IntroductionPsoriatic arthritis (PsA) is a chronic inflammatory arthritis characterized by bone erosion mediated by osteoclasts (OC). Our previous studies showed an elevated frequency of OC precursors (OCP) in PsA patients. Here, we examined if OC arise from CD16-positive monocytes in PsA.MethodsPeripheral blood mononuclear cells (PBMC) or monocytes were isolated from human peripheral blood and sorted based on CD16 expression. Sorted cells were cultured alone or with bone wafers in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Enumeration and bone erosion activity of OC were examined after culture. The effects of tumor necrosis factor-alpha (TNFα), OC-promoting (M-CSF plus RANKL), and dendritic cell (DC)-promoting (GM-CSF plus interleukin (IL)-4) cytokines on CD16 surface expression were examined by flow cytometry.ResultsPsA and psoriasis (Ps) subjects had a higher percentage of circulating inflammatory CD14+CD16+ cells than healthy controls (HC). Exposure of cells to OC-promoting, but not DC-promoting media, was associated with CD16 up-regulation. PBMC of Ps and PsA had a higher frequency of cells expressing intermediate levels of CD16. OC were mainly derived from CD16+ cells in PsA. Increased CD16 expression was associated with a higher bone erosion activity in PsA.ConclusionsAn increased frequency of circulating CD14+CD16+ cells was noted in PsA compared to controls, and intermediate levels of CD16 may suggest a transitional state of OCP during osteoclastogenesis. Intriguingly, TNFα blocked CD16 expression on a subset of CD14+ monocytes. Collectively, our data suggest that CD16 has the potential to serve as an OCP marker in inflammatory arthritis.
Osteoclasts (OC) are bone-resorbing, multinucleated cells that are generated via fusion of OC precursors (OCP). The frequency of OCP is elevated in patients with erosive inflammatory arthritis and metabolic bone diseases. Although many cytokines and cell surface receptors are known to participate in osteoclastogenesis, the molecular mechanisms underlying the regulation of this cellular transformation are poorly understood. Herein, we focused our studies on the dendritic cell-specific transmembrane protein (DC-STAMP), a seven-pass-transmembrane receptor-like protein known to be essential for cell-to-cell fusion during osteoclastogenesis. We identified an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail of DC-STAMP, and developed an anti-DC-STAMP monoclonal antibody 1A2 that detected DC-STAMP expression on human tumor giant cells, blocked OC formation in vitro, and distinguished four patterns of human PBMC with a positive correlation to OC potential. In freshly isolated monocytes, DC-STAMPhigh cells produced a higher number of OC in culture than DC-STAMPlow cells and the surface expression of DC-STAMP gradually declined during osteoclastogenesis. Importantly, we showed that DC-STAMP is phosphorylated on its tyrosine residues and physically interacts with SHP-1 and CD16, an SH2-domain-containing tyrosine phosphatase and an ITAM-associated protein, respectively. Taken together, these data show that DC-STAMP is a potential OCP biomarker in inflammatory arthritis. Moreover, in addition to its effect on cell fusion, DC-STAMP dynamically regulates cell signaling during osteoclastogenesis.
Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL-induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC-STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation-western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC-STAMP in non-denaturing and denaturing conditions respectively, with greater sensitivity vs. rabbit anti-sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100-fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC-STAMP lo and DC-STAMP hi cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC-STAMP hi cells to purified DC-STAMP lo cultures produced larger, more nucleated OC vs. pure DC-STAMP lo cultures. RT-qPCR analysis of these two populations showed that OC markers (Trap and Oc-stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC-STAMP lo vs. DC-STAMP hi cells. Collectively, these results demonstrate that DC-STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC-STAMP internalization in some OCP, and these DC-STAMP lo cells display the "master fusogen" phenotype. In contrast, DC-STAMP hi OCP can only act as mononuclear donors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.