protein. This deficiency was completely rescued by the introduction of a constitutively active form of NFATc1 into bone marrow-derived macrophages. Our data provide strong evidence that Btk plays a critical role in osteoclast multinucleation by modulating the activity of NFATc1.

Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can recapitulate the fusion process in vitro. In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo. These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites.

Expression of the self-marker CD47 on dendritic cells governs their trafficking to secondary lymphoid organs

Dendritic cells (DCs) capture and process Ag in the periphery. Thus, traffic through lymphatic vessels is mandatory before DCs relocate to lymph nodes where they are dedicated to T-cell priming. Here, we show that the ubiquitous self-marker CD47 selectively regulates DC, but not T and B cell trafficking across lymphatic vessels and endothelial barriers in vivo. We find an altered skin DC migration and impaired T-cell priming in CD47-deficient mice at steady state and under inflammatory conditions. Competitive DC migration assays and active immunization with myeloid DCs demonstrate that CD47 expression is required on DCs but not on the endothelium for efficient DC trafficking and T-cell responses. This migratory defect correlates with the quasi-disappearance of splenic marginal zone DCs in nonmanipulated CD47-deficient mice. Nonetheless, CCR7 expression and CCL19-driven chemotaxis remain intact. Our data reveal that CD47 on DCs is a critical factor in controlling migration and efficient initiation of the immune response.

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.

SHPS-1, a multifunctional transmembrane glycoprotein

Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein (SIRP) family. The amino-terminal immunoglobulin-like domain of SHPS-1 is necessary for interaction with CD47, a ligand for SHPS-1, which plays an important role in cell–cell interaction. The intracellular region of SHPS-1, on the other hand, may act as a scaffold protein, binding to various adapter proteins. Interestingly, increasing evidence has shown that SHPS-1 is involved in various biological phenomena, including suppression of anchorage-independent cell growth, negative regulation of immune cells, self-recognition of red blood cells, mediation of macrophage multinucleation, skeletal muscle differentiation, entrainment of circadian clock, neuronal survival and synaptogenesis. Recent progress has been made in attributing these novel exciting functions. Here we discuss how this interesting molecule works and consider its true role in biology.

Multinucleated giant cell appearance after whole body microwave irradiation of rats

Abstract Multinucleated giant cells are common for some chronic inflammatory processes in the lung. These cells are formed by fusion of macrophages, but how the process relates to the kinetics of alveolar macrophage generation is not clear. This study investigated the influence of 2450 MHz microwave irradiation on alveolar macrophage kinetics and formation of multinucleated giant cells after whole body irradiation of rats. The range of electromagnetic radiation was selected as 2450 MHz microwaves at a power density of 5 – 15mW/cm 2. A group of experimental animals was divided in four subgroups that received 2, 8, 13 and 22 irradiation treatments of two hours each. The animals were killed on experimental days 1, 8, 16, and 30. Free lung cell population was obtained by bronchoalveolar lavage. Cell response to the selected irradiation level was followed quantitatively, qualitatively and morphologically using standard laboratory methods. Total cell number retrieved by lavage slightly decreased in treated animals showing time- and dose-dependence. Cell viability did not significantly change in the irradiated animal group (G2) as compared with the control group (G1). Multinucleated cells significantly increased (p<0.01) in treated animals. The elevation of the number of nuclei per cell was time- and dose-dependent. Macrophages with two nucleoli were more common in animals treated twice or eight times. Polynucleation, that is three and more nucleoli in a single cell, was frequently observed after 13 or 22 treatments. Binucleation and multinucleation of alveolar macrophages were sensitive time- and dose-dependent morphological indicators of pulmonary stress.

CD47, a Ligand for the Macrophage Fusion Receptor, Participates in Macrophage Multinucleation

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.

Osteoclasts and giant cells: macrophage-macrophage fusion mechanism.

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.

CD44 occupancy prevents macrophage multinucleation.

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.