The chicken immunoregulatory receptor families SIRP, TREM, and CMRF35/CD300L

Mammalian immunoregulatory families of genes encoding activating and inhibitory Ig-like receptor pairs have been located on distinct chromosomes. In chicken, a single Ig-like receptor family with many members had been described so far. By looking at sequence similarity and synteny conservations in the chicken genome, the signal-regulatory protein (SIRP), triggering receptor expressed on myeloid cells (TREM), and CMRF35/CD300L Ig-like gene families were identified on chromosomes 20, 26, and 3, respectively. Further analysis of the three corresponding genomic regions and partial bacterial artificial chromosome sequencing were used to identify more members and to realign several contigs. All putative genomic sequences were monitored by investigating existing expressed sequence tag and cloning cDNA. This approach yielded a single pair of activating and inhibitory SIRP, two inhibitory, and one activating TREM as well as one inhibitory CMRF35/CD300L with a potentially soluble variant and an additional member lacking categorizing motifs. The CMRF35/CD300L and TREM receptors were composed of one or two V-set Ig domains, whereas in SIRP, either a single Ig V domain was present or a combination of a V and C1 domains. Like in many Ig superfamily members, separate exons encode individual Ig domains. However, in two CMRF35/CD300L genes, the signal peptide and the distal Ig domain were encoded by a single exon. In conclusion, the mammalian diversity of immunoregulatory molecules is present the chicken suggesting an important role for TREM, SIRP, and CMRF35/CD300L in a functionally conserved network.     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

Expression of and role for ovarian cancer G-protein-coupled receptor 1 (OGR1) during osteoclastogenesis

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1(tl)/csf1(tl)) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.     

The role of Mac-1 (CD11b/CD18) in osteoclast differentiation induced by receptor activator of nuclear factor-kappaB ligand

Multinuclear osteoclasts are derived from CD11b-positive mononuclear cells in bone marrow and in circulation. FACS sorting experiments showed impaired osteoclastogenesis in RAW264.7 cells with low CD11b expression. Neutralizing antibodies and siRNA against CD11b inhibited osteoclastogenesis induced by RANKL. Although primary cultured mouse bone marrow macrophages expressed CD11a and CD11b, osteoclastogenesis induced by M-CSF and RANKL was inhibited in the presence of anti-CD11b or anti-CD18 but not anti-CD11a antibodies. Furthermore, anti-CD11b antibodies inhibited NFATc1 expression induced by M-CSF and RANKL in BMMs. These findings suggest, at least partly, an important role of CD11b in osteoclastogenesis.     

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.     

Crystal structure of human triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.47 A

The triggering receptor expressed on myeloid cells (TREM) family of single extracellular immunoglobulin receptors includes both activating and inhibitory isoforms whose ligands are unknown. TREM-1 activation amplifies the Toll-like receptor initiated responses to invading pathogens allowing the secretion of pro-inflammatory chemokines and cytokines. Hence, TREM-1 amplifies the inflammation induced by both bacteria and fungi, and thus represents a potential therapeutic target. We report the crystal structure of the human TREM-1 extracellular domain at 1.47 A resolution. The overall fold places it within the V-type immunoglobulin domain family and reveals close homology with Ig domains from antibodies, T-cell receptors and other activating receptors, such as NKp44. With the additional use of analytical ultracentrifugation and 1H NMR spectroscopy of both human and mouse TREM-1, we have conclusively demonstrated the monomeric state of this extracellular ectodomain in solution and, presumably, of the TREM family in general.     

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

Tetracycline antibiotics, including doxycycli\e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.     

Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.     

The CD200 receptor is a novel and potent regulator of murine and human mast cell function

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcepsilonRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcepsilonRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.     

Crystal structure of the extracellular domain of mouse RANK ligand at 2.2-A resolution.

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Receptor activator of NF-kappa B ligand (RANKL, also known as ODF and OPGL), a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with its receptor, RANK. We have determined the crystal structure of the extracellular domain of mouse RANKL at 2.2-A resolution. The structure reveals that the RANKL extracellular domain is trimeric, which was also shown by analytical ultracentrifugation, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, reveals conserved and specific features of RANKL in the TNF superfamily and suggests the presence of key residues of RANKL for receptor binding.     

Multimerization of the receptor activator of nuclear factor-kappaB ligand (RANKL) isoforms and regulation of osteoclastogenesis

The receptor activator of nuclear factor-kappaB ligand (RANKL), a member of the tumor necrosis factor family, is a transmembrane protein, which is known as an essential initiation factor of osteoclastogenesis. Previously, we identified three RANKL isoforms. RANKL1 was identical to the originally reported RANKL. RANKL2 had a shorter intracellular domain. RANKL3 did not have the intracellular or transmembrane domains and was suggested to act as a soluble form protein. Here, we show that RANKL forms homo- or heteromultimers. NIH3T3 cells transfected with RANKL1 or RANKL2 form mononuclear tartrate-resistant acid phosphatase-positive preosteoclasts in an in vitro osteoclastogenesis assay system. Coexpression of RANKL1 and RANKL2 induces multinucleated osteoclasts. RANKL3 has no effect on the formation of preosteoclasts or osteoclasts but significantly inhibits fusion of preosteoclasts when coexpressed with RANKL1 and RANKL2. These findings imply the presence of multiple multimeric structures of RANKL, which may regulate bone metabolism.     

DIgR1, a novel membrane receptor of the immunoglobulin gene superfamily, is preferentially expressed by antigen-presenting cells

A novel membrane receptor of immunoglobulin gene superfamily (IgSF) has been identified from mouse dendritic cells (DC) and designated as DC-derived Ig-like receptor 1 (DIgR1). It encodes a 228-amino-acid (aa) residue polypeptide with a 21-aa signal peptide, a 20-aa transmembrane region, a 189-aa extracellular region, and a 19 aa intracellular region. Its extracellular region contains a single V domain of Ig. So it is a novel type I transmembrane glycoprotein of IgSF. DIgR1 shows significant homologies to human CMRF-35 antigens and polymeric immunoglobulin receptors (pIgR). The mRNA expression of DIgR1 was highly abundant in mouse spleen. The preferential expression of DIgR1 mRNA is observed in the known antigen-presenting cells (APC) including DC, monocytes/macrophages, and B lymphocytes. A 40 kDa of protein in NIH/3T3 cells transfected with the DIgR1 cDNA was detected by Western blot analysis using anti-DIgR1 polyclonal antibodies. The expression of DIgR1 protein on DC is not regulated by LPS stimulation. Further study should be conducted to investigate what were biological functions of DIgR1 in the immunobiology of APC.     

Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-kappaB activation. Curcumin inhibited the pathway leading from activation of IkappaBalpha kinase and IkappaBalpha phosphorylation to IkappaBalpha degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-kappaB was also confirmed independently, as RANKL failed to activate NF-kappaB in cells stably transfected with a dominant-negative form of IkappaBalpha and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-kappaB, and treatment with curcumin inhibits both the NF-kappaB activation and osteoclastogenesis induced by RANKL.     

Silibinin inhibits osteoclast differentiation mediated by TNF family members

Silibinin is a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), with known hepatoprotective, anticarcinogenic, and antioxidant effects. Herein, we show that silibinin inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 cells as well as from bone marrow-derived monocyte/macrophage cells in a dose-dependent manner. Silibinin has no effect on the expression of RANKL or the soluble RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts. However, we demonstrate that silibinin can block the activation of NF-κB, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK) in osteoclast precursors in response to RANKL. Furthermore, silibinin attenuates the induction of nuclear factor of activated T cells (NFAT) c1 and osteoclast-associated receptor (OSCAR) expression during RANKL-induced osteoclastogenesis. We demonstrate that silibinin can inhibit TNF-α-induced osteoclastogenesis as well as the expression of NFATc1 and OSCAR. Taken together, our results indicate that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL and TNF-α.     

RANKL-stimulated TNFalpha production in osteoclast precursor cells promotes osteoclastogenesis by modulating RANK signaling pathways

Although TNFalpha is known to be an important factor for bone resorption, particularly in inflammatory bone diseases, the relevance between RANKL and TNFalpha in osteoclastogenesis remains unclear. In this study we examined the mechanism of TNFalpha induced osteoclastogenesis and its downstream signaling. We show that osteoclastogenesis is suppressed by anti-TNFalpha- and anti-TNF receptor type I (TNFRI)-antibodies and in TNFalpha- and TNFRI-deficient mice using in vitro culture systems: (1) co-culture of mouse spleen derived osteoclast precursor cells (pOCs) with osteoblasts, (2) pure pOC culture and (3) RAW264.7 cells in presence of RANKL. Furthermore, TNFalpha production in pOCs was stimulated by RANKL. Endogenous TNFalpha in pOCs induced c-Fos and NFATc1. Expression rates of NFATc1 and c-Fos were significantly decreased in TNFalpha- and TNFRI-deficient pOCs during osteoclastogenesis. These results indicate that TNFalpha is induced by RANKL in pOCs and serves as an autocrine factor promoting osteoclastogenesis through c-Fos and NFATc1 activation.